Discovery of 2-(4-Substituted-piperidin/piperazine-1-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)-quinazoline-2,4-diamines as PAK4 inhibitors with potent A549 cell proliferation, migration, and invasion inhibition activity

Article abstract of DOI:10.3390/molecules23020417

A series of novel 2,4-diaminoquinazoline derivatives were designed, synthesized, and evaluated as p21-activated kinase 4 (PAK4) inhibitors. All compounds showed significant inhibitory activity against PAK4 (half-maximal inhibitory concentration IC50 < 1 ?M). Among them, compounds 8d and 9c demonstrated the most potent inhibitory activity against PAK4 (IC50 = 0.060 ?M and 0.068 ?M, respectively). Furthermore, we observed that compounds 8d and 9c displayed potent antiproliferative activity against the A549 cell line and inhibited cell cycle distribution, migration, and invasion of this cell line. In addition, molecular docking analysis was performed to predict the possible binding mode of compound 8d. This series of compounds has the potential for further development as PAK4 inhibitors for anticancer activity.

Full text of DOI:10.3390/molecules23020417

molecules
Article
Discovery of 2-(4-Substituted-piperidin/piperazine-1-
yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)-quinazoline-
2
,4-diamines as PAK4 Inhibitors with Potent A549
Cell Proliferation, Migration, and Invasion
Inhibition Activity
Tianxiao Wu, Yu Pang, Jing Guo, Wenbo Yin, Mingyue Zhu, Chenzhou Hao, Kai Wang, Jian Wang,
ID
Dongmei Zhao * and Maosheng Cheng
Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education,
Shenyang Pharmaceutical University, Shenyang 110016, China; 15330802221@163.com (T.W.);
pangyu038@163.com (Y.P.); guojingspu@163.com (J.G.); yinwenbo1994@163.com (W.Y.);
1
3555832736@163.com (M.Z.); spuhcz@163.com (C.H.); 15702442831@163.com (K.W.);
Jianwang@email.com (J.W.); mscheng@263.net (M.C.)
Correspondence: medchemzhao@163.com; Tel.: +86-24-4352-0219
*
Received: 29 January 2018; Accepted: 11 February 2018; Published: 14 February 2018
Abstract: A series of novel 2,4-diaminoquinazoline derivatives were designed, synthesized,
and evaluated as p21-activated kinase 4 (PAK4) inhibitors. All compounds showed significant
inhibitory activity against PAK4 (half-maximal inhibitory concentration IC50 < 1
compounds 8d and 9c demonstrated the most potent inhibitory activity against PAK4 (IC50 = 0.060
and 0.068 M, respectively). Furthermore, we observed that compounds 8d and 9c displayed potent
µM). Among them,
µM
µ
antiproliferative activity against the A549 cell line and inhibited cell cycle distribution, migration,
and invasion of this cell line. In addition, molecular docking analysis was performed to predict
the possible binding mode of compound 8d. This series of compounds has the potential for further
development as PAK4 inhibitors for anticancer activity.
Keywords: p21-activated kinase; PAK4 inhibitor; 2,4-diaminoquinazoline; anticancer
1
. Introduction
The p21-activated kinases (PAKs), which are members of the serine/threonine protein kinases,
0
are activated by the Rho family of small guanosine-5 -triphosphate (GTP)-binding proteins Rac and
Cdc42 [
1
,2]. Based on sequence and structural homology, six different PAKs members are classified
into two groups (PAK1–3 in group I, and PAK4–6 in group II) [
3–5
]. PAKs are key effectors of the
Rho family GTPases Rac and Cdc42, and their expression and activity are thought to play critical
roles in cellular function, including promoting cell growth, inhibiting cell apoptosis, and regulating
cytoskeleton functions [1,6–9]. Focus is currently shifting from group I PAKs to group II PAKs [10],
because of the acute cardiovascular toxicity resulting from the inhibition of PAK2 [11] and the unique
biological function of PAK4. It was reported in 2017 that although PAK1 and PAK4 are highly expressed
in HeLa cervical cancer cells, only PAK4 knockdown attenuates expression of HIF-1
In addition, recent studies have shown that PAK4 played an indispensable part in effective migration
and invasion of prostate, ovarian, pancreatic, and glioma cancer cell lines [13 14]. These studies
confirmed the scientific rationale for further development of PAK4 inhibitors as anticancer agents.
α in hypoxia [12].
,
2
4
In our prior study [15], N -(3-methoxyphenyl)-N -((tetrahydrofuran-2-yl)methyl)quinazoline-
2,4-diamine (LCH-7749944) was identified as a novel and potent inhibitor of PAK4 (Figure 1A).
Molecules 2018, 23, 417; doi:10.3390/molecules23020417
www.mdpi.com/journal/molecules

Molecules 2018, 23, 417
2 of 12
LCH-7749944 moderately inhibited PAK4 activity with a half-maximal inhibitory concentration (IC )
5
0
of 14.93
µM and effectively suppressed the proliferation, migration, and invasion of human gastric
cancer cells through downregulation of several PAK4-dependent pathways.
Figure 1. (A) Chemical structure of LCH-7749944; (B) Binding mode of LCH-7749944 within the PAK4
binding site; (C) Design strategy of the target compounds.
Molecular docking studies showed that the furan fragment at the C4-position of LCH-7749944
can form two hydrogen bonding interactions with PAK4 hinge residues Tyr397 and Leu398, and the
C2-position substituent extended to the P-loop pocket (Figure 1B). Given that LCH-7749944 was
not tightly filled with the P-loop pocket and that larger cavities were still left unoccupied, it may
result in increased potency that incorporating functional motifs of suitable length and H-bonding
potential at 2-position of quinazoline ring offered access to amino acid residues in the P-loop
pocket. The hinge-binder fragment was also the focus of our investigation, by replacing the original
group with a 3-amino-5-cyclopropyl-pyrazole fragment, which can form the classic three-point
donor-acceptor-donor hydrogen bond interactions with the PAK4 hinge residues Glu396 and
Leu398 (Figure 1C). On the basis of the above strategy, we designed and synthesized 19 novel
2
,4-diaminoquinazoline derivatives.

