Design and synthesis of 1H-pyrazolo[3,4-b]pyridines targeting mitogen-activated protein kinase kinase 4 (MKK4) - A promising target for liver regeneration

Article abstract of DOI:10.1016/j.ejmech.2021.113371

Currently, the therapeutic options for treatment of liver failure are very limited. As mitogen-activated protein kinase kinase 4 (MKK4) has recently been identified by in vivo RNAi experiments to be a major regulator in hepatocyte regeneration, we pursued the development of a small molecule targeting this protein kinase. Starting from the approved BRAF^V600E inhibitor vemurafenib (8), that showed a high off-target affinity to MKK4 in an initial screening, we followed a scaffold-hopping approach, changing the core heterocycle from 1H-pyrrolo[2,3-b]pyridine to 1H-pyrazolo[2,3-b]pyridine (10). Affinity to MKK4 could be conserved while the selectivity against off-target protein kinases was slightly improved. Further modifications led to 58 and 59 showing high affinity to MKK4 in the low nanomolar range and excellent selectivity profile from mandatory multiparameter-optimization for the essential anti-targets (MKK7, JNK1) and off-targets (BRAF, MAP4K5, ZAK) in the MKK4 pathway. Herein we report the first selective MKK4 inhibitors in this class.

Full text of DOI:10.1016/j.ejmech.2021.113371

European Journal of Medicinal Chemistry 218 (2021) 113371
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design and synthesis of 1H-pyrazolo[3,4-b]pyridines targeting
mitogen-activated protein kinase kinase 4 (MKK4) - A promising
target for liver regeneration
a
a
a
b
€
Bent Pfaffenrot , Philip Klovekorn , Michael Juchum , Roland Selig ,
Wolfgang Albrecht b, Lars Zender c e, Stefan A. Laufer a
Department of Pharmaceutical/Medicinal Chemistry an Tuebingen Center for Academic Drug Discovery (TüCAD2), Eberhard Karls Universitat, Auf der
,
d
,
, d, e, *
a
€
Morgenstelle 8, 72076, Tübingen, DE, Germany
b HepaRegenix GmbH, Eisenbahnstraße 63, 72072, Tübingen, DE, Germany
c Department of Medical Oncology and Pneumology (Internal Medicine VIII), University Hospital Tuebingen, 72076, Tuebingen, DE, Germany
d Cluster of Excellence ‘Image Guided and Functionally Instructed Tumor Therapies’ (iFIT), Eberhard Karls University of Tübingen, 72076, Tübingen,
Germany
e German Consortium for Translational Cancer Research (DKTK), Partner Site Tübingen, German Cancer Research Center (DKFZ), 69120, Heidelberg,
Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Currently, the therapeutic options for treatment of liver failure are very limited. As mitogen-activated
protein kinase kinase 4 (MKK4) has recently been identified by in vivo RNAi experiments to be a ma-
jor regulator in hepatocyte regeneration, we pursued the development of a small molecule targeting this
protein kinase. Starting from the approved BRAFV600E inhibitor vemurafenib (8), that showed a high off-
target affinity to MKK4 in an initial screening, we followed a scaffold-hopping approach, changing the
core heterocycle from 1H-pyrrolo[2,3-b]pyridine to 1H-pyrazolo[2,3-b]pyridine (10). Affinity to MKK4
could be conserved while the selectivity against off-target protein kinases was slightly improved. Further
modifications led to 58 and 59 showing high affinity to MKK4 in the low nanomolar range and excellent
selectivity profile from mandatory multiparameter-optimization for the essential anti-targets (MKK7,
JNK1) and off-targets (BRAF, MAP4K5, ZAK) in the MKK4 pathway. Herein we report the first selective
MKK4 inhibitors in this class.
Received 22 October 2020
Received in revised form
23 February 2021
Accepted 4 March 2021
Available online 17 March 2021
Keywords:
MKK4
Kinase inhibition
NAFLD
Liver failure
Scaffold hopping
1H-pyrazolo[2,3-b]pyridine
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
kinase kinase 4 (MKK4) to play a pivotal role in liver regeneration
using RNAi experiments. Silencing MKK4 expression with short-
About 1 million deaths per year are associated with chronic or
acute liver failure with main causes being viral infections (hepatitis
B/C), non-alcoholic fatty liver disease (NAFLD) or metabolic syn-
drome. Since there is no medical treatment for liver failure
currently available, the identification of a molecular target is being
pursued. Wuestefeld et al. discovered mitogen-activated protein
hairpin RNA (shRNA) resulted in an increase in robustness and
regenerative potential of the liver via amplified hepatocyte prolif-
eration. MKK4 silencing is expected to lead to a higher activation of
mitogen-activated protein kinase kinase 7 (MKK7) and thus to a
higher phosphorylation and activation of the downstream c-Jun-N-
terminal protein kinase 1 (JNK1). Therefore, phosphorylation of ETS
transcription factor (ELK1) and activating transcription factor 2
(ATF2) is increased, which appears to be the reason for the elevated
hepatocyte proliferation. Because of their opposite role, MKK7 and
JNK1 are considered as anti-targets in the development of small
molecule MKK4 inhibitors (see Fig. 1) [1,2].
Abbreviations: MKK4/7, mitogen-activated protein kinase kinase 4/7; BRAF, v-
Raf murine sarcoma viral oncogene homolog B1; JNK1, c-Jun-N-terminal protein
kinase 1; MAP4K5, MEK kinase kinase 5; ZAK, sterile alpha motif and leucine zipper
containing kinase.
* Corresponding author. Department of Pharmaceutical/Medicinal Chemistry an
Tuebingen Center for Academic Drug Discovery (TüCAD2), Eberhard Karls Uni-
To date, only few MKK4 inhibitors have been published (Fig. 2).
In 2004 Bayer described 4-phenyl-pyrimido[4,5-b]indole (1) with
€
versitat, Auf der Morgenstelle 8, 72076, Tübingen, DE, Germany.
IC50 values ꢀ 1
mM for both MKK4 and MKK7. In 2013 Krishna et al.
E-mail address: stefan.laufer@uni-tuebingen.de (S.A. Laufer).
https://doi.org/10.1016/j.ejmech.2021.113371
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

€
B. Pfaffenrot, P. Klovekorn, M. Juchum et al.
European Journal of Medicinal Chemistry 218 (2021) 113371
small molecule MKK4 inhibitor derived from the approved v-Raf
murine sarcoma viral oncogene homolog B1 (BRAFV600E) kinase
inhibitor vemurafenib (8) by variation of the core heterocycle
(hinge-binding scaffold) and further optimizations. We used the
commercially available KINOMEscan technology by DiscoverX,
Fremont CA to determine binding affinities (expressed as percent of
control (POC) values). A POC of 100 refers to no binding of the
compound to the kinase, whereas 0 refers to complete binding to
the kinase [7]. Besides BRAFwt, we also chose MEK kinase kinase 5
(MAP4K5) and sterile alpha motif and leucine zipper containing
kinase (ZAK) as off targets, as these kinases are assumed to reduce
JNK activity via suppression of the MKK4/7 pathway if inhibited [8].
2. Results and discussion
Biological evaluation. In our first approach, we compared
binding affinities of different hinge-binding scaffold derived from
1H-pyrrolo[2,3-b]pyridine (8) to 5H-pyrrolo[2,3-b]pyrazin 9 and
1H-pyrazolo[2,3-b]pyridin 10 (Table 1). Pyrrolopyrazin 9 showed
almost no affinity towards MKK4 at a concentration of 100 nM,
while pyrazolopyridine 10 had similar affinity to MKK4 and a
slightly lower affinity to BRAF when related with 8. Also, almost no
binding of JNK1 and MKK7 was observed for 10. Consequently, we
chose 10 as a new compound class for the further development.
First, we designed a set of compounds - based on the pyr-
azolopyridine as hinge-binding scaffold - that differed in the sub-
stitution pattern of the aromatic residue in the 5-position. For the
substitution of the para, ortho and meta position, wo chose chloro-
(10, 12, 13), fluoro- (14e16), methyl- (17e19), methoxy- (20e22),
hydroxy- (23e25) and amino (26e28) residues. We also included
dimethylamino- (29, 30), trifluoromethyl- (40, 41) and nitrogroups
(42, 43) for the meta and para position. Additionally, we selected
bulky and multiple-substituted (31e39) as well as sulfone- and
sulfamoyl- (44, 45) substituted residues to generate a first profile.
For evaluation of the binding data, the compounds were grouped
based on the residues’ properties.
Regarding MKK4, ortho and para substitution of the phenyl
residue with methoxy and nonpolar residues (fluorine (14, 16),
chlorine (8, 12) and methyl (17, 19)) lead to a higher affinity when
compared to meta substitution regardless of the substituent
(Table 2). The phenyl derivative 11 showed similar affinity to MKK4
like ortho and para substituted compounds. The corresponding
chloro derivatives 8, 12 and 13 showed lower affinity in the binding
assays. From these findings, effects of methoxy/nonpolar substitu-
tion of the phenyl ring on MKK4 affinity can be ordered from
Fig. 1. Illustration of the MKK4 silencing pathway modified from Wuestefeld et al. [1].
Genetic silencing of MKK4 leads to an increased activity of MKK7 via increased in-
duction of MKK7 and apoptosis signal-regulating kinase 1 (ASK1). This results in an
amplification of the MKK7 pathway, a higher activity of JNK1 and a higher phos-
phorylation of ELK1 and ATF2.
reported trihydroxyisoflavones (2, exemplary) as inhibitors of
MKK4, but no selectivity was given. In 2014 the protoberberin de-
rivative HWY336 (3) was published by Kim et al. with low micro-
molar IC50 values for both MKK4 and MKK7. Deibler et al. modified
published kinase inhibitors AST-487 (6), PLX4720 (5) and pazopa-
nib (7) for the development of MKK4 inhibitors generating de-
rivatives with IC50 values in the low micromolar range against
MKK4. Most recently (2019) Deibler et al. reported 3-arylindazoles
(4, exemplary) as selective MKK4 inhibitors having two- to three-
digit IC50 values in the nanomolar range [3e6].
In this study, we describe the development of a highly affine
Fig. 2. Recently published MKK4 inhibitors according to references.
2

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B. Pfaffenrot, P. Klovekorn, M. Juchum et al.
European Journal of Medicinal Chemistry 218 (2021) 113371
Table 1
Comparison of different hinge-binding scaffold derived from 8.
No.
X ¼
Y ¼
BRAFa
JNK1a
MAP4K5a
MKK4a
MKK7a
ZAKa
8
9
10
CH
N
CH
CH
CH
N
16
91
25
n.d.
96
89
48
100
32
14
90
12
100
100
100
n.d.
100
29
a
Binding affinities from KINOMEscan assay at a compound concentration of 100 nM. Values are shown as POC determined in duplicate.
ortho ꢁ para > meta. Selectivity over BRAF and ZAK was highest for
ortho-substituted compounds having selectivity factors ranging
from 7 to 70 for BRAF and 6e18 for ZAK at 100 nM, whereas fluoro,
methyl and methoxy substitution in the para position increased
affinity to both kinases. The affinity to JNK1 and MAP4K5 was low
in case of compounds with higher affinity to MKK4. MKK7 had not
been addressed in this series.
Compounds bearing polar hydroxy and amino residues in the
meta (24, 27) or para (25, 28) position showed high affinity to MKK4
with POC values in the range of 0.1e2.2 at 100 nM (Table 3), with
para substitutions having the highest impact on increased affinity.
For the less polar dimethylamino compounds 29 and 30 the affinity
to MKK4 was lower (POC 26 and 2.5 at 100 nM) when compared to
the unsubstituted amines. Besides high affinity to MKK4, the polar
meta- and para-substituted compounds showed low selectivity
over BRAF and ZAK (POC values in the range of 1.3e11 for BRAF and
0e9.7 for ZAK at 100 nM), while selectivity over MKK7 and JNK1
was high, except for 28 having a POC of 28 at 100 nM. In summary,
the compounds with polar substituents (Table 3) with compounds
25, 27 and 28 as most potent structures showed high affinities for
MKK4 but were less selective.
For compounds having bulky or multiple substituents (see
Table 4) a drop in affinity to all tested kinases was observed,
especially for the chloro compounds 31e33 when compared to the
mono-substituted compounds (see Table 2). Addition of another
methyl group (34) or increasing the size of the alkyl substituent to
iso-propyl (35) or tert-butyl (36) led to a decrease in affinity to all
tested kinases. Although the overall affinity of 35 is reduced
compared to the p-methyl compound 19, 35 showed a similar af-
finity towards MKK4 as 8 but with an improved selectivity profile.
The substitution pattern given from a combination of compounds
16 and 17 lead to compound 38 which showed a good profile but a
lower affinity to MKK4 than both parent molecules. 39 on the other
hand (a combination of 22 and 12), showed a similar affinity to-
wards MKK4 as 22 with an excellent selectivity profile comparable
with 12 and is the most potent compound within this set.
For compounds having electron withdrawing groups, no clear
trend could be observed (Table 5). The trifluoromethyl compounds
40 and 41 showed low affinity to the tested kinases. The 4-nitro
compound 43 exhibited a higher affinity to MKK4 by factor 2
than the corresponding 3-nitro compound 42 (POC value of 4.2 vs.
15 at 100 nM). Compounds 44 and 45 with sulfur-based residues
had very low POC values of 1.5 and 0.25 at 100 nM for MKK4, but
also high affinity to BRAF with POC values in the range of
0.45e0.9 at 100 nM. Compound 28 and 45 showed the lowest POC
values for MKK4 at 100 nM, a high selectivity over MAP4K5 and
MKK7, and a moderate selectivity over JNK1. Moreover, 45 was the
first compound to show a very high affinity to MKK4 (POC 0.25 at
100 nM) with superior ZAK selectivity (POC 91 at 100 nM). As a
result, we chose 45 as a new starting point for the further devel-
opment of selective MKK4 inhibitors.
For 28 and 45 POC values for MKK4 were determined again at a
lower concentration of 10 nM for better comprehension. It was
observed that both compounds were still binding MKK4 with a
high affinity reflected in POC values in the range of 3.7e5.1 at 10 nM
(see Table 6).
Modifications of the sulfamoylphenyl residue. To further
investigate the influence of the sulfaomyl residue, we chose to vary
the amino function by N-alkylation (46, 47, loss of a hydrogen bond
donor) and N-acylation (48, loss of hydrogen bond donor and in-
crease of acidity). We also inverted the sulfamoyl group (49) and
employed other more acidic residues like tetrazol (50) and car-
boxylic acid (51), as well as the neutral amide (52) (see Table 7).
From the previous testing, we observed that a loss of the amino
function (44, Table 5) resulted in reduced affinity to MKK4 and
selectivity for MAP4K5 and ZAK. The N-alkyl compounds 46 and 47
gave similar results, while the affinity to MKK4 was reduced with
increasing chain lengths. The results for the “inverted sulfonamide”
(49) were comparable to the N-alkylated compounds, but with an
increased affinity towards MAP4K5 and ZAK. The acidic N-acetyl
compound 48 had a higher affinity to MKK4 when compared to 45
with a POC value of 0.5 at 10 nM, with a very high affinity to BRAF
(POC value of 0 at 100 nM) and a slightly higher affinity to ZAK. The
data for the tetrazole 50 and the carboxylic acid compound 51 is in
line with the data generated from the acidic N-acetylated sulfon-
amide 48 having a high affinity to MKK4 (POC values from 0.2 to
1.1 at 100 nM). These acidic compounds also had low POC values for
BRAF (1.2e1.4 at 100 nM) and a moderate affinity to ZAK (POC
7.5e20 at 100 nM). Also, significant binding to MKK7 at 100 nM was
observed for compound 51. The amide 52 had a profile comparable
to the acid 51 but with a higher affinity to MAP4K5. From these
data, we decided to pursue the further development of a selective
MKK4 inhibitor based on compound 45 as 45 showed a better
selectivity profile with high binding to MKK4 and extraordinary
ZAK selectivity. In the next step we took the information from
Table 2 that ortho substitution of the phenyl residue with nonpolar
substituents leads to a reduced binding to BRAF generating the
equivalent sulfamoyl derivatives as depicted in Table 8.
As expected, ortho substitution of 45 (53e55, Table 8) leads to a
decrease in BRAF affinity (POC values from 2.2 to 21 vs. 0.9 (45) at
100 nM). The affinity to MKK4 was also decreased, with POC values
ranging from 1 to 3.4 vs. 0.25 (45) at 100 nM, while maintaining
high selectivity over MKK7 and ZAK. Also, the selectivity for JNK1
was improved for all compounds. Due to the decreased binding
affinity to MKK4 by ortho-substitution, we used another approach
to increase the selectivity of 45.
3

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B. Pfaffenrot, P. Klovekorn, M. Juchum et al.
European Journal of Medicinal Chemistry 218 (2021) 113371
Table 2
Binding affinities for single nonpolar and hydrogen accepting substituted derivatives.
No.
R ¼
BRAFa
29
JNK1a
70
MAP4K5a
24
MKK4a
MKK7a
100
ZAKa
1.9
11
2.4
12
13
70
72
100
90
96
69
9.3
40
100
100
64
16
10
25
89
32
12
100
29
14
15
40
97
85
59
65
2
83
97
32
12
4.9
8.2
16
17
18
28
65
38
83
90
90
59
67
18
2.1
7
100
100
100
4.8
41
15
5.5
19
20
16
70
73
54
25
34
5.4
1.9
100
100
5.9
36
21
23
73
69
24
28
6.3
4.2
100
100
23
22
5.9
1.9
a
Binding affinities from KINOMEscan assay at a compound concentration of 100 nM. Values are shown as POC determined in duplicate.
4

