Development of 2, 4-diaminoquinazoline derivatives as potent PAK4 inhibitors by the core refinement strategy

Article abstract of DOI:10.1016/j.ejmech.2017.02.063

Upon analysis of the reported crystal structure of PAK4 inhibitor KY04031 (PAK4 IC₅₀?=?0.790?μM) in the active site of PAK4, we investigated the possibility of changing the triazine core of KY04031 to a quinazoline. Using KY04031 as a starting

Full text of DOI:10.1016/j.ejmech.2017.02.063

European Journal of Medicinal Chemistry 131 (2017) 1e13
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Development of 2, 4-diaminoquinazoline derivatives as potent PAK4
inhibitors by the core refinement strategy
a
,
1
a
,
d
,
1
, Xiaodong Li , Jing Guo , Meng Chen c,
b
,
c
a
b,
Chenzhou Hao , Wanxu Huang
a
a
a
a
a
a, *
Zizheng Yan , Kai Wang , Xiaolin Jiang , Shuai Song , Jian Wang , Dongmei Zhao
,
b,
c
,
**
a, ***
Feng Li
a
, Maosheng Cheng
Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China
Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, China Medical University, Shenyang 110001, China
Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang 110001, China
b
c
d
School of Life Science and Bio-pharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 January 2017
Received in revised form
Upon analysis of the reported crystal structure of PAK4 inhibitor KY04031 (PAK4 IC50 ¼ 0.790
mM) in the
active site of PAK4, we investigated the possibility of changing the triazine core of KY04031 to a qui-
nazoline. Using KY04031 as a starting compound, a library of 2, 4-diaminoquinazoline derivatives were
designed and synthesized. These compounds were evaluated for PAK4 inhibition, leading to the iden-
24 February 2017
Accepted 27 February 2017
tification of compound 9d (PAK4 IC50 ¼ 0.033
mM). Compound 9d significantly induced the cell cycle in
the G1/S phase and inhibited migration and invasion of A549 cells that over-express PAK4 via regulation
of the PAK4-LIMK1 signalling pathway. A docking study of compound 9d was performed to elucidate its
possible binding modes and to provide a structural basis for further structure-guided design of PAK4
inhibitors. Compound 9d may serve as a lead compound for anticancer drug discovery and as a valuable
research probe for further biological investigation of PAK4.
Keywords:
p21-activated kinase
PAK4 inhibitor
Synthesis
Anti-cancer
© 2017 Published by Elsevier Masson SAS.
2,4-diaminoquinazoline
1. Introduction
pancreas, thyroid, and ovaries [3e5]. It has been linked to many
hallmarks of tumorigenesis, including anchorage-independent
p21-activated kinases (PAKs) are serine/threonine (Ser/Thr)
protein kinases that are downstream signalling effectors of the Rho/
Rac family of GTPases [1]. The six known mammalian PAK isoforms
are grouped into two subgroups, PAK1-3 (group I) and PAK4-6
growth [3], angiogenesis [6], increased cell survival [7], migration
[8], and invasion [8,9]. Therefore, PAK4 is a promising target for the
development of cancer therapeutics [1,10].
A number of groups have previously reported inhibitors of PAK4
as cancer treatments (Fig. 1) [11,12]. Among this studies, only one
pan-PAK inhibitor, PF-3758309 (PAK4 IC50 ¼ 19 nM, PAK1
IC50 ¼ 14 nM), has been advanced to clinical development [13]. PF-
3758309 inhibits the growth of many tumour cell lines and has
shown potent anticancer properties in xenografts and in a KRAS-
driven transgenic mouse model of skin cancer [13,14]. This com-
pound progressed to phase I clinical trials in patients with
advanced/metastatic solid tumours, but these clinical trials were
halted due to low oral bioavailability (~1%) and adverse events [15].
Compound 2 (GEN-2861), a highly potent group II PAK-selective
inhibitor with exquisite kinome selectivity, was reported by Gen-
entech [16]. This compound has been used as a tool compound to
evaluate group II PAK biology. Nonetheless, an obstacle to its use is
its poor cellular activity. Recently, a few PAK4 small molecule in-
hibitors have been reported in the literature with diverse
(
[
group II), based on their structural and functional characteristics
2]. PAKs have been shown to be important for regulation of the
actin cytoskeleton, focal adhesion contacts and cell motility and are
positioned at the nexus of several oncogenic signalling pathways.
Among the group II PAKs, PAK4 is overexpressed at a high fre-
quency in several malignancies including those of the colon, breast,
*
Corresponding author.
** Corresponding author. Department of Cell Biology, Key Laboratory of Cell
Biology, Ministry of Public Health, China Medical University, Shenyang 110001,
China.
*** Corresponding author.
E-mail addresses: dongmeiz-67@163.com (D. Zhao), fli@mail.cmu.edu.cn (F. Li),
mscheng@263.net (M. Cheng).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2017.02.063
0223-5234/© 2017 Published by Elsevier Masson SAS.

2
C. Hao et al. / European Journal of Medicinal Chemistry 131 (2017) 1e13
Fig. 1. Known PAK4 inhibitors.
chemotypes and affinities [17e19]. There is a growing need for
novel and potent PAK4 inhibitors that can be developed into ther-
apeutic candidates for cancer treatment.
Previously, we described the discovery of anilinoquinazoline
PAK4 inhibitor 3 (LCH-7749944) as a moderate PAK4 inhibitor using
HTS screening in 2012 [20]. Recently, Ryu et al. reported the dis-
covery of the 1, 3, 5-triazine-based PAK4 inhibitor KY04031 by high
throughput HTRF-based screening [21]. This compound inhibits
PAK4 kinase with moderate activity (PAK4 IC50 ¼ 0.790
mM). A co-
crystal structure of KY04031 with PAK4 showed that the indole and
indazole rings provide three hydrogen bonds to the kinase hinge
region and the triazine moiety enters a lipophilic phosphate-
binding pocket (Fig. 2) [21]. However, only this compound was
reported, thereby leaving this chemotype largely unexplored.
Compound 2 does not fully occupy the large and flexible ATP-
binding cleft of PAK4, so we hypothesize that extending the scaf-
fold of 2 into the hydrophobic subsite would provide an opportu-
nity to increase its potency against PAK4. In an attempt to design
compounds with enhanced potency against the kinase activity of
PAK4, we explored the heterocyclic core using bicyclic systems,
intending to induce an interaction with the phosphate-binding
region of the ATP-binding pocket (Fig. 3).
Fig. 3. Core refinement strategy for the development of a novel series of PAK4
inhibitors.
2. Chemistry
The synthesis of the 2, 4-diaminoquinazoline scaffold is
described in Scheme 1. Starting from substituted 2,4-
dichloroquinazolines 5a-g, the first chlorine was displaced by
ꢀ
nucleophilic substitution with 1H-indazol-5-amine at 0 C to pro-
duce the mono-substituted intermediates 6a-g. The displacement
of the second chlorine by tryptamine was accomplished by an HCl-
catalysed amination procedure, affording compounds 7a-g in good
yields. Likewise, the second series of 2, 4-diaminoquinazoline
regioisomers 9a-g were synthesized from compounds 5a-g in a
modified reaction sequence.
To study the structure and activity relationship (SAR) of the
indazole ring, some of the required hinge-binding moieties (e.g., 13,
17a-b and 23) were not commercially available and were prepared
as follows. As shown in Scheme 2, (Z)-2, 3-dibromo-4-oxobut-2-
This paper describes the design, synthesis, biological evaluation,
and molecular modeling of 2, 4-diaminoquinazoline derivatives as
potent PAK4 inhibitors. These compounds potently suppressed the
enzyme activity, and structure-activity relationships are deter-
mined. The most potent compound, 9d, inhibited PAK4 kinase ac-
tivity with an IC50 value of 0.033 mM, which was 24-fold greater
than that of KY04031. Furthermore, compound 9d inhibited the
PAK4-driven cell proliferation in a dose-dependent manner. In
addition, mechanistic studies demonstrate that compound 9d in-
hibits the migration and invasion of A549 cells via regulation of the
PAK4-LIMK1 signalling pathway. A molecular modeling study of
compound 9d was performed to elucidate its possible binding
modes and to provide a structural basis for the further structure-
guided design of PAK4 inhibitors.
2
enoic acid 10 was treated with NaNO in ethanol to give com-
pound 11, which was then converted to 12 by condensation with
1H-pyrazol-3-amine according to a literature procedure [22]. Sub-
sequent reduction of the nitro group of 12 afforded heteroaryl
amine 13. As shown in Scheme 3, halogenation of 2-methyl-4-
nitroaniline 14 in the presence of NCS or NBS gave halogenated
aryls 15a-b, which were cyclized and reduced to afford 17a and 17b.
Fig. 2. (A). X-ray crystal structure of the reported 1, 3, 5-triazine inhibitor KY04031 bound to PAK4 (PDB code 4NJD) [21]. Note that C-7 of the indazol ring interacts with the
gatekeeper residue Met395. (B). Space-filling diagram of the co-crystal structure of PAK4 with KY04031, highlighting the solvent accessibility of KY04031. Ample space is available
within the ATP-binding site allowing for the possibility of a ring fusion to form a quinazoline core, and highlighted by a dashed red oval. (C). Rotated relative to (B). (For inter-
pretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

C. Hao et al. / European Journal of Medicinal Chemistry 131 (2017) 1e13
3
ꢀ
ꢀ
ꢀ
Scheme 1. Reagents and conditions: (a) 1H-indazol-5-amine, DIEA, DMF, 0 C, 2 h (b) tryptamine, HCl, EtOH, 120 C, 6 h (c) tryptamine, DIEA, DMF, 0 C, 2 h (d) 1H-indazol-5-amine,
HCl, EtOH, 120 C, 6 h.
ꢀ
ꢀ
ꢀ
Scheme 2. Reagents and conditions: (a) NaNO
2
, H
2
O, EtOH, 54 C, 1 h (b) 1H-pyrazol-3-amine, H
2
O, 90 C, 16 h (c) H
2
, MeOH, Pd/C, r.t, 16 h.
ꢀ
Scheme 3. Reagents and conditions: (a) CH
3
CN, NH
4
OAc, NCS or NBS, 60 C, 3 h (b) AcOH, NaNO
2
, H
2
O, r.t, 12 h (c) SnCl
2
, EtOH, reflux, 18 h.
Similarly, methyl substituted analogue 23 was prepared from 2, 6-
dimethylaniline (Scheme 4). Finally, S Ar displacement of aryl
chloride 8d by various amines afforded the corresponding target
compounds 24a-h (Scheme 5).
and 24a-h were preliminarily evaluated via a well-established
homogeneous time-resolved fluorescence (HTRF) assay according
to the manufactory's instructions (Cisbio). The results are shown in
Tables 1 and 2. The well-characterized PAK4 inhibitors PF3758309
and staurosporine were included as positive controls to validate the
screening conditions. Under the experimental conditions, both of
these compounds displayed strong inhibition against PAK4 with
IC50 values of 7.0 and 1.9 nM, respectively, which are similar to the
previously reported data [11].
N
3
. Results and discussion
3.1. In vitro activity against PAK4 kinase
As shown in Table 1, a comparison of the A and B regioisomers of
PAK4 inhibitory activities of the designed compounds 7a-g, 9a-g
ꢀ
Scheme 4. Reagents and conditions: (a) p-TsCl, pyridine, r.t. (b) HNO
2 h.
, AcOH, NaNO , H
3 2 2
O, reflux. 4 h. (c) H
2
SO
4
, 50 C, 1 h (d) AcOH, NaNO
2
2 2
, H O, r.t. 12 h. (e) H , Pd-C, MeOH, r.t,
1

