Design and synthesis of 1H-indazole-3-carboxamide derivatives as potent and selective PAK1 inhibitors with anti-tumour migration and invasion activities

Article abstract of DOI:10.1016/j.ejmech.2020.112517

Aberrant activation of p21-activated kinase 1 (PAK1) is associated with tumour progression, and PAK1 has been recognized as a promising target for anticancer drug discovery. However, the development of potent PAK1 inhibitors with satisfactory kinase selec

Full text of DOI:10.1016/j.ejmech.2020.112517

Journal Pre-proof
Design and synthesis of 1H-indazole-3-carboxamide derivatives as potent and
selective PAK1 inhibitors with anti-tumour migration and invasion activities
Mingliang Zhang, Xiaobao Fang, Cong Wang, Chen Huang, Meng Wang, Lu Zhu,
Zixu Wang, Yuhan Gao, Tianyi Zhang, Haichun Liu, Yanmin Zhang, Shuai Lu, Tao
Lu, Yadong Chen, Hongmei Li
PII:
S0223-5234(20)30489-X
DOI:
https://doi.org/10.1016/j.ejmech.2020.112517
EJMECH 112517
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 January 2020
Revised Date: 15 May 2020
Accepted Date: 29 May 2020
Please cite this article as: M. Zhang, X. Fang, C. Wang, C. Huang, M. Wang, L. Zhu, Z. Wang, Y.
Gao, T. Zhang, H. Liu, Y. Zhang, S. Lu, T. Lu, Y. Chen, H. Li, Design and synthesis of 1H-indazole-3-
carboxamide derivatives as potent and selective PAK1 inhibitors with anti-tumour migration and invasion
activities, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112517.
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Graphical Abstract

Design and synthesis of 1H-indazole-3-carboxamide derivatives as potent and
selective PAK1 inhibitors with anti-tumour migration and invasion activities
Mingliang Zhanga, Xiaobao Fanga, Cong Wanga, Chen Huanga, Meng Wanga, Lu Zhua,
Zixu Wanga, Yuhan Gaoa, Tianyi Zhanga, Haichun Liua, Yanmin Zhanga, Shuai Lua,
Tao Lua,b*, Yadong Chena,b*, Hongmei Lia*
a
Laboratory of Molecular Design and Drug Discovery, China Pharmaceutical
University, Nanjing 211198, P.R. China.
b
State Key Laboratory of Natural Medicines, China Pharmaceutical University,
Nanjing 210009, P.R. China
Abstract
Aberrant activation of p21-activated kinase 1 (PAK1) is associated with tumour
progression, and PAK1 has been recognized as a promising target for anticancer drug
discovery. However, the development of potent PAK1 inhibitors with satisfactory
kinase selectivity and favourable physicochemical properties remains a daunting
challenge. Herein, we identified the 1H-indazole-3-carboxamide derivatives as
potential PAK1 inhibitors using a fragment-based screening approach. The
representative compound 30l exhibited excellent enzyme inhibition (PAK1 IC50 = 9.8
nM) and high PAK1 selectivity toward a panel of 29 kinases. The Structure-activity
relationship (SAR) analysis showed that substituting of an appropriate hydrophobic
ring in the deep back pocket and introducing a hydrophilic group in the bulk solvent
region were critical for PAK1 inhibitory activity and selectivity. Additionally, the
hERG channel activity of 30l demonstrated its low risk of hERG toxicity.
Furthermore, it significantly suppressed the migration and invasion of MDA-MB-231
cells by downregulating Snail expression without affecting the tumour growth. These
results provide a new type of chemical scaffolds targeting PAK1 and suggested that
1H-indazole-3-carboxamide derivatives may serve as lead compounds for the
development of potential and selective PAK1 inhibitors.
Keywords: PAK1 inhibitor, kinase selectivity, 1H-indazole-3-carboxamide scaffold,
anti-tumour metastasis

1. Introduction
The p21-activated kinases (PAKs) are Ser/Thr kinases in the STE20 kinase
family that function as downstream signalling effectors of the Rho/Rac family of
GTPases [1-3]. This family consists of six subtypes and is subdivided into two groups
based on their structural homology and regulatory functional characteristics: group I,
including PAK1, PAK2 and PAK3, and group II, including PAK4, PAK5 and PAK6.
Although these kinases show a high level of structural homology, their tissue
expression, subcellular localization, activation mechanism and downstream substrate
specificity are not consistent in various physiological processes. As key components
of the Rho family, PAKs govern many fundamental cellular process, including
cytoskeletal remodelling, morphological variation and cell migration during cancer
progression [4].
Among the group I PAK family, PAK1 has been implicated in multiple
oncogenic signalling pathways and is frequently overexpressed in malignancies such
as breast, colon and bladder cancers, leading to the poor prognosis [5-7].
Phosphorylation and subsequent activation of PAK1 regulate the PI3K/Akt and
Raf/MEK/ERK pathways [8, 9], which transduces the signal downstream and thus
contributes to tumour progression. Importantly, increased expression and/or activation
of PAK1 induce the epithelial-mesenchymal transition (EMT) by activating the zinc
transcription factor [10, 11]. PAK1 predominantly promotes the transcriptional
repression activity of Snail from E-cadherin promoters and results in cancer
metastasis [12]. Therefore, PAK1 has emerged as a promising target for cancer
therapeutics due to its liaison roles in tumor malignancy [13, 14].
Researchers have expressed considerable interest in the development of
small-molecule inhibitors targeting PAK1 in recent years (Fig. 1). PF-3578309 (1), a
pan-PAK inhibitor, was the first compound advanced to phase I trials (NCT00932126),
but was not progressed due to undesirable pharmacokinetic characteristics and
adverse events [15]. Compound 2 and compound 3 (FRAX1036) containing
aminopyrazole or pyrido[2,3-d]pyrimidine-7-one scaffolds, respectively, displayed
potential as group I PAK-selective inhibitors with exquisite kinase selectivity (more
than 100-fold) [3, 16]. Since selective inhibitors of PAK1 might exert advantageous
effects compared with pan inhibitors from a certain medicinal perspective, these
compounds have been used as probe compounds to evaluate group I PAK biology [17-
19]. Nevertheless, the poor anti-tumour effectiveness at the cellular level has
restrained the extensive application of these inhibitors [17]. Recently, Genentech

modified the pyrido[2,3-d]pyrimidine-7-one scaffold by introducing basic amine
group to obtain the compounds G5555 (4) [20] and G9791 (5) [21], which showed
high PAK1 selectivity and satisfactory cellular potency. However, their
pharmacokinetics and anti-tumour efficiency in vivo have not yet been disclosed. With
the exception of the aminopyrazole and pyrido[2,3-d]pyrimidine-7-one scaffolds,
other chemotypes have rarely been reported. The similar sequences between PAK
isoforms also prevent the development of a PAK1-selective inhibitor that does not
block PAK4 activity. Thus, the discovery of alternative chemotypes of potent and
highly selective inhibitors are urgently needed to exploit the functions of PAK1 and
treat its related disease.
Fig. 1. Structures of representative PAK1 inhibitors and general structure of the compounds
designed in this study.
In the present study, we reported a new structural chemotype containing the
6-(2-chlorophenyl)-1H-indazole-3-carboxamide scaffold for inhibitory activity and
isoform selectivity of PAK1 by a fragment-based virtual screening strategy. The
design, synthesis and structure-activity relationship studies that led to the discovery of
representative compound 30l are described. Furthermore, we also explored the
anti-tumour effects of the 30l on the proliferation and migration/invasion, as well as
the possible mechanism of action. It is expected that 30l derivates can be promising
scaffolds for development of PAK1 selective inhibitors.

2. Chemistry
The synthesis route of the THP-protected 1H-indazole-3-carboxylate scaffold is
described in Scheme 1. Compound 8 was obtained by treating the available
compound 7 using a three-step procedure: cleavage with aqueous sodium hydroxide,
diazotization with aqueous sodium nitrite in hydrochloric acid, and finally the
reduction and cyclization of the intermediate diazonium salts with a cooled solution of
tin(II) chloride dihydrate in hydrochloric acid. Then, the product was esterified to
obtain compound 9 under reflux conditions in methanol, which was converted to the
THP-protected intermediate 10 [22, 23].
Scheme 1. Reagents and conditions: (a) (i) NaOH, H2O, 50 °C, 1 h; (ii) HCl, NaNO2, H2O, 0 °C, 1
h; (iii) SnCl2, HCl, 25 °C, 2 h. (b) SOCl2, MeOH, 65 °C, 5 h. (c) TsOH, CH3CN, 25 °C, 2 h.
The synthesis route for compound 6 is shown in Scheme 2. Compound 12 was
obtained from the starting material through the Suzuki coupling reaction [24]. Then, it
was converted to the amide 14 through hydrolysis and amide coupling reactions.
Deprotection using HCl cleavage of the hemiaminal intermediate afforded the final
compound 6 [25].
Scheme 2. Reagents and conditions: (a) (2-chlorophenyl)boronic acid, Pd(dppf)Cl2, K2CO3,
dioxane, H2O, 80 °C, 2 h; (b) NaOH, MeOH, H2O, 65 °C, 2 h; (c) CH3NH2, HATU, DIEA, DMF,
25 °C, 12 h; (d) HCl, MeOH, 65 °C, 12 h.
The synthesis routes of compounds 23a-d are described in Scheme 3. Compound
15 was used to produce compound 17 in a 2-step Suzuki coupling reaction, which was
then diazotized and brominated to yield compound 18 [26]. Then, compound 19 was
prepared from compound 18 through the Suzuki coupling reaction. The final