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B. Pfaffenrot, P. Klovekorn, M. Juchum et al.
European Journal of Medicinal Chemistry 218 (2021) 113371
Table 3
Binding affinities for single polar and hydrogen accepting/donating and weakly basic substituted derivatives.
No.
R ¼
BRAFa
71
JNK1a
79
MAP4K5a
39
MKK4a
MKK7a
100
ZAKa
9.7
23
9
24
25
11
63
65
8
2.2
100
87
0.95
0
8.7
9.4
0.75
26
27
99
80
65
73
23
95
86
40
3.5
3.8
1.6
2.3
28
29
1.3
22
28
86
1.3
11
0.1
26
93
2.7
9.7
100
30
1.9
100
31
2.5
100
2.2
a
Binding affinities from KINOMEscan assay at a compound concentration of 100 nM. Values are shown as POC determined in duplicate.
Variation of the sulfonamide sidechain. The n-propane-
sulfonamide sidechain in 8 is known to be optimized to fit the raf-
selectivity-pocket which is unique to the Raf kinase family [9]. We
tried to decrease the BRAF binding of 45 by varying the length and
bulkiness of the sulfonamide sidechain. In a focused library, we
chose methyl, n-butyl and benzyl residues to investigate the effects
on BRAF affinity. The results are given in Table 9. Regarding BRAF
affinity, the longer n-butyl sidechain (57) yielded only a slight
reduction in affinity to BRAF, while shortening the sidechain to
methyl (56) led to a higher reduction in affinity (POC values 2.1 and
16 at 100 nM). Also, selectivity over JNK1 was slightly increased for
56 and 57 compared with 45. With benzyl (58) we achieved an
excellent selectivity profile showing POC values of 76 for BRAF and
0.4 for MKK4 at 100 nM and 10 at 10 nM. The selectivity over JNK1
could be further increased when compared to 45, 56 and 57.
Moreover, affinities to MKK7 and ZAK were not detectable at
100 nM.
values of 0.1 at 100 nM and 11 at 10 nM (see Table 10.). Only the
affinities for ZAK and MAP4K5 were still equally high compared to
the parent compound.
For a further compound characterization, we determined Kd-
values for 58 and 59 (see. Table 11). The Kd-values are in line
with the POC values given in Table 10. Both compounds exhibit a
one-digit nanomolar Kd-value for MKK4. For 59 the selectivity over
the anti-targets JNK1 and MKK7 is relatively high for JNK1 with two
orders of magnitude and excellent for MKK7 with a factor of >3000.
Nevertheless, the selectivity for MAP4K5 and ZAK is low, expressed
by selectivity factors of only around 5 and 9, respectively. The
remarkable selectivity of 58 over the off- and anti-targets is rep-
resented by the factors of >100, >250, >600 and almost 3000
against JNK1, BRAF, MAP4K5, MKK7 and ZAK respectively. 58 was
also screened against a panel of 97 kinases using scanEDGE assay at
a concentration of 1 mM, which is over 500 times higher than the
Kd-value for MKK4. At this concentration, only 2 kinases (AURKB
and SNARK) showed a POC-value <35%, resulting in a selectivity
factor of S(35) ¼ 0.022 (see supporting information for details).
To determine the inhibitory activity, we performed 33PanQinase
assay [10] with the highly selective and most promising compound
58, the n-propyl parent compound 45 as well as with compound 48,
The benzylsulfonamide moiety was then applied to the high
affinity compound 28 from the first set of compounds (vide supra,
Tables 3 and 6). The combination compound 59 showed a drasti-
cally improved selectivity profile when compared to 28, especially
for BRAF and MAP4K5, retaining a high affinity for MKK4 with POC
5

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B. Pfaffenrot, P. Klovekorn, M. Juchum et al.
European Journal of Medicinal Chemistry 218 (2021) 113371
Table 4
Binding affinities for bulky or multiple-substituted derivatives.
No.
R ¼
BRAFa
82
JNK1a
100
MAP4K5a
100
MKK4a
MKK7a
96
ZAKa
99
31
22
32
99
94
99
73
100
66
33
34
35
57
21
65
100
84
100
20
45
20
17
100
100
98
85
24
92
78
89
36
37
92
18
73
78
97
16
46
15
100
100
77
20
38
39
97
73
98
92
55
53
14
100
100
65
74
5.3
a
Binding affinities from KINOMEscan assay at a compound concentration of 100 nM. Values are shown as POC determined in duplicate.
one the compounds with the highest affinity towards MKK4
(Table 12). Consistent with the determination of POC values, a
decrease in potency from an IC50 of 89 nMe146 nM for MKK4 was
observed when the sulfonamide side chain was varied from n-
propyl (45) to the selectivity driving benzyl residue (58). Com-
pound 48 with a POC value of 0.5 at 10 nM (10-fold lower than 45)
exhibited a low two-digit nanomolar IC50 value of 29 nM for MKK4.
Due to the different nature of the assays, the results cannot be
compared quantitively, but their outcomes are qualitatively in line.
CHEMISTRY. Compound 9 was prepared according to the pro-
cedures described in the literature [11]. Although the preparation of
a precursor of the pyrazolopyridine compounds was partially
described by Ibrahim et al. [12], the published route was found to
be unreliable and unreproducible. A new synthetic route for the
preparation of the pyrazolopyridine compounds was established.
aReagents and conditions: (a) I2, KOH, DMF, RT (92%); (b) 1.)
CO(g), Xantphos, Pd(OAc)2, Et3N, MeOH, DMF, 60 ꢂC, 2.) NaOH, H2O,
95 ꢂC e RT (quant.); (c) CDI, N,O-dimethylhydroxylamine hydro-
chloride, 60 ꢂC (85%); (d) 1.) 2,4-difluoroaniline, n-BuLi, TMS-Cl,
NaH, ꢃ78 ꢂC to ꢃ15 ꢂC, 2.) conc. HCl, RT (70%); (e) appropriate
sulfonyl chloride, 4-DMAP, pyridine, 65 ꢂC (38e65%); (f) appro-
priate boronic acid or pinacol boronate, Pd(PPh3)4, XPhos Pd G3/G4
or P(t-Bu)3 Pd G3, K2CO3 or K3PO4, 1,4-dioxane, H2O,
mw,
100e130 ꢂC (24e87%); (g) DHP, p-TsOH, DCM, reflux (75%); (h)
B2Pin2, AcOK, Pd(dppf)Cl2, 1,4-dioxane, 85 ꢂC (91%); (i) 1.) appro-
priate aryl bromide, Pd(PPh3)4 or XPhos Pd G3, K2CO3, 1,4-dioxane,
H2O, 55 ꢂC, 2.) HCl, i-PrOH, 70 ꢂC (31e58%, two steps).
Scheme 1 depicts the synthesis used in the preparation of the
testing compounds. In the first step, the 3-iodo compound 61 was
generated by iodination of 60 with elemental iodine and potassium
hydroxide in N,N-dimethylformamide following a modified litera-
ture procedure [13]. The conversion to the corresponding carbox-
ylic acid (62) was performed via palladium-catalyzed carbonylation
using carbon monoxide gas and Xantphos as ligand [14], followed
by hydrolysis of the intermediate methyl ester. In the next step, the
Weinreb-amide 63 was generated by activation of the carboxylic
acid using carbonyldiimidazol (CDI) and subsequent reaction with
N,O-dimethylhydroxylamine hydrochloride [15]. For the ketone
synthesis, the amino function of 2,4-difluoroaniline was masked
with trimethylsilyl (TMS) and the aromatic ring was lithiated with
6

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B. Pfaffenrot, P. Klovekorn, M. Juchum et al.
European Journal of Medicinal Chemistry 218 (2021) 113371
Table 5
Binding affinities for derivatives substituted with electron withdrawing groups.
No.
R ¼
BRAFa
39
JNK1a
100
MAP4K5a
98
MKK4a
MKK7a
100
ZAKa
44
40
57
41
42
68
29
85
77
76
93
29
15
100
95
75
52
43
44
25
79
63
61
14
4.2
1.5
100
100
23
21
0.45
45
0.9
32
100
0.25
98
91
a
Binding affinities from KINOMEscan assay at a compound concentration of 100 nM. Values are shown as POC determined in duplicate.
Table 6
Comparison of binding affinities of 28 and 45 on MKK4 at different concentrations.
No.
28
R ¼
MKK4a [100 nM]
0.1
MKK4a [10 nM]
3.7
45
0.25
5.1
a
Binding affinities from KINOMEscan assay. Values are shown as POC determined in duplicate.
n-butyllithium (n-BuLi) to generate the nucleophile in a modified
literature procedure [16]. 62 was converted to the corresponding
sodium salt using sodium hydride. Reaction of both components
and acidic deprotection of the TMS groups yielded the ketone 64.
The conversion to the corresponding sulfonamides 65a e d was
carried out by heating 64 in pyridine with the appropriate sulfonyl
chloride and catalytic amounts of 4-dimethylaminopyridine (4-
DMAP). For most of the final compounds the Suzuki coupling was
performed as the last step under microwave irradiation at
100e120 ꢂC, while 54 and 55 were prepared from 67 and corre-
sponding aryl bromides. For the synthesis of 67, tetrahydropyranyl
(THP) protection of the core heterocycle of 65b was performed in
dichloromethane catalyzed with para-toluenesulfonic acid yielding
66 which was converted to the borylated species 67 under classical
Suzuki-Miyaura-borylation conditions [17]. Preparations of aryl
bromide and boronic acids/pinacol ester that were not commer-
cially available are described in the supporting information.
3. Conclusion
In this work we reported the development of novel compounds
selectively addressing MKK4, derived from the approved BRAFV600E
inhibitor 8, by variation of the core heterocycle from 1H-pyrrolo
[2,3-b]pyridine to 1H-pyrazolo[2,3-b]pyridine and further iterative
modifications. With compound 58 we were able to dramatically
shift selectivity from BRAF to MKK4 with a high affinity for MKK4 in
the low one-digit nanomolar range and high kinome selectivity. We
developed distinct structure-activity relationships (SARs) in the
multiparameter optimization process. The crucial off- and anti-
targets in the MKK4 inhibition pathway that might limit the
regeneration of hepatocytes were not addressed either. To the best
of our knowledge, this work is the first report of 1H-pyrazolo[2,3-b]
pyridine-based MKK4 inhibitors having such a selectivity profile.
4. Experimental section
General. All commercially available reagents and solvents were
7

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B. Pfaffenrot, P. Klovekorn, M. Juchum et al.
European Journal of Medicinal Chemistry 218 (2021) 113371
Table 7
Derivatives of 45 with different hydrogen bond/acceptor properties and pKa values.
No.
46
R ¼
BRAFa [100 nM]
JNK1a [100 nM]
MAP4K5a [100 nM]
MKK4a [100 nM]
1.2
MKK4a [10 nM]
14
MKK7a [100 nM]
ZAKa [100 nM]
0.5
74
17
100
33
47
48
49
50
0.4
0
59
47
59
38
26
67
1.1
81
3
41
100
71
32
38
4.3
20
0.35
0.85
0.15
0.5
16
8.2
1.4
100
64
1.1
51
52
1.2
28
48
75
10
0.1
0.2
4
27
99
7.5
8.1
0.65
0.15
a
Binding affinities from KINOMEscan assay. Values are shown as POC determined in duplicate.
Table 8
Ortho substitution of the sulfamoylphenyl residue.
No.
R ¼
BRAFa [100 nM]
2.2
JNK1a [100 nM]
100
MAP4K5a [100 nM]
77
MKK4a [100 nM]
MKK4a [10 nM]
MKK7a [100 nM]
100
ZAKa [100 nM]
100
53
1
9.4
54
55
16
21
100
100
n.d.
n.d.
3.1
3.4
24
23
100
100
94
100
a
Binding affinities from KINOMEscan assay. Values are shown as POC determined in duplicate.
used as received. Reactions sensitive to air or moisture were per-
formed under an atmosphere of argon and/or in anhydrous sol-
vents. Anhydrous solvents were purchased from Acros Organics
(AcroSeal). Unless stated otherwise, extracts were dried over so-
dium sulfate prior to filtration. Thin layer chromatography was
performed on TLC Silica Gel 60 F254 aluminum sheets provided by
Phenomenex Luna C8 150 ꢄ 4.6 mm, 5-
m
m column with gradient
elution (MeOH/0.01 M KH2PO4 buffer, pH 2.3, flow rate 1.5 mL/min)
and detection at
¼ 230 and 254 nm. All final compounds were
l
determined with >95% purity if not stated otherwise. Mass spectra
were recorded on Advion DCMS interface (ESI voltage: 3.50 kV,
capillary voltage: 187 V, source voltage: 44 V, capillary tempera-
ture: 250 ꢂC, desolvation gas temperature: 250 ꢂC, gas flowrate: 5L/
min N2), elution of the spots with MeOH. High resolution mass
spectra (ESI) of the final compounds were obtained from the Mass
Spectrometry Department, Eberhard Karls Universitaet Tuebingen.
NMR-spectra were measured on Bruker Avance 200 or 400 NMR
Merck, detection at
was carried out on Interchim PuriFlash XS420 flash-
chromatography system and Grace Davison Davisil LC60A 20e45
l
¼ 254 nm und 366 nm. Flash chromatography
m
m silica. Purity of the compounds was determined by HPLC
analysis on Agilent 1100 Series Liquid Chromatograph using a
8

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B. Pfaffenrot, P. Klovekorn, M. Juchum et al.
European Journal of Medicinal Chemistry 218 (2021) 113371
Table 9
Variations of the sulfonamide sidechain derived from 45.
No.
R ¼
BRAFa [100 nM]
16
JNK1a [100 nM]
42
MAP4K5a [100 nM]
99
MKK4a [100 nM]
MKK4a [10 nM]
MKK7a [100 nM]
99
ZAKa [100 nM]
100
56
0.6
12
57
58
2.1
76
58
86
100
96
0.25
0.4
6.9
10
100
99
100
100
a
Binding affinities from KINOMEscan assay. Values are shown as POC determined in duplicate.
spectrometers. The spectra were calibrated on the deuterated sol-
vents and chemical shifts ( ) are stated relative to tetramethylsilane
After a short induction period the exothermic reaction began. After
1 h about 1 g of iodine was added and the mixture stirred at 45 ꢂC
for 1 h. The mixture was poured into a dilute solution of Na2SO3 and
then acidified with 2N HCl. The solids were collected by suction
filtration, washed with water and dried in an oven at 110 ꢂC. Yield:
d
in ppm. Prepared intermediate reagents that are listed in the sup-
porting information are named R with consecutive numbering.
5-Bromo-3-iodo-1H-pyrazolo[3,4-b]pyridine (61). To a stirred
mixture of 5-bromo-1H-pyrazolo[3,4-b]pyridine (6.81 g,
34.4 mmol) and KOH (6.75 g, 120.4 mmol) in DMF (45 mL) iodine
was added (9.60 g, 37.8 mmol) in one portion at room temperature.
10.92 g (92%). 1H NMR (200 MHz, DMSO)
d
14.29 (s,1H), 8.62 (s,1H),
150.53, 150.17, 131.86,
8.17 (s, 1H); 13C NMR (50 MHz, DMSO)
d
120.58, 112.43, 91.95; [M-H]- ¼ 322.0/324.0.
Table 10
Comparison of benzylsulfonamide compounds 58 and 59.
No.
R ¼
BRAFa [100 nM]
76
JNK1a [100 nM]
86
MAP4K5a [100 nM]
96
MKK4a [100 nM]
MKK4a [10 nM]
MKK7a [100 nM]
ZAKa [100 nM]
100
58
0.4
10
99
59
75
49
1.4
0.1
11
100
1.8
a
Binding affinities from KINOMEscan assay. Values are shown as POC determined in duplicate.
Table 11
Determination of Kd-values.
No.
R ¼
BRAFa
450
JNK1a
210
MAP4K5a
1100
MKK4a
MKK7a
5000
ZAKa
5000
58
1.7
59
610
140
5.8
1.5
5000
9.5
a
Kd-values (nM) determined with KINOMEscan assay in duplicates.
9

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B. Pfaffenrot, P. Klovekorn, M. Juchum et al.
European Journal of Medicinal Chemistry 218 (2021) 113371
Table 12
Determination of inhibitory activity.
No.
R1
¼
R2
¼
IC50 MKK4 [nM]a
89
45
48
58
29
146
aIC50-values (nM) determined with 33PanQinase assay.
5-Bromo-1H-pyrazolo[3,4-b]pyridine-3-carboxylic acid (62).
filtration and washed with water (note: since the precipitate is very
voluminous, a large suction funnel has to be used). The solids were
dried in an oven at 110 ꢂC. After complete drying, the product was
sonicated in 100 mL of toluene for 5 min and stirred for 30 min. The
product was filtered, washed with an additional 20 mL of toluene
and dried at 110 ꢂC. Yield: 7.92 g (quant.). 1H NMR (200 MHz,
5-Bromo-3-iodo-1H-pyrazolo[3,4-b]pyridine
(61)
(10.44
g,
32.2 mmol) was dissolved in DMF, MeOH and triethylamine (75 mL
each). The vessel was evacuated and backfilled with argon (4x).
XantPhos (1.12 g, 1.93 mmol) and Pd(OAc)2 (270 mg, 0.97 mmol)
were added and carbon monoxide (generated from formic acid and
sulfuric acid) was bubbled through the solution while heating to
60 ꢂC. The mixture was stirred under an atmosphere of carbon
monoxide (balloon) for 8 h. Every 1.5 h carbon monoxide was
bubbled through the solution for 5 min. The mixture was concen-
trated under reduced pressure, and the residue was triturated with
2N HCl. The solids were heated at 95 ꢂC in about 100 mL 1N NaOH
overnight. After cooling to room temperature, the mixture was
acidified with conc. HCl, and the precipitate collected by suction
DMSO)
DMSO)
d
8.64 (d, J ¼ 7.9 Hz, 2H), 5.69 (bs, 1H); 13C NMR (50 MHz,
d
163.27, 150.97, 149.67, 136.69, 132.65, 115.73, 113.6; [M-
H]- ¼ 239.9/241.9.
5-Bromo-N-methoxy-N-methyl-1H-pyrazolo[3,4-b]pyridine-
3-carboxamide (63). 5-bromo-1H-pyrazolo[3,4-b]pyridine-3-
carboxylic acid (62) (7.91 g, 32.7 mmol) and 1,10-carbon-
yldiimidazole (5.83 g, 35.9 mmol) were stirred in 200 mL of DMF at
60 ꢂC for 45 min. N,O-dimethylhydroxylamine hydrochloride
Scheme 1. Synthesis of the pyrazolopyridine compounds.a.
10