4
C. Hao et al. / European Journal of Medicinal Chemistry 131 (2017) 1e13
ꢀ
Scheme 5. Reagents and conditions: (a) amine, HCl, EtOH, 120 C, 6 h.
the 2,4-diaminoquinazoline derivatives (7a-g and 9a-g) revealed
that, in general, the B regioisomer is a more active PAK4 inhibitor.
inhibitory activity was retained, but the potency decreased.
To further study the relationship between the indazol ring and
PAK4 inhibition, additional compounds, 24a-h, were prepared,
based on compound 9d and evaluated for their activity (Table 2). As
mentioned above, the indazol ring makes a two-point interaction
with Leu398 and Glu396 in the hinge region. Rearrangement of the
donor-acceptor array on the indazol ring led to a loss of potency
(24a-d), highlighting the necessity of the hydrogen bond donor and
acceptor in this region.
As observed in the PAK4-compound 4 co-crystal structure (PDB
ID 4NJD, Fig. 2), C-7 of the indazol ring interacts with the gate-
keeper residue Met395. Compound 24e-h were synthesized to
probe the effects of substitution on the inhibitory potency of PAK4.
Compounds with C-H (9d), N (24e), C-Cl (24f), C-Br (24g), and C-
methyl (24h) at the 7 position of indazole ring displayed IC50 values
For example, compound 9a (IC50 ¼ 0.406
higher affinity to PAK4 than 7a (IC50 ¼ 3.516
the triazine core to a quinazoline resulted in a clear increase in
PAK4 inhibition activity (9a, IC50 ¼ 0.46 M), compared to the 1,3,5-
triazinic lead compound KY04031 (IC50 ¼ 0.79 M), suggesting a
mM) showed 8.7-fold
mM). Morphing from
m
m
specific contribution by the quinazoline heterocyclic core. We
further investigated the impact of quinazoline ring substitution on
PAK4 inhibitory activity and found that the position of the sub-
stituent on the ring significantly affected the biological activity of
the resulting compounds. Among the substituents at the C-6 po-
sition of the quinazoline core, the 6-Cl substituted derivative 9d
demonstrated the greatest potency (IC50 ¼ 0.033
atom substitution of the C-7 position led to a loss of potency (9e,
M). Further investigation showed that the 6-chloro
mM). Chlorine
IC50 ¼ 0.360
m
of 0.033, 0.63, 0.94, 1.06, and 2.33
mM, respectively. As the size of
group in 9d could be replaced by 6-Br (9f) or 6-F (9g) and PAK4
the C7-substituents increased from H to Cl to Br and CH
3
, the
Table 1
In vitro enzymatic activities of isomeric 2,4-diaminoquinazoline derivatives 7a-g and 9a-g.
M)a b
,
No.
R
R
1
R
2
Regioisomer
IC50
(
m
7
7
7
7
7
7
7
9
9
9
9
9
9
9
1
a
b
c
d
e
f
g
a
b
c
d
e
f
H
1H-indazol-5-yl
1H-indazol-5-yl
1H-indazol-5-yl
1H-indazol-5-yl
1H-indazol-5-yl
1H-indazol-5-yl
1H-indazol-5-yl
(1H-indol-3-yl)ethyl
(1H-indol-3-yl)ethyl
(1H-indol-3-yl)ethyl
(1H-indol-3-yl)ethyl
(1H-indol-3-yl)ethyl
(1H-indol-3-yl)ethyl
(1H-indol-3-yl)ethyl
e
(1H-indol-3-yl)ethyl
(1H-indol-3-yl)ethyl
(1H-indol-3-yl)ethyl
(1H-indol-3-yl)ethyl
(1H-indol-3-yl)ethyl
(1H-indol-3-yl)ethyl
(1H-indol-3-yl)ethyl
1H-indazol-5-yl
1H-indazol-5-yl
1H-indazol-5-yl
1H-indazol-5-yl
1H-indazol-5-yl
1H-indazol-5-yl
1H-indazol-5-yl
e
A
A
A
A
A
A
A
B
B
B
B
B
B
B
e
e
3.516
1.709
3.956
0.093
4.470
0.788
1.736
0.406
1.506
0.483
0.033
0.360
0.177
0.920
0.007
0.0019
6-OMe
7-OMe
6-Cl
7-Cl
6-Br
6-F
H
6-OMe
7-OMe
6-Cl
7-Cl
6-Br
6-F
g
c
e
Staurosporinec
e
e
e
a
IC50 values are calculated based on the homogeneous time-resolved fluorescence (HTRF) assay.
Reported data are the mean values from two independent experiments.
Used as a positive control.
b
c

C. Hao et al. / European Journal of Medicinal Chemistry 131 (2017) 1e13
5
Table 2
SAR of the indazol ring.
bulkier quinazoline core of 9d addresses the lipophilic phosphate-
binding pocket to a greater degree than the triazine core of com-
pound 4, making hydrophobic contact with Val335. These
hydrogen-bond interactions, together with the interactions made
by the quinazoline ring, may be responsible for the increased the
binding affinity of 9d to compared to KY04031.
3.3. Compound 9d induces cell cycle arrest in G2/M phase
PAK4 is an important oncogene whose expression is increased in
various human cancers, and many of its functions are dependent on
its kinase activity. Based on the enzymatic assay results, we tested
the ability of compound 9d to inhibit the growth of the PAK4-
dependent tumour cell line A549 (human lung adenocarcinoma)
No.
R
IC50
(
m
M)a,b No.
R
IC50 (m
M)a,b
24a
1.163
24e
0.633
[8] and cell line. Meanwhile, the tumour cell line NCI-H460 (human
large cell lung cancer cell) [24], whose growth was not dependent
on PAK4, was used to test the potential off-target effects. The
cytotoxicity in HEK-293 cells (human embryonic kidney 293 cell)
was determined as an indicator of toxicity. In the MTT assay,
compound 9d inhibited the proliferation of A549 cells in a
concentration-dependent manner (Fig. 5A). Of note, the weak
cytotoxicity in HEK-293 cells suggested the potential safety of
compound 9d. Next, we determined whether the 9d-induced
decrease in PAK4 phosphorylation would result in cell cycle arrest
in the A549 cell line. Cell cycle analysis was performed using flow
2
4b
3.071
24f
0.937
2
2
4c
4d
d
1.757
2.040
0.033
24g
1.056
2.334
0.007
24h
cytometry (Fig. 5B). When A549 cells were treated with 9d at 1
or 5 M, the number of cells in the G1 phase increased from 73.42%
to 73.57% and 84.43%, whereas cells in the S phase decreased from
9.83% to 19.71% and 10.98%, respectively, compared with the
mM
m
1
1c
controls. To further dissect the molecular mechanisms underlying
the cell proliferation regulation, we investigated the expression of
some genes related to cell cycle progression. Western blot analysis
9
e
showed that exposure of the A549 cells to 10 mmol/L 9d for 24 h
markedly increased protein expression of CDK inhibitors (p21, p18)
and decreased the protein expression of cyclin D3 and CDK6
a
IC50 values are calculated based on the homogeneous time-resolved fluores-
cence (HTRF) assay.
b
Reported data are the mean values from two independent experiments.
Used as a positive control.
(Fig. 5C). This result indicates that 9d arrested cells at the G1 phase
c
in a PAK4 kinase-dependent manner, as a result, suppressed lung
adenocarcinoma cell growth via cyclin D3 and CDK6.
activity against PAK4 gradually decreased. The X-ray crystal struc-
ture of 4 in PAK4 indicates that the distance between the C7 atom of
the indazol ring and Met395 is 3.9 Å (d C-7 … S), which is approxi-
mately the sum of van der Waals radii of both atoms. This compact
packing might explain why groups with larger van der Waals radii,
such as chlorine (24f), bromine (24g) or methyl (24h), are not well-
tolerated at this position. These results confirmed that compound
3.4. Effect of compound 9d on cell migration and invasion
Metastasis is a multistep process that results from the conver-
gence of a variety of cellular processes. These processes include cell
migration, invasion, adhesion to the surrounding stroma and
through vessel walls, and survival in a foreign environment. As
previously mentioned, PAK4 is critical for migration and invasion.
The inhibitory effects of 9d on cell migration and invasion of
A549 cells cancer cell lines were analysed using a Transwell assay
(with or without Matrigel) [25]. As shown in Fig. 6A and B, in
A549 cells, a dose-dependent decrease in cell migration and inva-
sion was observed following treatment with compound 9d.
9
d binds in the ATP binding site with C-7 of the indazol ring
interacting with the gatekeeper residue Met395.
3.2. Molecular docking study of compound 9d
In an attempt to gain insight into the putative binding mode of
optimized inhibitor 9d with the PAK4 kinase compared to the lead
compound KY04031, molecular docking of this compound into the
ATP binding site of the PAK4 kinase (PDB ID 4NJD) was performed.
The program AutoDock 4 with default parameters was used to
perform this simulation. The resulting binding mode of compound
Previous studies showed that LIMK and its downstream cofilin
activated by PAK4. This pathway plays an important role in pro-
moting actin polymerization and defining the direction of cell
motility [26]. To further illustrate the mechanism of our compound
with respect to these effects, we investigated the effects of com-
pound 9d on the PAK4/LIMK1/cofilin signalling pathway by west-
ern blot analysis. As expected, exposure of A549 cells to different
9
d is depicted in Fig. 4. As expected, compound 9d is able to bind to
the ATP-binding site of PAK4 in a similar way as KY04031, sand-
wiched in a narrow cleft formed by the C and N lobes. The three
hydrogen bonds to the kinase hinge region (i.e., residues, Glu396
and Leu398) made by the indole and indazole rings of KY04031 are
concentrations of compound 9d for 24 h inhibited the levels of
pꢁSer474
activated PAK4 (PAK4
(LIMK
)
[27] and phosphor-LIMK1
pꢁThr508
) [28] in a dose-dependent manner (Fig. 6C), indi-
4
conserved in the docked pose of 9d. The N -NH of the quinazoline
cating that the mechanism of 9d repressing breast cancer cell
migration and invasion correlates with 9d inhibiting PAK4 kinase
activity and its downstream substrates.
core leads to a hydrogen bond contact with the neighboring
charged residue Asp458 (DFG-motif). Compared to KY04031, the