Molecules 2018, 23, 417
3 of 12
2
. Results and Discussion
2
.1. Chemistry
Synthesis of the designed 2,4-diaminoquinazoline derivatives 6a
,
b,
7a
–
i
,
8a
–
e,
9a–
c
employed
2
,6-dichloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)-quinazolin-4-amine (
5
) as the key intermediate.
) through the synthetic
This intermediate was obtained from 2-amino-5-chlorobenzoic acid (
1
◦
1 with urea at 200 C provided a good yield
methodology depicted in Scheme 1. Treatment of
of . Then, the resulting intermediate was subjected to chlorination with phosphoryl trichloride
to produce . Under basic conditions, the 4-position chlorine of was selectively substituted with
-amino-5-cyclopropylpyrazole to give the key intermediate 16]. Finally, the chlorine at the 2-position
was substituted with available piperazine or piperidine derivatives in the presence of potassium
2
2
3
3
3
of
5 [
5
iodide to provide the target compounds 6a
proton nuclear magnetic resonance ( H-NMR), carbon-13 nuclear magnetic resonance ( C-NMR) and,
High-resolution accurate mass determinations (HRMSs). (Supplementary Materials).
,
b
,
7a
–
i
,
8a
–
e
,
9a
–
c
. These compounds were confirmed by
1
13
Scheme 1. Synthesis of target compounds 6a
,
b
,
7a
–
i
,
8a
–
e
,
9a
–c. Reagent and conditions: (a) Urea,
◦
2
00 C, 98%; (
b
) POCl , N,N-diethylaniline, reflux, 80.2%; (
c) N,N-Diisopropylethylamine (DIEA),
3
◦
4, dimethylformamide (DMF), 0 C, 74.6%.
(d) DIEA, KI, DMF, appropriate substituted
◦
piperidine/piperazine, 65 C, overnight, 57.2–70.6%.
2
.2. In Vitro Activity against PAK4 Kinase and Structure-Activity Relationships
As mentioned earlier, docking studies indicated that extending the length of the molecules may be
helpful in increasing their activity. Hence, we fixed the quinazoline scaffold as well as the 4-substituted
-amino-5-cyclopropylpyrazole moiety and introduced piperazines or piperidines at the 2-position
3
to increase the length of the molecule. At the end of this linker, a hydrophobic aromatic ring or
alicyclic ring was introduced to explore the P-loop region. In total, 19 compounds against PAK4 were
determined by using the homogeneous time-resolved fluorescence (HTRF) kinase assay.
The enzyme inhibitory activities are summarized in Table 1. Introducing an alicyclic ring at the
end of the linkage to increase the length of the molecules (compounds 6a
,
b) resulted in better inhibitory
activity than LCH-7749944. Replacing the alicyclic ring with a phenyl ring (compounds 7a
–
i) increased

Molecules 2018, 23, 417
4 of 12
PAK4 potency, except for 7a, indicating that flat geometric configuration was beneficial. Phenyl rings
containing substituents (compounds 7b ) exhibited higher kinase inhibition than non-substituted
derivatives (compound 7a), and the para-substituted phenyl analogs (7e ) showed higher enzyme
activity than the meta-substituted analogs 7f . Furthermore, we examined the effects of different
substituents at the same position (para-substituted phenyl ring derivatives 7b ). The activities were
–
i
,
g
,h
–e,i
in the order 4-Br > 4-Cl > 4-F > 4-Me > 4-CF . Analog 7c displayed strong inhibition against PAK4,
3
suggesting that bromine atoms had a moderate spatial structure to occupy the hydrophobic cavity of
the P-loop pocket. Upon investigation of other aromatic rings, better activity was seen when the N-4
of piperazine was substituted with a pyridine (compounds 8a
a−c) groups compared with phenyl substituted derivatives 7a−c. A possible explanation is
that the nitrogen atom of the pyridine and pyrimidine group form a stronger hydrogen-bonding
interaction with the hydrophobic region of PAK4. Br-substituted pyridine 8d (IC50 = 0.060 M) and
,c,d) and pyrimidine (compounds
9
µ
pyrimidine 9c (IC50 = 0.068 µM), like Br-substituted phenyl 7c, showed better potency than other
analogs 8a,c and 9a,b.
Table 1. IC50 values (µM) of the synthesized compounds against PAK4.
PAK4
IC50 a
PAK4
IC50
PAK4
IC50
Compound
Compound
Compound
6
a
0.44
0.56
0.85
0.24
0.10
0.65
0.16
7f
7g
7h
7i
8a
8b
8c
0.49
0.30
0.69
0.20
0.31
0.38
0.087
8d
8e
9a
9b
9c
0.060
0.22
0.38
0.11
0.068
6
b
7
a
7
b
7
c
PF-3758309 b
7
d
0.016
7
e
a
IC50 values are calculated based on homogeneous time-resolved fluorescence (HTRF) assay; b PF-3758309 was
used as a positive control.
2
.3. Effects of Compounds 8d and 9c on Cell Proliferation
Based on the enzymatic assay, compounds 8d and 9c were selected for cellular assay on the
A549 cell line, in which PAK4 has been found to be overexpressed. Meanwhile, the tumor cell line
HT1080, whose growth is not dependent on PAK4, was used to test the potential off-target effects
of the potent PAK4 inhibitors. Both 8d and 9c demonstrated potent inhibition against the A549 cell
line (IC50 = 4.685 µM, and 4.751 µM, respectively), and negligible activity was observed for HT1080
(Table 2).