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B. Pfaffenrot, P. Klovekorn, M. Juchum et al.
European Journal of Medicinal Chemistry 218 (2021) 113371
(3.51 g, 35.9 mmol) was added to the resulting suspension, and the
mixture was stirred for 4 h at 65 ꢂC. Most of the solvent was
removed under vacuum, and half sat. NaHCO3-solution was added
to the residue. The solids were collected by suction filtration,
washed with water and dried at 110 ꢂC. Yield: 7.94 g (85%). 1H NMR
warm solution was added to about 80 mL 2N HCl, the solids
collected and washed with water. The solids were taken up in EtOAc
and washed with 2N HCl and brine, dried over sodium sulfate and
evaporated. The product was purified by flash chromatography
(SiO2, DCM/EtOAc 0%e20%) and triturated with n-hexane. Yield:
(200 MHz, DMSO)
d
14.46 (s, 1H), 8.62 (d, J ¼ 20.4 Hz, 2H), 3.76 (s,
1.68 g (65%). 1H NMR (200 MHz, DMSO) d 9.86 (s, 1H), 8.79 (dd,
3H), 3.44 (s, 3H), [M-H]- ¼ 283.0/285.0.
J ¼ 5.3, 2.0 Hz, 3H), 7.64 (td, J ¼ 9.0, 6.0 Hz, 1H), 7.30 (t, J ¼ 8.9 Hz,
(3-Amino-2,6-difluorophenyl)
pyridin-3-yl)methanone (64).
(5-bromo-1H-pyrazolo[3,4-b]
1H), 3.17e2.96 (m, 3H), 1.87e1.62 (m, 2H), 0.96 (t, J ¼ 7.4 Hz, 4H);
13C NMR (50 MHz, DMSO)
d
182.75, 157.39 (dd, J ¼ 177.1, 7.4 Hz),
To a solution of 2,4-difluoroaniline (7.99 g, 61.9 mmol, 2.2 equiv.)
in THF (55 mL), 2N n-BuLi in n-hexane (24.8 mL, 61.9 mmol, 2.2
equiv.) was added dropwise at ꢃ78 ꢂC, followed by the dropwise
addition of trimethylsilylchloride (7.85 mL, 61.9 mmol) in THF
(10 mL). The mixture was warmed to ꢃ50 ꢂC and stirred for 10 min.
After cooling to ꢃ78 ꢂC, 2N n-BuLi in n-hexane (24.8 mL, 61.9 mmol,
2.2 equiv.) and trimethylsilylchloride (7.85 mL, 61.9 mmol) in THF
(10 mL) were added consecutively dropwise, and the reaction was
stirred for 30 min at room temperature. The mixture was cooled
to ꢃ78 ꢂC, 2N n-BuLi in n-hexane (24.8 mL, 61.9 mmol, 2.2 equiv.)
was added dropwise, and the mixture was stirred for 30 min. This is
considered reaction solution A. In a separate flask, 60% NaH in
mineral oil (1.24 g, 30.9 mmol, 1.05 equiv.) was added in portions to
a suspension of 62 in THF (55 mL) at 0 ꢂC, and the mixture was
stirred for 30 min at room temperature. This is considered reaction
solution B. Reaction solution B was added to reaction solution A,
warmed to ꢃ15 ꢂC and stirred for 15 min. Conc. HCl (12 mL) was
carefully added, and the mixture stirred for 5 min at room tem-
perature. Saturated NaHCO3-solution was slowly added until phase
separation occurred. The phases were separated, the aqueous phase
extracted with THF, and the extracts were dried and filtered. i-PrOH
(150 mL) was added to the extract, and the extract was concen-
trated to a volume of about 100 mL. The product was collected by
suction filtration, washed with i-PrOH and dried. Yield:7.14 g (70%).
152.42 (dd, J ¼ 180.3, 7.3 Hz), 151.56, 151.34, 141.39, 132.59,
130.78e130.05 (m), 122.20 (dd, J ¼ 13.5, 3.6 Hz), 117.19 (dd, J ¼ 23.0,
20.9 Hz), 116.14, 115.18, 112.59 (dd, J ¼ 22.2, 3.8 Hz), 54.14, 17.22
12.97; [M-H]- ¼ 457.1/459.1.
N-[3-(5-bromo-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,4-
difluorophenyl]butane-1-sulfonamide
(65c).
(3-amino-2,6-
difluorophenyl)-(5-bromo-1H-pyrazolo[3,4-b]pyridin-3-yl)meth-
anone (64) (350 mg, 0.991 mmol) and 4-DMAP (6.05 mg,
0.0496 mmol) were dissolved in pyridine (2 mL) and heated to
65 ꢂC. Butane-1-sulfonyl chloride (0.36 mL, 1.49 mmol, 1.5 equiv.)
was added, and the mixture stirred overnight. 0.25 equiv. butane-1-
sulfonyl chloride were added, and stirring continued for 2h at 65 ꢂC.
The mixture was poured into aqueous 2N HCl and extracted with
EtOAc. The organic phase was washed with 2N HCl and brine, dried
over sodium sulfate, filtered, and the solvent removed under
reduced pressure. The residue was purified by flash chromatog-
raphy (n-hexane/EtOAc) 10%e50% and triturated with n-hexane.
Yield: 180 mg (38%) 1H NMR (200 MHz, DMSO) d 15.03 (s, 1H), 9.83
(s, 1H), 9.02e8.62 (m, 2H), 3.21e3.03 (m, 2H), 1.70 (dt, J ¼ 15.0,
7.5 Hz, 2H), 1.37 (dq, J ¼ 14.5, 7.3 Hz, 2H), 0.84 (t, J ¼ 7.2 Hz, 3H); 13C
NMR (50 MHz, DMSO)
d 182.4, 151.1, 151.0, 141.1, 132.3, 115.8, 114.8,
51.8, 25.1, 20.7, 13.4; [M-1]- ¼ 470.8.
N-[3-(5-bromo-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,4-
difluorophenyl]-1-phenylmethanesulfonamide (65d). (3-amino-
2,6-difluorophenyl)-(5-bromo-1H-pyrazolo[3,4-b]pyridin-3-yl)
methanone (64) (350 mg, 0.99 mmol) and 4-DMAP (6.05 mg,
0.05 mmol) were dissolved in pyridine (2 mL) and heated to 65 ꢂC.
Phenylmethanesulfonyl chloride (283 mg, 1.49 mmol, 1.5 equiv.)
was added, and the mixture stirred for 2h. The mixture was poured
into aqueous 2N HCl and extracted with EtOAc. The organic phase
was washed with 2N HCl and brine, dried over sodium sulfate,
filtered, and the solvent removed under reduced pressure. The
residue was purified by flash chromatography (n-hexane þ EtOAc)
0%e50% and triturated with n-hexane. Yield: 315 mg (63%). 1H
1H NMR (200 MHz, DMSO)
2H), 7.18e6.59 (m, 2H), 5.25 (s, 2H); 13C NMR (50 MHz, DMSO)
d
14.91 (s, 1H), 8.77 (dd, J ¼ 5.4, 2.1 Hz,
d
183.95, 151.04, 150.79, 150.27 (dd, J ¼ 161.0, 6.8 Hz), 145.50 (dd,
J ¼ 167.3, 6.8 Hz), 141.34, 133.35 (dd, J ¼ 12.8, 2.6 Hz), 132.28, 117.45
(dd, J ¼ 8.4, 6.5 Hz), 116.24 (dd, J ¼ 22.7, 19.1 Hz), 115.55, 114.81,
111.26 (dd, J ¼ 21.7, 3.5 Hz); [M-H]- ¼ 351.1/353.1.
N-[3-(5-bromo-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,6-
difluorophenyl]methanesulfonamide
(65a).
(3-amino-2,6-
difluorophenyl)-(5-bromo-1H-pyrazolo[3,4-b]pyridin-3-yl)meth-
anone (64) (350 mg, 0.991 mmol) and 4-DMAP (6.05 mg,
0.0496 mmol, 5%) were dissolved in pyridine (2 mL) and heated to
65 ꢂC. Methanesulfonyl chloride (0.36 mL, 1.49 mmol, 1.5 equiv.)
was added, and the mixture stirred for 2 h. The mixture was poured
into aqueous 2N HCl and extracted with EtOAc. The organic phase
was washed with 2N HCl and brine, dried over sodium sulfate,
filtered, and the solvent removed under reduced pressure. The
residue was purified by flash chromatography (DCM þ EtOAc) 0%e
30% and triturated with n-hexane. Yield: 164 mg, 0,38 mmol (38%).
NMR (200 MHz, CDCl3)
J ¼ 2.2 Hz, 1H), 8.59 (d, J ¼ 2.1 Hz, 1H), 7.52e7.25 (m, 6H), 6.87 (td,
182.7,
d 14.22 (s, 1H), 8.96 (s, 1H), 8.79 (d,
J ¼ 9.1, 1.6 Hz, 1H), 4.32 (s, 2H); 13C NMR (101 MHz, CDCl3)
d
156.6 (dd, J ¼ 251, 6.8 Hz), 153.03 (d, J ¼ 7.7 Hz), 151.2, 150.6, 150.5,
141.6, 132.9, 130.8, 127.75 (dd, J ¼ 151.3, 7.9 Hz), 126.9, 121.87 (dd,
J ¼ 13.1, 3.9 Hz), 117.12 (dd, J ¼ 22.8, 20.7 Hz), 115.7, 115.5, 111.61 (dd,
J ¼ 22.5, 3.8 Hz), 58.8. [M-1]- ¼ 504.7.
N-[3-[5-bromo-1-(oxan-2-yl)pyrazolo[3,4-b]pyridine-3-
carbonyl]-2,4-difluorophenyl]propane-1-sulfonamide. (66). To a
suspensions of N-[3-(5-bromo-1H-pyrazolo[3,4-b]pyridine-3-
1H NMR (200 MHz, DMSO)
J ¼ 4.8, 2.1 Hz, 2H), 7.65 (td, J ¼ 9.0, 5.9 Hz, 1H), 7.32 (td, J ¼ 8.9,
1.5 Hz, 1H), 3.07 (s, 3H); 13C NMR (101 MHz, DMSO) 182.3, 156.4
d 15.07 (s, 1H), 9.82 (s, 1H), 8.80 (dd,
d
carbonyl)-2,4-difluorophenyl]propane-1-sulfonamide
(65b)
(dd, J ¼ 248.8, 6.6 Hz), 152.92 (dd, J ¼ 251.8, 8.1 Hz), 151.0, 150.9,
141.0, 132.2, 130.1, 130.0, 121.80 (dd, J ¼ 13.2, 3.7 Hz), 116.84 (dd,
J ¼ 23.0, 20.9 Hz), 115.7, 114.7, 112.16 (dd, J ¼ 22.4, 3.7 Hz), 40.4; [M-
1]- ¼ 428.7.
(0.33 g, 0.73 mmol) in dichloromethane (3 mL) dihydropyran
(0.13 mL, 1.45 mmol, 2equiv.) and p-toluenesulfonic acid mono-
hydrate (27 mg, 0.145 mmol, 0.2 equiv.) were added, and the
mixture was heated to reflux temperature for 45 min. After cooling,
the mixture was washed with sat. NaHCO3-solution, dried over
sodium sulfate, filtered, and the solvent was removed under
reduced pressure. The residue was dissolved in a minimum amount
of DCM and added dropwise to n-hexane with stirring. After 5 min
the solids were collected by suction filtration and dried. Yield:
297 mg (75%), used without further purification. 1H NMR
N-(3-(5-Bromo-1H-pyrazolo[3,4-b]pyridine-3-carbonyl)-2,4-
difluorophenyl)propane-1-sulfonamide (65b). (3-amino-2,6-
difluorophenyl) (5-bromo-1H-pyrazolo[3,4-b]pyridin-3-yl)meth-
anone (64) (2.00 g, 5.66 mmol) and 4-DMAP (35 mg, 0.28 mmol,
5%) were heated in 9 mL pyridine to 65 ꢂC and 1-propanesulfonyl
chloride (1.21 g, 0.96 mL, 8.50 mmol) was added. After 2 h
another 0.19 mL of 1-propanesulfonyl chloride were added. The
(200 MHz, CDCl3)
d
8.83 (s, 1H), 8.65 (s, 1H), 7.70 (dd, J ¼ 13.6,
11