6
C. Hao et al. / European Journal of Medicinal Chemistry 131 (2017) 1e13
Fig. 4. Predicted binding mode of 9d in the ATP-binding site of PAK4. (A). Overlay of 9d (green) and lead compound 4 (KY04031, yellow) bound in the kinase active site (PDB code
NJD for KY04031). The bulkier quinazoline core of 9d addresses the lipophilic phosphate-binding pocket to a greater degree than the triazine core of compound 4. Hydrogen bond
4
distances are in Å. (B). Compound 9d interaction diagram (2D representation) by LigandScout software [23]. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
Fig. 5. Compound 9d affects the cell cycle distribution in A549 cancer cells. (A) A549, NCI-H460, and HEK-293 cells were cultured with the indicated concentrations of compound
9d for the indicated hours. Compound 9d suppresses the proliferation of lung adenocarcinoma cells. (B) Untreated cells (control) and cells treated for 24 h with different con-
centrations of compound 9d. (C) Proteins from the cells in (B) were extracted to examine the expression of the indicated proteins by western blot analysis. Compound 9d up-
pꢁSer474
regulates the expression levels of CDK inhibitors (p21, p18) and down-regulates the expression levels of PAK4
, cyclin D3, and CDK6.
4
. Conclusions
5. Experimental
In summary, based on a scaffold morphing strategy and
5.1. Chemistry
structure-guided SAR design, we developed a series of 2,4-
diaminoquinazoline derivatives as a new class of PAK4 inhibitors.
The compounds potently suppressed the enzymatic activities of
PAK4 with IC50 values in the 10 -10 M range. SAR and biological
evaluations of this series of compounds were discussed. The opti-
mized compound 9d suppressed the proliferation of human A549
cancer cells, in which PAK4 expression was high. Furthermore,
compound 9d potently suppressed cancer cell migration and in-
vasion, indicating its potential to serve as a new lead compound for
further anticancer drug discovery.
1H NMR and 13C NMR spectral data were recorded in CDCl
DMSO-d on Bruker ARX-400 NMR or Bruker ARX-600 NMR spec-
or
3
6
ꢁ
6
ꢁ8
trometers. High resolution accurate mass spectrometry de-
terminations (HRMS) for all final target compounds were obtained
on a Bruker Micromass Time of Flight mass spectrometer equipped
with an electrospray ionisation (ESI) detector. Unless otherwise
noted, all commercial reagents and solvents were purchased from
vendors and were used without further purification or distillation.
All tested compounds had a purity ꢂ95%. Column chromatography

C. Hao et al. / European Journal of Medicinal Chemistry 131 (2017) 1e13
7
Fig. 6. Compound 9d suppresses the migration and invasion of A549 cells and inhibits the PAK4/LIMK1 pathways. (A) (B) The migratory and invasive capacities of A549 cells were
evaluated using a Transwell assay (with or without Matrigel). After 24 h of treatment with the indicated concentrations of compound 9d, the invaded cells were fixed and stained
and 10 random fields were counted. (C) Western blot analysis showing the protein level of the proteins involved in migration and invasion. Compound 9d inhibited the levels of
activated PAK4 and phosphor-LIMK1 in a dose-dependent manner.
ꢀ
was carried out on silica gel (200e300 mesh). All reactions were
monitored using thin layer chromatography (TLC) on silica gel
plates.
7a as white solid (103 mg, 43%). Mp: decomp. above 265 C. Rf: 0.20
1
(DCM/MeOH,10/1, v/v). H NMR (400 MHz, DMSO-d
12.88 (br, 1H), 11.04e10.70 (m, 2H), 8.64e8.63 (m, 1H), 8.18e8.09
m, 2H), 7.92e7.82 (m, 2H), 7.64e7.45 (m, 4H), 7.33e7.28 (m, 2H),
6
): 13.22 (s,1H),
(
2
13
5
.1.1. Representative procedure for the synthesis of N -(2-(1H-
7.03e6.75 (m, 3H), 3.75e3.59 (m, 2H), 3.03e2.92 (m, 2H). C NMR
(100 MHz, DMSO-d ): 159.4, 153.7, 140.2, 138.5, 136.7, 135.8, 134.2,
130.3, 127.5, 125.2, 124.6, 123.3, 123.0, 121.4, 118.7, 117.4, 116.2, 111.8,
4
indol-3-yl)ethyl)-N -(1H-indazol-5-yl)quinazoline -2,4-diamine
7a)
.1.1.1. 2-Chloro-N-(1H-indazol-5-yl)quinazolin-4-amine
To a flask charged with 2, 4-dichloroquinazoline (5a, 0.20 g,
mmol) was added DMF (5 mL) and DIEA (0.17 mL, 3 mmol). The
6
(
5
þ
(6a).
111.5, 110.5, 42.0, 25.6. HRMS (ESI ): m/z calcd for C25
H
21
N ,
7
420.1931, found, 420.1933.
1
2
4
mixture was cooled in an ice water bath prior to the addition of 1H-
5.1.2. N -(2-(1H-indol-3-yl)ethyl)-N -(1H-indazol-5-yl)-6-
indazol-5-amine (0.15 g, 1.1 mmol). The reaction mixture was stir-
red at 0 C. After completion of the reaction (as determined by TLC
methoxyquinazoline-2,4-diamine (7b)
ꢀ
Following the general procedure mentioned above, using 5b
(0.23 g, 1 mmol) as the starting material, compound 7b was ob-
tained as a light yellow solid (99 mg, 22%, two steps). Mp:
analysis), the mixture was poured into ice-cold water. The resulting
solid was collected on a glass filter to give the crude product. The
filtrate was subjected to silica gel column chromatography using
dichloromethane/acetone (15:1) as the mobile phase to afford the
compound 6a as a pale-yellow solid (168 mg, 0.57 mmol, 57% yield).
ꢀ
1
265e268 C. Rf: 0.17 (DCM/MeOH, 10/1, v/v). H NMR (400 MHz,
DMSO-d ): 13.21 (s, 1H), 12.65 (br, 1H), 10.99e10.73 (m, 2H),
8.13e7.70 (m, 4H), 7.66e7.50 (m, 4H), 7.33e7.24 (m, 2H), 7.03e6.77
6
ꢀ
1
13
Mp: decomp. above 300 C. Rf: 0.40 (DCM/MeOH, 15/1, v/v). H
NMR (400 MHz, DMSO-d ): 13.16 (s, 1H), 10.31 (s, 1H), 8.58 (d,
(m, 3H), 3.92 (s, 3H), 3.68e3.58 (m, 2H), 3.01e2.91 (m, 2H).
C
NMR (100 MHz, DMSO-d ): 159.1, 156.5, 152.9, 138.6, 136.6, 134.2,
6
6
J ¼ 8.2 Hz, 1H), 8.14 (s, 1H), 8.10 (s, 1H), 7.88 (t, J ¼ 8.0 Hz, 1H), 7.71
130.3, 127.5, 124.8, 121.4, 118.7, 116.4, 111.8, 111.5, 110.5, 106.2, 56.7,
þ
þ
þ
(
d, J ¼ 8.1 Hz, 1H), 7.67e7.60 (m, 3H). MS (ESI ) m/z: 295.9 [MþH] ,
42.1, 25.6. HRMS (ESI ): m/z calcd for C26
23
H N
7
O, 450.2037, found,
þ
3
18.0 [MþNa] .
450.2034.
2
4
2
4
5
.1.1.2. N -(2-(1H-indol-3-yl)ethyl)-N -(1H-indazol-5-yl)quinazo-
5.1.3. N -(2-(1H-indol-3-yl)ethyl)-N -(1H-indazol-5-yl)-7-
methoxyquinazoline-2,4-diamine (7c)
line-2,4-diamine (7a). To a vessel charged with 2-chloro-N-(1H-
indazol-5-yl)quinazolin-4-amine (6a, 168 mg, 0.57 mmol) was
added ethanol(5 mL), hydrochloric acid (10
91 mg, 0.57 mmol). The vessel was sealed and stirred for 6 h at
20 C. The resulting mixture was dried under reduced pressure
Following the general procedure mentioned above, using 5c
(0.23 g, 1 mmol) as the starting material, compound 7c was ob-
tained as a white solid (117 mg, 26%, two steps). Mp: 299e300 C.
mL), and tryptamine
ꢀ
(
1
ꢀ
1
Rf: 0.28 (DCM/MeOH, 10/1, v/v). H NMR (400 MHz, DMSO-d
6
):
and subjected to silica gel column chromatography using DCM/
methanol (90:10) as mobile phase to afford the desired compound
13.17 (s, 1H), 12.68 (br, 1H), 10.86e10.48 (m, 2H), 8.54e8.49 (m, 1H),
8.49e7.90 (m, 3H), 7.61e7.51 (m, 2H), 7.32e7.26 (m, 2H), 7.08e6.77