compounds 23a-d were prepared using a method similar to the procedure described
for compound 6.
Scheme 3. Reagents and conditions: (a) bis(pinacolato)diboron, AcOK, Pd(dppf)Cl2, dioxane,
110 °C, 12 h; (b) Pd(PPh3)4, K2CO3, dioxane, H2O, 80 °C, 2 h; (c) (i) HBr, NaNO2, 0 °C, 1 h and
(ii) CuBr, HBr, 70 °C 2 h; (d) bis(pinacolato)diboron, AcOK, Pd(dppf)Cl2, dioxane, 110 °C, 12 h;
(e) Pd(PPh3)4, K2CO3, dioxane, H2O, 80 °C, 2 h; (f) NaOH, MeOH, H2O, 65 °C, 2 h; (g) R1NH2,
HATU, DIEA, DMF, 25 °C, 12 h; (h) HCl, MeOH, 65 °C, 12 h.
The synthesis routes of compounds 30a-q are shown in Scheme 4. Compound 24
was synthesized from compound 15 using the Ullmann coupling reaction [27]. Then,
it was diazotized and brominated to obtain compound 25, which was installed using
Suzuki coupling to obtain compound 26 [28, 29]. The final compounds 30a-r were
prepared using a method similar to the procedure used for compound 6.

Scheme 4. Reagents and conditions: (a) CuI, N,N-dimethyl-1,2-ethanediamine, K3PO4, dioxane,
120 °C, 24 h; (b) (i) HBr, NaNO2, 0 °C, 1 h (ii) CuBr, HBr, 70 °C, 2 h; (c) AcOK, Pd(dppf)Cl2,
bis(pinacolato)diboron, dioxane, 110 °C, 12 h; (d) Pd(dppf)Cl2, K2CO3, dioxane, H2O, 80 °C, 2 h;
(e) NaOH, MeOH, H2O, 65 °C, 2 h; (f) R1NH2, HATU, DIEA, DMF, 25 °C, 12 h; (g) HCl, MeOH,
65 °C, 12 h.
3. Results and Discussion
3.1. Hit identification and binding mode analysis

Fig. 2. (A) Predicted binding modes of FRAX1036 (3) and compound 6 in the ATP-binding site of
PAK1. Overlay of compound 6 (cyan) and FRAX1036 (3) (yellow) bound in the PAK1 kinase
active site (PDB code 5DEY). The ligands and important residues are shown in stick form. The
hydrogen bonds are shown as red dashed lines. (B) A schematic illustrating the interaction
between compound 3 and the surrounding residues within the binding pocket of PAK1. (C)
Strategy used to design the series of 1H-indazole-3-carboxamide derivatives of PAK1 inhibitors.
An in-house hit fragment library consisting of 235 diverse fragment molecules
was subjected to a virtual screening schedule for generating the starting molecule
targeting PAK1 (Fig. S1). After a cluster analysis and visual inspection, 21
representative compounds (for details see Supplementary Material Table S2) with
reasonable poses, Glide-Score value, physicochemical properties (i.e. molecular
weight, tipological polar surface areas, numbers of hydrogen bond acceptor and donor
groups) and structure accessible were selected for further biological evaluation. In
addition, these compounds passed the pan-assay interference compound (PAINS)
substructure filtration. Among them, Hit 5, Hit 10 and Hit 12 showed potent inhibition
activity against PAK1 with the inhibition ratio of higher than 80% at 10 μM and were
regarded as active scaffolds for PAK1. However, the structural modification space of
Hit 5 is limited as pyrrole group is not convenient for introducing complementary
chemical groups. B. Yang has reported the structural analogs of Hit 10 as PAK1
inhibitor [30]. Therefore, the 6-(2-chlorophenyl)-1H-indazole-3-carboxamide indazole
scaffold (6) was a suitable hit compound due to its novel structure and large chemical
space for modification. From the docking prediction, compound 6 formed conserved
hydrogen bonds with the PAK1 kinase hinge residues Glu345 and Leu347 (Fig. 2A),
while the aromatic ring of the chlorophenyl moiety was refined in the hydrophobic
side chains consisting of the Met344 gatekeeper residue and Lys299 catalytic residue.
These results prompted us to choose compound 6 as a starting point for optimization.
3.2. Scaffold modification and analysis of the structure-activity relationship
Table 1. Optimization of the substitutions on the hydrophobic moiety.
PAK1
Compound
R1
R2
H
clogP b
3.6
tPSA(Å) c
53.5
IC50 (μM) a
-NHCH3
6
5

-NHCH3
-NHCH3
23a
23b
23c
23d
30a
30b
30c
30d
0.159
0.052
0.777
0.229
0.016
0.330
0.034
0.590
3.7
4.7
4.7
5.7
3.9
3.4
4.9
4.4
78.2
65.9
78.2
65.9
73.8
73.8
73.8
73.8
-NHCH3
-NHCH3
FRAX1036
-
-
-
-
0.023 e
(0.0006) f
0.052/
3.6
3.7
53.5
69.8
Staurosporine d
PF-3758309 d
-
-
4.3
98.3
(0.036) f
a IC50 values were determined using Kinase HotSpot Profiling assays. b The clogP values were
calculated using the Qikprop software with the default settings (pH = 7.0). c Topological polar
surface area. d used as a positive control [31]. e Ki values (nM). f reported data [31].
To design more potent derivatives, the binding modes of compound 6 and
FRAX1036 in PAK1 were overlaid. We discerned that the hydrophobic moiety at the
6-position on the pyrido[2,3-d] pyrimidin-7-one of FRAX1036 was important for its
binding to PAK1. Hence, we introduced similar moieties in the pyrazine moiety of
FRAX1036 into compound 6 and the modified hydrophobic group was subjected to
SAR analyses (Table1) to improve the inhibitory potency against PAK1 kinase (Fig.
2C).

We first incorporated hydrophobic moieties onto the chlorophenyl group of
compound 6 to enlarge the fragment on the right side. The introduction of a
5-methyl-2-pyrazinyl group (23a) significantly increased the enzymatic potency (IC50
= 159 nM) compared with compound 6 (Table 1), consistent with our hypothesis
about the importance of a hydrophobic group at the 4-position of the 2-chloro-phenyl
ring (Fig. 2A and 2B). Subsequently, substitutions at the position including
5-methyl-2-pyridinyl (23b) and 5-methyl-2-pyridone (30a) groups slightly increased
the enzymatic activities (IC50 = 52 and 16 nM, respectively), implying that terminal
hydrophobic ring might contribute to the PAK1 activity. Among these compounds,
30a showed the most effective inhibition, with an IC50 value of 16 nM. The methyl
substituent showed improved activity by filling a small hydrophobic pocket beside the
ATP binding area during the optimization of the FRAX1036 structure [20]. We then
removed the methyl group at the 3-position of pyridone to confirm the importance of
the potential pocket. As expected, the elimination of the methyl group resulted in a
slight decrease in the inhibitory potency against PAK1 (IC50 = 330 nM) compared to
compound 30a. While investigating substitutions of the R1 position with a
cyclopropylmethylene amine group, the compounds displayed similar or slightly
decreased potency compared to that with the methyl amine group. Therefore, the
3-methylpyridone group was deemed best suited to reach a desirable enzyme potency
during the optimization of hydrophobic groups in the indazole derivatives. We
retained the 3-methylpyridone for further structural modification.
We docked compound 30a into the ATP binding site of PAK1 to obtain insights
into the putative binding mode of this inhibitor with the PAK1 kinase. As shown in
Fig. 3A, obvious hydrogen bonds between the kinase hinge residues Glu345 and
Leu347 of PAK1 were observed and identified as important interactions contributing
to the inhibition of PAK1 activity. The chlorophenyl moiety fits suitably in the cleft
formed by the hydrophobic chains of the Met344 gatekeeper residue, and the
3-methylpyridone moiety protrudes into the deep back pocket of the ATP binding site
surrounded by Glu315, Ile316, and Val342. Additionally, the 3-methylpyridone group
also forms a hydrogen bond with the catalytic Ly299 residue, thus improving the
inhibitory potency against PAK1. Since the amide group is directed toward the bulk
solvent pocket, the activity may be modulated through the optimization of this
position for further drug development. Therefore, a group extending from the
1H-indazole-3-carboxamide indazole scaffold would be exposed to the solvent area
and balance the inhibitory activity and drug-like properties.