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European Journal of Medicinal Chemistry 218 (2021) 113371
8.1 Hz,1H), 7.02 (d, J ¼ 9.6 Hz, 2H), 6.14 (d, J ¼ 9.2 Hz,1H), 4.14e3.64
(m, 2H), 3.22e2.90 (m, 2H), 2.61e2.31 (m, 1H), 2.15e1.16 (m, 10H);
7.31 (t, J ¼ 8.7 Hz, 1H), 3.18e3.04 (m, 2H), 1.87e1.63 (m, 2H), 0.97 (t,
J ¼ 7.4 Hz, 3H); 13C NMR (101 MHz, DMSO)
d 182.6, 152.3, 149.8,
13C NMR (50 MHz, CDCl3)
d
182.4, 151.0, 149.7, 140.7, 133.7, 127.33
141.9,137.2,133.0,129.9,129.8,129.3,128.4,128.1,127.6,127.5,126.7,
(d, J ¼ 8.6 Hz), 121.36 (dd, J ¼ 13.1, 3.8 Hz), 116.8, 116.6, 112.44 (dd,
J ¼ 22.6, 3.7 Hz), 83.3, 77.2, 68.2, 54.1, 28.9, 24.8, 22.4, 17.3, 12.9; [M-
1]- ¼ 540.7.
113.6, 53.9, 16.8, 12.6; ESI-HRMS: m/z ¼ 455.10002, calcd for
C
22H18F2N4O3S m/z ¼ 455.09949 [M-H]-. IR (ATR) [cmꢃ1] 1486,
1145, 974, 791, 700, 558, 408.
N-[2,4-difluoro-3-[1-(oxan-2-yl)-5-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]
phenyl]propane-1-sulfonamide (67). A vessel was charged with
N-[3-[5-bromo-1-(oxan-2-yl)pyrazolo[3,4-b]pyridine-3-carbonyl]-
N-(3-(5-(2-Chlorophenyl)-1H-pyrazolo[3,4-b]pyridine-3-
carbonyl)-2,4-difluorophenyl)propane-1-sulfonamide
(12).
Compound 12 was prepared according to general procedure A
using 65b (50 mg, 0.11 mmol), 2-chlorophenylboronic acid (22 mg,
0.14 mmol, 1.3 equiv.), Pd(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3
(0.25 mL, 0.38 mmol, 3.5 equiv.) in 1,4-dioxane (0.5 mL) at 120 ꢂC
for 0.5 h. Flash-chromatography (SiO2, DCM/EtOAc 0e30%) and
trituration with DCM and n-hexane. Yield: 23 mg (43%). HPLC-
2,4-difluorophenyl]propane-1-sulfonamide
(66)
(271
mg,
0.499 mmol), bis(pinacolato)diboron (139 mg, 0.55 mmol, 1.1
equiv.) and anhydrous potassium acetate (147 mg, 1.50 mmol, 3
equiv.). Degassed, dry 1,4-dioxane (5 mL) was added, and the vessel
was evacuated and refilled with argon (3x). Pd(dppf)Cl2 (9.12 mg,
0.0125 mmol) was added, and the mixture stirred at 85 ꢂC over-
night. After cooling to room temperature, the mixture was diluted
with EtOAc, filtered over celite, and the solvent was removed. The
residue was dissolved in DCM, petrol ether (60/90) was added, and
DCM removed under reduced pressure. After cooling for 1 h at 4 ꢂC
the solids were collected by suction filtration and dried. Yield:
267 mg (91%), which was used without further purification. 1H
purity: 99%. 1H NMR (200 MHz, DMSO)
d 15.00 (s, 1H), 9.82 (s,
1H), 8.79 (d, J ¼ 2.1 Hz, 1H), 8.62 (d, J ¼ 2.1 Hz, 1H), 7.73e7.44 (m,
5H), 7.31 (td, J ¼ 9.0, 1.6 Hz, 1H), 3.19e2.94 (m, 2H), 1.87e1.61 (m,
2H), 0.97 (t, J ¼ 7.4 Hz, 3H). ESI-HRMS: m/z ¼ 489.06052, calcd for
C
22H17ClF2N4O3S m/z ¼ 489.06052 [M-H]- IR (ATR) [cmꢃ1] 1686,
.
1478, 1436, 1328, 1240, 1132, 987, 916, 762, 789, 621, 500.
N-(3-(5-(3-Chlorophenyl)-1H-pyrazolo[3,4-b]pyridine-3-
carbonyl)-2,4-difluorophenyl)propane-1-sulfonamide
(13).
NMR (200 MHz, CDCl3)
1H), 7.12e6.83 (m, 2H), 6.22 (d, J ¼ 9.0 Hz, 1H), 4.19e3.60 (m, 2H),
3.07 (s, 2H), 2.57e1.11 (m, 23H); 13C NMR (50 MHz, CDCl3) 182.5,
155.5, 152.4, 141.9, 139.3, 127.3 (d, J ¼ 9.0 Hz), 121.23 (dd, J ¼ 13.1,
4.0 Hz), 114.9, 112.33 (dd, J ¼ 23.2, 2.9 Hz), 84.4, 82.8, 77.2, 68.1, 54.1,
29.0, 24.9, 22.5, 17.2, 12.9; [M-1]- ¼ 588.9.
d
9.11 (s, 1H), 8.94 (s, 1H), 7.68 (d, J ¼ 5.9 Hz,
Compound 13 was prepared according to general procedure A
using 65b (50 mg, 0.11 mmol), 3-chlorophenylboronic acid (19 mg,
0.14 mmol, 1.1 equiv.), Pd(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3
(0.25 mL, 0.59 mmol, 3.5 equiv.) in 1,4-dioxane (0.3 mL) at 120 ꢂC
for 0.5 h. Flash-chromatography (SiO2, DCM/EtOAc 0e35%). Yield:
d
30 mg (56%). HPLC-purity: 99%. 1H NMR (200 MHz, DMSO)
d 9.05 (s,
Suzuki-coupling reaction under microwave irradiation
(General procedure A). A microwave vessel is charged with a
magnetic stir bar, corresponding 5-bromo-1H-pyrazolo[3,4-b]pyr-
idine (65a-d), appropriate boronic acid or pinacol boronate, cata-
lyst, and purged with argon. Degassed 1,4-dioxane and degassed
aqueous 1.5 M K2CO3 are added, and the mixture is heated to the
stated temperature (100e120 ꢂC) under microwave irradiation
until complete conversion (usually 30 min). After cooling, the
mixture is diluted with EtOAc and neutralized with sat. NH4Cl so-
lution. The aqueous phase is removed, the organic phase is
concentrated, and the product isolated by flash chromatography
and triturated if necessary.
1H), 8.80 (s, 1H), 8.06e7.19 (m, 6H), 3.18e3.03 (m, 2H), 1.91e1.61
(m, 2H), 0.97 (t, J ¼ 7.2 Hz, 3H); 13C NMR (101 MHz, DMSO)
d 182.5,
152.4, 149.8, 142.0, 139.4, 133.9, 131.5, 130.9, 129.9, 129.8, 128.1,
127.9, 127.2, 126.2, 121.8 (dd, J ¼ 13.0, 3.1 Hz), 113.4, 112.1 (dd,
J ¼ 22.8, 3.4 Hz), 53.8, 16.8, 12.5; ESI-HRMS: m/z ¼ 489.006115,
calcd for C22H17ClF2N4O3S m/z ¼ 489.06052 [M-H]-.IR (ATR) [cmꢃ1
1686, 1495, 1432, 1149, 979, 816, 558, 508.
]
N-(2,4-Difluoro-3-(5-(2-fluorophenyl)-1H-pyrazolo[3,4-b]
pyridine-3-carbonyl)phenyl)propane-1-sulfonamide (14). Com-
pound 14 was prepared according to general procedure A using
65b (60 mg, 0.17 mmol), 2-fluorophenylboronic acid (24 mg,
0.17 mmol, 1.3 equiv.), Pd(PPh3)4 (8 mg, 5%) and 1.5 M K2CO3
(0.25 mL, 0.38 mmol) in 1,4-dioxane (0.5 mL) at 120 ꢂC for 0.5 h.
Flash-chromatography (SiO2, DCM/EtOAc 0e30%). Yield: 38 mg
N-(3-(5-(4-Chlorophenyl)-1H-pyrazolo[3,4-b]pyridine-3-
carbonyl)-2,4-difluorophenyl)propane-1-sulfonamide
(10).
Compound 10 was prepared according to general procedure A
using 65b (77 mg, 0.17 mmol), 4-chlorophenylboronic acid (29 mg,
0.18 mmol, 1.1 equiv.), Pd(PPh3)4 (10 mg, 5%) and 1.5 M K2CO3
(0.39 mL, 0.59 mmol, 3.5 equiv.) in 1,4-dioxane (0.5 mL) at 120 ꢂC
for 0.5 h. Flash-chromatography (SiO2, DCM/diethyl ether 5e35%).
Yield: 46 mg (56%). HPLC-purity: 99%. 1H NMR (200 MHz, DMSO)
(61%). HPLC-purity: 99%. 1H NMR (200 MHz, DMSO)
d 14.97 (s, 1H),
9.85 (s, 1H), 8.91 (t, J ¼ 1.9 Hz, 1H), 8.77e8.61 (m, 1H), 7.80e7.25 (m,
6H), 3.18e3.02 (m, 2H), 1.86e1.64 (m, 2H), 0.97 (t, J ¼ 7.5 Hz, 3H);
ESI-HRMS: m/z
¼
473.09043, calcd for C22H17F3N4O3S m/
z ¼ 473.09007 [M-H]- IR (ATR) [cmꢃ1] 1682, 1599, 1482, 1253, 1145,
.
941, 804, 762, 712, 566, 500, 475.
d
9.04 (s, 1H), 8.78 (s, 1H), 7.89 (d, J ¼ 8.5 Hz, 2H), 7.71e7.54 (m, 3H),
N-(2,4-Difluor-3-(5-(3-Fluorphenyl)-1H-pyrazolo[3,4-b]pyr-
idin-3-carbonyl)phenyl)propan-1-sulfonamide (15). Compound
15 was prepared according to general procedure A using 65b
(50 mg, 0.11 mmol), 3-fluorophenylboronic acid (17 mg, 0.12 mmol,
1.1 equiv.), Pd(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3 (0.25 mL,
0.38 mmol, 3.5 equiv.) in 1,4-dioxane (0.5 mL) at 120 ꢂC for 0.5 h.
Flash-chromatography (SiO2, DCM/EtOAc 0e35%). Yield: 31 mg
7.31 (t, J ¼ 8.7 Hz, 1H), 3.19e3.05 (m, 2H), 1.75 (dd, J ¼ 14.8, 7.6 Hz,
2H), 0.97 (t, J ¼ 7.2 Hz, 3H); 13C NMR (50 MHz, DMSO)
d 182.9,152.8,
150.0, 142.3, 136.4, 133.5, 132.0, 130.3 (dd, J ¼ 10, 2 Hz), 129.7, 129.6,
128.1, 122.2 (dd, J ¼ 14, 4 Hz), 117.5 (dd, J ¼ 23, 21 Hz), 113.9, 112.6
(dd, J ¼ 22, 4 Hz), 54.1, 17.2, 13.0; ESI-HRMS: m/z ¼ 489.06149, calcd
for C22H17ClF2N4O3S m/z ¼ 489.06052 [M-H]-. IR (ATR) [cmꢃ1] 1673,
1486, 1432, 1140, 895, 825, 500.
(60%). HPLC-purity: 100%. 1H NMR (400 MHz, DMSO)
d 14.96 (s,1H),
N-(2,4-Difluoro-3-(5-phenyl-1H-pyrazolo[3,4-b]pyridine-3-
9.82 (s,1H), 9.06 (d, J ¼ 2.1 Hz,1H), 8.81 (d, J ¼ 2.1 Hz,1H), 7.79e7.53
carbonyl)-2,4-difluorophenyl)propane-1-sulfonamide
(11).
(m, 4H), 7.37e7.24 (m, 2H), 3.16e3.07 (m, 2H), 1.82e1.68 (m, 2H),
Compound 11 was prepared according to general procedure A
using 65b (50 mg, 0.11 mmol), phenylboronic acid (15 mg,
0.12 mmol, 1.1 equiv.), Pd(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3
(0.25 mL, 0.59 mmol, 3.5 equiv.) in 1,4-dioxane (0.3 mL) at 120 ꢂC
for 0.5 h. Flash-chromatography (SiO2, DCM/EtOAc 0e35%) and
trituration with n-hexane. Yield: 27 mg (56%). HPLC-purity: 97%.1H
0.97 (t, J ¼ 7.4 Hz, 3H); 13C NMR (101 MHz, DMSO)
d 182.5, 163.9,
161.5, 152.3, 149.8, 142.0, 139.6, 139.5, 131.6, 131.6, 131.2, 131.1, 129.9,
129.8, 128.0, 123.6, 121.9, 121.8, 121.7, 121.7, 114.9, 114.7, 114.4, 114.2,
113.4, 112.2, 112.2, 112.0, 112.0, 53.8, 16.8, 12.5; ESI-HRMS: m/
z ¼ 473.09063, calcd for C22H17F3N4O3S m/z ¼ 473.09007 [M-H]- IR
.
(ATR) [cmꢃ1] 1682, 1490, 1320, 1153, 979, 900, 820, 778, 554, 504.
NMR (200 MHz, DMSO)
d
7.84 (d, J ¼ 7.2 Hz, 2H), 7.71e7.40 (m, 4H),
N-(2,4-Difluoro-3-(5-(4-fluorophenyl)-1H-pyrazolo[3,4-b]
12

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B. Pfaffenrot, P. Klovekorn, M. Juchum et al.
European Journal of Medicinal Chemistry 218 (2021) 113371
pyridine-3-carbonyl)phenyl)propane-1-sulfonamide (16). Com-
pound 16 was prepared according to general procedure A using
65b (50 mg, 0.11 mmol), 4-fluorophenylboronic acid (17 mg,
0.12 mmol, 1.1 equiv.), Pd(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3
(0.25 mL, 0.38 mmol, 3.5 equiv.) in 1,4-dioxane (0.3 mL) at 120 ꢂC
for 0.5 h. Flash-chromatography (SiO2, DCM/EtOAc 0e35%). Yield:
0.12 mmol, 1.1 equiv.), Pd(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3
(0.25 mL, 0.38 mmol, 3.5 equiv.) in 1,4-dioxane (0.3 mL) at 120 ꢂC
for 0.5 h. Flash-chromatography (SiO2, DCM/EtOAc 0e40%). Yield:
46 mg (87%). HPLC-purity: 100%. 1H NMR (200 MHz, DMSO)
d 14.90
(s, 1H), 9.83 (s, 1H), 8.81 (d, J ¼ 2.0 Hz, 1H), 8.61 (d, J ¼ 2.0 Hz, 1H),
7.63 (td, J ¼ 9.0, 5.9 Hz, 1H), 7.45 (dd, J ¼ 12.8, 4.6 Hz, 2H), 7.38e7.05
(m, 3H), 3.82 (s, 3H), 3.17e3.04 (m, 2H), 1.87e1.61 (m, 2H), 0.97 (t,
33 mg (64%). HPLC-purity: 100%.1H NMR (200 MHz, DMSO)
d 14.91
(s, 1H), 9.89 (s, 1H), 9.02 (d, J ¼ 2.1 Hz, 1H), 8.74 (d, J ¼ 2.1 Hz, 1H),
7.89 (dd, J ¼ 8.6, 5.4 Hz, 2H), 7.64 (td, J ¼ 9.0, 5.9 Hz, 1H), 7.45e7.21
(m, 3H), 3.21e3.02 (m, 2H), 1.87e1.61 (m, 2H), 0.97 (t, J ¼ 7.4 Hz,
J ¼ 7.5 Hz, 3H); 13C NMR (101 MHz, DMSO)
d 182.6, 156.3, 151.6,
151.5, 141.7, 130.9, 130.7, 129.8, 129.8, 126.2, 121.1, 113.1, 111.9, 55.7,
53.8, 16.8, 12.5; ESI-HRMS: m/z
C
1315, 1244, 1136, 753, 720, 503.
¼
485.11061, calcd for
3H); 13C NMR (101 MHz, DMSO)
d
182.5, 163.5, 161.0, 157.5, 157.5,
23H20F2N4O4S m/z ¼ 485.11006 [M-H]-. IR (ATR) [cmꢃ1] 1481,1432,
155.1, 155.0, 154.0, 153.9, 152.1, 151.5, 151.5, 149.7, 141.9, 133.6, 133.6,
132.0, 129.8, 129.7, 129.6, 129.5, 127.6, 121.9, 121.8, 121.7, 121.7, 117.3,
117.3, 117.1, 117.1, 116.9, 116.9, 116.1, 115.9, 113.4, 112.2, 112.2, 112.0,
112.0, 53.8, 16.8, 12.5; ESI-HRMS: m/z ¼ 473.09057, calcd for
N-(2,4-Difluoro-3-(5-(3-methoxyphenyl)-1H-pyrazolo[3,4-b]
pyridine-3-carbonyl)phenyl)propane-1-sulfonamide (21). Com-
pound 21 was prepared according to general procedure A using
65b (50 mg, 0.11 mmol), 3-methoxyphenylboronic acid (18 mg,
0.12 mmol, 1.1 equiv.), Pd(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3
(0.25 mL, 0.38 mmol, 3.5 equiv.) in 1,4-dioxane (0.3 mL) at 120 ꢂC
for 0.5 h. Flash-chromatography (SiO2, DCM/EtOAc 0e40%). Yield:
C
22H17F3N4O3S m/z ¼ 473.09007 [M-H]-. IR (ATR) [cmꢃ1] 1677,1490,
1440, 1315, 1228, 1144, 974, 811, 537, 503.
N-(2,4-Difluoro-3-(5-(o-tolyl)-1H-pyrazolo[3,4-b]pyridine-3-
carbonyl)phenyl)propane-1-sulfonamide (17). Compound 17 was
prepared according to general procedure A using 65b (50 mg,
0.11 mmol), 2-methylphenylboronic acid (19 mg, 0.14 mmol, 1.3
equiv.), Pd(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3 (0.2 mL) in 1,4-
dioxane (0.8 mL) at 120 ꢂC for 0.5 h. Flash-chromatography (SiO2,
DCM/EtOAc 0e30%) and trituration with DCM and n-hexane. Yield:
33 mg (62%). HPLC-purity: 100%. 1H NMR (200 MHz, DMSO)
d 14.94
(s, 1H), 9.83 (s, 1H), 9.04 (d, J ¼ 2.2 Hz, 1H), 8.76 (d, J ¼ 2.2 Hz, 1H),
7.64 (td, J ¼ 9.1, 6.0 Hz, 1H), 7.51e7.26 (m, 4H), 7.09e6.99 (m, 1H),
3.87 (s, 3H), 3.20e2.99 (m, 2H),1.86e1.61 (m, 2H), 0.97 (t, J ¼ 7.5 Hz,
3H); 13C NMR (50 MHz, DMSO)
d 182.6, 159.9, 152.3, 150.0, 141.9,
32 mg (62%). HPLC-purity: 99%. 1H NMR (200 MHz, DMSO)
d
8.75
138.7, 132.9, 130.4, 127.7, 121.8 (dd, J ¼ 13, 4 Hz), 119.8, 113.9, 113.5,
(d, J ¼ 2.0 Hz, 1H), 8.52 (d, J ¼ 2.0 Hz, 1H), 7.67 (td, J ¼ 9.0, 5.9 Hz,
1H), 7.48e7.23 (m, 5H), 3.24e3.00 (m, 2H), 2.31 (s, 3H), 1.89e1.65
(m, 2H), 1.00 (t, J ¼ 7.4 Hz, 3H); ESI-HRMS: m/z ¼ 469.11528, calcd
for C23H20F2N4O3S m/z ¼ 469.11514 [M-H]-. IR (ATR) [cmꢃ1] 1677,
1477, 1432, 1323, 1244, 1312, 982, 907, 757, 724, 636, 620, 503.
N-(2,4-Difluoro-3-(5-(m-tolyl)-1H-pyrazolo[3,4-b]pyridine-
3-carbonyl)phenyl)propane-1-sulfonamide (18). Compound 18
was prepared according to general procedure A using 65b (50 mg,
0.11 mmol), 3-methylphenylboronic acid (16 mg, 0.12 mmol, 1.1
equiv.), Pd(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3 (0.25 mL, 0.38 mmol,
3.5 equiv.) in 1,4-dioxane (0.8 mL) at 120 ꢂC for 0.5 h. Flash-
chromatography (SiO2, DCM/EtOAc 0e30%). Yield: 34 mg (66%).
112.9, 55.3, 53.8, 16.9, 12.6; ESI-HRMS: m/z ¼ 485.10872, calcd for
C
23H20F2N4O4S m/z ¼ 485.11006 [M-H]-. IR (ATR) [cmꢃ1] 1678,
1594, 1495, 1436, 1141, 783, 691, 558, 504.
N-(2,4-Difluoro-3-(5-(4-methoxyphenyl)-1H-pyrazolo[3,4-b]
pyridine-3-carbonyl)phenyl)propane-1-sulfonamide (22). Com-
pound 22 was prepared according to general procedure A using
65b (50 mg, 0.11 mmol), 4-methoxyphenylboronic acid (18 mg,
0.12 mmol, 1.1 equiv.), Pd(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3
(0.25 mL, 0.38 mmol, 3.5 equiv.) in 1,4-dioxane (0.3 mL) at 120 ꢂC
for 0.5 h. Flash-chromatography (SiO2, DCM/EtOAc 0e40%). Yield:
33 mg (62%). HPLC-purity: 100%. 1H NMR (200 MHz, DMSO)
d 14.87
(s, 1H), 9.79 (s, 1H), 9.00 (d, J ¼ 2.2 Hz, 1H), 8.69 (d, J ¼ 2.2 Hz, 1H),
7.78 (d, J ¼ 8.8 Hz, 1H), 7.63 (td, J ¼ 9.0, 5.9 Hz, 1H), 7.31 (td, J ¼ 9.0,
1.3 Hz, 1H), 7.11 (d, J ¼ 8.7 Hz, 1H), 3.83 (s, 1H), 3.22e3.03 (m, 1H),
1.86e1.63 (m, 1H), 0.97 (t, J ¼ 7.4 Hz, 1H); 13C NMR (101 MHz,
HPLC-purity: 100%. 1H NMR (200 MHz, DMSO)
d 14.92 (s, 1H), 9.86
(s, 1H), 9.05 (d, J ¼ 2.1 Hz, 1H), 8.77 (d, J ¼ 2.1 Hz, 1H), 7.74e7.56 (m,
3H), 7.55e7.23 (m, 3H), 3.22e3.00 (m, 2H), 2.45 (s, 3H), 1.92e1.58
(m, 2H), 1.00 (t, J ¼ 7.4 Hz, 3H); 13C NMR (50 MHz, DMSO)
d
182.6,
DMSO) d 182.6, 159.5, 152.0, 149.6, 141.8, 132.8, 129.9, 129.4, 128.7,
152.2, 149.9, 141.9, 138.6, 137.1, 133.1, 129.2, 128.8, 128.1, 127.5, 124.6,
126.7, 114.8, 113.7, 55.3, 53.9, 16.9, 12.6; ESI-HRMS: m/
z ¼ 485.11009, calcd for C23H20F2N4O4S m/z ¼ 485.11006 [M-H]-. IR
(ATR) [cmꢃ1] 1503, 1253, 1137, 899, 837, 795, 708, 496.
113.6, 53.8, 21.1, 16.9, 12.6; ESI-HRMS: m/z ¼ 469.11596, calcd for
C
23H20F2N4O3S m/z ¼ 469.11514 [M-H]-. IR (ATR) [cmꢃ1] 1674, 1482,
1241, 1128, 766, 695, 646, 496.
N-(2,4-Difluoro-3-(5-(2-hydroxyphenyl)-1H-pyrazolo[3,4-b]
pyridine-3-carbonyl)phenyl)propane-1-sulfonamide (23). Com-
pound 23 was prepared according to general procedure A using
65b (50 mg, 0.11 mmol), 2-hydroxyphenylboronic acid (17 mg,
0.12 mmol, 1.1 equiv.), Pd(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3
(0.25 mL, 0.38 mmol, 3.5 equiv.) in 1,4-dioxane (0.3 mL) at 120 ꢂC
for 0.5 h. Flash-chromatography (SiO2, DCM/EtOAc 10e50%). Yield:
N-(2,4-Difluoro-3-(5-(p-tolyl)-1H-pyrazolo[3,4-b]pyridine-3-
carbonyl)phenyl)propane-1-sulfonamide (19). Compound 19 was
prepared according to general procedure A using 65b (50 mg,
0.11 mmol), 4-methylphenylboronic acid (16 mg, 0.12 mmol, 1.1
equiv.), Pd(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3 (0.25 mL, 0.38 mmol,
3.5 equiv.) in 1,4-dioxane (0.3 mL) at 120 ꢂC for 0.5 h. Flash-
chromatography (SiO2, DCM/EtOAc 0e30%). Yield: 37 mg (72%).
29 mg (56%). HPLC-purity: 94%. 1H NMR (200 MHz, DMSO)
d 14.89
HPLC-purity: 100%. 1H NMR (200 MHz, DMSO)
d
14.91 (s, 1H), 9.85
(s, 1H), 9.88 (s, 2H), 8.87 (d, J ¼ 2.1 Hz, 1H), 8.71 (d, J ¼ 2.1 Hz, 1H),
7.63 (td, J ¼ 9.0, 6.0 Hz, 1H), 7.45 (dd, J ¼ 7.3, 1.4 Hz, 1H), 7.38e7.21
(m, 2H), 7.10e6.89 (m, 2H), 3.20e3.02 (m, 2H), 1.86e1.64 (m, 2H),
(s, 1H), 9.01 (d, J ¼ 1.9 Hz, 2H), 8.72 (d, J ¼ 2.0 Hz, 2H), 7.82e7.55 (m,
5H), 7.45e7.23 (m, 4H), 3.21e3.04 (m, 3H), 2.38 (s, 4H), 1.86e1.64
(m, 3H), 0.97 (t, J ¼ 7.3 Hz, 4H); 13C NMR (50 MHz, DMSO)
d
182.6,
0.97 (t, J ¼ 7.4 Hz, 3H); 13C NMR (50 MHz, DMSO)
d 182.7, 154.6,
152.1, 149.8, 141.8, 137.6, 134.3, 132.9, 129.9, 127.3, 127.1, 121.8 (dd,
J ¼ 13, 4 Hz), 113.6, 112.2 (dd, J ¼ 22, 4 Hz), 53.8, 20.7, 16.9, 12.6; ESI-
HRMS: m/z ¼ 469.11596, calcd for C23H20F2N4O3S m/z ¼ 469.11514
[M-H]-. IR (ATR) [cmꢃ1] 1686, 1486, 1432, 1324, 1153, 970, 804, 554,
512.
151.6, 151.5, 141.7, 131.1, 130.8, 130.0 129.8, 129.7, 129.6, 124.2, 121.8
(dd, J ¼ 13, 4 Hz), 119.9, 116.2, 113.2, 112.2 (dd, J ¼ 23, 4 Hz), 53.8,
16.9, 12.6; ESI-HRMS: m/z ¼ 471.09516, calcd for C22H18F2N4O4S m/
z ¼ 471.09441 [M-H]-. IR (ATR) [cmꢃ1] 1486, 1436, 1253, 1141, 895,
755, 500.
N-(2,4-Difluoro-3-(5-(2-methoxyphenyl)-1H-pyrazolo[3,4-b]
pyridine-3-carbonyl)phenyl)propane-1-sulfonamide (20). Com-
pound 20 was prepared according to general procedure A using
65b (50 mg, 0.11 mmol), 2-methoxyphenylboronic acid (18 mg,
N-(2,4-Difluoro-3-(5-(3-hydroxyphenyl)-1H-pyrazolo[3,4-b]
pyridine-3-carbonyl)phenyl)propane-1-sulfonamide (24). Com-
pound 24 was prepared according to general procedure A using
65b (50 mg, 0.11 mmol), 3-hydroxyphenylboronic acid (17 mg,
13