Table 2. IC50 a (µM) of potent compounds on cell proliferation.
Cells
Compound
PAK4
A549
HT1080
8
9
d
c
0.060
0.068
4.685
4.751
21.519
39.303
a
IC50: concentration of the compound displaying 50% cell growth inhibition after 24 h of drug exposure, as determined
by MTT assay.
2
.4. Effects of Compound 8d on Cell Cycle Progression, Cell Migration, and Cell Invasion
We next examined the effects of compound 8d on the cycle progression of A549. Cells were treated
with compound 8d at varying concentrations (1.0, 3.0, 9.0
µ
M) and vehicle (dimethyl sulfoxide DMSO)
−
for 24 h, and subjected to flow cytometry analysis to determine the distribution of cells in various phases
of the cell cycle. Compound 8d was found to induce a dose-dependent decrease in the percentage of
cells in the G1 phase and an increase in the S phase compared with the vehicle control (Figure 2A,B),

Molecules 2018, 23, 417
5 of 12
indicating that compound 8d arrested A549 cells at the S phase of the cell cycle. PAK4 activity is involved
in cytoskeleton dynamics and cancer cell migration and invasion; therefore, the inhibitory effect of 8d on
A549 cell migration and invasion were analyzed. As displayed in Figure 2C–F, 8d decreased the migration
and invasion potential of the A549 cells in a dose-dependent manner. These results demonstrate that 8d
efficiently exhibits antimetastatic potential against cancer cells.
Figure 2.
after treatment with various concentrations of compound 8d for 24 h; (
cell cycle; ( ) Compound 8d inhibited migration and invasion of A549 cells in transwell assays.
The images were captured using phase contrast microscopy after 24 h of treatment. Scale bar, 20 M;
D,F) Quantitative analysis of migration and invasion.
(A
) Compound 8d induced S cell cycle arrest of A549 cells. A549 cells were harvested
B
) Quantitative analysis of
C,E
µ
(
2
.5. Binding Mode Analysis
To investigate potential binding modes, compound 8d was docked into the ATP-binding site of
PAK4 (Protein Data Bank ID: 2X4Z) using Autodock 4.2 (Figure 3).
Figure 3. Model of compound 8d bound to PAK4, generated by energy minimization of the elaborated
structure of 8d within the PAK4 protein using the MMFF94 force field within LigandScout. (
interactions with the protein residues. Hydrogen bonds are rendered as dotted red lines; (
A
) Detailed
) 2D
B
interaction diagram showing compound 8d docking pose interactions with the key amino acids in the
PAK4 active site by LigandScout.

Molecules 2018, 23, 417
6 of 12
Compound 8d binds to the PAK4 catalytic domain in the ATP binding site and makes multiple
contacts with the hinge region through hydrogen-bond interactions with the pyrazole motif and the
amine linker to the quinazoline ring. The piperidine ring of 8d adopted a twist-boat conformation.
This led to projection of the equatorial pyridine substituent along a more direct vector to the P-loop,
which sat within a hydrophobic environment created by the P-loop backbone and the side chain
of Phe461.
3
. Experimental
3
.1. Chemicals and Instruments
Unless otherwise noted, all materials were obtained from commercially available sources and
were used without purification. Thin-layer chromatography (TLC) was performed on silica gel plates
with F-254 indicator (Shanghai jingke industrial Co. Ltd., Shanghai, China) and visualized by UV
1
light. Proton nuclear magnetic resonance ( H-NMR, 400 MHz) and carbon-13 nuclear magnetic
13
resonance ( C-NMR, 151 MHz) spectra were recorded using a Bruker 400 MHz NMR spectrometer
Bruker, Karlsruhe, Germany) in DMSO-d , and the chemical shifts were recorded in parts per million
(
6
downfield from tetramethylsilane. High-resolution accurate mass determinations (HRMSs) for all final
target compounds were obtained on a Bruker Micromass Time of Flight mass spectrometer (Bruker
Bioscience, Billerica, MA, USA) equipped with electrospray ionization (ESI). Column chromatography
was performed on silica gel (200–300 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd.
(Qingdao, China).
3
.2. Synthesis
6-Chl.oroquinazoline-2,4-diol (2). This compound was synthesized according to previously reported
methods [17].
,4,6-Trichloroquinazoline (
methods [18].
,6-Dichloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)quinazolin-4-amine (
pyrazol-3-amine (0.267 g, 2.17 mmol) and 2,4,6-trichloroquinazoline (0.432 g, 2.17 mmol) in
2
3). This compound was synthesized according to previously reported
2
5). A solution of 5-cyclopropyl-1H-
dimethylformamide (DMF) (10 mL) was treated with EtN(i-Pr) (0.49 mL, 2.82 mmol) and stirred for
2
◦
4
h at 0 C. The reaction mixture was poured over ice water. The solid was collected by filtration and
dried in air to provide compound 5 without further purification [16]. The yield was 74.6%.
General Procedure for Synthesis of Compounds 6a,b, 7a–i, 8a–e, 9a–c
The corresponding piperazine or piperidine (1.1 mmol), KI (0.18 g, 1.1 mmol), and DIEA (0.21 mL,
1
.3 mmol) was added to a solution of compound
5
(0.32 g, 1 mmol) in DMF (10 mL). The reaction
◦
mixture was stirred at 60 C overnight. After completion of the reaction, the reaction mixture was
cooled to room temperature and poured over ice water. The solid was collected by filtration and
purified by chromatography on silica gel (dichloromethane/methanol, 30:1) to give the title compounds
6
a,b, 7a–i, 8a–e, 9a–c in moderate yields.