Fig. 3. (A) Predicted binding mode of compound 30a in the ATP-pocket of PAK1 (PDB code
5DEY). The ligands and important residues are shown in stick form. The hydrogen bonds are
shown as red dashed lines. (B) A schematic illustrating the interactions between compound 30a
and the surrounding residues within the binding site of PAK1.
Based on compound 30a, we next turned our attention to further study the SAR
of amide groups (Table 2). Various amines were utilized to replace the methyl amine
in compound 30a. Compounds 30e-30g with ethyl amide, 2-hydroxyethy amide or
2-methoxyethyl amide groups were 3-5-fold less potent inhibitors of PAK1 than
compound 30a. However, the PAK1 inhibitory potency nearly completely disappeared
in compounds containing the cyclic secondary amines (30h-30i). We speculated that
the amide NH group would function as a crucial hydrogen bond donor and interact
with Leu347 in the hinge region of PAK1. As shown in Fig. S2, compounds 30h-30i
lacked a key hydrogen bond in the hinge region, resulting a direct loss of activity of
these compounds. Interestingly, compounds 30n and 30o regained their enzyme
inhibitory activity (IC50 = 744 nM and 143 nM, respectively) upon the reintroduction
of a primary amine group. Based on these results, the amide NH group was beneficial
for increasing the potency, and the solvent area of the compound also participated in
PAK1 activity. Subsequently, the length of the linker was explored by comparing
compounds 30p and 30q. The inclusion of the methylpiperidine group appeared to
increase the PAK1 inhibitory activity (IC50 = 22 nM), suggesting that the flexibility of
the attached group might improve PAK1 inhibition upon the introduction of the cyclic
hydrophilic moiety. The saturated alkyl ring was replaced with pyridyl amine groups
in compounds 30k-30m to further explore the effect of the aromatic substituent.These
compounds exhibited similar or improved inhibitory potencies (IC50 = 16 nM, 9.8 nM
and 21 nM, respectively) compared to 30a. However, the replacement of the pyridine
group with a phenyl hydrophilic group led to 10-fold decrease in potency (30j). The
potency of this analogue was lost due to the requirement for a highly polar group in

the PAK1 solvent pocket. Finally, compounds 30a, 30c, 30e and 30l displayed
excellent enzyme potency and would be candidates for a detailed evaluation.
Table 2. Studies of the structure-activity relationship after replacing the methyl amine group with
various amine derivatives in the hydrophilic moiety
PAK1
IC50 (μM) a
Compound
R
clogP b
tPSA (Å) c
-NHC2H5
30e
30f
0.045
4.5
3.4
4.1
4.1
73.8
94.0
83.0
65.0
-NHC2H5OH
-NHC2H5OCH3
0.066
0.071
＞10
30g
30h
30i
30n
30o
30p
30q
30k
30l
＞10
0. 744
0.143
0.042
0.022
0.016
0.0098
0.021
3.6
3.6
4.0
4.2
4.6
4.7
4.4
4.5
74.2
83.0
77.0
83.0
77.0
86.2
86.2
86.2
30m
30j
0.092
0.023 e
(0.0006) f
0.052
5.9
3.6
3.7
4.3
73.8
53.5
69.8
98.3
FRAX1036
Staurosporine d
PF-3758309 d
-
-
-

(0.036) f
a IC50 values were determined using Kinase HotSpot Profiling assays. b The clogP values were
calculated using Qikprop software with the default settings (pH = 7.0). c Topological polar surface
area. d used as a positive control. e Ki values (nM). f reported data [31].
3.3. PAK subtype selectivity and hERG inhibition
Fig. 4. Sequence alignment of the kinase domains of PAK1 and PAK4. Identical residues between
the two kinases are highlighted in green boxes. Different residues are shown with a white
background. The red stars label key residues of the hinge regions in PAK1 and PAK4. The helices
are shown in red and β strands are shown in cyan.
Sequences anslysis of the PAK1 and PAK4 kinase domains revealed that PAK1
shared 72% sequence similarity with PAK4 in the kinase domain (Fig. 4). As was
mentioned before, PAK1 subtype-selective inhibitors possess pharmacological
advantages over pan-PAK inhibitors. We then examined the kinase subtype selectivity
for potent compounds. As shown in Table 3, these compounds were highly active
PAK1 inhibitors, but did not inhibit PAK4 (IC50 > 10 μM). The four compounds
exhibited comparable subtype selectivity to FRAX1036, while compound 30l
displayed a more favourable PAK1 specificity (1000-fold greater over PAK4).
Table 3. Selectivity and hERG profiles of selected compounds.
hERG
PAK1
IC50(μM) a
PAK4
PAK1
tPSA
(Å) d
%inh.
Compound
clogP c
IC50(μM) a Select.index b
@ 10 μM
/IC50(μM)
30a
30c
30e
30p
0.016
0.034
0.045
0.042
＞10
＞10
＞10
ND e
＞625x
＞290x
＞200x
ND
21.0
82.3
90.4
42.1
3.9
4.9
4.5
4.2
73.8
73.8
73.8
83.0

30l
0.010
＞10
2.4 f
＞1000x
105x g
10x
16.0
89% h
ND
4.4
3.6
3.7
86.2
53.5
69.8
FRAX1036
0.023 f
Staurosporine i (0.0006) h
(0.06) h
0.038/
(0.015) h
0.052/
PF-3758309 i
0.73x
0.42x
41.1
4.3
98.3
(0.036) h
a IC50 values were determined using Kinase HotSpot Profiling assays. b PAK4 IC50/ PAK1 IC50, x =
fold. c The clogP values were calculated using Qikprop software with the default settings (pH =
d
f
g
7.0). Topological polar surface area. e ND = not determined. Ki values (nM) [31]. PAK4 Ki/
PAK1 Ki, x = fold. h reported data [31]. i used as a positive control.
3.4. Kinase selectivity assessment
We further screened the selectivity of 30l against a panel of 29 kinases including
PAK1 signal pathways-related, the high structural homology of PAK1 and wide-used
marketed drug-related kinase using the Kinase HotSpot Profiling assays (Table 4). It
was found that compound 30l did not show obvious inhibition on these tested kinases
except that it displayed moderate inhibition on PAK2, PAK3 at 0.1 μM, which were
also the group I PAK family. The data indicated that 30l had good selectivity profile.
Taken together, the data demonstrated that 30l was a potent and selective PAK1
inhibitor.
Table 4. Kinase selectivity profile of compound 30l.
% Enzyme Activity a
% Enzyme Activitya
Kinase
Kinase
(0.1 μM)
(0.1 μM)
ABL1
AKT1
ALK
106.39
98.85
96.99
87.80
88.41
84.41
99.13
99.18
JNK1
KDR/VEGFR2
LCK
72.75
92.41
88.64
115.26
84.39
63.96
77.96
27.55
Aurora A
AXL
LIMK1
MEK1
BRAF
BTK
MST3/STK24
P38a/MAPK14
PAK1
c-Kit

CDK6/cyclin D1
EGFR
111.75
89.31
91.90
98.96
98.77
89.62
88.65
PAK2
PAK3
PAK4
PAK5
PAK6
SYK
27.98
33.90
103.71
116.44
80.02
94.16
EPHA1
ERK1
FLT3
HPK1/MAP4K1
JAK1
a Values are reported as the averages of two independent experiments.
3.5. In vitro assessment of the antiproliferative activities
Following the SAR analysis of newly synthesized compounds and in vitro kinase
assay, those compounds with IC50 values for PAK1 that were less 150 nM were tested
for their antiproliferative activity against the MDA-MB-231 and HCT-116 cell lines
(Table 5), which were PAK1 overexpressed tumour cells [5, 15]. Unfortunately, these
compounds exhibited limited inhibitory activity, with an inhibition ratio less than 20%
at 1 μM. The poor antiproliferative activity of the potent PAK1 compound might be
attributed to minor contribution of PAK1 to tumour proliferation [3, 32]. Further
analyses of the anti-tumour efficiency for this series of PAK1 inhibitors were
performed by examining the anti-tumour metastatic effect.
Table 5. Antiproliferative activity against MDA-MB-231and HCT-116 cells in vitro.
MDA-MB-231
Inhibition %
(1 μM) a
HCT-116
MDA-MB-231
Inhibition %
(1 μM) a
HCT-116
Inhibition %
(1 μM) a
Compound
Inhibition % Compound
(1μM) a
30a
30c
30e
30f
13.18±0.57
6.87±0.81
10.20±0.87
17.36±0.69
3.07±0.26
19.19±0.77
9.45±0.23
17.23±0.64
10.75±0.47
9.37±0.51
19.23±1.27
10.29±2.36
7.91±1.52
16.18±0.45
30q
30k
30l
18.40±0.26
6.59±0.65
6.31±0.57
8.42±0.32
9.66±0.46
15.26±0.59
8.12±0.37
20.12±2.35
4.41±0.56
8.73±0.52
15.29±1.47
13.15±3.26
11.49±1.57
7.16±0.17
30m
30j
30g
30n
30p
23a
23b