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B. Pfaffenrot, P. Klovekorn, M. Juchum et al.
European Journal of Medicinal Chemistry 218 (2021) 113371
0.12 mmol, 1.1 equiv.), Pd(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3
(0.25 mL, 0.38 mmol, 3.5 equiv.) in 1,4-dioxane (0.3 mL) at 120 ꢂC
for 0.5 h. Flash-chromatography (SiO2, DCM/EtOAc 10e50%) and
trituration with DCM. Yield: 32 mg (62%). HPLC-purity: 100%. 1H
dioxaborolan-2-yl)aniline (26 mg, 0.12 mmol, 1.1 equiv.),
Pd(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3 (0.25 mL, 0.38 mmol, 3.5
equiv.) in 1,4-dioxane (0.3 mL) at 120 ꢂC for 0.5 h. Flash-
chromatography (SiO2, DCM/(DCM/EtOAc (9 þ 1) 0e60%). Yield:
NMR (200 MHz, DMSO)
d
15.21 (s, 1H), 10.04 (s, 2H), 9.32 (d,
28 mg (54%). HPLC-purity: 98%. 1H NMR (200 MHz, DMSO)
d 8.93 (s,
J ¼ 2.1 Hz, 1H), 9.03 (d, J ¼ 2.1 Hz, 1H), 7.98 (td, J ¼ 9.1, 6.0 Hz, 1H),
1H), 8.58 (s, 1H), 7.72e7.43 (m, 3H), 7.30 (t, J ¼ 9.0 Hz, 1H), 6.72 (d,
J ¼ 7.9 Hz, 2H), 5.39 (s, 2H), 3.21e2.95 (m, 2H), 1.74 (dd, J ¼ 13.9,
5.0 Hz, 2H), 0.96 (t, J ¼ 7.5 Hz, 3H); ESI-HRMS: m/z ¼ 470.11079,
7.76e7.43 (m, 4H), 7.29e7.14 (m, 1H), 3.54e3.34 (m, 2H), 2.19e1.96
(m, 2H), 1.31 (t, J ¼ 7.4 Hz, 3H); 13C NMR (101 MHz, DMSO)
d 182.6,
158.0, 152.2, 149.6, 141.8, 138.4, 133.0, 130.3, 127.3, 118.1, 115.1, 114.1,
calcd for C22H19F2N5O3S m/z ¼ 470.11039 [M-H]-. IR (ATR) [cmꢃ1
]
113.5, 53.8, 16.8, 12.5; ESI-HRMS: m/z ¼ 471.09437, calcd for
1490, 1440, 1324, 1278, 1253, 1149, 1008, 899, 816, 571, 504.
C
22H18F2N4O4S m/z ¼ 471.09441 [M-H]-. IR (ATR) [cmꢃ1] 1678,1594,
N-(3-(5-(3-(Dimethylamino)phenyl)-1H-pyrazolo[3,4-b]pyri-
dine-3-carbonyl)-2,4-difluorophenyl)propane-1-sulfonamide
(29). Compound 29 was prepared according to general procedure
A using 65b (50 mg, 0.11 mmol), 3-dimethylaminophenylboronic
acid (20 mg, 0.12 mmol, 1.1 equiv.), Pd(PPh3)4 (6 mg, 5%) and
1.5 M K2CO3 (0.25 mL, 0.38 mmol, 3.5 equiv.) in 1,4-dioxane
(0.3 mL) at 120 ꢂC for 0.5 h. Flash-chromatography (SiO2, DCM/
EtOAc 0e40%), trituration with DCM/n-hexane (2 þ 1). Yield: 29 mg
1495, 1436, 1141, 783, 691, 558, 504.
N-(2,4-Difluoro-3-(5-4-hydroxyphenyl)-1H-pyrazolo[3,4-b]
pyridine-3-carbonyl)phenyl)propane-1-sulfonamide (25). Com-
pound 25 was prepared according to general procedure A using
65b (50 mg, 0.11 mmol), 3-hydroxyphenylboronic acid (17 mg,
0.12 mmol, 1.1 equiv.), XPhos Pd G3 (6 mg) and 1.5 M K2CO3
(0.25 mL, 0.38 mmol, 3.5 equiv.) in 1,4-dioxane (0.3 mL) at 120 ꢂC
for 0.5 h. Flash-chromatography (SiO2, DCM/EtOAc 10e55%) and
trituration with DCM. Yield: 33 mg (64%). HPLC-purity: 96%. 1H
(53%). HPLC-purity: 97%. 1H NMR (200 MHz, DMSO)
d 9.11e8.84 (m,
1H), 8.70 (d, J ¼ 2.1 Hz, 1H), 7.64 (td, J ¼ 9.0, 6.0 Hz, 1H), 7.32 (q,
J ¼ 8.1 Hz, 2H), 7.11e6.99 (m, 2H), 6.81 (dd, J ¼ 8.1, 1.8 Hz, 1H),
3.18e3.04 (m, 2H), 2.99 (s, 6H), 1.87e1.63 (m, 2H), 0.97 (t, J ¼ 7.4 Hz,
NMR (200 MHz, DMSO) d 14.86 (s, 1H), 9.82 (s, 1H), 9.73 (s, 1H), 8.97
(d, J ¼ 2.1 Hz, 1H), 8.65 (d, J ¼ 2.0 Hz, 1H), 7.72e7.52 (m, 3H), 7.30 (t,
J ¼ 8.8 Hz, 1H), 6.93 (d, J ¼ 8.5 Hz, 2H), 3.19e3.00 (m, 2H), 1.85e1.63
3H); 13C NMR (101 MHz, DMSO)
d 182.5, 152.3, 151.0, 150.0, 141.8,
(m, 2H), 0.97 (t, J ¼ 7.4 Hz, 3H); 13C NMR (101 MHz, DMSO)
d
182.5,
137.9, 134.0, 129.7, 127.4, 115.3, 113.5, 112.2, 112.2, 112.1, 112.0, 112.0,
157.7, 151.8, 149.5, 141.7, 133.1, 128.6, 127.7, 126.2, 116.1, 113.6, 53.8,
16.8, 12.5; ESI-HRMS: m/z ¼ 471.09510, calcd for C22H18F2N4O4S m/
z ¼ 471.09441 [M-H]-. IR (ATR) [cmꢃ1] 1649, 1495, 1132, 833, 566,
504.
111.2, 53.8, 16.8, 12.5; ESI-HRMS: m/z ¼ 498.14204, calcd for
C
24H23F2N5O3S m/z ¼ 498.14169 [M-H]-. IR (ATR) [cmꢃ1] 1682,1486,
1336, 1153, 991, 912, 762, 566.
N-(3-(5-(4-(Dimethylamino)phenyl)-1H-pyrazolo[3,4-b]pyri-
dine-3-carbonyl)-2,4-difluorophenyl)propane-1-sulfonamide
(30). Compound 30 was prepared according to general procedure
N-(3-(5-(2-Aminophenyl)-1H-pyrazolo[3,4-b]pyridine-3-
carbonyl)-2,4-difluorophenyl)propane-1-sulfonamide
(26).
Compound 26 was prepared according to general procedure A
using 65b (50 mg, 0.11 mmol), 2-aminophenylboronic acid (17 mg,
0.12 mmol, 1.1 equiv.), Pd(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3
(0.25 mL, 0.38 mmol, 3.5 equiv.) in 1,4-dioxane (0.3 mL) at 120 ꢂC
for 0.5 h. Flash-chromatography (SiO2, DCM/EtOAc 10e60%). Yield:
A using 65b (50 mg, 0.11 mmol), N,N-dimethyl-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (30 mg, 0.12 mmol,
1.1 equiv.), XPhos Pd G4 (4 mg, 5%) and 1.5 M K2CO3 (0.25 mL,
0.38 mmol, 3.5 equiv.) in 1,4-dioxane (0.3 mL) at 115 ꢂC for 45 min.
Flash-chromatography (SiO2, DCM/EtOAc 0e50%). Yield: 38 mg
31 mg (60%). HPLC-purity: 100%. 1H NMR (200 MHz, DMSO)
d
14.85
(70%). HPLC-purity: 100%. 1H NMR (200 MHz, DMSO)
d 14.83 (s,1H),
(s, 1H), 9.87 (s, 1H), 8.69 (d, J ¼ 2.1 Hz, 1H), 8.56 (d, J ¼ 2.0 Hz, 1H),
7.63 (td, J ¼ 9.1, 6.0 Hz, 1H), 7.31 (t, J ¼ 8.7 Hz, 1H), 7.20e7.03 (m,
2H), 6.82 (d, J ¼ 7.8 Hz, 1H), 6.69 (t, J ¼ 7.5 Hz, 1H), 5.02 (s, 2H),
3.18e3.03 (m, 2H), 1.85e1.58 (m, 2H), 0.97 (t, J ¼ 7.4 Hz, 3H); 13C
9.82 (s, 1H), 8.98 (d, J ¼ 2.2 Hz, 1H), 8.63 (d, J ¼ 2.2 Hz, 1H),
7.73e7.54 (m, 3H), 7.31 (td, J ¼ 8.9, 1.5 Hz, 1H), 6.87 (d, J ¼ 8.9 Hz,
2H), 3.19e3.05 (m, 2H), 2.97 (s, 6H), 2.97 (s, 6H), 1.86e1.64 (m, 2H),
0.97 (t, J ¼ 7.4 Hz, 3H); 13C NMR (101 MHz, DMSO)
d 182.5,156.3 (dd,
NMR (101 MHz, DMSO)
d
182.6,151.8,151.3,145.9,141.7,132.4,130.7,
J ¼ 249, 6 Hz), 152.7 (dd, J ¼ 251, 9 Hz), 151.6, 150.2, 149.3, 141.6,
133.3, 129.7 (d, J ¼ 9 Hz), 127.8, 125.3, 124.2,121.74 (dd, J ¼ 14, 3 Hz),
113.8, 112.8, 112.07 (dd, J ¼ 23, 3 Hz), 53.8, 16.8, 12.5; ESI-HRMS: m/
z ¼ 498.14208, calcd for C24H23F2N5O3S m/z ¼ 498.14169 [M-H]-. IR
(ATR) [cmꢃ1] 1599, 1490, 1432, 1366, 1316, 1141, 812, 558.
129.8, 129.7, 129.0, 122.2, 116.9, 115.5, 113.6, 53.8, 16.8, 12.5; ESI-
HRMS: m/z ¼ 470.11087, calcd for C22H19F2N5O3S m/z ¼ 470.11039
[M-H]-. IR (ATR) [cmꢃ1] 1675, 1618, 1487, 1336, 1136, 993, 899, 756,
653, 490.
N-(3-(5-(3-Aminophenyl)-1H-pyrazolo[3,4-b]pyridine-3-
N-(3-(5-(2,4-Dichlorophenyl)-1H-pyrazolo[3,4-b]pyridine-3-
carbonyl)-2,4-difluorophenyl)propane-1-sulfonamide
(27).
carbonyl)-2,4-difluorophenyl)propane-1-sulfonamide
(31).
Compound 27 was prepared according to general procedure A
using 65b (50 mg, 0.11 mmol), 3-aminophenylboronic acid (19 mg,
0.14 mmol, 1.3 equiv.), Pd(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3
(0.2 mL) in 1,4-dioxane (0.8 mL) at 120 ꢂC for 0.5 h. Flash-
chromatography (SiO2, DCM/EtOAc 10e60%), trituration with
DCM and n-hexane. Yield: 28 mg (54%). HPLC-purity: 98%. 1H NMR
Compound 31 was prepared according to general procedure A
using 65b (50 mg, 0.11 mmol), 2,4-dichlorophenylboronic acid
(27 mg, 0.14 mmol, 1.3 equiv.), Pd(PPh3)4 (6 mg, 5%) and
1.5 M K2CO3 (0.2 mL) in 1,4-dioxane (0.8 mL) at 120 ꢂC for 0.5 h.
Flash-chromatography (SiO2, DCM/(DCM/EtOAc (9 þ 1) 0e60%).
Yield: 28 mg (49%). HPLC-purity: 97%. 1H NMR (200 MHz, DMSO)
(400 MHz, DMSO)
d
14.91 (s, 1H), 9.83 (s, 1H), 8.94 (s, 1H), 8.66 (s,
d
15.03 (s,1H), 9.83 (s,1H), 8.78 (d, J ¼ 2.1 Hz,1H), 8.64 (d, J ¼ 1.9 Hz,
1H), 7.63 (s, 1H), 7.37e7.10 (m, 2H), 7.04e6.86 (m, 2H), 6.66 (d,
J ¼ 6.2 Hz, 1H), 5.30 (s, 2H), 3.12 (s, 2H), 1.75 (d, J ¼ 5.9 Hz, 2H), 0.97
1H), 7.84 (d, J ¼ 1.7 Hz, 1H), 7.71e7.55 (m, 3H), 7.35e7.22 (m, 1H),
3.17e3.05 (m, 2H), 1.86e1.58 (m, 2H), 0.97 (t, J ¼ 7.4 Hz, 3H); ESI-
(s, 3H); 13C NMR (101 MHz, DMSO)
d
182.6, 152.2, 149.5, 149.4, 141.7,
HRMS: m/z
¼
523.02184, calcd for C22H16Cl2F2N4O3S m/
137.6, 133.7, 129.7, 126.9, 114.7, 113.7, 113.5, 112.5, 53.8, 16.8, 12.5;
z ¼ 523.02155 [M-H]-. IR (ATR) [cmꢃ1] 1682, 1482, 1328, 1323, 1124,
ESI-HRMS: m/z
¼
470.11076, calcd for C22H19F2N5O3S m/
1053, 1020, 983, 920, 808, 708, 504.
z ¼ 470.11039 [M-H]-. IR (ATR) [cmꢃ1] 1594, 1486, 1432,1336, 1149,
N-(3-(5-(3,5-Dichlorophenyl)-1H-pyrazolo[3,4-b]pyridine-3-
987, 912, 870, 783, 566, 504.
carbonyl)-2,4-difluorophenyl)propane-1-sulfonamide
(32).
N-(3-(5-(4-Aminophenyl)-1H-pyrazolo[3,4-b]pyridine-3-
Compound 32 was prepared according to general procedure A
using 65b (50 mg, 0.11 mmol), 3,5-dichlorophenylboronic acid
(23 mg, 0.12 mmol, 1.1 equiv.), Pd(PPh3)4 (6 mg, 5%) and
1.5 M K2CO3 (0.25 mL, 0.38 mmol, 3.5 equiv.) in 1,4-dioxane
carbonyl)-2,4-difluorophenyl)propane-1-sulfonamide
(28).
Compound 27 was prepared according to general procedure A
using 65b (50 mg, 0.11 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-
14