0 0
[1,4 -Bipiperidin]-1 -yl)-6-chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)quinazolin-4-amine (6a). White solid,
(
◦
1
yield 65.3%, m.p.: 208.3–209.6 C. H-NMR (DMSO-d )
7
δ
12.22 (s, 1H), 10.10 (s, 1H), 8.52 (s, 1H),
.52 (dd, J = 8.9, 1.9 Hz, 1H), 7.30 (d, J = 8.9 Hz, 1H), 6.35 (s, 1H), 4.78 (d, J = 12.7 Hz, 2H), 3.17 (d,
J = 5.2 Hz, 1H), 2.83 (t, J = 12.1 Hz, 2H), 2.48~2.40 (m, 4H), 1.97~1.86 (m, 1H), 1.76 (d, J = 11.5 Hz,
6
13
2
δ
2
H), 1.49~1.44 (m, 4H), 1.40~1.32 (m, 4H), 0.99~0.94 (m, 2H), 0.7~0.66 (m, 2H). C-NMR (DMSO-d6)
158.79, 157.14, 151.58, 147.96, 145.91, 133.28, 127.47, 124.81, 123.03, 111.36, 94.55, 62.72, 50.13, 43.79,
+
8.03, 26.57, 25.05, 8.42, 7.30. HRMS calcd. for C H ClN , [M + H] , 452.2331; found 452.2331.
24
30
7

Molecules 2018, 23, 417
7 of 12
5
-Chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(4-morpholinopiperidin-1-yl)quinazolin-4-amine
(
6b).
12.23 (s, 1H), 10.11 (s, 1H), 8.53
s, 1H), 7.53 (dd, J = 8.9, 1.8 Hz, 1H), 7.30 (d, J = 8.9 Hz, 1H), 6.35 (s, 1H), 4.75 (d, J = 13.0 Hz, 2H),
◦
1
White solid, yield 67.4%, m.p.: 214.3–216.6 C. H-NMR (DMSO-d )
(
δ
6
3
2
.57–3.55 (m, 4H), 2.89 (t, J = 12.0 Hz, 2H), 2.49~2.43 (m, 4H), 1.98~1.89 (m, 1H), 1.84 (d, J = 10.9 Hz,
H), 1.38–1.28 (m, 2H), 0.97 (d, J = 7.3 Hz, 2H), 0.70~0.66 (m, 2H). 13C-NMR (DMSO-d6)
δ
158.80,
1
8
57.18, 151.56, 147.93, 145.90, 133.31, 127.48, 124.85, 123.04, 111.39, 94.60, 67.05, 62.10, 49.85, 43.39, 28.25,
+
.44, 7.30. HRMS calcd. for C H ClN O, [M + H] , 454.2119; found 454.2119.
23
28
7
6
-Chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(4-phenylpiperazin-1-yl)quinazolin-4-amine (7a). White solid,
◦
1
yield 69.0%, m.p.: 239.5–240.8 C. H-NMR (DMSO-d )
δ
12.97 (s, 1H), 10.26 (s, 1H), 8.55 (s, 1H),
6
7
7
3
.71 (d, J = 7.5 Hz, 2H), 7.52 (d, J = 8.5 Hz, 1H), 7.44 (t, J = 7.4 Hz, 2H), 7.31 (d, J = 4.2 Hz, 2H),
.16 (t, J = 7.9 Hz, 2H), 7.06 (s, 1H), 6.94 (d, J = 8.1 Hz, 2H), 6.73 (t, J = 7.2 Hz, 1H), 3.96~3.87 (m, 4H),
.22~3.06 (m, 4H). 13C-NMR (DMSO-d ) δ 158.98, 157.25, 151.60, 151.34, 147.84, 133.42, 129.41, 127.59,
6
1
52.21, 123.12, 119.62, 116.34, 111.64, 94.91, 48.90, 44.02, 8.49, 7.39. HRMS calcd. for C H ClN ,
24
24
7
+
[M + H] , 446.1859; found 446.1859.
6
-Chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(4-(p-tolyl)piperazin-1-yl)quinazolin-4-amine
(
7b).
◦
1
White solid, yield 67.5%, m.p.: 204.9–207.5 C. H-NMR (DMSO-d )
δ
12.24 (s, 1H), 10.16 (s,
H), 8.57 (s, 1H), 7.57 (dd, J = 1.84, 9.00 Hz, 1H), 7.35 (d, J = 9.04 Hz, 1H), 7.05 (d, J = 8.40 Hz, 2H), 6.92
d, J = 8.56 Hz, 2H), 6.39 (s, 1H), 3.94–3.91 (m, 4H), 3.16–3.14 (m, 4H), 2.22 (s, 3H), 1.97~1.93 (m, 1H),
6
1
(
13
0.99~0.97 (m, 2H), 0.73~0.72 (m, 2H). C-NMR (DMSO-d6) δ 158.92, 157.23, 151.24, 149.53, 147.75,
1
8
45.95, 133.47, 129.86, 128.52, 127.52, 125.26, 123.12, 116.67, 111.62, 94.85, 52.50, 49.42, 45.92, 44.09, 20.54,
+
.99, 8.47, 7.65. HRMS calcd. for C H ClN , [M + H] , 460.2011; found 460.2011.
24
24
7
2
-(4-(4-Bromophenyl)piperazin-1-yl)-6-chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)quinazolin-4-amine (7c).