PF-3758309 b
62.60±1.43
83.20±4.26
a Each compound was tested in duplicate and the data are presented as the means ± SD;b used as a
positive control.
3.6. Effects of compound 30l on cell migration and invasion
The relatively low cell growth inhibitory activities suggested that PAK1 might
not be the “driving force” for tumorigenesis and tumour growth, consistent with the
oncogenic function mentioned above. Based on accumulating evidence, PAK1 is
definitely required for tumour metastasis [5, 33-35], which remains a substantial
challenge for the treatment of tumours. As compound 30l represents a new chemical
type of PAK1 kinase inhibitor with a high level of PAK1 inhibitory activity, an
evaluation of its anti-metastasis effect would be interesting. We then performed
wound healing assays in MDA-MD-231 and HCT-116 cells treated with different
concentrations of compounds. The distance migrated was measured using ImageJ
software. As shown in Fig. 5A and Fig. S3, wound healing was suppressed by
treatments with the compound at 1 μM in a dose-dependent manner.
Fig. 5. (A) The migration and invasion capacities of 30l in MDA-MB-231 cells were tested using
wound healing assays and Transwell assays, respectively, after 24 h of treatment with the indicated
concentrations. (B) The distance migrated in the wound healing assay and the number of
penetrated cells in the Transwell assay were counted, respectively. The results are presented as
means ± standard deviations. (C) The levels of proteins involved in PAK1 related signalling

pathways in MDA-MB-231 cells after treatment with compound 30l for 1 h or 24 h. β-actin served
as the loading control.
Transwell invasion assays were performed to analyse the capacity of cell motility
and invasiveness toward a 30l attractant gradient. The numbers of tumour cells that
penetrated through the matrigel and migrated to the other side of the filter membrane
were quantified. A significant decrease in the number of penetrated cells (less than
40%) was observed after 24 h of treatment with different concentrations of compound
30l (Fig. 5B). Together with the low level of cytotoxicity in the cell proliferation
assay at the same concentration, the anti-tumour effect of compound 30l was mainly
due to the inhibition of cell migration. Collectively, compound 30l possessed potent
anti-metastatic ability against PAK1-related tumour cells and provided a potential
application for tumour treatment.
3.7. Analysis of the Mechanism of Action of compound 30l
After confirming the on-target activity of the 1H-indazole-3-carboxamide
derivative 30l against the PAK1 protein and potential anti-tumour metastasis profile,
we further investigated whether compound 30l inhibited PAK1 in cells. As expected,
it decreased the levels of activated PAK1 (PAKp-Ser144) in a dose-dependent manner
(Fig. 5C) when MDA-MB-231 cells were exposed to different concentrations of
compound 30l, confirming the on-target behaviour of 30l in tumour cells. Recent
studies have shown the modulation of the downstream EMT-related signal pathways
upon PAK1 deactivation [5, 10]. The zinc finger protein Snail is one of the critical
indicators of the EMT, allowing cells to detach from their neighbours and migrate
[10]. PAK1 phosphorylation promotes Snail expression, thereby regulating the
metastatic potential of tumor cells [10, 36]. We monitored the Snail expression in
MDA-MB-231 cells treated with compound 30l to clarify the cellular activity of
compound 30l in inhibiting the PAK1-induced EMT. As evident from Fig. 5C, the
level of the Snail protein was reduced in cells treated with 2.5, 5, 10 and 20 μM
compound 30l for both 1 h and 24ꢀh compared to untreated cells, indicating that the
EMT process is indeed impaired, thereby decreasing the migration and invasion of
MDA-MB-231 cells. Importantly, the potency of compound 30l in reducing tumour
invasion was evaluated at a relatively low concentration that does not inhibit cell
proliferation. Based on these results, compound 30l functions as promising PAK1
inhibitor by blocking tumour metastasis through Snail downregulation and could
serve as a lead compound for drug discovery.
3.8. In silico binding mode of 30l

Fig. 6. (A) Predicted docking model of compound 30l in the ATP-binding pocket of PAK1 or (B)
PAK4. Protein residues are shown as green sticks (PAK1) or grey sticks (PAK4) with labelled
names. The hydrogen bonds are shown as red dashed lines.
Molecular docking of the representative compound 30l in the PAK1 and PAK4
kinase domains was performed to exploit the structural basis of the isoform selectivity.
As shown in Fig. 6A and 6B, compound 30l forms conserved hydrogen bond
interactions with the kinase hinge residues Glu345 and Leu347 of PAK1, which is
important for PAK1 inhibition, but it does not interact with Glu396 and Leu398 in the
hinge region of PAK4. Despite the similar architecture of the ATP binding pockets
between the two proteins, the back pocket of PAK1 is more extensive than in PAK4,
which enables the 2-chloro-4-(3-methyl-2-oxopyridin-1(2H)-yl) phenyl moiety of
compound 30l to occupy the pocket with its extended hydrophobic groups.
Additionally, the carbonyl group in the 3-methylpyridone moiety of compound 30l
forms an additional hydrogen bond interaction with the catalytic Lys299 residue of
PAK1 that improves the inhibitory potency against PAK1.
To better understand the stability of 30l with PAK family, we performed
molecular dynamics (MD)simulation for monitoring its binding with PAK1 and PAK4
at 50 ns (nanosecond) scale. RMSF and Molecular mechanics generalized
born/surface area (MM_GBSA) binding free energy were calculated to investigate the
selective binding mechanism of 30l to the ATP-binding site of PAK1 over PAK4. As
shown in Fig. 7, the PAK1 or PAK4 protein reached equilibrium stage after 10 ns of
simulation trajectory. The RMSD values eventually became stable and were about
2.71 Å and 1.96 Å, respectively, for PAK1 and PAK4 (Fig. 7A). The conformation of
30l also exhibited stability with the values of 2.1 Å and 0.99 ± 0.19Å (Fig. 7B) in the
ATP-binding site of PAK1 and PAK4 thus indicating that the MD simulation model
was reliable. The RMSF values of PAK1 key residues (Leu347, Glu345, Met344,
Val342, Lys299, Glu315 and Ile316) contributing to the binding interaction were
significantly smaller than the corresponding PAK4 residues (Fig. 7C), implied the
compound 30l possessed more potent on stabilizing PAK1 protein. By comparative
analysis on the GB binding-free energies (∆G prep), we found that the predicted ∆G

of 30l in PAK1 were calculated to be -57.62 kcal/mol, which was also lower than that
of PAK4 (-32.12 kcal/mol, Table S2). These results suggested compound 30l had high
binding affinity to PAK1 than PAK4 and were consist with the actual bioactivities.
Collectively, these differences in the binding to PAK1 and PAK4 provide an
explanation for the high potency and selectivity of compound 30l toward PAK1.
Figure. 7. The time dependence of RMSDs for PAK1 (black) and PAK4 (red) (A); and 30l in the
ATP- binding site of PAK1(black) and PAK4 (red) (B) during the 50 ns MD simulations; The
RMSF (C) of each residue of the protein for the complex obtained from PAK1/30l (black)and the
PAK4/30l (red). One nm is equal to 10 Å.
4. Conclusions
In summary, we designed, synthesized and evaluated the biological effects of a
series of 1H-indazole-3-carboxamide derivatives as an unexplored chemotype for
PAK1 inhibitors. The molecular modelling suggested that PAK1 contains a large
hydrophobic region in the deep back pocket surrounded by Glu315, Ile316, Val342,
Met344 and Lys299, which maybe is crucial for its selectivity. Further SAR studies
revealed that the incorporation of a large hydrophobic group particularly improved the
inhibitory activity and selectivity toward PAK1. We successfully identified 30l as
PAK1 selective inhibitor with powerful PAK1 inhibition (IC50 = 9.8 nM) and high
selectivity. The binding modes were also proposed through molecular docking and
MD simulations and rationalized the preceding SAR. Notably, 30l displayed low
hERG channel activity, which implied the safety of the compound. Its specificity and
safety were further illustrated by low inhibitory effect on the tumour cell viability.
However, this selected compound 30l effectively suppressed the migration and
invasion of human MDA-MB-231 and HCT116 cancer cells due to its ability to
decrease the level of phosphorylated PAK1 and then attenuated the expression of
Snail. Our findings provided molecular insights into PAK1 drug design with the new
structure type of the 1H-indazole-3-carboxamide moiety. 30l with its favourable
biological performance might serve as a qualified anti-tumour metastasis lead
compound for further investigation.

5. Experimental
5.1. Chemistry
All reagents and solvents were purchased from commercial suppliers and used without
further purification. The reactions were monitored by TLC (Merck Kieselgel GF254) and spots
were visualized with UV light; Isolation and purification of the compounds were performed by
flash column chromatography on silica gel 60 (200-400 mesh). Melting points were determined by
X-4 digital display micro-melting point apparatus (Beijing Tech Instrument Co., Ltd.); The
structures of synthesized compounds were characterized by 1H NMR, 13C NMR, MS and HRMS.
1
NMR spectra were recorded on Bruker AVANCE AV-400 spectrometer (400 MHz for H, 101
MHz for 13C) or Bruker AVANCE AV-300 spectrometer (300 MHz for 1H, 75 MHz for 13C); Mass
spectra were obtained on the Agilent 1100 LC / MSD mass spectrometer (Agilent, USA) or
Q-tofmicro MS (micromass company).
6-Bromo-1H-indazole-3-carboxylic acid (8) To a stirring solution of NaOH (0.386 mg, 19.3
mmol) in H2O (10 mL) was added 6-bromoindoline-2,3-dione 7 (2.00 g, 17.7 mmol). The
resulting mixture was heated to 50 °C and stirred for 1 h. HCl (3.2 mL) was added under
ice-cooling, then NaNO2 (0.610 g, 17.7 mmol) in H2O (2 mL) was added dropwise for 0.5 h.
Reaction was stirred under ice-cooling for 1 h. SnCl2·2H2O (4.98 g, 44.24 mmol) in HCl (6 mL)
was added dropwise for 10min. The mixture was stirred for 2 h at 25 °C. Upon completion, the
mixture was filtered and rinsed with H2O, then the precipitate was collected as crude product
which was used for next step directly. Yield: 91%.
Methyl 6-bromo-1H-indazole-3-carboxylate (9) To the solution of compound 8 (2.00 g) in
methanol (20 mL) were added SOCl2 (5 mL). Reaction mixture was heated to reflux and stirred
for 6 h. Upon completion, the residue was taken up in ice water (60 mL). The pH was adjusted to
9-10 with saturated sodium carbonate solution. The aqueous phase was extracted with ethyl
acetate (80 mL × 3). The combined organic extracts were washed with brine (40 mL × 3), dried
over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by silica gel column
chromatography (n-hexane/ethyl acetate, 5/1) to afford title compound as a yellow solid. Yield:
79%. Rf: 0.46 (n-hexane/ethyl acetate, 4/1, v/v). 1H NMR (300 MHz, DMSO-d6) δ: 14.10 (br, 1H),
8.01 (d, J = 8.5 Hz, 1H), 7.93 (d, J = 1.8 Hz, 1H), 7.46 (dd, J = 8.5, 1.8 Hz, 1H), 3.93 (s, 3H). MS
(m/z): [M+H]+ 255.1.
Methyl 6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3-carboxylate (10) To the
solution of compound 9 (2.00 g, 7.84 mmol) in anhydrous ACN (50 mL) were added DHP (1.32 g,
15.69 mmol) and TsOH (0.135 g, 0.784 mmol). Reaction mixture was stirred at 25 °C for 2 h.
Upon completion, the solvent was removed in vacuo, and the residue was extracted with ethyl
acetate (60 mL × 3). The combined organic extracts were washed with brine (30 mL × 3), dried