€
B. Pfaffenrot, P. Klovekorn, M. Juchum et al.
European Journal of Medicinal Chemistry 218 (2021) 113371
(0.3 mL) at 120 ꢂC for 0.5 h. Flash-chromatography (SiO2, DCM/
(101 MHz, DMSO)
d
182.5, 156.3 (dd, J ¼ 248, 7 Hz), 152.7 (dd,
EtOAc 0e30%). Yield: 31 mg (54%). HPLC-purity: 95%. 1H NMR
J ¼ 251, 8 Hz), 152.1, 150.6, 149.7, 141.8, 134.2, 132.8, 127.1, 126.0,
(200 MHz, DMSO)
d
9.06 (d, J ¼ 2.2 Hz, 1H), 8.86 (d, J ¼ 2.2 Hz, 1H),
121.8 (dd, J ¼ 14, 3 Hz),113.6,112.1 (dd, J ¼ 23, 4 Hz), 53.8, 39.5, 34.3,
7.96 (d, J ¼ 1.8 Hz, 2H), 7.72e7.55 (m, 2H), 7.31 (td, J ¼ 9.0, 1.5 Hz,
31.0, 16.8, 12.5; ESI-HRMS: m/z
¼
511.16261, calcd for
1H), 3.20e3.00 (m, 2H), 1.87e1.64 (m, 2H), 0.97 (t, J ¼ 7.5 Hz, 3H);
C
26H26F2N4O3S m/z ¼ 511.16209 [M-H]-. IR (ATR) [cmꢃ1] 1686, 1495,
13C NMR (101 MHz, DMSO)
d
182.4, 152.7, 149.7, 142.1, 140.9, 134.8,
1432, 1149, 979, 816, 558, 508.
130.1, 128.7, 127.4, 126.3, 113.3, 53.8, 16.8, 12.5; ESI-HRMS: m/
z ¼ 523.02155, calcd for C22H16Cl2F2N4O3S m/z ¼ 523.02155 [M-H]-.
IR (ATR) [cmꢃ1] 1686, 1590, 1486, 1141, 999, 904, 800, 558, 504.
N-(3-(5-(3,4-Dichlorophenyl)-1H-pyrazolo[3,4-b]pyridine-3-
N-(3-(5-(4-Chloro-3-(trifluoromethyl)phenyl)-1H-pyrazolo
[3,4-b]pyridine-3-carbonyl)-2,4-difluorophenyl)propane-1-
sulfonamide (37). Compound 37 was prepared according to gen-
eral procedure A using 65b (50 mg, 0.11 mmol), 4-chloro-3-
trifluoromethylphenylboronic acid (21 mg, 0.12 mmol, 1.1 equiv.),
Pd(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3 (0.25 mL, 0.38 mmol, 3.5
equiv.) in 1,4-dioxane (0.3 mL) at 110 ꢂC for 1.5 h. Flash-
chromatography (SiO2, DCM/EtOAc 0e25%). Yield: 31 mg (51%).
carbonyl)-2,4-difluorophenyl)propane-1-sulfonamide
(33).
Compound 33 was prepared according to general procedure A
using 65b (50 mg, 0.11 mmol), 3,4-dichlorophenylboronic acid
(27 mg, 0.14 mmol, 1.3 equiv.), Pd(PPh3)4 (6 mg, 5%) and
1.5 M K2CO3 (0.2) in 1,4-dioxane (0.8 mL) at 120 ꢂC for 0.5 h. Flash-
chromatography (SiO2, DCM/EtOAc 0e30%), trituration with n-
pentane. Yield: 34 mg (58%). HPLC-purity: 98%. 1H NMR (200 MHz,
HPLC-purity: 98%. 1H NMR (200 MHz, DMSO)
d 15.00 (s,1H), 9.83 (s,
1H), 9.10 (d, J ¼ 2.2 Hz, 1H), 8.89 (d, J ¼ 2.0 Hz, 1H), 8.33e8.10 (m,
2H), 7.89 (d, J ¼ 8.3 Hz, 1H), 7.64 (td, J ¼ 9.0, 6.0 Hz, 1H), 7.31 (td,
J ¼ 8.8, 1.2 Hz, 1H), 3.21e3.02 (m, 2H), 1.87e1.62 (m, 2H), 0.97 (t,
DMSO)
d 9.05 (s, 1H), 8.83 (s, 1H), 8.17 (s, 1H), 7.92e7.53 (m, 3H),
7.31 (t, J ¼ 8.8 Hz, 1H), 3.26e2.91 (m, 2H), 1.90e1.58 (m, 2H), 0.96 (t,
J ¼ 7.4 Hz, 3H); 13C NMR (101 MHz, DMSO)
d 182.5, 152.4, 149.8,
J
C
¼
7.3 Hz, 3H); ESI-HRMS: m/z
¼
523.02171, calcd for
142.0, 136.9, 133.1, 132.3, 130.4, 128.6, 126.9, 126.8, 113.3, 53.8, 16.8,
12.5; ESI-HRMS: m/z ¼ 557.04836, calcd for C23H16ClF5N4O3S m/
z ¼ 557.04790 [M-H]-. IR (ATR) [cmꢃ1] 1686, 1482, 1424, 1316, 1141,
974, 816, 554, 508.
22H16Cl2F2N4O3S m/z ¼ 523.02155 [M-H]-. IR (ATR) [cmꢃ1] 1678,
1590, 1482, 1424, 1316, 1257, 1124, 1024, 941, 895, 825, 704, 566,
487.
N-(3-(5-(3,4-Dimethylphenyl)-1H-pyrazolo[3,4-b]pyridine-3-
N-(2,4-Difluoro-3-(5-(4-fluoro-2-methylphenyl)-1H-pyr-
azolo[3,4-b]pyridine-3-carbonyl)phenyl)propane-1-
carbonyl)-2,4-difluorophenyl)propane-1-sulfonamide
(34).
Compound 34 was prepared according to general procedure A
using 65b (50 mg, 0.11 mmol), 3,4-dimethylphenylboronic acid
(18 mg, 0.12 mmol,1.1 equiv.), Pd(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3
(0.25 mL, 0.38 mmol, 3.5 equiv.) in 1,4-dioxane (0.3 mL) at 120 ꢂC
for 0.5 h. Flash-chromatography (SiO2, DCM/EtOAc 0e30%). Yield:
sulfonamide (38). Compound 38 was prepared according to gen-
eral procedure A using 65b (62 mg, 0.13 mmol), 4-fluoro-2-
methylphenylboronic acid (25 mg, 0.16 mmol, 1.2 equiv.),
Pd(PPh3)4 (8 mg, 5%) and 1.5 M K2CO3 (0.31 mL, 0.47 mmol, 3.5
equiv.) in 1,4-dioxane (0.5 mL) at 120 ꢂC for 0.5 h. Flash-
chromatography (SiO2, DCM/EtOAc 0e30%). Yield: 49 mg (74%).
34 mg (64%). HPLC-purity: 99%. 1H NMR (200 MHz, DMSO)
d 14.44
(s, 1H), 9.37 (s, 1H), 8.54 (d, J ¼ 2.1 Hz, 1H), 8.25 (d, J ¼ 2.1 Hz, 1H),
7.25e7.04 (m, 3H), 6.93e6.76 (m, 2H), 2.73e2.56 (m, 2H), 1.87 (s,
3H), 1.83 (s, 3H), 1.41e1.17 (m, 2H), 0.51 (t, J ¼ 7.4 Hz, 3H); 13C NMR
HPLC-purity: 100%. 1H NMR (200 MHz, DMSO)
d 14.95 (s, 1H), 9.85
(s, 1H), 8.70 (d, J ¼ 1.8 Hz, 1H), 8.48 (d, J ¼ 1.8 Hz, 1H), 7.64 (td,
J ¼ 9.1, 6.0 Hz, 1H), 7.48e7.10 (m, 4H), 3.21e3.03 (m, 2H), 2.28 (s,
3H), 1.85e1.65 (m, 2H), 0.97 (t, J ¼ 7.3 Hz, 3H); 13C NMR (101 MHz,
(101 MHz, DMSO)
d 182.5, 152.1, 149.7, 141.8, 137.1, 136.3, 134.5,
133.0, 130.3,128.3,126.9,124.6, 113.5, 53.8,19.3, 19.0, 16.8, 12.5; ESI-
HRMS: m/z ¼ 483.13124, calcd for C24H22F2N4O3S m/z ¼ 483.13079
[M-H]-. IR (ATR) [cmꢃ1] 1486, 1432, 1321, 1145, 971, 900, 814, 559,
508.
DMSO) d 182.6, 163.1, 160.6, 151.9, 151.1, 141.7, 138.5, 138.4, 134.0,
134.0, 132.7, 132.2, 132.1, 129.9, 129.8, 121.9, 121.7, 117.0, 116.8, 113.1,
113.0, 112.8, 112.2, 112.0, 53.8, 20.1, 16.8, 12.6; ESI-HRMS: m/
z ¼ 487.10665, calcd for C23H19F3N4O3S m/z ¼ 487.10572 [M-H]-. IR
(ATR) [cmꢃ1] 1665, 1474, 1428, 1316, 1249, 1137, 916, 712, 500.
N-(2,4-Difluoro-3-(5-(3-(trifluoromethyl)phenyl)-1H-pyr-
azolo[3,4-b]pyridine-3-carbonyl)phenyl)propane-1-
N-(3-(5-(4-Isopropylphenyl)-1H-pyrazolo[3,4-b]pyridine-3-
carbonyl)-2,4-difluorophenyl)propane-1-sulfonamide
(35).
Compound 35 was prepared according to general procedure A
using 65b (50 mg, 0.11 mmol), 4-isopropylphenylboronic acid
(23 mg, 0.14 mmol, 1.3 equiv.), Pd(PPh3)4 (6 mg, 5%) and
1.5 M K2CO3 (0.2 mL) in 1,4-dioxane (0.8 mL) at 120 ꢂC for 0.5 h.
Flash-chromatography (SiO2, DCM/EtOAc 0e30%). Yield: 21 mg
sulfonamide (39). Compound 39 was prepared according to gen-
eral procedure A using 65b (53 mg, 0.12 mmol), 2-chloro-4-
methoxyphenylboronic acid (22 mg, 0.14 mmol, 1.2 equiv.),
Pd(PPh3)4 (7 mg, 5%) and 1.5 M K2CO3 (0.27 mL, 0.4 mmol, 3.5
equiv.) in 1,4-dioxane (0.5 mL) at 120 ꢂC for 0.5 h. Flash-
chromatography (SiO2, DCM/EtOAc 10e40%). Yield: 44 mg (73%).
(39%). HPLC-purity: 97%. 1H NMR (400 MHz, DMSO)
d 14.92 (s, 1H),
9.83 (s, 1H), 9.02 (s, 1H), 8.73 (s, 1H), 7.75 (d, J ¼ 7.9 Hz, 2H),
7.69e7.59 (m, 1H), 7.42 (d, J ¼ 8.0 Hz, 2H), 7.31 (t, J ¼ 8.8 Hz, 1H),
3.19e3.07 (m, 2H), 2.96 (dt, J ¼ 13.6, 6.8 Hz, 1H), 1.82e1.67 (m, 2H),
1.25 (d, J ¼ 6.9 Hz, 6H), 0.97 (t, J ¼ 7.4 Hz, 3H); ESI-HRMS: m/
z ¼ 497.14710, calcd for C25H24F2N4O3S m/z ¼ 497.14644 [M-H]-. IR
(ATR) [cmꢃ1] 1669, 1486, 1428, 1320, 1149, 1037, 970, 895, 812, 691,
558, 508.
HPLC-purity: 97%. 1H NMR (200 MHz, DMSO)
d 14.93 (s, 1H), 9.83 (s,
1H), 8.74 (s, 1H), 8.58 (s, 1H), 7.75e7.47 (m, 2H), 7.37e7.03 (m, 3H),
3.86 (s, 3H), 3.18e3.02 (m, 2H), 1.88e1.61 (m, 2H), 0.97 (t, J ¼ 7.2 Hz,
3H); 13C NMR (50 MHz, DMSO)
d 182.6, 160.0, 151.9, 151.3, 141.8,
132.8, 132.5, 131.1, 130.4, 128.5, 121.9 (dd, J ¼ 13, 4 Hz), 115.1, 114.0,
113.0, 55.8, 53.8, 16.9, 12.6; ESI-HRMS: m/z ¼ 519.07206, calcd for
N-(3-(5-(4-tert-Butylphenyl)-1H-pyrazolo[3,4-b]pyridine-3-
C
23H19ClF2N4O4S m/z ¼ 519.07108 [M-H]-. IR (ATR) [cmꢃ1] 1686,
carbonyl)-2,4-difluorophenyl)propane-1-sulfonamide
(36).
1607, 1482, 1432, 1328, 1141, 1016, 720, 504.
Compound 36 was prepared according to general procedure A
using 65b (50 mg, 0.11 mmol), 4-tert-butylphenylboronic acid
(21 mg, 0.12 mmol,1.1 equiv.), Pd(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3
(0.25 mL, 0.38 mmol, 3.5 equiv.) in 1,4-dioxane (0.3 mL) at 120 ꢂC
for 0.5 h. Flash-chromatography (SiO2, DCM/EtOAc 0e25%). Yield:
N-(2,4-Difluoro-3-(5-(3-(trifluoromethyl)phenyl)-1H-pyr-
azolo[3,4-b]pyridine-3-carbonyl)phenyl)propane-1-
sulfonamide (40). Compound 40 was prepared according to gen-
eral procedure
A
using 65b (50 mg, 0.11 mmol), 3-
trifluoromethylphenylboronic acid (27 mg, 0.14 mmol, 1.3 equiv.),
Pd(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3 (0.2 mL) in 1,4-dioxane
(0.8 mL) at 120 ꢂC for 0.5 h. Flash-chromatography (SiO2, DCM/
EtOAc 0e25%). Yield: 33 mg (58%). HPLC-purity: 98%. 1H NMR
41 mg (74%). HPLC-purity: 100%. 1H NMR (200 MHz, DMSO)
d 14.92
(s, 1H), 9.83 (s, 1H), 9.03 (d, J ¼ 1.9 Hz, 1H), 8.73 (d, J ¼ 1.7 Hz, 1H),
7.84e7.50 (m, 5H), 7.31 (t, J ¼ 8.7 Hz, 1H), 3.22e3.01 (m, 2H),
1.85e1.63 (m, 2H), 1.34 (s, 9H), 0.97 (t, J ¼ 7.4 Hz, 3H); 13C NMR
(200 MHz, DMSO)
d
14.99 (s,1H), 9.83 (s,1H), 9.10 (d, J ¼ 2.2 Hz,1H),
15