◦
1
White solid, yield 59.2%, m.p.: 248.7–251.9 C. H-NMR (DMSO-d ) δ 12.23 (s, 1H), 10.16 (s, 1H), 8.57
6
(
s, 1H), 7.57 (dd, J = 8.8, 1.6 Hz, 1H), 7.37 (t, J = 9.0 Hz, 3H), 6.98 (d, J = 9.1 Hz, 2H), 6.39 (d, J = 0.5 Hz,
H), 3.92~3.94 (m, 4H), 3.25~3.22 (m, 4H), 1.96~1.94 (m, 1H), 0.99~0.97 (m, 2H), 0.74~0.73 (m, 2H).
1
1
3
C-NMR (DMSO-d6)
δ
158.93, 157.24, 151.29, 150.69, 133.45, 131.95, 127.58, 125.24, 118.16, 111.63,
+
1
10.69, 48.45, 43.83, 8.48, 7.39. HRMS calcd. for C H BrClN , [M + H] , 526.0969; found 526.0969.
24
23
7
6
-Chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)quinazolin-4-amine
◦
1
(7d). White solid, yield 67.4%, m.p.: 256.4–257.8 C. H-NMR (DMSO-d )
δ
12.24 (s, 1H), 10.18 (s, 1H),
.58 (d, J = 1.1 Hz, 1H), 7.57 (dd, J = 9.1, 1.9 Hz, 1H), 7.54 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 8.9 Hz, 1H),
.14 (d, J = 8.8 Hz, 2H), 6.41 (s, 1H), 4.04~3.85 (m, 4H), 3.49~3.37 (m, 4H), 2.08~1.86 (m, 1H), 1.09~0.92
158.95, 157.28, 153.72, 151.30, 147.82, 145.94, 133.45,
6
8
7
(
m, 2H), 0.86~0.66 (m, 2H). 13C-NMR (DMSO-d6)
δ
1
27.58, 126.66, 126.62, 125.28, 123.14, 118.55, 118.23, 114.82, 111.68, 94.93, 47.36, 43.69, 8.49, 7.40. HRMS
+
calcd. for C H ClF N , [M + H] , 514.1744; found 514.1744.
25
23
3
7
6
-Chloro-2-(4-(4-chlorophenyl)piperazin-1-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)quinazolin-4-amine (7e).
◦
1
White solid, yield 61.6%, m.p.: 250–251.8 C. H-NMR (DMSO-d )
δ
12.22 (s, 1H), 10.17 (s, 1H),
.56 (s, 1H), 7.57 (dd, J = 8.9, 2.2 Hz, 1H), 7.35 (d, J = 8.9 Hz, 1H), 7.26 (d, J = 9.0 Hz, 2H), 7.03
d, J = 9.1 Hz, 2H), 6.38 (d, J = 0.8 Hz, 1H)., 4.19~3.67 (m, 4H), 3.29~3.08 (m, 4H), 1.97~1.93 (m, 1H),
.99~0.97 (m, 2H), 0.77~0.70 (m, 2H). 13C-NMR (DMSO-d6)
158.96, 157.26, 151.31, 150.38, 145.92,
6
8
(
0
δ
1
33.44, 129.10, 127.59, 122.25, 123.06, 117.73, 111.65, 94.89, 48.61, 43.86, 8.48, 7.39. HRMS calcd. for
+
C H Cl N , [M + H] , 480.1471; found 480.1471.
24
23
2
7
6
-Chloro-2-(4-(3-chlorophenyl)piperazin-1-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)quinazolin-4-amine (7f).
◦
1
White solid, yield 57.2%, m.p.: 229.9–232.6 C. H-NMR (DMSO-d ) δ 12.23 (s, 1H), 10.17 (s, 1H), 8.57
6
(
(
1
1
4
s, 1H), 7.57 (dd, J = 8.8, 1.7 Hz, 1H), 7.36 (d, J = 8.9 Hz, 1H), 7.24 (t, J = 8.1 Hz, 1H), 7.03 (s, 1H), 6.98
dd, J = 8.4, 1.8 Hz, 1H), 6.81 (dd, J = 7.8, 1.2 Hz, 1H), 6.40 (s, 1H), 3.92~3.91 (m, 4H), 3.31~3.16 (m, 4H),
.99~1.90 (m, 1H), 1.01~0.95 (m, 2H), 0.76~0.72 (m, 2H). 13C-NMR (DMSO-d6)
δ 158.95, 157.25, 152.79,
51.31, 147.81, 145.93, 134.30, 133.44, 130.88, 127.59, 125.26, 123.12, 118.73, 115.37, 114.46, 111.66, 94.95,
+
8.20, 43.83, 8.49, 7.42. HRMS calcd. for C H Cl N , [M + H] , 480.1465; found 480.1461.
24
23
2
7

Molecules 2018, 23, 417
8 of 12
6
-Chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(4-(4-methoxyphenyl)piperazin-1-yl)quinazolin-4-amine (7g).
◦
1
White solid, yield 70.1%, m.p.: 237.1–239.5 C. H-NMR (DMSO-d ) δ 12.23 (s, 1H), 10.16 (s, 1H), 8.57
6
(d, J = 1.5 Hz, 1H), 7.56 (dd, J = 8.9, 2.0 Hz, 1H), 7.35 (d, J = 8.9 Hz, 1H), 6.98 (d, J = 9.1 Hz, 2H), 6.84
(
0
d, J = 9.1 Hz, 2H), 6.39 (d, J = 0.4 Hz, 1H), 3.93~3.92 (m, 4H), 3.15~3.01 (m, 4H), 2.04~1.88 (m, 1H),
.99~0.97 (m, 2H), 0.73~0.72 (m, 2H). C-NMR (DMSO-d6) δ 158.97, 157.25, 153.69, 151.36, 145.98,
13
1
33.42, 127.57, 125.18, 123.11, 118.49, 114.73, 111.62, 94.87, 55.65, 50.43, 44.16, 8.46, 7.36. HRMS calcd.