over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by silica gel column
chromatography (n-hexane/ethyl acetate, 10/1) to afford title compound as a yellow solid. Yield:
85%. Rf: 0.32 (n-hexane/ethyl acetate, 10/1, v/v). 1H NMR (300 MHz, DMSO-d6) δ: 8.22 (s, 1H),
8.01 (d, J = 8.7, 1H), 7.52 (d, J = 8.6 Hz, 1H), 6.03 (d, J = 9.4, 1H), 3.94 (s, 3H), 3.84 (m, 2H),
2.34 (m, 1H), 2.00 (m, 2H), 1.73 (m, 1H), 1.61 (m, 2H). MS (m/z): [M+H]+ 339.1.
Methyl 6-(2-chlorophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3-carboxylate (12) To
the solution of compound 10 (0.500 g, 1.47 mmol) in dioxane (20 mL) and H2O (20 mL) were
added compound 11 (0.276 g, 1.77 mmol), K2CO3 (0.611 g, 4.42 mmol) and Pd(dppf)Cl2 (0.0538 g,
0.0735 mmol). The resulting mixture was heated to 80 °C under a nitrogen atmosphere for 4 h.
Upon completion, the solvent was removed in vacuo, and the residue was extracted with ethyl
acetate (60 mL × 3). The combined organic extracts were washed with brine (30 mL × 3), dried
over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by silica gel column
chromatography (n-hexane/ethyl acetate, 10/1, v/v) to afford title compound as a yellow solid.
Yellow solid was afforded as the crude product which was used in next step.
6-(2-Chlorophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3-carboxylic acid (13) To the
solution of compound 12 (0.300 g, 0.811 mmol) in MeOH (10 mL) and H2O (10 mL) were added
NaOH (0.162 g, 4.05 mmol). Reaction mixture was stirred at 65 °C for 12 h. Upon completion, the
solvent was removed in vacuo. The pH was adjusted to 4-5 with AcOH solution. The aqueous
phase was extracted with ethyl acetate (80 mL × 3). The combined organic extracts were washed
with brine (40 mL × 3), dried over anhydrous Na2SO4 and concentrated in vacuo. Yellow solid was
afforded as the crude product which was used in next step.
6-(2-Chlorophenyl)-N-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3- carboxamide (14)
To the solution of compound 13 (0.200 g, 0.562 mmol) in dry DMF (20 mL) were added HATU
(0.427 g, 1.12 mmol), DIEA (0.217 g, 1.69 mmol) and 2 M CH3NH2-MeOH (0.5 mL, 32.2 mmol).
Reaction mixture was stirred at 25 °C for 12 h. Upon completion, the reaction mixture was
quenched with ethyl acetate (80 mL), which was washed with water (40 mL × 3) and brine (40 mL
× 3) successively, dried over anhydrous Na2SO4, filtered and concentrated in vacuo. Yellow solid
was afforded as the crude product which was used in next step.
6-(2-Chlorophenyl)-N-methyl-1H-indazole-3-carboxamide (6) To the solution of compound 14
(0.100 g, 0.271 mmol) in MeOH (10 mL) and 2 M HCl (6 mL). The resulting mixture was heated
to 65 °C for 4 h. Upon completion, the solvent was removed in vacuo. The pH was adjusted to
9-10 with saturated sodium carbonate solution. The aqueous phase was extracted with ethyl
acetate (30 mL × 3). The combined organic extracts were washed with brine (20 mL × 3), dried
over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by silica gel column
chromatography (n-hexane/ethyl acetate, 1/1, v/v) to afford title compound as a yellow solid.

1
Yield: 79%. mp: 228-233 °C. Rf: 0.23 (n-hexane/ethyl acetate, 1/1, v/v). H NMR (300 MHz,
DMSO-d6) δ: 13.69 (s, 1H), 8.40 (q, J = 5.0 Hz, 1H), 8.23 (d, J = 8.4 Hz, 1H), 7.60 (m, 2H), 7.54
– 7.40 (m, 3H), 7.29 (d, J = 8.4 Hz, 1H), 2.84 (d, J = 4.5 Hz, 3H). HRMS-EI m/z [M+H]+ calcd
for C15H13ClN3O: 286.0669, found: 286.0742.
2-Chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (16) To the solution of
compound 15 (2.00 g, 9.76 mmol) in dry dioxane (50 mL) were added Bis(pinacolato)diboron
(2.97 g, 11.71 mmol), Potassium Acetate(2.87 g, 29.28 mmol) and Pd(dppf)Cl2 (0.358g, 0.488
mmol) under an argon atmosphere. Reaction mixture was stirred at 100 °C for 12 h. Upon
completion, the solvent was removed in vacuo, and the residue was extracted with ethyl acetate
(60 mL × 3). The combined organic extracts were washed with brine (30 mL × 3), dried over
anhydrous Na2SO4 and concentrated in vacuo. Black solid was afforded as the crude product
which was used in next step.
2-Chloro-4-(6-methylpyrazin-2-yl)aniline (17a) To the solution of compound 16 (2.00 g, 7.91
mmol) in dioxane (50 mL) and H2O (40 mL) were added 2-chloro-6-methylpyrazine (1.20 g, 9.49
mmol), K2CO3 (3.27 g, 23.7 mmol), Pd(PPh3)4 (0.457 g, 0.396 mmol) under an argon atmosphere.
Reaction mixture was stirred at 80 °C for 6 h. Upon completion, the solvent was removed in vacuo,
and the residue was extracted with ethyl acetate (60 mL × 3). The combined organic extracts were
washed with brine (30 mL × 3), dried over anhydrous Na2SO4 and concentrated in vacuo. The
residue was purified by silica gel column chromatography (n-hexane/ethyl acetate, 4/1, v/v) to
afford title compound as a yellow solid. Yield: 90%. Rf: 0.26 (n-hexane/ethyl acetate, 2/1, v/v). 1H
NMR (300 MHz, DMSO-d6) δ: 8.91 (s, 1H), 8.34 (s, 1H), 8.01 (d, J = 2.0 Hz, 1H), 7.84 (dd, J =
8.5, 2.1 Hz, 1H), 6.89 (d, J = 8.5 Hz, 1H), 5.83 (s, 2H), 2.51 (s, 3H). MS (m/z): [M+H]+ 220.2.
2-Chloro-4-(6-methylpyridin-2-yl)aniline (17b) The preparation of compound 17b was similar
with that of compound 17a to afford title compound as a yellow solid. Yield: 86%. Rf: 0.23
(n-hexane/ethyl acetate, 1/1, v/v). 1H NMR (300 MHz, DMSO-d6) δ: 7.97 (m, 1H), 7.78 (m, 1H),
7.63 (m, 2H), 7.07 (d, J = 7.4 Hz, 1H), 6.86 (dd, J = 8.4, 1.8 Hz, 1H), 5.67 (s, 2H), 2.49 (s, 3H).
MS (m/z): [M+H]+ 219.3.
2-(4-Bromo-3-chlorophenyl)-6-methylpyrazine (18a) To a stirring solution of 48% of HBr (5
mL) and H2O (5 mL) were added compound 17a (2.80 g, 12.84 mmol). The resulting mixture was
colded to 0 °C and NaNO2 (0.895 g, 12.97 mmol) in H2O (5 mL) was added dropwise for 0.5 h.
Reaction was stirred under ice-cooling for 1 h. Then CuBr (2.75 g, 19.26mmol) in 48% of HBr (5
mL) was added dropwise for 5 min. The mixture was stirred for 2 h at 70 °C. Upon completion,
the pH was adjusted to 9-10 with saturated sodium carbonate solution. The reaction mixture was
extracted with ethyl acetate (60 mL × 3), which was washed with water (30 mL × 3) and brine (30
mL × 3) successively, dried over NaSO4, filtered and concentrated in vacuo. The residue was