8
C. Hao et al. / European Journal of Medicinal Chemistry 131 (2017) 1e13
(
m, 4H), 3.90 (s, 3H), 3.72e3.57 (m, 2H), 3.02e2.91 (m, 2H). 13C
5.1.8. Representative procedure for the synthesis of N4-(2-(1H-
NMR (100 MHz, DMSO-d
6
): 164.9, 159.0, 153.7, 142.4, 138.4, 136.7,
indol-3-yl)ethyl)-N2-(1H -indazol-5-yl)quinazoline -2,4-diamine
(9a)
5.1.8.1. N-(2-(1H-indol-3-yl)ethyl)-2-chloroquinazolin-4-amine (8a).
To a flask charged with 2, 4-dichloroquinazoline (5a, 0.20 g,
134.1, 130.4, 127.5, 127.1, 124.6, 123.3, 123.0, 121.4, 118.7, 116.1, 113.9,
þ
111.8, 111.5, 110.4, 103.9, 99.3, 42.0, 25.6. HRMS (ESI ): m/z calcd for
26 23 7
C H N
O, 450.2037, found, 450.2051.
1.0 mmol) was added DMF (5 mL) and DIEA (0.17 mL,1.5 mmol). The
mixture was cooled in an ice water bath prior to the addition of
2
4
5
.1.4. N -(2-(1H-indol-3-yl)ethyl)-6-chloro-N -(1H-indazol-5-yl)
tryptamine (0.18 g, 1.1 mmol). The reaction mixture was stirred at
quinazoline-2,4-diamine (7d)
Following the general procedure mentioned above, using 5d
0.23 g, 1 mmol) as the starting material, compound 7d was ob-
ꢀ
0
C. After completion of the reaction (as determined by TLC
analysis), the mixture was poured into ice-cold water. The resulting
solid was collected on a glass filter to give the crude product. The
filtrate was subjected to silica gel column chromatography using
dichloromethane/acetone (15:1) as mobile phase to afford the
compound 8a as a pale-yellow solid (232 mg, 0.72 mmol, 72%
(
tained as an off-white solid (191 mg, 42%, two steps). Mp:
ꢀ
1
2
45e246 C. Rf: 0.28 (DCM/MeOH, 10/1, v/v). H NMR (100 MHz,
DMSO-d ): 13.02 (s, 1H), 10.81 (s, 1H), 9.67e9.49 (m, 1H), 8.48 (d,
J ¼ 2.0 Hz, 1H), 8.37 (s, 1H), 8.04 (s, 1H), 7.70 (dd, J ¼ 1.4, 8.9 Hz, 1H),
.58 (dd, J ¼ 2.0, 8.9 Hz, 1H), 7.53 (s, 1H), 7.38 (s, 1H), 7.34 (d,
J ¼ 8.2 Hz, 1H), 7.18e7.00 (m, 4H), 3.64e3.59 (m, 2H), 3.00e2.96 (m,
6
ꢀ
1
yield). Mp: 171e172 C. Rf: 0.60 (DCM/MeOH, 15/1, v/v). H NMR
400 MHz, DMSO-d
7
(
6
): 10.85 (s, 1H), 8.94 (t, J ¼ 5.4 Hz, 1H), 8.25 (d,
J ¼ 8.0 Hz, 1H), 7.82e7.78 (m, 1H), 7.72 (d, J ¼ 7.8 Hz, 1H), 7.64 (d,
J ¼ 8.0 Hz, 1H), 7.56e7.52 (m, 1H), 7.35 (d, J ¼ 8.1 Hz, 1H), 7.22 (d,
J ¼ 2.0 Hz, 1H), 7.05 (t, J ¼ 7.1 Hz, 1H), 6.99 (d, J ¼ 7.3 Hz, 1H),
13
2
1
1
H). C NMR (400 MHz, DMSO-d
6
):159.5, 158.1, 151.4, 157.6, 136.7,
33.9, 133.2, 132.7, 127.8, 124.6, 123.6, 123.3, 123.0, 122.8, 121.3,
19.8, 118.6, 113.5, 112.7, 111.8, 110.2, 42.1, 25.7. HRMS (ESI ): m/z
calcd for C25H20ClN , 454.1541, found, 454.1542.
7
þ
þ
þ
3
.81e3.76 (m, 2H), 3.12e3.06 (m, 2H). MS (ESI ) m/z: 323.1[MþH] .
4
2
5
.1.8.2. N -(2-(1H-indol-3-yl)ethyl)-N -(1H-indazol-5-yl)quinazo-
line-2,4-diamine (9a). To a vessel charged with N-(2-(1H-indol-3-
yl)ethyl)-2-chloroquinazolin-4-amine (8a, 232 mg, 0.72 mmol)
2
4
5
.1.5. N -(2-(1H-indol-3-yl)ethyl)-7-chloro-N -(1H-indazol-5-yl)
quinazoline-2,4-diamine (7e)
Following general the procedure mentioned above, using 5e
0.23 g, 1 mmol) as the starting material, compound 7e was ob-
tained as a white solid (163 mg, 36%, two steps). Mp: 298e301 C.
6
Rf: 0.28 (DCM/MeOH, 10/1, v/v). H NMR (400 MHz, DMSO-d ):
3.22 (s, 1H), 12.89 (br, 1H), 11.11e10.81 (m, 2H), 8.66e8.60 (m, 1H),
.34e7.92 (m, 3H), 7.62e7.53 (m, 4H), 7.33e7.28 (m, 2H), 7.06e6.75
was added ethanol (5 mL), hydrochloric acid (10
mL), and 1H-
indazol-5-amine (96 mg, 0.72 mmol). The vessel was sealed and
(
ꢀ
stirred for 6 h at 120 C. The resulting mixture was dried under
ꢀ
reduced pressure and subjected to silica gel column chromatog-
raphy using dichloromethane/methanol (90:10) as the mobile
1
1
8
(
phase to afford the desired compound 9a as a white solid (184 mg,
ꢀ
0
.87 mmol, 61% yield). Mp: decomp. above 260 C. Rf: 0.17 (DCM/
13
m, 3H), 3.78e3.58 (m, 2H), 3.04e2.91 (m, 2H). C NMR (100 MHz,
DMSO-d ): 158.9, 153.5, 141.1, 140.1, 138.5, 136.6, 134.2, 130.1, 127.5,
27.3,124.8,124.6,124. 4,123.3,123.0,121.4,118.7,116.6,116.2,111.8,
1
6
MeOH, 10/1, v/v). H NMR (400 MHz, DMSO-d ): 13.21 (s, 1H), 12.57
6
(
1
br, 1H), 10.92 (s, 1H), 10.50 (s, 1H), 10.01 (s, 1H), 8.39 (d, J ¼ 8.1 Hz,
1
1
4
H), 7.98 (s, 1H), 7.91 (s, 1H), 7.81 (t, J ¼ 7.7 Hz, 1H), 7.55 (d,
þ
7
11.4, 110.5, 109.4, 42.1, 25.5. HRMS (ESI ): m/z calcd for C25H20ClN ,
54.1541, found, 454.1564.
J ¼ 8.0 Hz,1H), 7.47e7.43 (m, 3H), 7.35 (d, J ¼ 8.1 Hz,1H), 7.15 (s,1H),
7
3
.04 (t, J ¼ 7.3 Hz, 1H), 6.84 (t, J ¼ 7.1 Hz, 1H), 3.86e3.85 (m, 2H),
13
.12e3.08 (m, 2H).
6
C NMR (100 MHz, DMSO-d ): 160.4, 152.5,
2
4
139.3, 138.4, 136.7, 135.6, 134.0, 129.8, 127.5, 125.0, 124.7, 123.6,
5
.1.6. N -(2-(1H-indol-3-yl)ethyl)-6-bromo-N -(1H-indazol-5-yl)
1
23.4, 123.2, 124.5, 118.7, 117.8, 111.9, 111.7, 111.1, 110.8, 42.8, 24.7.
quinazoline-2,4-diamine (7f)
Following general the procedure mentioned above, using 5f
0.28 g, 1 mmol) as the starting material, compound 7f was ob-
þ
HRMS (ESI ): m/z calcd for C25
H
21
N
7
, 420.1931, found, 420.1931.
(
4
2
5
.1.9. N -(2-(1H-indol-3-yl)ethyl)-N -(1H-indazol-5-yl)-6-
tained as a white solid (195 mg, 39%, two steps). Mp: decomp.
ꢀ
1
methoxyquinazoline-2,4-diamine (9b)
above 284 C. Rf: 0.28 (DCM/MeOH, 10/1, v/v). H NMR (400 MHz,
DMSO-d ): 13.23 (s, 1H), 13.07 (br, 1H), 11.03 (s, 1H), 10.89 (s, 1H),
.93e8.87 (m, 1H), 8.27e8.23 (m, 1H), 8.11 (s, 1H), 8.00e7.91 (m,
H), 7.65e7.27 (m, 6H), 7.13e6.76 (m, 3H), 3.75e3.60 (m, 2H),
Following the general procedure mentioned above, using 5b
0.23 g, 1 mmol) as the starting material, compound 9b was ob-
6
(
8
1
3
1
1
tained as a light yellow solid (144 mg, 32%, two steps). Mp:
ꢀ
1
.03e2.93 (m, 2H). 1 C NMR (100 MHz, DMSO-d
3
258e260 C. Rf: 0.17 (DCM/MeOH, 10/1, v/v). H NMR (400 MHz,
DMSO-d ): 13.19 (s, 1H), 12.25 (br, 1H), 10.91 (s, 1H), 10.27 (s, 1H),
6
): 158.3, 153.8,
38.5, 138.2, 136.7, 134.1, 130.3, 127.5, 124.2, 123.3, 123.0, 121.4,
20.0, 118.7, 116.1, 115.9, 112.1, 111.8, 111.5, 110.5, 42.1, 25.6. HRMS
ESI ): m/z calcd for C25
6
9
.85 (s, 1H), 7.94 (s, 2H), 7.89 (d, J ¼ 2.4 Hz, 1H), 7.57e7.53 (m, 2H),
þ
7.47e7.41 (m, 3H), 7.35 (d, J ¼ 8.1 Hz, 1H), 7.15 (s, 1H), 7.05 (t,
J ¼ 7.3 Hz, 1H), 6.86 (t, J ¼ 7.2 Hz, 1H), 3.87 (s, 3H), 3.85e3.83 (m,
(
H20BrN
7
, 500.1016, found, 500.1019.
13
6
2H), 3.12e3.08 (m, 2H). C NMR (100 MHz, DMSO-d ): 160.1, 156.6,
2
4
5
.1.7. N -(2-(1H-indol-3-yl)ethyl)-6-fluoro-N -(1H-indazol-5-yl)
152.0,138.3,136.7,134.0,127.6,125.1,123.7,123.4,123.2,121.5,119.4,
þ
quinazoline-2,4-diamine (7g)
118.7, 111.9, 111.8, 111.4, 111.1, 106.0, 56.7, 42.8, 24.7. HRMS (ESI ): m/
Following the general procedure mentioned above, using 5g
z calcd for C26
H
23
N
7
O, 450.2037, found, 450.2053.
(
0.22 g, 1 mmol) as the starting material, compound 7g was ob-
4
2
tained as a white solid (118 mg, 27%, two steps). Mp: decomp. above
2
5.1.10. N -(2-(1H-indol-3-yl)ethyl)-N -(1H-indazol-5-yl)-7-
methoxyquinazoline-2,4-diamine (9c)
ꢀ
1
62 C. Rf: 0.28 (DCM/MeOH, 10/1, v/v). H NMR (400 MHz, DMSO-
d
8
6
): 13.20 (s, 1H), 12.77 (br, 1H), 10.88 (s, 2H), 8.53e8.47 (m, 1H),
.25e8.13 (m, 2H), 7.90e7.60 (m, 2H), 7.66e7.57 (m, 3H), 7.34e7.25
Following the general procedure mentioned above, using 5c
(0.23 g, 1 mmol) as the starting material, compound 9c was ob-
tained as a white solid (167 mg, 37%, two steps). Mp: decomp. above
(
m, 2H), 7.05e6.79 (m, 2H), 3.70e3.62 (m, 2H), 3.02e2.94 (m, 2H).
1
3
ꢀ
1
C NMR (100 MHz, DMSO-d
6
): 158.9, 154.3, 138.3, 136.7, 134.1,
288 C. Rf: 0.20 (DCM/MeOH, 10/1, v/v). H NMR (600 MHz, DMSO-
): 13.18 (s, 1H), 12.33 (br, 1H), 10.89 (s, 1H), 10.44 (s, 1H), 9.68 (s,
130.6, 127.6, 124.2, 123.3, 123.1, 121.4, 118.7, 115.7, 111.8, 110.5, 42.1,
d
6
þ
2
4
5.6. HRMS (ESI ): m/z calcd for C25
7
H20FN , 438.1837, found,
1H), 8.27 (d, J ¼ 9.1 Hz, 1H), 7.97 (s, 1H), 7.91 (s, 1H), 7.55 (d,
38.1826.
J ¼ 7.5 Hz,1H), 7.44e7.42 (m, 2H), 7.34 (d, J ¼ 8.1 Hz,1H), 7.14 (s,1H),