+
for C H ClN O, [M + H] , 476.1966; found 476.1966.
25
26
7
6
-Chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(4-(3-methoxyphenyl)piperazin-1-yl)quinazolin-4-amine (7h).
◦
1
White solid, yield 70.5%, m.p.: 199.9–201.8 C. H-NMR (DMSO-d ) δ 12.22 (s, 1H), 10.14 (s, 1H), 8.56 (s,
6
1
H), 7.56 (d, J = 8.8 Hz, 1H), 7.35 (d, J = 8.9 Hz, 1H), 7.13 (t, J = 8.2 Hz, 1H), 6.60 (dd, J = 8.2, 1.6 Hz, 1H)
,
6
(
.53 (s, 1H), 6.40 (dd, J = 7.9, 1.8 Hz, 2H), 4.10~3.84 (m, 4H), 3.73 (s, 3H), 3.28~3.10 (m, 4H), 2.00~1.84
160.68, 158.97, 157.26, 152.97,
m, 1H), 0.99~0.96 (m, 2H), 0.79~0.52 (m, 2H). 13C-NMR (DMSO-d6)
δ
1
4
51.34, 147.85, 145.90, 133.41, 130.09, 127.57, 125.21, 123.13, 111.64, 108.96, 104.88, 102.47, 94.96, 55.36,
+
8.85, 43.99, 8.48, 7.41. HRMS calcd. for C H ClN O, [M + H] , 476.1992; found 476.1992.
25
26
7
6
-Chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(4-(4-fluorophenyl)piperazin-1-yl)quinazolin-4-amine
(
7i).
12.24 (s, 1H), 10.16 (s, 1H), 8.57 (d,
J = 0.9 Hz, 1H), 7.57 (dd, J = 9.1, 1.7 Hz, 1H), 7.35 (d, J = 8.9 Hz, 1H), 7.14~6.98 (m, 4H), 6.39 (s, 1H),
.99~3.88 (m, 4H), 3.23~3.11 (m, 4H), 2.06~1.87 (m, 1H), 1.02~0.94 (m, 2H), 0.78~0.69 (m, 2H). 13C-NMR
◦
1
White solid, yield 66.2%, m.p.: 228.8–231 C. H-NMR (DMSO-d )
δ
6
3
(
1
DMSO-d6)
δ
158.96, 157.27, 151.33, 148.53, 147.82, 145.92, 145.09, 133.44, 127.58, 125.22, 123.12, 118.24,
+
18.17, 115.87, 115.66, 111.64, 94.90, 49.72, 44.03, 8.48, 7.37. HRMS calcd. for C H ClFN , [M + H] ,
24
23
7
4
64.1763; found 464.1763.
6
-Chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(4-(pyridin-2-yl)piperazin-1-yl)quinazolin-4-amine
◦
1
(
1
(
8a). White solid, yield 59.3%, m.p.: 184.4–186.7 C. H-NMR (DMSO-d6)
0.13 (d, J = 22.7 Hz, 1H), 8.56 (s, 1H), 8.14 (dd, J = 4.8, 1.4 Hz, 1H), 7.56~7.54 (m 2H), 7.35
d, J = 8.9 Hz, 1H), 6.89 (d, J = 8.6 Hz, 1H), 6.66 (dd, J = 6.9, 5.0 Hz, 1H), 6.40 (s, 1H), 3.99~3.77 (m,
δ 12.23 (s, 1H),
13
4
δ
4
H), 3.67~3.52 (m, 4H), 1.97~1.92 (m, 1H), 0.99~0.97 (m, 2H), 0.78~0.68 (m, 2H). C-NMR (DMSO-d6)
159.45, 159.02, 157.22, 151.34, 148.03, 138.02, 133.45, 127.57, 125.20, 123.10, 113.58, 111.63, 107.73, 94.81,
+
4.96, 43.81, 8.48, 7.42. HRMS calcd. for C H ClN , [M + H] , 447.1808; found 447.1808.
23
23
8
6
-Chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(4-(4-methoxypyridin-2-yl)piperazin-1-yl)quinazolin-4-amine
◦
1
(
8b). White solid, yield 68.8%, m.p.: 210.3–211.6 C. H-NMR (DMSO-d )
δ
12.24 (s, 1H), 10.17 (s, 1H),
.57 (s, 1H), 7.97 (d, J = 5.7 Hz, 1H), 7.57 (dd, J = 8.7, 1.9 Hz, 1H), 7.36 (d, J = 8.9 Hz, 1H), 6.41 (s, 1H),
.37 (d, J = 1.7 Hz, 1H), 6.33 (dd, J = 5.7, 2.0 Hz, 1H), 3.91~3.87 (m, 4H), 3.80 (s, 3H), 3.62~3.57 (m, 4H),
.00~1.92 (m, 1H), 1.0~0.98 (m, 2H), 0.75~0.72 (m, 2H). 13C-NMR (DMSO-d6)
167.40, 161.34, 159.02,
6
8
6
2
δ
1
4
57.23, 151.33, 149.09, 147.90, 145.95, 133.44, 127.57, 125.20, 123.12, 111.63, 101.90, 94.92, 92.11, 55.49,
+
5.17, 43.82, 8.47, 7.38. HRMS calcd. for C H ClN O, [M + H] , 477.1914; found 477.1914.