purified by silica gel column chromatography (n-hexane/ethyl acetate, 10/1, v/v) to afford title
compound as a yellow solid. Yield: 50%. Rf: 0.36 (n-hexane/ethyl acetate, 5/1, v/v). 1H NMR (300
MHz, DMSO-d6) δ: 9.14 (s, 1H), 8.57 (s, 1H), 8.35 (d, J = 2.1 Hz, 1H), 8.04 (dd, J = 8.4, 2.1 Hz,
1H), 7.93 (d, J = 8.4 Hz, 1H), 2.58 (s, 3H). MS (m/z): [M+H]+ 283.2.
2-(4-Bromo-3-chlorophenyl)-6-methylpyridine (18b) The preparation of compound 18b was
similar with that of compound 18a to afford title compound as a yellow solid. Yield: 46%. Rf: 0.36
(n-hexane/ethyl acetate, 2/1, v/v). 1H NMR (300 MHz, DMSO-d6) δ: 8.31 (m, 1H), 7.98 (m, 1H),
7.84 (m, 3H), 7.27 (m, 1H), 2.54 (s, 3H). MS (m/z): [M+H]+ 282.1.
2-(3-Chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-6-methyl-pyrazine (19a)
To the solution of compound 18a (2.00 g, 7.12 mmol) in dry dioxane (50 mL) were added
Bis(pinacolato)diboron (2.17 g, 8.54 mmol), Potassium Acetate(2.09 g, 21.36 mmol) and
Pd(dppf)Cl2 (0.261 g, 0.356 mmol) under an argon atmosphere. Reaction mixture was stirred at
100 °C for 12 h. Upon completion, the solvent was removed in vacuo, and the residue was
extracted with ethyl acetate (60 mL × 3). The combined organic extracts were washed with brine
(30 mL × 3), dried over anhydrous Na2SO4 and concentrated in vacuo. Black solid was afforded as
the crude product which was used in next step.
2-(3-Chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-6-methyl-pyridine (19b)
The preparation of compound 19b was similar with that of compound 19a to afford title
compound as a yellow solid.
Methyl 6-(2-chloro-4-(6-methylpyrazin-2-yl)phenyl)-1-(tetrahydro-2H-pyran-2- yl)-1H-
indazole-3-carboxylate (20a) To the solution of compound 19a (1.90 g, 5.80 mmol) in dioxane
(50 mL) and H2O (40 mL) were added compound 10 (1.97 g, 5.80 mmol), K2CO3 (2.40 g, 17.4
mmol), Pd(dppf)Cl2 (0.246 g, 0.290 mmol) under an argon atmosphere. Reaction mixture was
stirred at 80 °C for 6 h. Upon completion, the solvent was removed in vacuo, and the residue was
extracted with ethyl acetate (60 mL × 3). The combined organic extracts were washed with brine
(30 mL × 3), dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by
silica gel column chromatography (n-hexane/ethyl acetate, 4/1, v/v) to afford title compound as a
1
yellow solid. Yield: 90%. Rf: 0.25 (n-hexane/ethyl acetate, 2/1, v/v). H NMR (300 MHz,
DMSO-d6) δ: 9.19 (s, 1H), 8.59 (s, 1H), 8.38 (s, 1H), 8.22 (m, 3H), 8.01 (s, 1H), 7.68 (m, 1H),
7.52 (m, 1H), 6.09 (m, 1H), 3.98 (s, 3H), 3.90 (m, 1H), 3.81 (m, 2H), 2.62 (s, 3H), 2.41 (m, 1H),
2.05 (m, 2H), 1.77 (m, 1H), 1.61 (m, 3H). MS (m/z): [M+H]+ 463.3.
Methyl 6-(2-chloro-4-(6-methylpyridin-2-yl)phenyl)-1-(tetrahydro-2H-pyran-2- yl)-1H-
indazole-3-carboxylate (20b) The preparation of compound 20b was similar with that of
compound 20a to afford title compound as a yellow solid. Yield: 82%. Rf: 0.21 (n-hexane/ethyl
acetate, 1/1, v/v). 1H NMR (300 MHz, DMSO-d6) δ: 8.32 (d, J = 1.7 Hz, 1H), 8.18 (d, J = 3.1 Hz,

1H), 8.15 (d, J = 2.3 Hz, 1H), 8.00 (s, 1H), 7.90 (d, J = 7.8 Hz, 1H), 7.82 (t, J = 7.7 Hz, 1H), 7.62
(d, J = 8.0 Hz, 1H), 7.49 (dd, J = 8.4, 1.3 Hz, 1H), 7.29 (d, J = 7.5 Hz, 1H), 6.09 (dd, J = 9.6, 2.3
Hz, 1H), 3.98 (s, 3H), 3.90 (m, 1H), 3.81 (m, 1H), 2.58 (s, 3H), 2.41 (m, 1H), 2.04 (m, 2H), 1.74
(m, 1H), 1.60 (m, 2H). MS (m/z): [M+H]+ 462.4.
6-(2-Chloro-4-(6-methylpyrazin-2-yl)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H- indazole-3-
carboxylic acid (21a) To the solution of compound 20a (1.00 g, 2.17 mmol) in MeOH (30 mL)
and H2O (30 mL) were added NaOH (0.434 g, 10.85 mmol). Reaction mixture was stirred at 65 °C
for 2 h. Upon completion, the solvent was removed in vacuo. The pH was adjusted to 9-10 with 2
M HCl. The aqueous phase was extracted with ethyl acetate (60 mL × 3). The combined organic
extracts were washed with brine (30 mL × 3), dried over anhydrous Na2SO4 and concentrated in
vacuo. Yellow solid was afforded as the crude product which was used in next step.
6-(2-Chloro-4-(6-methylpyridin-2-yl)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-3-
carboxylic acid (21b) The preparation of compound 21b was similar with that of compound 21a
to afford title compound as the crude product which was used in next step.
6-(2-Chloro-4-(6-methylpyrazin-2-yl)phenyl)-N-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-in-
dazole-3-carboxamide (22a) To the solution of compound 21a (0.145 g, 0.324 mmol) in dry
DMF (20 mL) were added HATU (0.246 g, 0.648 mmol), DIEA (0.125 g, 0.972 mmol) and 2 M
CH3NH2-MeOH (0.5 mL, 32.2 mol). Reaction mixture was stirred at 25 °C for 12 h. Upon
completion, the reaction mixture was quenched with ethyl acetate (30 mL), which was washed
with water (20 mL × 3) and brine (20 mL × 3) successively, dried over Na2SO4, filtered and
concentrated in vacuo. The residue was purified by silica gel column chromatography
(n-hexane/ethyl acetate, 2/1, v/v) to afford title compound as a yellow solid. Yellow solid was
afforded as the crude product which was used in next step.
6-(2-Chloro-4-(6-methylpyridin-2-yl)phenyl)-N-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-
indazole-3-carboxamide (22b) The preparation of compound 22b was similar with that of
compound 22a to afford title compound as the crude product which was used in next step.
6-(2-Chloro-4-(6-methylpyrazin-2-yl)phenyl)-N-(cyclopropylmethyl)-1-(tetrahydro-2H-
pyran -2-yl)-1H-indazole-3-carboxamide (22c) The preparation of compound 22c was similar
with that of compound 22a to afford title compound as the crude product which was used in next
step.
6-(2-Chloro-4-(6-methylpyridin-2-yl)phenyl)-N-(cyclopropylmethyl)-1-(tetrahy-dro-2H-
pyran-2-yl)-1H-indazole-3-carboxamide (22d) The preparation of compound 22d was similar
with that of compound 22a to afford title compound as the crude product which was used in next
step.
6-(2-Chloro-4-(6-methylpyrazin-2-yl)phenyl)-N-methyl-1H-indazole-3-carboxamide (23a) To