C. Hao et al. / European Journal of Medicinal Chemistry 131 (2017) 1e13
9
þ
7
.05e7.03 (m, 2H), 7.00 (s, 1H), 6.85 (m, 1H), 3.87 (s, 3H), 3.83e3.82
114.5, 111.9, 111.7, 111.1, 110.2, 110.0, 42.8, 24.6. HRMS (ESI ): m/z
calcd for C25 , 438.1837, found, 438.1842.
13
(
m, 2H), 3.09e3.07 (m, 2H). C NMR (100 MHz, DMSO-d
6
): 164.6,
H20FN
7
1
60.0, 136.7, 134.0, 127.6, 126.5, 123.2, 121.4, 118.7, 114.2, 111.9, 111.8,
þ
104.3, 99.9, 56.4, 42.6, 24.8. HRMS (ESI ): m/z calcd for C26H N O,
23 7
5
.1.15. Sodium nitromalonaldehyde monohydrate (11)
4
50.2037, found, 450.2044.
To a solution of NaNO (20.0 g, 0.29 mol) in water (20 mL) was
2
dropwise added a solution of mucobromic acid (20.0 g, 0.078 mol)
4
2
5
.1.11. N -(2-(1H-indol-3-yl)ethyl)-6-chloro-N -(1H-indazol-5-yl)
ꢀ
in ethanol at 54 C over 45 min, and the mixture was stirred at the
quinazoline-2,4-diamine (9d)
Following the general procedure mentioned above, using 5d
0.23 g, 1 mmol) as the starting material, compound 9d was ob-
ꢀ
same temperature for 15 min. The reaction was cooled to 0 C and
the solid was filtered to give sodium nitromalonaldehyde mono-
(
ꢁ
hydrate (11) as an off-white solid (3.33 g, 27%). MS (ESI ) m/z:115.4
tained as a light yellow solid (249 mg, 55%, two steps). Mp:
-
[
MꢁH] .
ꢀ
1
2
89e292 C. Rf: 0.27 (DCM/MeOH, 10/1, v/v). H NMR (400 MHz,
DMSO-d ):13.17 (s, 1H), 12.76 (br, 1H), 10.86 (s, 1H), 10.71 (s, 1H),
.99 (s, 1H), 8.54 (d, J ¼ 2.0 Hz, 1H), 8.08 (s, 1H), 7.88e7.85 (m, 2H),
.76 (d, J ¼ 8.6 Hz, 2H), 7.60 (d, J ¼ 8.9 Hz,1H), 7.45 (d, J ¼ 7.9 Hz,1H),
.33 (d, J ¼ 8.1 Hz,1H), 7.26 (dd, J ¼ 1.3, 8.6 Hz,1H), 7.14 (d, J ¼ 1.4 Hz,
6
5.1.16. 5-Nitro-1H-pyrazolo[3,4-b]pyridine (12)
9
7
7
1
3
1
1
To a solution of sodium nitromalonaldehyde monohydrate
(
(
1.23 g, 7.81 mmol) in water was added 1H-pyrazol-3-amine
0.84 g, 7.43 mmol). The mixture was stirred at 90 C for 16 h and
ꢀ
H), 7.03 (t, J ¼ 7.4 Hz,1H), 6.83 (t, J ¼ 7.4 Hz,1H), 3.91e3.86 (m, 2H),
.15e3.11 (m, 2H). 13 C NMR (100 MHz, DMSO-d
): 159.6, 152.7,
38.4, 136.7, 135.4, 134.0, 128.8, 127.5, 124.2, 123.3, 121.5, 120.0,
then cooled to room temperature. The reaction solution was
6
adjusted to pH ¼ 5 and extracted by ethyl acetate (200 mL). The
2 4
organic layer was dried over Na SO and evaporated. The residue
þ
18.7, 118.6, 112.1, 111.9, 111.6, 111.1, 42.9, 24.5. HRMS (ESI ): m/z
was purified by silica gel chromatography (1% MeOH in dichloro-
methane) to give 5-nitro-1H-pyrazolo [4,3-b]pyridine as a white
solid (118 mg, 27%). Rf: 0.23 (DCM/MeOH, 100/1, v/v). Mp:
7
calcd for C25H20ClN , 454.1541, found, 454.1550.
4
2
5
.1.12. N -(2-(1H-indol-3-yl)ethyl)-7-chloro-N -(1H-indazol-5-yl)
ꢀ
1
2
06e208 C. H NMR (DMSO-d
6
, 400 MHz)
d
14.40 (s, 1H), 9.35 (d,
quinazoline-2,4-diamine (9e)
Following the general procedure mentioned above, using 5e
0.23 g, 1 mmol) as the starting material, compound 9e was ob-
ꢁ
J ¼ 2.5 Hz, 1H), 9.21 (d, J ¼ 2.5 Hz, 1H), 8.46 (s, 1H). MS (ESI ) m/
-
z:162.8 [MꢁH] .
(
tained as a yellow solid (231 mg, 51%, two steps). Mp: decomp.
ꢀ
1
5.1.17. 1H-Pyrazolo[3,4-b]pyridin-5-amine (13)
above 267 C. Rf: 0.27 (DCM/MeOH, 10/1, v/v). H NMR (400 MHz,
DMSO-d ): 13.23 (s, 1H), 12.62 (br, 1H), 10.91 (s, 1H), 10.56 (s, 1H),
0.02 (s, 1H), 8.39 (d, J ¼ 8.7 Hz, 1H), 7.95 (s, 1H), 7.65 (s, 1H),
.58e7.52 (m, 2H), 7.44e7.42 (m, 2H), 7.34 (d, J ¼ 8.1 Hz, 1H), 7.15 (s,
To a solution of 5-nitro-1H-pyrazolo [4,3-b]pyridine (7.3 g,
6
3
3
8.0 mmol) in MeOH (240 mL) was added 10% wt Pd/C (4.03,
.8 mmol). The reaction mixture was hydrogenated under
1
7
hydrogen (1 atm) for 16 h. The Pd/C was removed by filtration, and
the filtrate was concentrated to give 1H-pyrazolo [4,3-b]pyridin-5-
amine (5.1 g, 82% yield) as a light brown solid. Rf: 0.33 (DCM/MeOH,
1
3
H), 7.05 (t, J ¼ 7.4 Hz, 1H), 6.85 (s, 1H), 3.90e3.77 (m, 2H),
.17e3.02 (m, 2H). 1 C NMR (100 MHz, DMSO-d
6
): 159.9, 139.8,
3
1
36.7, 134.1, 127.5, 126.8, 125.0, 123.3, 121.5, 118.7, 118.6, 117.6, 111.9,
ꢀ
1
þ
19/1, v/v). Mp: 178 C. H NMR (DMSO-d , 400 MHz) d 13.12 (s, 1H),
6
111.7, 111.2, 109.8, 42.8, 24.6. HRMS (ESI ): m/z calcd for C25H20ClN ,
7
8
2
.05 (d, J ¼ 2.4 Hz, 1H), 7.81 (s, 1H), 7.18 (d, J ¼ 2.3 Hz, 1H), 5.04 (s,
4
54.1541, found, 454.1545.
þ
þ
þ
þ
H). MS (ESI ) m/z:134.8 [MþH] ,156.7 [MþNa] , 301.0 [2MþNa] .
4
2
5
.1.13. N -(2-(1H-indol-3-yl)ethyl)-6-bromo-N -(1H-indazol-5-yl)
5.1.18. 2-Chloro-6-methyl-4-nitroaniline (15a)
quinazoline-2,4-diamine (9f)
To a solution of 2-methyl-4-nitroaniline (2.0 g, 13.2 mmol) in
Following the general procedure mentioned above, using 5f
0.28 g, 1 mmol) as the starting material, compound 9f was ob-
acetonitrile (40 mL) was added N-chlorosuccinimide (2.1 g,
(
ꢀ
1
5.8 mmol) at 60 C. The mixture was heated to reflux for 3 h and
tained as a light yellow solid (245 mg, 49%, two steps). Mp: decomp.
ꢀ
1
then cooled to room temperature. The mixture was evaporated and
diluted with dichloromethane (50 mL). The organic layer was
above 283 C. Rf: 0.23 (DCM/MeOH, 10/1, v/v). H NMR (400 MHz,
DMSO-d ): 13.23 (s, 1H), 12.80 (br, 1H), 10.93 (s, 1H), 10.59 (s, 1H),
0.03 (s, 1H), 8.68 (s, 1H), 7.96e7.94 (m, 3H), 7.56 (d, J ¼ 8.3 Hz, 1H),
.51 (d, J ¼ 8.8 Hz,1H), 7.43 (d, J ¼ 8.1 Hz, 2H), 7.35 (d, J ¼ 8.1 Hz,1H),
6
washed with 2.5 M NaOH and brine, dried over Na
evaporated. The residue was purified by silica gel chromatography
n-hexane/ethyl acetate, 4/1, v/v) to give 2-chloro-6-methyl-4-
nitroaniline as a yellow solid (2.33 g, 95%). Mp: 172e174 C. Rf:
.40 (n-hexane/ethyl acetate, 5/1, v/v). H NMR (DMSO-d
00 MHz)
2
SO4, filtered, and
1
7
7
(
.16 (s, 1H), 7.05 (t, J ¼ 7.3 Hz, 1H), 6.87e6.84 (m, 1H), 3.84e3.83 (m,
1
3
ꢀ
2
H), 3.11e3.07 (m, 2H). C NMR (100 MHz, DMSO-d
6
): 159.4,152.7,
1
0
4
6
,
139.0,138.3,138.0,136.7,134.0,129.7,127.5,127.1,123.3,121.5,120.2,
d
8.01 (d, J ¼ 2.6 Hz, 1H), 7.89 (d, J ¼ 2.1 Hz, 1H), 6.61 (s,
1
18.8, 118.7, 116.6, 114.4, 112.5, 111.9, 111.7, 111.1, 42.9, 24.5. HRMS
ꢁ
-
þ
2H), 2.22 (s, 3H). MS (ESI ) m/z:184.5 [MꢁH] .
(
ESI ): m/z calcd for C25
H20BrN
7
, 500.1016, found, 500.1023.
4
2
5
.1.14. N -(2-(1H-indol-3-yl)ethyl)-6-fluoro-N -(1H-indazol-5-yl)
5.1.19. 2-Bromo-6-methyl-4-nitroaniline (15b)
quinazoline-2,4-diamine (9g)
To a solution of 2-methyl-4-nitroaniline (2.0 g, 13.2 mmol) in
acetonitrile (40 mL) was added N-bromosuccinimide (2.8 g,
15.8 mmol) at 60 C. The mixture was heated to reflux for 3 h and
Following the general procedure mentioned above, using 5g
ꢀ
(
0.22 g, 1 mmol) as the starting material, compound 9g was ob-
tained as a light yellow solid (140 mg, 32%, two steps). Mp:
then cooled to room temperature. The mixture was evaporated and
diluted with dichloromethane (50 mL). The organic layer was
washed with 2.5 M NaOH and brine, dried over Na SO4, filtered, and
2
ꢀ
1
2
64e267 C. Rf: 0.20 (DCM/MeOH, 10/1, v/v). H NMR (400 MHz,
DMSO-d ): 13.19 (s, 1H), 12.52 (br, 1H), 10.92 (s, 1H), 10.38 (s, 1H),
.88 (s, 1H), 8.33 (d, J ¼ 8.1 Hz, 1H), 7.99 (s, 1H), 7.92 (s, 1H),
.73e7.70 (m, 1H), 7.63e7.59 (m, 1H), 7.55 (d, J ¼ 8.7 Hz,1H), 7.44 (d,
6
9
7
evaporated. The residue was purified by silica gel chromatography
(n-hexane/ethyl acetate, 4/1, v/v) to give 2-bromo-6-methyl-4-
ꢀ
J ¼ 8.1 Hz, 2H), 7.35 (d, J ¼ 8.1 Hz, 1H), 7.16 (s, 1H), 7.04 (t, J ¼ 7.4 Hz,
nitroaniline as a yellow solid (2.75 g, 91%). Mp: 178e179 C. Rf:
1
3
1
1
H), 6.85 (t, J ¼ 7.2 Hz, 1H), 3.85e3.84 (m, 2H), 3.11e3.08 (m, 2H).
0.40 (n-hexane/ethyl acetate, 5/1, v/v). H NMR (DMSO-d
6
,
C NMR (100 MHz, DMSO-d
6
): 160.0, 159.7, 157.3, 153.1, 138.4, 136.7,
400 MHz)
d
8.15 (d, J ¼ 2.5 Hz, 1H), 7.93 (d, J ¼ 2.1 Hz, 1H), 6.53 (s,
ꢁ
-
133.9, 130.1, 127.5, 123.9, 123.6, 123.4, 123.3, 121.5, 121.0, 118.7, 118.7,
2H), 2.23 (s, 3H). MS (ESI ) m/z:228.5 [MꢁH] .