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B. Pfaffenrot, P. Klovekorn, M. Juchum et al.
European Journal of Medicinal Chemistry 218 (2021) 113371
8.86 (d, J ¼ 2.2 Hz, 1H), 8.26e8.02 (m, 2H), 7.87e7.54 (m, 3H), 7.32
(td, J ¼ 8.7, 1.4 Hz, 1H), 3.18e2.99 (m, 2H), 1.86e1.54 (m, 2H), 0.97 (t,
7.32 (td, J ¼ 9.0, 1.5 Hz, 1H), 3.30 (s, 3H), 3.21e3.03 (m, 2H),
1.85e1.64 (m, 2H), 0.97 (t, J ¼ 7.4 Hz, 3H); 13C NMR (50 MHz, DMSO)
J
¼
7.4 Hz, 3H); ESI-HRMS: m/z
¼
523.08771, calcd for
d 182.6, 152.6, 150.0, 142.2, 142.1, 140.2, 131.3, 128.7, 128.5, 127.8,
C
23H17F5N4O3S m/z ¼ 523.08688 [M-H]-. IR (ATR) [cmꢃ1] 1499,
121.8 (dd, J ¼ 14, 4 Hz), 113.5, 53.8, 43.5, 16.9, 12.6; ESI-HRMS: m/
z ¼ 533.07774, calcd for C23H20F2N4O5S m/z ¼ 533.07704 [M-H]-. IR
(ATR) [cmꢃ1] 1669, 1594, 1486, 1291, 1153, 895, 783, 533, 496.
4-(3-(2,6-Difluoro-3-(propylsulfonamido)benzoyl)-1H-pyr-
azolo[3,4-b]pyridine-5-yl)benzensulfonamide (45). Compound
45 was prepared according to general procedure A using 65b
(50 mg, 0.11 mmol), 4-sulfamoylphenylboronic acid (24 mg,
0.12 mmol, 1.1 equiv.), P(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3
(0.25 mL, 0.38 mmol, 3.5 equiv.) in 1,4-dioxane (0.3 mL) at 120 ꢂC
for 0.5 h. Flash-chromatography (SiO2, DCM/EtOAc 10e60%). Yield:
1436, 1320, 1291, 1241, 1145, 1091, 904, 804, 700, 566, 500.
N-(2,4-Difluoro-3-(5-(4-(trifluoromethyl)phenyl)-1H-pyr-
azolo[3,4-b]pyridine-3-carbonyl)phenyl)propane-1-
sulfonamide (41). Compound 41 was prepared according to gen-
eral procedure
A
using 65b (50 mg, 0.11 mmol), 4-
trifluoromethylphenylboronic acid (23 mg, 0.12 mmol, 1.1 equiv.),
Pd(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3 (0.25 mL, 0.38 mmol, 3.5
equiv.) in 1,4-dioxane (0.3 mL) at 120 ꢂC for 0.5 h. Flash-
chromatography (SiO2, DCM/EtOAc 0e30%). Yield: 29 mg (51%).
HPLC-purity: 100%. 1H NMR (200 MHz, DMSO)
d
15.03 (s, 1H), 9.85
39 mg (67%). HPLC-purity: 99%. 1H NMR (200 MHz, DMSO)
d 14.53
(s, 1H), 9.10 (d, J ¼ 2.2 Hz, 1H), 8.86 (d, J ¼ 2.2 Hz, 1H), 8.10 (d,
J ¼ 8.0 Hz, 2H), 7.90 (d, J ¼ 8.4 Hz, 2H), 7.64 (td, J ¼ 9.0, 5.9 Hz, 1H),
7.32 (td, J ¼ 8.9, 1.4 Hz, 1H), 3.24e2.96 (m, 2H), 1.89e1.60 (m, 2H),
(s, 1H), 9.37 (s, 1H), 8.64 (d, J ¼ 2.2 Hz, 1H), 8.40 (d, J ¼ 2.1 Hz, 1H),
7.62 (d, J ¼ 8.5 Hz, 2H), 7.51 (d, J ¼ 8.5 Hz, 2H), 7.28e7.06 (m, 2H),
7.02 (s, 2H), 6.86 (td, J ¼ 8.8, 1.3 Hz, 1H), 2.76e2.53 (m, 2H),
1.40e1.18 (m, 2H), 0.51 (t, J ¼ 7.4 Hz, 3H); 13C NMR (101 MHz,
0.97 (t, J ¼ 7.4 Hz, 3H); 13C NMR (101 MHz, DMSO)
d 182.5, 152.5,
149.8, 142.0, 141.2, 131.4, 128.4, 128.3, 125.9, 125.9, 113.4, 53.8, 16.8,
12.5; ESI-HRMS: m/z ¼ 523.08726, calcd for C23H17F5N4O3S m/
z ¼ 523.08688 [M-H]-. IR (ATR) [cmꢃ1] 1674, 1486, 1324, 1245, 1108,
837, 500.
DMSO) d 182.5, 152.4, 149.8, 143.5, 142.0, 140.3, 131.5, 131.5, 131.4,
128.7, 128.6, 128.2, 127.9, 126.4, 113.4, 53.8, 16.8, 12.5; ESI-HRMS: m/
z ¼ 534.07295, calcd for C22H19F2N5O5S2 m/z ¼ 534.07229 [M-H]-.
IR (ATR) [cmꢃ1] 1486, 1432, 1324, 1153, 891, 691, 554.
N-(2,4-Difluoro-3-(5-(3-nitrophenyl)-1H-pyrazolo[3,4-b]pyr-
idine-3-carbonyl)phenyl)propane-1-sulfonamide (42). Com-
pound 42 was prepared according to general procedure A using
65b (50 mg, 0.11 mmol), 3-nitrophenylboronic acid (20 mg,
0.12 mmol, 1.1 equiv.), XPhos Pd G4 (6 mg) and 1.5 M K2CO3
(0.25 mL, 0.38 mmol, 3.5 equiv.) in 1,4-dioxane (0.3 mL) at 120 ꢂC
for 0.5 h. Flash-chromatography (SiO2, DCM/EtOAc 10e50%). Yield:
4-(3-(2,6-Difluoro-3-(propylsulfonamido)benzoyl)-1H-pyr-
azolo[3,4-b]pyridine-5-yl)-N-methylbenzenesulfonamide (46).
Compound 46 was prepared according to general procedure A
using 65b (50 mg, 0.11 mmol), R1 (26 mg, 0.12 mmol, 1.1 equiv.),
P(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3 (0.25 mL, 0.38 mmol, 3.5
equiv.) in 1,4-dioxane (0.3 mL) at 120 ꢂC for 45 min. Flash-
chromatography (SiO2, DCM/EtOAc 20e60%), trituration with
DCM. Yield: 33 mg (55%). HPLC-purity: 100%. 1H NMR (400 MHz,
40 mg (73%). HPLC-purity: 98%. 1H NMR (200 MHz, DMSO)
d 14.99
(s, 1H), 9.84 (s, 1H), 9.13 (d, J ¼ 2.2 Hz, 1H), 8.91 (d, J ¼ 2.2 Hz, 1H),
8.64 (t, J ¼ 1.9 Hz, 1H), 8.37e8.25 (m, 1H), 7.84 (t, J ¼ 8.0 Hz, 1H),
7.64 (td, J ¼ 9.0, 6.0 Hz, 1H), 7.32 (td, J ¼ 9.0, 1.3 Hz, 1H), 3.21e3.04
(m,1H),1.88e1.61 (m,1H), 0.97 (t, J ¼ 7.4 Hz,1H); 13C NMR (50 MHz,
DMSO)
d
15.01 (s, 1H), 9.83 (s, 1H), 9.11 (d, J ¼ 2.1 Hz, 1H), 8.87 (d,
J ¼ 2.0 Hz, 1H), 8.11 (d, J ¼ 8.3 Hz, 2H), 7.93 (d, J ¼ 8.3 Hz, 2H), 7.64
(td, J ¼ 9.0, 5.9 Hz,1H), 7.58 (q, J ¼ 5.0 Hz,1H), 7.32 (t, J ¼ 8.7 Hz,1H),
3.19e3.02 (m, 2H), 1.80e1.70 (m, 2H), 0.97 (t, J ¼ 7.5 Hz, 3H); 13C
DMSO)
d
182.6, 152.5, 149.9, 148.5, 142.1, 138.9, 134.2, 130.8, 130.7,
NMR (101 MHz, DMSO) d 182.6, 152.5, 149.9, 142.0, 141.0, 138.8,
128.8, 122.8, 122.2, 113.4, 53.8, 16.9, 12.6; ESI-HRMS: m/
z ¼ 500.08487, calcd for C22H17F2N5O5S m/z ¼ 500.08457 [M-H]-. IR
(ATR) [cmꢃ1] 1653, 1486, 1336, 1320, 1141, 987, 891, 841, 741, 616,
496.
131.4, 128.4, 128.2, 127.5, 113.4, 53.8, 28.6, 16.8, 12.5; ESI-HRMS: m/
z ¼ 548.08842, calcd for C23H21F2N5O5S2 m/z ¼ 548.28794 [M-H]-.
IR (ATR) [cmꢃ1] 1490, 1324, 1137, 899, 814, 566, 496.
4-(3-(2,6-Difluoro-3-(propylsulfonamido)benzoyl)-1H-pyr-
N-(2,4-Difluoro-3-(5-(4-nitrophenyl)-1H-pyrazolo[3,4-b]pyr-
idine-3-carbonyl)phenyl)propane-1-sulfonamide (43). Com-
pound 43 was prepared according to general procedure A using
65b (50 mg, 0.11 mmol), 4-nitrophenylboronic acid (20 mg,
0.12 mmol, 1.1 equiv.), P(t-Bu)3 Pd G4 (3 mg, 5%) and 1.5 M K2CO3
(0.25 mL, 0.38 mmol, 3.5 equiv.) in 1,4-dioxane (0.3 mL) at 115 ꢂC for
45 min. Flash-chromatography (SiO2, DCM/EtOAc 0e50%). Yield:
azolo[3,4-b]pyridine-5-yl)-N-ethylbenzenesulfonamide
(47).
Compound 47 was prepared according to general procedure A
using 65b (50 mg, 0.11 mmol), R2 (25 mg, 0.11 mmol, 1.0 equiv.),
P(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3 (0.25 mL, 0.38 mmol, 3.5
equiv.) in 1,4-dioxane (0.3 mL) at 120 ꢂC for 45 min. Flash-
chromatography (SiO2, DCM/EtOAc 10e50%), trituration with
DCM. Yield: 33 mg (55%). HPLC-purity: 100%. 1H NMR (400 MHz,
21 mg (38%). HPLC-purity: 100%. 1H NMR (200 MHz, DMSO)
d
24.19
DMSO)
d
14.97 (s, 1H), 9.82 (s, 1H), 9.10 (d, J ¼ 2.1 Hz, 1H), 8.86 (d,
(s, 1H),18.97 (s,1H),18.29 (d, J ¼ 2.0 Hz,1H),18.06 (d, J ¼ 2.1 Hz,1H),
17.53 (d, J ¼ 8.7 Hz, 2H), 17.32 (d, J ¼ 8.7 Hz, 2H), 16.79 (td, J ¼ 9.0,
5.8 Hz, 1H), 16.47 (t, J ¼ 8.7 Hz, 1H), 12.35e12.17 (m, 2H),
11.01e10.78 (m, 2H), 10.12 (t, J ¼ 7.4 Hz, 3H); 13C NMR (50 MHz,
J ¼ 2.1 Hz, 1H), 8.09 (d, J ¼ 8.3 Hz, 2H), 7.94 (d, J ¼ 8.5 Hz, 2H),
7.73e7.58 (m, 2H), 7.31 (t, J ¼ 8.7 Hz, 1H), 3.16e3.06 (m, 2H),
2.89e2.80 (m, 2H), 1.81e1.69 (m, 2H), 1.05e0.94 (m, 6H); 13C NMR
(101 MHz, DMSO)
d 182.5, 152.5, 149.8, 142.0, 140.8, 140.0, 131.4,
DMSO)
d
182.6, 159.7, 152.7, 149.9, 147.1, 143.8, 142.1, 130.7, 128.9,
128.3, 128.2, 127.3, 113.4, 53.8, 37.6, 16.8, 14.8, 12.5; ESI-HRMS: m/
z ¼ 562.10400, calcd for C24H23F2N5O5S2 m/z ¼ 562.10359 [M-H]-.
IR (ATR) [cmꢃ1] 1499, 1328, 1149, 895, 695, 558, 496.
128.8, 124.2, 113.4, 53.8, 16.9, 12.6; ESI-HRMS: m/z ¼ 500.08512,
calcd for C22H17F2N5O5S m/z ¼ 500.08457 [M-H]-. IR (ATR) [cmꢃ1
]
1670, 1515, 1490, 1345, 1328, 1245, 1137, 991, 850, 492.
N-((4-(3-(2,6-Difluoro-3-(propylsulfonamido)benzoyl)-1H-
pyrazolo[3,4-b]pyridine-5-yl)phenyl)sulfonyl)acetamide (48).
Compound 48 was prepared according to general procedure A
using 65b (50 mg, 0.11 mmol), R3 (46 mg, 0.14 mmol, 1.3 equiv.),
XPhos Pd G3 (5 mg, 5%) and 1.5 M K3PO4 (0.25 mL, 0.38 mmol, 3.5
equiv.) in 1,4-dioxane (0.5 mL) at 120 ꢂC for 30 min. Flash-
chromatography (SiO2, DCM/MeOH 5%). Yield: 27 mg (43%).
N-(2,4-Difluoro-3-(5-(4-(methylsulfony)phenyl)-1H-pyrazolo
[3,4-b]pyridine-3-carbonyl)phenyl)propane-1-sulfonamide
(44). Compound 44 was prepared according to general procedure
A using 65b (50 mg, 0.11 mmol), 4-methylsulfonylphenylboronic
acid (24 mg, 0.12 mmol, 1.1 equiv.), P(PPh3)4 (6 mg, 5%) and
1.5 M K2CO3 (0.25 mL, 0.38 mmol, 3.5 equiv.) in 1,4-dioxane
(0.3 mL) at 120 ꢂC for 0.5 h. Flash-chromatography (SiO2, DCM/
EtOAc 0e50%). Yield: 34 mg (58%). HPLC-purity: 100%. 1H NMR
HPLC-purity: 99%. 1H NMR (400 MHz, DMSO)
d 15.01 (s, 1H), 12.21
(s, 1H), 9.83 (s, 1H), 9.11 (d, J ¼ 2.1 Hz, 1H), 8.87 (d, J ¼ 2.1 Hz, 1H),
8.12 (d, J ¼ 8.5 Hz, 2H), 8.05 (d, J ¼ 8.5 Hz, 2H), 7.64 (td, J ¼ 9.0,
5.9 Hz, 1H), 7.32 (t, J ¼ 8.5 Hz, 1H), 3.11 (dd, J ¼ 5.7, 3.8 Hz, 2H), 1.96
(200 MHz, DMSO)
8.88 (d, J ¼ 2.2 Hz, 1H), 8.12 (q, J ¼ 8.7 Hz, 4H), 7.72e7.55 (m, 1H),
d
15.03 (s,1H), 9.83 (s,1H), 9.12 (d, J ¼ 2.2 Hz,1H),
16