the solution of compound 22a (0.120 g, 0.261 mmol) in MeOH (10 mL) and 6 M HCl (8 mL). The
resulting mixture was heated to 65 °C for 12 h. Upon completion, the solvent was removed in
vacuo. The pH was adjusted to 9-10 with saturated sodium carbonate solution. The aqueous phase
was extracted with ethyl acetate (30 mL × 3). The combined organic extracts were washed with
brine (20 mL × 3), dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was
purified by silica gel column chromatography (DCM/MeOH, 100/1, v/v) to afford title compound
as a yellow solid. Yield: 57%. mp: 266-271 °C. Rf: 0.26 (DCM/MeOH, 50/1, v/v). 1H NMR (300
MHz, DMSO-d6) δ: 13.75 (s, 1H), 9.18 (s, 1H), 8.58 (s, 1H), 8.43 (q, J = 4.6 Hz, 1H), 8.35 (d, J =
1.8 Hz, 1H), 8.27 (dd, J = 8.4, 0.9 Hz, 1H), 8.21 (dd, J = 8.1, 1.8 Hz, 1H), 7.70 (t, J = 1.2 Hz, 1H),
7.67 (d, J = 8.1 Hz, 1H), 7.36 (dd, J = 8.4, 1.4 Hz, 1H), 2.86 (d, J = 4.8 Hz, 3H), 2.61 (s, 3H). 13
C
NMR (100 MHz, DMSO-d6) δ: 163.11, 153.66, 148.85, 144.19, 141.41, 141.34, 139.53, 139.00,
137.47, 136.87, 132.85, 132.81, 128.18, 126.02, 124.20, 121.88, 121.39, 111.61, 26.03, 21.80.
HRMS-EI m/z [M+H]+ calcd for C20H17ClN5O: 378.1091, found: 378.1116.
6-(2-Chloro-4-(6-methylpyridin-2-yl)phenyl)-N-methyl-1H-indazole-3-carboxamide
(23b)
The preparation of compound 23b was similar with that of compound 23a to afford title
1
compound as a white solid. Yield: 59%. mp: 284-288 °C. Rf: 0.38 (DCM/MeOH, 50/1, v/v). H
NMR (300 MHz, DMSO-d6) δ: 13.77 (s, 1H), 8.42 (q, J = 4.7 Hz, 1H), 8.30 (d, J = 1.6 Hz, 1H),
8.27 (d, J = 8.5 Hz, 1H), 8.13 (dd, J = 8.1, 1.7 Hz, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.80 (t, J = 7.7
Hz, 1H), 7.69 (s, 1H), 7.60 (d, J = 8.1 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.27 (d, J = 7.4 Hz, 1H),
2.86 (d, J = 4.6 Hz, 3H), 2.57 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ: 163.16, 158.50, 153.75,
141.45, 140.45, 140.22, 139.01, 138.13, 137.09, 132.59, 132.53, 128.02, 125.82, 124.28, 123.12,
121.85, 121.34, 118.05, 111.57, 26.04, 24.78. HRMS-EI m/z [M+H]+ calcd for C21H18ClN4O:
377.1091, found: 377.1164.
6-(2-Chloro-4-(6-methylpyrazin-2-yl)phenyl)-N-(cyclopropylmethyl)-1H-indazole-3-carbox-
amide (23c) The preparation of compound 23c was similar with that of compound 23a to afford
title compound as a white solid. Yield: 51%. mp: 228-233 °C. Rf: 0.28 (DCM/MeOH, 50/1, v/v).
1H NMR (300 MHz, DMSO-d6) δ: 13.68 (s, 1H), 9.05 (s, 1H), 8.45 (s, 1H), 8.40 (t, J = 6.0 Hz,
1H), 8.22 (d, J = 1.8 Hz, 1H), 8.15 (d, J = 8.4 Hz, 1H), 8.08 (dd, J = 8.1, 1.8 Hz, 1H), 7.59 (s, 1H),
7.53 (d, J = 8.1 Hz, 1H), 7.23 (dd, J = 8.4, 1.4 Hz, 1H), 3.10 (t, J = 6.4 Hz, 2H), 2.48 (s, 3H), 0.99
(m, 1H), 0.33 (m, 2H), 0.18 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 162.49, 153.66, 148.85,
144.19, 141.47, 141.33, 139.52, 139.00, 137.47, 136.89, 132.85, 132.81, 128.18, 126.02, 124.22,
121.91, 121.45, 111.64, 43.19, 21.80, 11.75, 3.77. HRMS-EI m/z [M+H]+ calcd for C23H21ClN5O:
418.1356, found: 418.1429.
6-(2-Chloro-4-(6-methylpyridin-2-yl)phenyl)-N-(cyclopropylmethyl)-1H-indazole-3-carbox-
amide (23d) The preparation of compound 23d was similar with that of compound 23a to afford

title compound as a white solid. Yield: 62%. mp: 217-222 °C. Rf: 0.40 (DCM/MeOH, 50/1, v/v).
1H NMR (300 MHz, DMSO-d6) δ: 13.80 (s, 1H), 8.49 (t, J = 6.0 Hz, 1H), 8.30 (d, J = 1.7 Hz, 1H),
8.27 (d, J = 8.4 Hz, 1H), 8.14 (dd, J = 8.1, 1.8 Hz, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.80 (t, J = 7.7
Hz, 1H), 7.70 (s, 1H), 7.61 (d, J = 8.0 Hz, 1H), 7.35 (dd, J = 8.4, 1.4 Hz, 1H), 7.27 (d, J = 7.4 Hz,
1H), 3.22 (t, J = 6.4 Hz, 2H), 2.57 (s, 3H), 1.11 (m, 1H), 0.44 (m, 2H), 0.29 (m, 2H). 13C NMR
(100 MHz, DMSO-d6) δ: 162.50, 158.52, 153.75, 141.51, 140.46, 140.23, 139.01, 138.18, 137.10,
132.63, 132.54, 128.03, 125.85, 124.31, 123.16, 121.87, 121.39, 118.08, 111.59, 43.18, 24.80,
11.75, 3.77. HRMS-EI m/z [M+H]+ calcd for C24H22ClN4O: 417.1404, found: 417.1477.
1-(4-Amino-3-chlorophenyl)pyridin-2(1H)-one (24a) In a sealed tube, compound 15(4.0 g, 19.4
mmol) was dissolved in anhydrous dioxane (50 mL), followed by the addition of
2-hydroxypyridine (2.77 g, 29.1 mmol), potassium phosphate(6.17 g, 29.13 mmol), cuprous iodide
(0.742 g, 3.88 mmol) and N,N-Dimethylethylene- diamine (0.740 g, 7.77 mmol) under an argon
atmosphere. Reaction was heated to 120 °C for 24 h. Upon completion, the solvent was removed
in vacuo, and the residue was extracted with ethyl acetate (70 mL × 3). The combined organic
extracts were washed with brine (30 mL × 3), dried over anhydrous Na2SO4 and concentrated in
vacuo. The residue was purified by silica gel column chromatography (n-hexane/ethyl acetate, 2/1,
v/v) to afford title compound as a yellow solid. Yield: 90%. Rf: 0.32 (n-hexane/ethyl acetate, 1/1,
v/v). 1H NMR (300 MHz, DMSO-d6) δ: 7.56 (dd, J = 6.9, 2.1 Hz, 1H), 7.45 (m, 1H), 7.26 (d, J =
2.4 Hz, 1H), 7.04 (dd, J = 8.6, 2.4 Hz, 1H), 6.86 (d, J = 8.6 Hz, 1H), 6.44 (d, J = 8.6 Hz, 1H), 6.25
(m, 1H), 5.61 (s, 2H). MS (m/z): [M+H]+ 221.1.
1-(4-Amino-3-chlorophenyl)-3-methylpyridin-2(1H)-one (24b) The preparation of compound
24b was similar with that of compound 24a to afford title compound as a yellow solid. Yield: 91%.
Rf: 0.38 (n-hexane/ethyl acetate, 1/1, v/v). 1H NMR (300 MHz, DMSO-d6) δ: 7.43 (dd, J = 6.8, 2.1
Hz, 1H), 7.35 (m, 1H), 7.24 (d, J = 2.4 Hz, 1H), 7.02 (dd, J = 8.6, 2.4 Hz, 1H), 6.84 (d, J = 8.6 Hz,
1H), 6.17 (t, J = 6.8 Hz, 1H), 5.61 (s, 2H), 2.01 (s, 3H). MS (m/z): [M+H]+ 235.1.
1-(4-Bromo-3-chlorophenyl)pyridin-2(1H)-one (25a) To a stirring solution of 48% of HBr (10
mL) and H2O (10 mL) were added compound 24a (3.00 g, 12.82 mmol). The resulting mixture
was colded to 0 °C and NaNO2 (0.855 g, 12.82 mmol) in H2O (5 mL) was added dropwise for 0.5
h. Reaction was stirred under ice-cooling for 1 h. Then CuBr (2.75 g, 19.23mmol) in 48% of HBr
(10 mL) was added dropwise for 5 min. The mixture was stirred for 4 h at 80 °C. Upon
completion, the reaction mixture was extracted with ethyl acetate (80 mL × 3), which was washed
with water (40 mL × 3) and brine (40 mL × 3) successively, dried over NaSO4, filtered and
concentrated in vacuo. The residue was purified by silica gel column chromatography
(n-hexane/ethyl acetate, 5/1, v/v) to afford title compound as a yellow solid. Yield: 90%. Rf: 0.26
1
(n-hexane/ethyl acetate, 4/1, v/v). H NMR (300 MHz, DMSO-d6) δ: 7.91 (d, J = 8.5 Hz, 1H),