24
25
8
6
-Chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(4-(5-methylpyridin-2-yl)piperazin-1-yl)quinazolin-4-amine
◦
1
(
8c). White solid, yield 70.3%, m.p.: 263–265.4 C. H-NMR (DMSO-d )
δ
12.24 (s, 1H), 10.16 (s, 1H),
.57 (s, 1H), 8.00 (s, 1H), 7.57 (dd, J = 8.9, 1.5 Hz, 1H), 7.42 (dd, J = 8.6, 1.9 Hz, 1H), 7.36 (d, J = 9.0 Hz,
H), 6.85 (d, J = 8.7 Hz, 1H), 6.41 (s, 1H), 3.92~3.87 (m, 4H), 3.57~3.50 (m, 4H), 2.16 (s, 3H), 2.02~1.91
m, 1H), 1.0~0.99 (m, 2H), 0.74~0.73 (m, 2H). 13C-NMR (DMSO-d6)
159.02, 158.04, 157.21, 151.33,
6
8
1
(
δ
1
7
47.60, 138.82, 133.43, 127.56, 125.19, 123.12, 123.10, 122.16, 111.62, 107.64, 94.82, 45.44, 43.81, 1734, 8.47,
+
.39. HRMS calcd. for C H ClN , [M + H] , 461.2023; found 461.2023.
24
25
8
2
-(4-(5-Bromopyridin-2-yl)piperazin-1-yl)-6-chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)quinazolin-4-amine (8d).
◦
1
White solid, yield 66.9%, m.p.: 270.5–272.8 C. H-NMR (DMSO-d ) δ 12.24 (s, 1H), 10.18 (s, 1H), 8.57
6
(d, J = 1.9 Hz, 1H), 8.21 (d, J = 2.5 Hz, 1H), 7.73 (dd, J = 9.1, 2.5 Hz, 1H), 7.57 (dd, J = 9.1, 2.2 Hz, 1H),
7
4
.36 (d, J = 8.9 Hz, 1H), 6.93 (d, J = 9.2 Hz, 1H), 6.40 (d, J = 2.0 Hz, 1H), 3.96~3.83 (m, 4H), 3.71~3.53 (m,
H), 2.02~1.91 (m, 1H), 1.01~0.98 (m, 2H), 0.74~0.73 (m, 2H). 13C-NMR (DMSO-d6)
δ
158.98, 158.10,

Molecules 2018, 23, 417
9 of 12
1
4
57.24, 151.32, 148.22, 145.94, 140.17, 133.44, 127.57, 125.24, 123.13, 111.65, 109.71, 107.21, 94.87, 44.90,
+
3.65, 8.48, 7.38. HRMS calcd. for C H BrClN , [M + H] , 527.0894; found 527.0894.
23
22
8
6
-Chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)quinazol-
◦
1
in-4-amine (8e). White solid, yield 63.7%, m.p.: 247.6–259.6 C. H-NMR (DMSO-d )
δ
12.24
6
(s, 1H), 10.19 (s, 1H), 8.58 (d, J = 1.9 Hz, 1H), 8.46 (s, 1H), 7.84 (dd, J = 9.1, 2.5 Hz, 1H),
7
4
.57 (dd, J = 9.0, 2.0 Hz, 1H), 7.36 (d, J = 8.9 Hz, 1H), 7.04 (d, J = 9.0 Hz, 1H), 6.41 (d, J = 1.4 Hz, 1H),
.02~3.86 (m, 4H), 3.78–3.77 (m, 4H), 2.09~1.85 (m, 1H), 1.01~0.98 (m, 2H), 0.77~0.72 (m, 2H). C-NMR
13
(
1
DMSO-d6)
δ
160.63, 158.92, 157.23, 151.29, 145.74, 134.96, 133.44, 127.56, 126.70, 125.26, 123.13, 113.90,
+
13.59, 106.84, 94.87, 44.44, 43.63, 8.47, 7.38. HRMS calcd. for C H ClF N , [M + H] , 515.1678; found
24
22
3
8
5
15.1678.
6
-Chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(4-(pyrimidin-2-yl)piperazin-1-yl)quinazolin-4-amine (9a).
◦
1
White solid, yield 67.2%, mp: 249.3–250.5 C. H-NMR (DMSO-d )
δ
ppm 12.25 (s, 1H), 10.19 (s,
H), 8.56 (s, 1H),8.41 (d, J = 4.68 Hz, 2H), 7.57 (dd, J = 1.92, 8.96 Hz, 1H), 7.36 (d, J = 8.88 Hz, 1H), 6.67
t, J = 4.68 Hz, 1H), 6.39 (s, 1H), 3.89~3.84 (m, 8H), 2.00~1.92 (m, 1H), 1.00~0.98 (m, 2H), 0.74~0.70 (m,
6
1
(
13
2
H). C-NMR (DMSO-d6)
δ
161.72, 158.98, 158.44, 157.22, 157.15, 151.36, 147.43, 133.47, 127.55, 125.24,
+
123.11, 111.63, 110.73, 94.71, 43.85, 43.66, 8.46, 7.43. HRMS calcd for C H ClN , [M + H] , 448.1860;
22
22
9
found 448.1760.
6
-Chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(4-(5-methylpyrimidin-2-yl)piperazin-1-yl)quinazolin-4-amine
◦
1
(9b). White solid, yield 66.5%, m.p.: 262.8–264.7 C. H-NMR (DMSO-d )
δ
12.25 (s, 1H), 10.18 (s,
6
1H), 8.57 (d, J = 1.8 Hz, 1H)., 8.27 (s, 2H), 7.56 (dd, J = 8.9, 2.0 Hz, 1H), 7.35 (d, J = 8.9 Hz, 1H), 6.40
(
2
d, J = 1.5 Hz, 1H), 3.87~3.85 (m, 4H), 3.83~3.77 (m, 4H), 2.10 (s, 3H), 2.01~1.91 (m, 1H), 1.01–0.98 (m,
H), 0.73~0.71 (m, 2H). 13C-NMR (DMSO-d6)
δ
160.71, 159.01, 158.22, 157.22, 151.35, 147.84, 145.97,
1
33.42, 127.53, 125.19, 123.13, 118.91, 111.64, 94.83, 43.96, 43.84, 14.54, 8.47, 7.33. HRMS calcd. for
+
C H ClN , [M + H] , 462.1926; found 462.1926.