10
C. Hao et al. / European Journal of Medicinal Chemistry 131 (2017) 1e13
5
.1.20. 7-Chloro-5-nitro-1H-indazole (16a)
To a 0 C solution of 2-chloro-6-methyl-4-nitroaniline (15a)
5.1.26. 2,6-dimethyl-4-nitroaniline (21)
ꢀ
N-Tosyl-2,6-dimethyl-4-nitroaniline (20) (3.2 g, 10.0 mmol) was
ꢀ
(
0.9 g, 4.9 mmol) in glacial acetic acid (18 mL) was added a solution
dissolved in H
2
SO
4
(32 mL) and warmed at 50 C for 1 h. The re-
of NaNO (0.40 g, 5.8 mmol) in 4 mL water dropwise over a period
2
action mixture was poured slowly into an ice/water mixture. The
precipitate was filtered and washed with water. The crude product
was purified by recrystallization (ethyl acetate) to give 21 as yellow
crystals (1.0 g, 53%). Rf: 0.34 (n-hexane/ethyl acetate, 5/1, v/v). Mp:
of 10 min with stirring. The mixture was allowed to warm to room
temperature and stirred for 12 h. The precipitate was removed by
filtration of the reaction mixture through a pad of Celite. The filtrate
ꢀ
1
was poured into H
and purified by silica gel column chromatography (DCM/MeOH
00:1 as eluent) to afford 16a as a light brown solid (0.50 g, 52%).
2
O (100 mL); the solid was collected by filtration
164e165 C. H NMR (DMSO-d
2H), 2.16 (s, 6H). MS (ESI ) m/z: 166.8 [MþH] , 188.8 [MþNa] .
6
, 400 MHz)
d
7.79 (s, 2H), 6.16 (s,
þ
þ
þ
1
ꢀ
1
Mp: 250e251 C. Rf: 0.47 (DCM/MeOH,100/1, v/v). H NMR (DMSO-
, 400 MHz)
14.33 (s,1H), 8.86 (d, J ¼ 1.3 Hz,1H), 8.53 (s,1H), 8.30
s, 1H). MS (ESI ) m/z:195.5 [M - H] .
5.1.27. 7-methyl-5-nitro-1H-indazole (22)
ꢀ
d
6
d
To a 0 C solution of 2,6-dimethyl-4-nitroaniline (21) (0.9 g,
ꢁ
-
(
5.4 mmol) in glacial acetic acid (18 mL) was added a solution of
2
NaNO (0.45 g, 6.5 mmol) in 5 mL water by dropwise addition over
a period of 10 min with stirring. The mixture was allowed to warm
to room temperature and stirred for 12 h. The precipitate was
removed by filtration of the reaction mixture through a pad of
Celite. The filtrate was poured into H O (100 ml); the solid was
2
collected by filtration and purified by silica gel column chroma-
tography (DCM/MeOH 100:1 as eluent) to afford 22 as a yellow
5
.1.21. 7-Bromo-5-nitro-1H-indazole (16b)
Compound 16b was prepared in a manner similar to that of 16a
as a light brown solid (49%). Rf: 0.53 (DCM/MeOH, 100/1, v/v). Mp:
2
ꢀ
1
50e251 C. H NMR (DMSO-d
6
, 400 MHz)
d
14.22 (s, 1H), 8.88 (d,
ꢁ
J ¼ 1.2 Hz, 1H), 8.55 (s, 1H), 8.42 (d, J ¼ 1.7 Hz, 1H). MS (ESI ) m/
-
z:239.5 [M - H] .
ꢀ
solid (0.50 g, 52%). Mp: 247e248 C. Rf: 0.24 (DCM/MeOH, 100/1, v/
1
v). H NMR (DMSO-d
6
, 400 MHz)
d
13.83 (s, 1H), 8.67 (d, J ¼ 1.8 Hz,
5
.1.22. 7-Chloro-1H-indazol-5-amine (17a)
A mixture of 7-chloro-5-nitro-1H-indazole (16a) (200 mg,
.94 mmol), SnCl -H O (1.0 g, 4.7 mmol) in EtOH (10 mL) was
2 2
ꢁ
1
H), 8.39 (d, J ¼ 1.1 Hz, 1H), 8.02 (s, 2H), 2.62 (s, 3H). MS (ESI ) m/z:
-
175.5 [MꢁH] .
0
heated at reflux for 18 h. After filtration, the filtrate was concen-
5
.1.28. 7-Methyl-1H-indazol-5-amine (23)
A mixture of 7-methyl-5-nitro-1H-indazole (22) (0.23 g,
trated to yield 164 mg (97%) of 1H-indazol-5-amine as a light
ꢀ
1
brown solid. Rf: 0.25 (DCM/MeOH, 100/1, v/v). Mp: 157e159 C. H
1.3 mmol) and 10% Pd/C (50 mg) in MeOH (4 mL) was stirred under
NMR (DMSO-d
6
, 400 MHz)
d
13.02 (s, 1H), 7.84 (d, J ¼ 1.0 Hz, 1H),
H
2
(1 atm) overnight. After filtration, the filtrate was concentrated
þ
6
.88 (d, J ¼ 1.6 Hz, 1H), 6.73 (s, 1H), 4.99 (s, 2H). MS (ESI ) m/z:167.7
to yield 7-methyl-1H-indazol-5-amine (23) as a brown solid
without further purification (188 mg, 97%). Rf: 0.40 (DCM/MeOH,
þ
[MþH] .
ꢀ
1
19/1, v/v). Mp: 134e136 C. H NMR (DMSO-d
6
, 400 MHz)
d
12.64 (s,
þ
1
H), 7.69 (s, 1H), 6.55 (s, 2H), 4.66 (s, 2H), 2.38 (s, 3H). MS (ESI ) m/
5.1.23. 7- Bromo-1H-indazol-5-amine (17b)
þ
z: 147.8 [MþH] .
Compound 17b was prepared in a manner similar to that for 17a
Compounds 24a-h were synthesized by the same procedure as
described for synthesis of compound 9a.
as a light brown solid (92%). Rf: 0.23 (DCM/MeOH, 100/1, v/v). Mp:
ꢀ
1
1
65e167 C. H NMR (DMSO-d
6
, 400 MHz)
d
12.93 (s, 1H), 7.86 (d,
þ
J ¼ 8.1 Hz, 1H), 7.02 (d, J ¼ 1.6 Hz, 1H). MS (ESI) m/z: 211.7[MþH] .
4
2
5
5
.1.29. N -(2-(1H-indol-3-yl)ethyl)-N -(1H-benzo[d] [1-3]triazol-
-yl)-6-chloroquinazoline-2,4-diamine (24a)
5.1.24. N-(2, 6-dimethylphenyl)-4-methylbenzenesulfonamide (19)
Using 8d (179 mg, 0.5 mmol) and 1H-benzo[d] [1-3]triazol-5-
A mixture of 2, 6-dimethylaniline (2.4 g, 20 mmol), 4-
amine (67 mg, 0.5 mmol) as starting materials, compound 24a
was prepared as an off-white solid (168 mg, 74%). Mp: 218e219 C.
Rf: 0.31 (DCM/MeOH, 10/1, v/v). H NMR (400 MHz, DMSO-d
d
toluenesulfonyl chloride (2.5 g, 13.2 mmol) and silica gel (14.4 g,
00e200 mesh) was stirred at room temperature. The progress of
ꢀ
1
1
6
):
the reaction was monitored by TLC. After completion of the reac-
tion, MeOH (100 mL) was added, and the mixture was filtered
through a sintered funnel. The solvent was evaporated and the
residue was purified by recrystallization (MeOH) to give the
product 19 as a white solid (4.79 g, 87%). Rf: 0.57 (n-hexane/ethyl
ppm 13.03 (br, 1H), 10.86 (s, 1H), 10.74 (s, 1H), 10.07 (s, 1H), 9.92 (s,
H), 8.57 (d, J ¼ 1.1 Hz, 1H), 7.90 (dd, J ¼ 1.3, 5.9 Hz, 1H), 7.75e7.65
1
(
m, 3H), 7.42 (t, J ¼ 5.2 Hz, 1H), 7.32 (d, J ¼ 5.4 Hz, 2H), 7.04 (t,
J ¼ 5.0 Hz,1H), 6.91 (t, J ¼ 4.9 Hz, 1H), 6.82 (d, J ¼ 5.1 Hz,1H), 3.76 (s,
13
2
H), 2.95 (s, 2H). C NMR (100 MHz, DMSO-d
6
): 159.7, 152.0, 138.1,
ꢀ
1
3
acetate, 5/1, v/v). Mp: 134e135 C. H NMR (CDCl , 400 MHz) d 7.61
136.7, 135.7, 129.4,127.4,124.4,123.2,121.4, 120.0,118.8,118.6,112.2,
(
1
d, J ¼ 8.3 Hz, 2H), 7.26 (d, J ¼ 8.5 Hz, 2H), 7.12e7.09 (m, 1H), 7.04 (s,
þ
8
111.9, 111.4, 43.0, 24.6. HRMS (ESI ): m/z calcd for C24H19ClN ,
þ
H), 7.02 (s, 1H), 6.57 (s, 1H), 2.44 (s, 3H), 2.05 (s, 6H). MS (ESI ) m/
4
55.1498, found, 455.1494.
þ
þ
z: 275.9 [MþH] , 298.0 [MþNa] .
4
2
5.1.30. N -(2-(1H-indol-3-yl)ethyl)-N -(1H-benzo[d]imidazol-5-
5.1.25. N-(2,6-dimethyl-4-nitrophenyl)-4-
yl)-6-chloroquinazoline-2,4-diamine (24b)
methylbenzenesulfonamide (20)
N-tosyl-2-6-dimethylaniline (19) (2.0 g, 7.3 mmol) was sus-
2 3
pended in 100 mL AcOH-H O-HNO (1:1:0.2) mixture. Sodium ni-
trite (1.0 g, 14.6 mmol) was added and the reaction mixture was
reflux for 4 h. The reaction was monitored via TLC and, upon
completion, was allowed to cool to room temperature overnight to
yield colourless crystals. The crystals were filtered and washed with
Using 8d (179 mg, 0.5 mmol) and 1H-benzo[d]imidazol-5-amine
(67 mg, 0.5 mmol) as starting materials, compound 24b was pre-
pared as an off-white solid (186 mg, 82%). Mp: decomp. above
ꢀ
1
214 C. Rf: 0.07 (DCM/MeOH, 10/1, v/v). H NMR (400 MHz, DMSO-
): ppm 10.87 (s, 1H), 10.43 (s, 1H), 9.60 (s, 1H), 8.99 (s, 1H), 8.49
d
6
d
(s, 1H), 8.21 (s, 1H), 7.81 (d, J ¼ 8.2 Hz, 2H), 7.70 (d, J ¼ 8.7 Hz, 1H),
7.59e7.56 (m, 2H), 7.44 (d, J ¼ 7.2 Hz, 1H), 7.33 (d, J ¼ 7.9 Hz, 1H),
7.18 (s, 1H), 7.04 (t, J ¼ 7.0 Hz, 1H), 6.84 (t, J ¼ 7.0 Hz, 1H), 3.87e3.86
ꢀ
1
water to afford 1.6 g (70%) of 20. Rf: 0.73 (DCM). Mp: 163e164 C. H
13
NMR (CDCl
3
, 400 MHz) 7.92 (s, 2H), 7.61 (d, J ¼ 8.2 Hz, 2H), 7.31 (s,
6
(m, 2H), 3.13e3.11 (m, 2H). C NMR (100 MHz, DMSO-d ): 159.7,
ꢁ
1
H), 7.29 (s,1H), 6.02 (s, 1H), 2.46 (s, 3H), 2.17 (s, 6H). MS (ESI ) m/z:
153.9,141.8,136.7,134.9,128.7,128.1,127.6,123.8,123.4,122.2,121.4,
-
þ
318.7 [M - H] .
119.7, 118.6, 115.5, 112.4, 111.9, 107.7, 42.8, 24.6. HRMS (ESI ): m/z