7.79 (d, J = 2.5 Hz, 1H), 7.67 (m, 1H), 7.52 (m, 1H), 7.37 (dd, J = 8.6, 2.5 Hz, 1H), 6.50 (m, 1H),
6.34 (m, 1H). MS (m/z): [M+H]+ 284.0.
1-(4-Bromo-3-chlorophenyl)-3-methylpyridin-2(1H)-one (25b) The preparation of compound
25b was similar with that of compound 25a to afford title compound as a yellow solid. Yield: 86%.
Rf: 0.30 (n-hexane/ethyl acetate, 4/1, v/v). 1H NMR (300 MHz, DMSO-d6) δ: 7.90 (d, J = 8.6 Hz,
1H), 7.78 (d, J = 2.4 Hz, 1H), 7.53 (dd, J = 7.2, 1.6 Hz, 1H), 7.40 (m, 1H), 7.36 (dd, J = 8.5, 2.5
Hz, 1H), 6.25 (t, J = 6.8 Hz, 1H), 2.04 (s, 3H). MS (m/z): [M+H]+ 298.0.
1-(3-Chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)pyridin-2(1H)-one (26a)
To the solution of compound 25a (2.00 g, 6.73 mmol) in dry dioxane (50 mL) were added
Bis(pinacolato)diboron (2.57 g, 10.10 mmol), Potassium Acetate (1.97 g, 20.13 mmol) and
Pd(dppf)Cl2 (0.246 g, 0.336 mmol) under an argon atmosphere. Reaction mixture was stirred at
100 °C for 12 h. Upon completion, the solvent was removed in vacuo, and the residue was
extracted with ethyl acetate (60 mL × 3). The combined organic extracts were washed with brine
(30 mL × 3), dried over anhydrous Na2SO4 and concentrated in vacuo. Black solid was afforded as
the crude product which was used in next step.
1-(3-Chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3-methyl-pyridin-2(1H)-
one (26b) The preparation of compound 26b was similar with that of compound 26a to afford title
compound as a black solid.
Methyl 6-(2-chloro-4-(2-oxopyridin-1(2H)-yl)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-inda-
zole-3-carboxylate (27a) To the solution of compound 26a (2.00 g, 5.80 mmol) in dioxane (50
mL) and H2O (40 mL) were added compound 9 (1.97 g, 5.80 mmol), K2CO3 (2.40 g, 17.4 mmol),
Pd(dppf)Cl2 (0.246 g, 0.290 mmol) under an argon atmosphere. Reaction mixture was stirred at
80 °C for 6 h. Upon completion, the solvent was removed in vacuo, and the residue was extracted
with ethyl acetate (60 mL × 3). The combined organic extracts were washed with brine (30 mL ×
3), dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by silica gel
column chromatography (n-hexane/ethyl acetate, 2/1, v/v) to afford title compound as a yellow
1
solid. Yield: 84%. Rf: 0.32 (n-hexane/ethyl acetate, 1/1, v/v). H NMR (300 MHz, DMSO-d6) δ:
8.09 (dd, J = 8.5, 4.0 Hz, 1H), 7.91 (d, J = 4.0 Hz, 1H), 7.74 – 7.34 (m, 6H), 6.44 (dd, J = 9.3, 3.9
Hz, 1H), 6.28 (q, J = 6.7, 4.9 Hz, 1H), 5.99 (dd, J = 8.2, 3.7 Hz, 1H), 3.88 (s, 3H), 3.83 – 3.66 (m,
2H), 2.30 (m, 1H), 2.01 – 1.87 (m, 2H), 1.66 (m, 2H), 1.51 (m, 2H). MS (m/z): [M+H]+ 464.3.
Methyl 6-(2-chloro-4-(3-methyl-2-oxopyridin-1(2H)-yl)phenyl)-1-(tetrahydro-2H-pyran-yl)-
1H-indazole-3-carboxylate (27b). The preparation of compound 27b was similar with that of
compound 27a to afford title compound as a yellow solid. Yield: 87%. Rf: 0.36 (n-hexane/ethyl
1
acetate, 1/1, v/v). H NMR (300 MHz, DMSO-d6) δ: 8.20 (m, 1H), 8.00 (s, 1H), 7.76 (m, 1H),
7.56 (m, 5H), 6.30 (m, 1H), 6.09 (d, J = 9.1 Hz, 1H), 3.96 (s, 3H), 3.84 (m, 2H), 2.41 (m, 1H),

2.07 (s, 3H), 2.03 (m, 2H), 1.74 (m, 1H), 1.60 (m, 2H). MS (m/z): [M+H]+ 478.2.
6-(2-Chloro-4-(2-oxopyridin-1(2H)-yl)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-
3-carboxylic acid (28a) To the solution of compound 27a (1.00 g, 2.09 mmol) in MeOH (30 mL)
and H2O (30 mL) were added NaOH (0.418 g, 10.46 mmol). Reaction mixture was stirred at 65 °C
for 2 h. Upon completion, the solvent was removed in vacuo. The pH was adjusted to 9-10 with 2
M HCl. The aqueous phase was extracted with ethyl acetate (60 mL × 3). The combined organic
extracts were washed with brine (30 mL × 3), dried over anhydrous Na2SO4 and concentrated in
vacuo. Yellow solid was afforded as the crude product which was used in next step.
6-(2-Chloro-4-(3-methyl-2-oxopyridin-1(2H)-yl)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-in-
dazole-3-carboxylic acid (28b) The preparation of compound 28b was similar with that of
compound 28a to afford title compound as a yellow solid. Yellow solid was afforded as the crude
product which was used in next step.
6-(2-Chloro-4-(3-methyl-2-oxopyridin-1(2H)-yl)phenyl)-N-methyl-1-(tetrahydro-2H-pyran-
2-yl)-1H-indazole-3-carboxamide (29a) To the solution of compound 28b (0.150 g, 0.324 mmol)
in dry DMF (20 mL) were added HATU (0.246 g, 0.648 mmol), DIEA (0.125 g, 0.972 mmol) and
2 M CH3NH2-MeOH (0.5 mL, 32.2 mmol). Reaction mixture was stirred at 25 °C for 12 h. Upon
completion, the reaction mixture was quenched with ethyl acetate (30 mL), which was washed
with water (20 mL × 3) and brine (20 mL × 3) successively, dried over Na2SO4, filtered and
concentrated in vacuo. The residue was purified by silica gel column chromatography
(n-hexane/ethyl acetate, 2/1, v/v) to afford title compound as a yellow solid. Yellow solid was
afforded as the crude product which was used in next step.
6-(2-Chloro-4-(2-oxopyridin-1(2H)-yl)phenyl)-N-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-
indazole-3-carboxamide (29b) The preparation of compound 29b was similar with that of
compound 29a to afford title compound as the crude product which was used in next step.
6-(2-Chloro-4-(3-methyl-2-oxopyridin-1(2H)-yl)phenyl)-N-(cyclopropylmethyl)-1-(tetrahy-
dro-2H-pyran-2-yl)-1H-indazole-3-carboxamide (29c) The preparation of compound 29c was
similar with that of compound 29a to afford title compound as the crude product which was used
in next step.
6-(2-Chloro-4-(2-oxopyridin-1(2H)-yl)phenyl)-N-(cyclopropylmethyl)-1-(tetrahy-dro-2H-
pyran-2-yl)-1H-indazole-3-carboxamide (29d) The preparation of compound 29d was similar
with that of compound 29a to afford title compound as the crude product which was used in next
step.
6-(2-Chloro-4-(3-methyl-2-oxopyridin-1(2H)-yl)phenyl)-N-ethyl-1-(tetrahydro-2H-pyran-2-
yl)-1H-indazole-3-carboxamide (29e) The preparation of compound 29e was similar with that of
compound 29a to afford title compound as the crude product which was used in next step.

6-(2-Chloro-4-(3-methyl-2-oxopyridin-1(2H)-yl)phenyl)-N-(2-hydroxyethyl)-1-(tetrahydro-
2H-pyran-2-yl)-1H-indazole-3-carboxamide (29f) The preparation of compound 29f was similar
with that of compound 29a to afford title compound as the crude product which was used in next
step.
6-(2-Chloro-4-(3-methyl-2-oxopyridin-1(2H)-yl)phenyl)-N-(2-methoxyethyl)-1-(tetrahydro-
2H-pyran-2-yl)-1H-indazole-3-carboxamide (29g) The preparation of compound 29g was
similar with that of compound 29a to afford title compound as the crude product which was used
in next step.
1-(3-Chloro-4-(3-(pyrrolidine-1-carbonyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)-
phenyl)-3-methylpyridin-2(1H)-one (29h) The preparation of compound 29h was similar with
that of compound 29a to afford title compound as the crude product which was used in next step.
1-(3-Chloro-4-(3-(morpholine-4-carbonyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)-
phenyl)-3-methylpyridin-2(1H)-one (29i) The preparation of compound 29i was similar with
that of compound 29a to afford title compound as the crude product which was used in next step.
N-benzyl-6-(2-chloro-4-(3-methyl-2-oxopyridin-1(2H)-yl)phenyl)-1-(tetrahydro-2H-pyran-2-
yl)-1H-indazole-3-carboxamide (29j) The preparation of compound 29j was similar with that of
compound 29a to afford title compound as the crude product which was used in next step.
6-(2-Chloro-4-(3-methyl-2-oxopyridin-1(2H)-yl)phenyl)-N-(pyridin-4-yl)-1-(tetrahydro-2H-
pyran-2-yl)-1H-indazole-3-carboxamide (29k) The preparation of compound 29k was similar
with that of compound 29a to afford title compound as the crude product which was used in next
step.
6-(2-Chloro-4-(3-methyl-2-oxopyridin-1(2H)-yl)phenyl)-N-(pyridin-4-ylmethyl)-1-(tetra-
hydro-2H-pyran-2-yl)-1H-indazole-3-carboxamide (29l) The preparation of compound 29l was
similar with that of compound 29a to afford title compound as the crude product which was used
in next step.
6-(2-Chloro-4-(3-methyl-2-oxopyridin-1(2H)-yl)phenyl)-N-(2-(pyridin-4-yl)ethyl)-1-(tetra-
hydro-2H-pyran-2-yl)-1H-indazole-3-carboxamide (29m) The preparation of compound 29m
was similar with that of compound 29a to afford title compound as the crude product which was
used in next step.
6-(2-Chloro-4-(3-methyl-2-oxopyridin-1(2H)-yl)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-N-
(tetrahydro-2H-pyran-4-yl)-1H-indazole-3-carboxamide (29n) The preparation of compound
29n was similar with that of compound 29a to afford title compound as the crude product which
was used in next step.
6-(2-Chloro-4-(3-methyl-2-oxopyridin-1(2H)-yl)phenyl)-N-(1-methylpiperidin-4-yl)-1-(tetra-
hydro-2H-pyran-2-yl)-1H-indazole-3-carboxamide (29o) The preparation of compound 29o