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B. Pfaffenrot, P. Klovekorn, M. Juchum et al.
European Journal of Medicinal Chemistry 218 (2021) 113371
(s, 3H), 1.83e1.69 (m, 2H), 0.97 (t, J ¼ 7.5 Hz, 3H); 13C NMR
1H), 3.18e3.06 (m, 2H), 1.83e1.68 (m, 2H), 0.97 (t, J ¼ 7.5 Hz, 3H);
(101 MHz, DMSO)
d
182.5, 168.9, 152.5, 149.9, 142.1, 138.8, 131.2,
13C NMR (101 MHz, DMSO)
d 182.6, 167.4, 152.4, 149.8, 142.0, 139.8,
128.6, 128.3, 128.1, 113.4, 53.8, 23.2, 16.8, 12.5; ESI-HRMS: m/
z ¼ 576.08346, calcd for C24H21F2N5O6S2 m/z ¼ 576.08286 [M-H]-.
IR (ATR) [cmꢃ1] 1728, 1457, 1320, 1141, 866, 825, 537.
133.7, 132.0, 128.4, 127.9, 127.2, 113.5, 53.8, 16.8, 12.6; ESI-HRMS: m/
z ¼ 498.10575, calcd for C23H19F2N5O4S m/z ¼ 498.10531 [M-H]-. IR
(ATR) [cmꢃ1] 1686, 1490, 1411, 1324, 1141, 895, 500.
N-(2,4-Difluoro-3-(5-(4-(methylsulfonamido)phenyl)-1H-
pyrazolo[3,4-b]pyridine-3-carbonyl)phenyl)propane-1-
4-(3-(2,6-Difluoro-3-(propylsulfonamido)benzoyl)-1H-pyr-
azolo[3,4-b]pyridine-5-yl)-3-fluorobenzenesulfonamide
(53).
sulfonamide (49). Compound 49 was prepared according to gen-
eral procedure A using 65b (50 mg, 0.11 mmol), R4 (36 mg,
0.12 mmol, 1.1 equiv.), Pd(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3
(0.25 mL, 0.38 mmol, 3.5 equiv.) in 1,4-dioxane (0.3 mL) at 120 ꢂC
for 30 min. Flash-chromatography (SiO2, DCM/MeOH 20e60%).
Yield: 42 mg (69%). HPLC-purity: 99%. 1H NMR (200 MHz, DMSO)
Compound 53 was prepared according to general procedure A
using 65b (89 mg, 0.19 mmol), R5 (233 mg, 0.78 mmol, 4.1 equiv.),
XPhos Pd G4 (17 mg, 5%) and 1.5 M K2CO3 (0.78 mL, 0.16 mmol, 3.5
equiv.) in 1,4-dioxane (1 mL) at 130 ꢂC for 20 min. Flash-
chromatography (SiO2, DCM/MeOH 3e10%), trituration with DCM.
Yield: 27 mg (24%). HPLC-purity: 94%. 1H NMR (200 MHz, DMSO)
d
14.93 (s, 1H), 9.98 (s, 1H), 9.83 (s, 1H), 9.02 (d, J ¼ 2.2 Hz, 1H), 8.73
d 15.06 (s, 1H), 9.83 (s, 1H), 8.95 (s, 1H), 8.81 (s, 1H), 7.99 (t,
(d, J ¼ 2.1 Hz, 1H), 7.84 (d, J ¼ 8.6 Hz, 2H), 7.64 (td, J ¼ 9.0, 6.1 Hz,
J ¼ 8.0 Hz, 1H), 7.88e7.74 (m, 2H), 7.73e7.55 (m, 3H), 7.40e7.23 (m,
1H), 7.43e7.26 (m, 3H), 3.19e3.03 (m, 5H), 1.86e1.63 (m, 2H), 0.97
1H), 3.22e2.98 (m, 2H), 1.88e1.59 (m, 2H), 0.97 (t, J ¼ 7.4 Hz, 3H);
(t, J ¼ 7.4 Hz, 3H); 13C NMR (101 MHz, DMSO)
d
182.5, 152.1, 149.6,
ESI-HRMS: m/z
¼
552.06320, calcd for C22H18F3N5O5S2 m/
141.8, 138.5, 132.4, 128.3, 127.0, 120.0, 113.5, 53.8, 16.8, 12.5; ESI-
z ¼ 552.06287 [M-H]-. IR (ATR) [cmꢃ1] 1490, 1320, 1153, 895, 587,
HRMS: m/z
¼
548.08820, calcd for
C23H21F2N5O5S2 m/
492.
z ¼ 548.08794 [M-H]-. IR (ATR) [cmꢃ1] 1490, 1457, 1336, 1307, 1441,
4-(3-(2,6-Difluoro-3-(propylsulfonamido)benzoyl)-1H-pyr-
azolo[3,4-b]pyridine-5-yl)-3-methylbenzenesulfonamide (54). A
vessel was charged with 67 (75 mg, 0.13 mmol), R6 (35 mg,
0.14 mmol, 1.1 equiv.), XPhos Pd G3 (3 mg, 2.5%) and purged with
argon. Degassed 1,4-Dioxan (0.4 mL) and degassed 1.5 M K2CO3
(0.25 mL, 0.38 mmol, 3.5 equiv.) were added and the reaction was
stirred at 55 ꢂC for 1.5 h. The reaction was diluted with i-PrOH,
strongly acidified with conc. HCl and stirred at 70 ꢂC over night. The
reaction was neutralized with NaHCO3-solution and extracted with
EtOAc. After removal of the solvent, the product was isolated by
flash-chromatography (SiO2, DCM/EtOAc 10e50%) and triturated
with DCM. Yield: 41 mg (58%). HPLC-purity: 99%. 1H NMR
887, 820, 500.
N-(3-(5-(4-(1H-Tetrazol-5-yl)phenyl)-1H-pyrazolo[3,4-b]pyr-
idine-3-carbonyl)-2,4-difluorphenyl)propane-1-sulfonamide
(50). Compound 50 was prepared according to general procedure
A using 65b (50 mg, 0.11 mmol), 4-(1H-tetrazol-5-yl)phenylboronic
acid (23 mg, 0.12 mmol, 1.1 equiv.), XPhos Pd G4 (5 mg, 5%) and
1.5 M K2CO3 (0.25 mL, 0.38 mmol, 3.5 equiv.) in 1,4-dioxane
(0.3 mL) at 120 ꢂC for 30 min. Flash-chromatography (SiO2, DCM/
MeOH (þ1% formic acid) 5%). Yield: 45 mg (79%). HPLC-purity:
100%. 1H NMR (200 MHz, DMSO)
d 14.99 (s, 1H), 9.83 (s, 1H), 9.14
(d, J ¼ 2.1 Hz, 1H), 8.88 (d, J ¼ 2.2 Hz, 1H), 8.17 (dd, J ¼ 19.9, 8.4 Hz,
4H), 7.65 (td, J ¼ 9.0, 6.1 Hz, 1H), 7.32 (td, J ¼ 9.0, 1.4 Hz, 1H),
3.20e3.05 (m, 2H), 1.90e1.59 (m, 2H), 0.97 (t, J ¼ 7.4 Hz, 3H); 13C
(400 MHz, DMSO)
d
15.00 (s, 1H), 9.82 (s, 1H), 8.76 (d, J ¼ 2.0 Hz,
1H), 8.55 (d, J ¼ 1.9 Hz, 1H), 7.84 (s, 1H), 7.78 (dd, J ¼ 8.0, 1.3 Hz, 1H),
7.67e7.56 (m, 2H), 7.44 (s, 2H), 7.31 (t, J ¼ 8.7 Hz, 1H), 3.16e3.07 (m,
2H), 1.80e1.69 (m, 2H), 0.97 (t, J ¼ 7.4 Hz, 3H); 13C NMR (101 MHz,
NMR (101 MHz, DMSO)
d
182.5, 156.29 (dd, J ¼ 248.3, 6.4 Hz), 152.8
(dd, J ¼ 251.2, 8.2 Hz),152.4, 149.8, 142.0, 139.7, 131.7, 129.9, 129.8,
128.3, 127.9, 127.7, 127.6, 123.8, 121.79 (dd, J ¼ 13.4, 3.4 Hz), 117.1,
113.5, 112.12 (dd, J ¼ 22.6, 4.3 Hz), 53.8, 16.8, 12.5; ESI-HRMS: m/
z ¼ 523.11225, calcd for C23H18F2N8O3S m/z ¼ 523.11179 [M-H]-. IR
(ATR) [cmꢃ1] 1481, 1436, 1153, 899, 845, 749, 499.
DMSO)
d 182.5, 152.0, 150.7, 143.7, 141.8, 141.0, 136.6, 132.4, 130.9,
129.9, 127.3, 123.3, 113.0, 53.8, 20.1, 16.8, 12.5; ESI-HRMS: m/
z ¼ 548.08844, calcd for C23H21F2N5O5S2 m/z ¼ 548.08794 [M-H]-.
IR (ATR) [cmꢃ1] 1495, 1320, 1128, 899, 583, 504.
4-(3-(2,6-Difluoro-3-(propylsulfonamido)benzoyl)-1H-pyr-
azolo[3,4-b]pyridine-5-yl)benzoic acid (51). Compound 51 was
prepared according to general procedure A using 65b (50 mg,
0.11 mmol), 4-carboxyphenylboronic acid (23 mg, 0.12 mmol, 1.1
equiv.), XPhos Pd G4 (5 mg, 5%) and 1.5 M K2CO3 (0.33 mL,
0.49 mmol, 4.5 equiv.) in 1,4-dioxane (0.4 mL) at 120 ꢂC for 30 min.
Flash-chromatography (SiO2, DCM/MeOH (þ1% formic acid) 5%).
Yield: 41 mg (75%). HPLC-purity: 99%. 1H NMR (200 MHz, DMSO)
3-Chloro-4-(3-(2,6-difluoro-3-(propylsulfonamido)benzoyl)-
1H-pyrazolo[3,4-b]pyridine-5-yl)benzensulfonamide (55).
A
vessel was charged with 67 (71 mg, 0.12 mmol), R7 (39 mg,
0.14 mmol, 1.1 equiv.), Pd(PPh3)4 (7 mg, 5%) and purged with argon.
Degassed 1,4-Dioxan (0.4 mL) and degassed 1.5 M K2CO3 (0.24 mL,
0.38 mmol, 3.5 equiv.) were added and the reaction was stirred at
55 ꢂC for 1 h. The reaction was diluted with i-PrOH, strongly acid-
ified with conc. HCl and stirred at 70 ꢂC over night. The reaction was
neutralized with NaHCO3-solution and extracted with EtOAc. After
removal of the solvent, the product was isolated by flash-
chromatography (SiO2, DCM/EtOAc 20e60%). Yield: 23 mg (31%).
d
14.99 (s,1H),13.14 (bs,1H), 9.83 (s,1H), 9.10 (d, J ¼ 2.1 Hz,1H), 8.84
(d, J ¼ 2.2 Hz, 1H), 8.04 (dd, J ¼ 22.3, 8.5 Hz, 4H), 7.64 (td, J ¼ 9.0,
5.9 Hz,1H), 7.31 (td, J ¼ 8.9,1.5 Hz,1H), 3.21e3.03 (m, 2H),1.86e1.61
(m, 2H), 0.97 (t, J ¼ 7.4 Hz, 3H); 13C NMR (101 MHz, DMSO)
d
182.5,
HPLC-purity: 94%. 1H NMR (200 MHz, DMSO)
d
8.82 (d, J ¼ 2.0 Hz,
167.0, 152.4, 149.8, 142.0, 141.3, 131.8, 130.2, 130.1, 128.1, 127.6, 113.5,
1H), 8.69 (d, J ¼ 1.8 Hz, 1H), 7.96e7.81 (m, 2H), 7.74e7.55 (m, 3H),
7.31 (t, J ¼ 8.7 Hz, 1H), 3.19e3.05 (m, 3H), 1.87e1.62 (m, 2H), 0.97 (t,
53.8, 16.8, 12.5; ESI-HRMS: m/z
C
1499, 1145, 895, 766, 571, 504.
¼
499.08935, calcd for
23H18F2N4O5S m/z ¼ 499.08932 [M-H]-. IR (ATR) [cmꢃ1] 1686,
J ¼ 7.5 Hz, 3H); 13C NMR (101 MHz, DMSO)
d 183.1, 152.7, 151.3,
145.9, 142.5, 140.2, 133.4, 132.9, 131.2, 130.6, 127.3, 125.2, 122.4,
4-(3-(2,6-Difluoro-3-(propylsulfonamido)benzoyl)-1H-pyr-
azolo[3,4-b]pyridine-5-yl)benzamide (52). Compound 52 was
prepared according to general procedure A using 65b (50 mg,
0.11 mmol), 4-carbamoylphenylboronic acid (20 mg, 0.12 mmol, 1.1
equiv.), XPhos Pd G4 (5 mg, 5%) and 1.5 M K2CO3 (0.25 mL,
0.38 mmol, 3.5 equiv.) in 1,4-dioxane (0.4 mL) at 120 ꢂC for 30 min.
Flash-chromatography (SiO2, DCM/MeOH 2e15%). Yield: 34 mg
113.3, 54.4, 17.3, 13.1; ESI-HRMS: m/z ¼ 568.03364, calcd for
C
22H18F2N5O5S2 m/z ¼ 568.03332 [M-H]-. IR (ATR) [cmꢃ1] 1486,
1324, 1141, 1095, 899, 492.
4-(3-(2,6-Difluoro-3-(methylsulfonamido)benzoyl)-1H-pyr-
azolo[3,4-b]pyridine-5-yl)benzenesulfonamide (56) Compound
56 was prepared according to general procedure A using 65a
(50 mg, 0.12 mmol), 4-sulfamoylphenylboronic acid (26 mg,
0.13 mmol, 1.1 equiv.), P(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3
(0.27 mL, 0.41 mmol, 3.5 equiv.) in 1,4-dioxane (0.4 mL) at 120 ꢂC
for 0.5 h. Flash-chromatography (SiO2, DCM/EtOAc 50e100%),
(60%). HPLC-purity: 96%. 1H NMR (400 MHz, DMSO)
d 14.97 (s, 1H),
9.83 (s, 1H), 9.10 (d, J ¼ 1.3 Hz, 1H), 8.83 (d, J ¼ 1.3 Hz, 1H), 8.13e7.92
(m, 5H), 7.64 (td, J ¼ 8.9, 6.2 Hz, 1H), 7.46 (s, 1H), 7.32 (t, J ¼ 8.7 Hz,
17

€
B. Pfaffenrot, P. Klovekorn, M. Juchum et al.
European Journal of Medicinal Chemistry 218 (2021) 113371
trituration with DCM. Yield: 37 mg (62%). HPLC-purity: 98%. 1H
Funding sources
NMR (200 MHz, DMSO)
d 14.99 (s, 1H), 9.85 (s, 1H), 9.10 (d,
J ¼ 2.1 Hz, 1H), 8.86 (d, J ¼ 2.1 Hz, 1H), 8.03 (dd, J ¼ 20.8, 8.4 Hz, 4H),
This work was supported by the Deutsche For-
schungsgemeinschaft (DFG, German Research Foundation) under
Germany’s excellence strategy e EXC 2180e390900677 [Image
Guided and Functionally Instructed Tumour Therapies“ (iFIT)]
TüCAD2 is funded by the Federal Ministry of Education and
Research (BMBF) and the Baden-Württemberg Ministry of Science
as part of the Excellence Strategy of the German Federal and State
Governments.
7.65 (td, J ¼ 9.1, 6.1 Hz, 1H), 7.48 (s, 2H), 7.33 (t, J ¼ 8.8 Hz, 1H), 3.08
(s, 3H); 13C NMR (101 MHz, DMSO)
d
182.5, 156.40 (dd, J ¼ 248.5,
6.7 Hz), 153.0 (dd, J ¼ 251.5, 8.0 Hz), 152.4, 149.8, 143.5, 142.0, 140.3,
131.5, 130.0, 129.9, 128.2, 127.9, 126.4, 121.8 (dd, J ¼ 13.3, 3.5 Hz),
117.13 (dd, J ¼ 23.0, 21.5 Hz), 113.4, 112.15 (dd, J ¼ 22.1, 3.1 Hz), 40.4;
ESI-HRMS: m/z
¼
506.04162, calcd for C20H15F2N5O5S2 m/
z ¼ 506.04099 [M-H]-.IR (ATR) [cmꢃ1] 1686, 1499, 1424, 1332, 1311,
1145, 904, 546, 500.
4-(3-(2,6-Difluoro-3-(butylsulfonamido)benzoyl)-1H-pyr-
azolo[3,4-b]pyridine-5-yl)benzenesulfonamide (57). Compound
57 was prepared according to general procedure A using 65c
(50 mg, 0.11 mmol), 4-sulfamoylphenylboronic acid (23 mg,
0.12 mmol, 1.1 equiv.), P(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3
(0.25 mL, 0.37 mmol, 3.5 equiv.) in 1,4-dioxane (0.4 mL) at 120 ꢂC
for 0.5 h. Flash-chromatography (SiO2, DCM/EtOAc 20e60%), trit-
uration with DCM. Yield: 37 mg (62%). HPLC-purity: 98%. 1H NMR
Declaration of competing interest
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.
Acknowledgment
(200 MHz, DMSO)
d
15.00 (s,1H), 9.82 (s,1H), 9.10 (d, J ¼ 2.1 Hz,1H),
We thank E. Rist for technical support and K. Schmidt for lan-
guage proof-reading.
8.86 (d, J ¼ 2.0 Hz, 1H), 8.13e7.93 (m, 4H), 7.64 (td, J ¼ 9.0, 6.2 Hz,
1H), 7.48 (s, 2H), 7.32 (td, J ¼ 8.9, 1.2 Hz, 1H), 3.21e3.07 (m, 2H),
1.82e1.61 (m, 2H), 1.50e1.27 (m, 2H), 0.85 (t, J ¼ 7.2 Hz, 3H); 13C
Appendix A. Supplementary data
NMR (101 MHz, DMSO)
d
182.5, 156.3 (dd, J ¼ 248, 6 Hz), 152.7 (dd,
J ¼ 251, 9 Hz), 152.4, 149.8, 143.5, 142.0, 140.3, 131.5, 129.9, 129.8,
128.2, 127.9, 127.5, 126.4, 126.3, 121.8 (dd, J ¼ 13, 4 Hz), 117.1 (dd,
J ¼ 23, 21 Hz), 113.4, 112.2, 112.2, 112.0, 51.8, 25.1, 20.7, 13.4. ESI-
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113371.
HRMS: m/z
¼
548.08853, calcd for
C23H21F2N5O5S2 m/
Supporting information
z ¼ 548.08794 [M-H]-. IR (ATR) [cmꢃ1] 1486, 1323, 1161, 1132, 890,
695, 545.
Experimental procedures for the synthesis of intermediates R1
e R12, results from scanEDGE kinase profiling for 58, representa-
tive NMR spectra for compounds 61e67 and 58.
4-(3-(2,6-Difluoro-3-((phenylmethyl)sulfonamido)benzoyl)-
1H-pyrazolo[3,4-b]pyridine-5-yl)benzenesulfonamide
(58).
Compound 58 was prepared according to general procedure A
using 65d (50 mg, 0.10 mmol), 4-sulfamoylphenylboronic acid
(22 mg, 0.11 mmol, 1.1 equiv.), P(PPh3)4 (6 mg, 5%) and 1.5 M K2CO3
(0.23 mL, 0.35 mmol, 3.5 equiv.) in 1,4-dioxane (0.4 mL) at 120 ꢂC
for 0.5 h. Flash-chromatography (SiO2, DCM/EtOAc 20e60%), trit-
uration with DCM. Yield: 30 mg (49%). HPLC-purity: 98%. 1H NMR
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Author contributions
All authors contributed to writing the article. All authors have
approved the final version of the article.
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