C. Hao et al. / European Journal of Medicinal Chemistry 131 (2017) 1e13
11
4
2
calcd for C25H19ClN
7
, 454.1541, found, 454.1536.
5.1.35. N -(2-(1H-indol-3-yl)ethyl)-N -(7-bromo-1H-indazol-5-
yl)-6-chloroquinazoline-2,4-diamine (24g)
Using 8d (179 mg, 0.5 mmol) and 17b (106 mg, 0.5 mmol) as
starting materials, compound 24g was prepared as an off-white
5
2
.1.31. 5-((4-((2-(1H-indol-3-yl)ethyl)amino)-6-chloroquinazolin-
-yl)amino)indolin-2-one (24c)
Using 8d (179 mg, 0.5 mmol) and 5-aminoindolin-2-one (74 mg,
ꢀ
solid (125 mg, 47%). Mp: decomp. above 289 C. Rf: 0.42 (DCM/
1
MeOH, 10/1, v/v). H NMR (400 MHz, DMSO-d
6
):
d
ppm 13.62 (s,
0
.5 mmol) as starting materials, compound 24c was prepared as an
1
8
H), 10.90 (s, 1H), 10.61 (s, 1H), 9.96 (s, 1H), 8.53 (d, J ¼ 1.8 Hz, 1H),
ꢀ
off-white solid (180 mg, 77%). Mp: decomp. above 212 C. Rf: 0.34
.13 (s, 1H), 7.94 (s, 1H), 7.87e7.85 (m, 2H), 7.62 (d, J ¼ 8.9 Hz, 1H),
1
(
DCM/MeOH, 10/1, v/v). H NMR (600 MHz, DMSO-d
6
): d ppm 12.65
7
6
.39 (s,1H), 7.34 (d, J ¼ 8.1 Hz,1H), 7.17 (s,1H), 7.04 (t, J ¼ 7.4 Hz,1H),
(
br, 1H), 10.90 (s, 1H), 10.46 (s, 1H), 10.38 (s, 1H), 9.92 (s, 1H), 8.51 (d,
13
.82 (s, 1H), 3.83e3.81 (m, 2H), 3.11e3.08 (m, 2H). C NMR
): 159.6, 152.6, 136.7, 135.4, 130.8, 128.9, 127.5,
J ¼ 2.0 Hz, 1H), 7.84 (dd, J ¼ 1.8, 8.9 Hz, 1H), 7.58 (d, J ¼ 8.8 Hz, 1H),
.43 (s, 1H), 7.40 (s, 1H), 7.34 (d, J ¼ 8.1 Hz, 1H), 7.27 (d, J ¼ 8.1 Hz,
H), 7.18 (s, 1H), 7.05 (t, J ¼ 7.4 Hz, 1H), 6.90 (t, J ¼ 7.3 Hz, 1H), 6.82
(100 MHz, DMSO-d
6
7
1
1
2
5
25.9, 124.2, 123.3, 121.4, 120.1, 118.7, 118.6, 112.1, 111.9, 111.6, 43.0,
þ
4.5. HRMS (ESI ): m/z calcd for C25
7
H19BrClN , 534.0626, found,
13
(
(
1
4
d, J ¼ 7.7 Hz, 1H),3.82e3.81 (m, 2H), 3.09e3.07 (m, 2H). C NMR
100 MHz, DMSO-d ): 159.4, 152.0, 138.0, 136.7, 135.5, 128.9, 127.5,
26.9, 124.3, 123.3, 121.5, 119.6, 118.7, 118.6, 111.9, 111.9, 111.7, 109.7,
34.0630.
6
4
2
5
.1.36. N -(2-(1H-indol-3-yl)ethyl)-6-chloro-N -(7-methyl-1H-
þ
6
3.0, 36.3, 24.5. HRMS (ESI ): m/z calcd for C26H20ClN O, 469.1538,
indazol-5-yl)quinazoline-2,4-diamine (24h)
found, 469.1538.
Using 8d (179 mg, 0.5 mmol) and 23 (74 mg, 0.5 mmol) as
starting materials, compound 24h was prepared as an off-white
4
2
ꢀ
5.1.32. N -(2-(1H-indol-3-yl)ethyl)-6-chloro-N -(1H-indazol-6-yl)
solid (150 mg, 64%). Mp: decomp. above 289 C. Rf: 0.40 (DCM/
1
quinazoline-2,4-diamine (24d)
MeOH,10/1, v/v). H NMR (100 MHz, DMSO-d
6
):
d
ppm 13.29 (s,1H),
Using 8d (179 mg, 0.5 mmol) and 1H-indazol-6-amine (67 mg,
10.93 (s, 1H), 10.52 (s, 1H), 9.96 (s, 1H), 8.53 (d, J ¼ 2.0 Hz, 1H), 7.94
(s, 1H), 7.85 (d, J ¼ 8.6 Hz, 1H), 7.76 (s, 1H), 7.59 (d, J ¼ 8.9 Hz, 1H),
7.43 (s, 1H), 7.35 (d, J ¼ 8.1 Hz, 1H), 7.25 (s, 1H), 7.17 (s, 1H), 7.05 (t,
J ¼ 7.3 Hz, 1H), 6.84 (s, 1H), 3.85e3.84 (m, 2H), 3.12e3.09 (m, 2H),
0
.5 mmol) as starting materials, compound 24d was prepared as an
ꢀ
off-white solid (152 mg, 76%). Mp: decomp. above 217 C. Rf: 0.42
DCM/MeOH, 10/1, v/v). H NMR (400 MHz, DMSO-d
1
(
(
6
): d ppm 13.17
13
s, 1H), 12.72 (br, 1H), 10.86 (s, 1H), 10.70 (s, 1H), 9.97 (s, 1H), 8.54 (d,
6
2.45 (s, 3H). C NMR (100 MHz, DMSO-d ): 159.5, 152.4, 138.6,
J ¼ 2.0 Hz, 1H), 8.08 (s, 1H), 7.88e7.85 (m, 2H), 7.76 (d, J ¼ 8.6 Hz,
138.2, 136.7, 135.4, 134.3, 128.8, 127.5, 124.3, 123.2, 123.0, 121.5,
121.2, 119.7, 118.7, 118.6, 111.9, 111.7, 42.9, 24.5, 17.3. HRMS (ESI ): m/
z calcd for C26H21ClN , 468.1698, found, 468.1695.
7
þ
2
H), 7.61 (d, J ¼ 8.9 Hz,1H), 7.46 (d, J ¼ 7.9 Hz,1H), 7.33 (d, J ¼ 8.1 Hz,
H), 7.26 (dd, J ¼ 1.3, 8.6 Hz, 1H), 7.14 (d, J ¼ 1.4 Hz, 1H), 7.03 (t,
1
J ¼ 7.4 Hz, 1H), 6.84 (t, J ¼ 7.5 Hz, 1H), 3.91e3.86 (m, 2H), 3.15e3.11
(
1
m, 2H). 13C NMR (100 MHz, DMSO-d
36.7, 135.6,135.1, 133.9, 129.1, 127.6, 124.3,123.5,121.5, 121.4, 120.9,
6
): 159.7, 152.2, 140.7, 138.1,
5.2. Pharmacological assay
5.2.1. PAK4 HTRF assays
1
19.9, 118.7, 117.1, 112.1, 112.1, 111.9, 111.6, 103.5, 43.2, 24.4. HRMS
þ
®
(
ESI ): m/z calcd for C25
H19ClN
7
, 454.1541, found, 454.1547.
The PAK4 kinase assay was performed using HTRF KinEASE™-
STK kit (Cisbio Bioassays, France) in 384-well low volume micro-
plate (Nunc, ThermoFisher Scientific). Purified enzyme of PAK4 was
purchased from Carna Biosciences (Japan) The kinase activity was
assessed under conditions determined experimentally. Specifically,
PAK4 0.0256 ng/uL was incubated with saturating concentration of
4
2
5
.1.33. N -(2-(1H-indol-3-yl)ethyl)-6-chloro-N -(1H-pyrazolo [3,4-
b]pyridin-5-yl)quinazoline-2,4-diamine (24e)
Using 8d (179 mg, 0.5 mmol) and 1H-pyrazolo [3,4-b]pyridin-5-
amine (13, 67 mg, 0.5 mmol) as starting materials, compound 24e
was prepared as an off-white solid (134 mg, 59%). Mp: decomp.
substrate S2 at 1
without compounds in 5 mM MgCl
enzymatic buffer at a total volume of 10
was started by adding kinase, incubated at R.T. for 40 min, and
terminated by adding 10 L EDTA-containing detection reagents,
m
M and Km concentration of ATP at 4
, 1 mM DTT and 1X KinEASE
L. The enzymatic reaction
mM, with or
ꢀ
1
2
above 278 C. Rf: 0.37 (DCM/MeOH, 10/1, v/v). H NMR (400 MHz,
DMSO-d ): ppm 13.81 (s, 1H), 10.90 (s, 1H), 10.57 (s, 1H), 9.99 (s,
H), 8.64 (d, J ¼ 2.2 Hz, 1H), 8.55 (s, 1H), 8.38 (s, 1H), 8.04 (s, 1H),
.87 (d, J ¼ 8.0 Hz, 1H), 7.64 (d, J ¼ 8.9 Hz, 1H), 7.39 (s, 1H), 7.34 (d,
J ¼ 8.1 Hz,1H), 7.13 (s,1H), 7.04 (t, J ¼ 7.2 Hz,1H), 6.83 (s,1H), 3.77 (s,
H), 3.05 (s, 2H). 13C NMR (100 MHz, DMSO-d
): 159.6, 153.2, 146.4,
36.7, 135.4, 133.7, 128.9, 127.5, 124.4, 123.3, 121.4, 118.7, 118.6, 114.6,
m
6
d
1
7
m
prepared according to kit instruction. Kinase activity was at the
linear range with protein amount and incubation time. HTRF signal
was obtained by reading the plate in Infinite® F500 microplate
reader (Tecan, Switzerland). Test compounds were prepared in
DMSO at stock solutions of 20 mM. For primary compound
screening, compounds were diluted in kinase reaction buffer and
2
1
6
þ
1
12.3, 111.9, 111.6, 42.8, 24.6. HRMS (ESI ): m/z calcd for C24H19ClN ,
8
4
55.1498, found, 455.1505.
tested at 100, 10 and 1.0
below or at 0.5%. For IC50 studies, compounds were 5X serial diluted
from 1000 M to 0.1 nM using kinase reaction buffer. IC50s were
mM in 10 mL kinase reaction with DMSO
4
2
5
.1.34. N -(2-(1H-indol-3-yl)ethyl)-6-chloro-N -(7-chloro-1H-
indazol-5-yl)quinazoline-2,4-diamine (24f)
m
Using 8d (179 mg, 0.5 mmol) and 17a (84 mg, 0.5 mmol) as
starting materials, compound 24f was prepared as an off-white
obtained by fitting data to sigmoidal dose-response curve.
ꢀ
solid (124 mg, 51%). Mp: decomp. above 297 C. Rf: 0.42 (DCM/
5.2.2. Anti-proliferative assay
1
MeOH, 10/1, v/v). H NMR (400 MHz, DMSO-d
6
):
d
ppm13.71 (s, 1H),
The human pulmonary carcinoma cell line A-549 was cultured
in RPMI-1640 medium containing 10% FBS, 100 U/mL streptomycin
1
2.54 (br, 1H), 10.89 (s, 1H), 10.55 (s, 1H), 9.89 (s, 1H), 8.50 (d,
J ¼ 1.6 Hz, 1H), 8.12 (s, 1H), 7.90 (s, 1H), 7.86 (d, J ¼ 9.0 Hz, 2H), 7.70
s, 1H), 7.62 (d, J ¼ 8.9 Hz, 1H), 7.40 (s, 1H), 7.34 (d, J ¼ 8.2 Hz, 1H),
.16 (s, 1H), 7.04 (t, J ¼ 7.5 Hz, 1H), 6.82 (s, 1H), 3.82e3.81 (m, 2H),
.10e3.07 (m, 2H). 13C NMR (100 MHz, DMSO-d
): 159.6, 152.9,
36.7, 136.1, 135.4, 130.8, 128.8, 127.5, 124.8, 124.2, 123.3, 122.9,
21.4, 120.4, 118.7, 118.7, 115.4, 112.2, 111.9, 111.7, 42.9, 24.5. HRMS
ꢀ
and 100 U/mL penicillin at 37 C in a humidified atmosphere con-
(
7
3
1
2
taining 5% CO .
4
A549 cells (1 ꢃ 10 /well) were plated in 0.1 mL of a medium
containing 10% FBS in 96-well Corning plates. After 24 h, the me-
dium was removed and replaced with 0.1 mL medium containing
the indicated concentrations of the compound 9d for 24, 48, or 72 h.
At the end of the incubation, the cellular proliferation was
6
1
þ
(
7
ESI ): m/z calcd for C25H19Cl2N , 488.1152, found, 488.1150.

12
C. Hao et al. / European Journal of Medicinal Chemistry 131 (2017) 1e13
measured by the modified tetrazolium salt 3-(4, 5-dimethylthiozol-
-yl)-2, 5-biphenyl-tetrazolium bromide (MTT, Sigma) assay. For
in number of grid points; grid spacing ¼ 0.375 Å) centred at the
ATP-binding pocket was created using AutoGrid4. Docking was
performed using the Lamarckian genetic algorithm in AutoDock4.
Each docking experiment was performed 10 times, yielding 10
docked conformations. The solutions were ranked by the calculated
binding free energy. Figures were drawn using PyMOL.
2
this, 0.01 mL MTT solution (5 mg/mL in PBS) was added to each
ꢀ
well. After 4 h incubation at 37 C, the medium was replaced by
ꢀ
0
.15 mL DMSO. After 15 min incubation at 37 C, the optical den-
sities at 490 nm were measured using a Microplate Reader (BIO-
RAD). The data were calculated and plotted as the percent viability
compared to the control. The 50% inhibitory concentration (IC50
)
Acknowledgments
was defined as the concentration that reduced the absorbance of
the untreated wells by 50% of the vehicle in the MTT assay.
We gratefully acknowledge the financial support from the Na-
tional Natural Science Foundation of China (Grant 81230077) and
Program for Innovative Research Team of the Ministry of Education,
and Program for Liaoning Innovative Research Team in University.
5
.2.3. Cell-cycle analysis by flow cytometry
A549 cells (8 ꢃ 10 cells) were incubated with the indicated
4
concentrations of 9d for 24 h. After incubation, cells were collected,
washed with PBS, and suspended in a staining buffer (10
mg/mL
Appendix A. Supplementary data
propidium iodide, 0.5% Tween-20, 0.1% RNase in PBS). The cells
were analysed using a FACSVantage flow cytometer with the Cell
Quest acquisition and analysis software program (Becton Dickinson
and Co., San Jose, CA).
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2017.02.
063. These data include MOL files and InChiKeys of the most
important compounds described in this article.
5.2.4. Western blot assay
To determine the expression of protein, whole cell extracts were
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