23
24
9
2
-(4-(5-Bromopyrimidin-2-yl)piperazin-1-yl)-6-chloro-N-(5-cyclopropyl-1H-pyrazol-3-yl)quinazolin-4-amine
◦
1
(9c). White solid, yield 70.6%, m.p.: 280.6–282.7 C. H-NMR (DMSO-d )
δ
12.24 (s, 1H), 10.19 (s, 1H),
.57 (d, J = 0.5 Hz, 1H), 8.51 (s, 2H), 7.57 (dd, J = 8.9, 1.7 Hz, 1H), 7.36 (d, J = 8.9 Hz, 1H), 6.39 (s, 1H),
.92~3.87 (m, 4H), 3.85~3.81 (m, 4H), 2.02~1.91 (m, 1H), 1.03~0.93 (m, 2H), 0.73~0.71 (m, 2H). 13C-NMR
6
8
3
(
1
DMSO-d6)
δ
160.02, 158.93, 158.48, 157.23, 155.87, 151.32, 145.99, 133.45, 127.56, 125.27, 123.11, 111.65,
+
05.99, 94.82, 43.93, 43.68, 8.46, 7.40. HRMS calcd. for C H BrClN , [M + H] , 528.0849; found
22
21
9
5
28.0849.
3
.3. PAK4 HTRF Assays
STK Substrate 1-, 2-, or 3-biotin (1 vial of 500
lyophilized), STK Antibody-Cryptate (1 vial of 20,000 tests lyophilized), 5
µ
g lyophilized), streptavidin-XL665 (1 vial of 3 mg
enzymatic buffer (1 vial
) were purchased from
Cis-Bio International (Tecan Infinite F200, Männedorf, Switzerland). PAK4, ATP (Sigma A7699), DTT
×
of 50 mL liquid 5 ), and HTRF detection buffer (1 vial of 200 mL liquid 1
×
×
(
Sigma D0632), and MgCl (Sigma M1028) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
2
First, PAK4, STK Substrate 1-, 2-, or 3-biotin, and ATP were diluted with kinase buffer (5 mM
MgCl , 1 mM DTT, 1 mM 5
×
buffer (HEPES 250 mM (pH 7.0), NaN 0.1%, BSA 0.05%, orthovanadate
3
2
0
.5 mM), H O) to prepare a working solution. In a typical Nunclon 384 flat-bottom white polystyrene
2
catalog plate assay (Thermo Fisher Scientific, Waltham, MA, USA) each well contained the following
reagents: 2 L PAK4 or PAK1 (4.1 g/ L), 2 L STK Substrate 1-, 2-, or 3-biotin (1 mol/L), 2 L ATP
2.08 mol/L), and 4 L test compound in kinase buffer were added to give a total assay volume of
L/well. Blank values were determined by the inclusion of 2 L STK Substrate 1-, 2-, or 3-biotin
mol/L), 2 L ATP (2.08 mol/L), and 6 L kinase buffer. After mixing, the reaction mixture was
incubated at room temperature for 50 min. Then 5 L Streptavidin-XL665 in EDTA diluted with
µ
µ
µ
µ
µ
µ
(
µ
µ
10
µ
µ
(1
µ
µ
µ
µ
µ

Molecules 2018, 23, 417
10 of 12
detection buffer and 5
µL STK Antibody-Cryptate in EDTA diluted with detection buffer were added.
After mixing, the reaction mixture was incubated at room temperature for 50 min.
Fluorescence were determined using a Infinite F500 multimodal plate reader (Tecan Infinite F200)
with excitation at 340 nm and simultaneous signal collection at 670 and 612 nm. The time-resolved
settings were set at 150
the equation 1000 (670 nm/612 nm). Data were processed by using GraphPad Prism 6 (GraphPad
Software Inc., San Diego, CA, USA) to obtain IC .
µs for the delay and 500 µs integration time. Date were calculated using
×
50
3
.4. Cell Proliferation Assay
The human pulmonary carcinoma cell line A549 and the human fibrosarcoma cell line HT1080
◦
were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), at 37 C in a humidified
atmosphere containing 5% CO2.
The in vitro antiproliferative activity of some of the target compounds was determined by the
4
MTT assay (Amresco, Seattle, WA, USA). A suspension of 100
µ
L human cancer cells (5
×
10 /mL)
◦
was plated in a 96-well cell culture plate and incubated with 5% CO at 37 C for 24 h. Then cells were
2
exposed to various concentrations of compounds and further cultured for 24 h. After that, MTT was
added and absorbance was measured at 490 nm using a EL
VT, USA). Dose-response curves were plotted to determine the IC50 values using GraphPad Prism 5.0.
×
800 microplate reader (BioTek, Winooski,
Each IC50 value was expressed as mean ± standard deviation.
3
.5. Cell Cycle Analysis by Flow Cytometry
Cells were seeded in a 6-well plate overnight and treated with different concentrations of
compound 8d the following day. After 24 h, the cells were collected by EDTA-free trypsinization,
◦
centrifuged at 2000 rpm for 5 min, washed with PBS, and fixed in 70% ethanol at 4 C for 2 h. The cells
◦
were then put in a water bath, and 100 µL RNase A was added at 37 C for 30 min. Finally, 400 µL PI
was added, they were incubated in the dark for 30 min at 4 C, and analyzed using flow cytometry
◦
(FACS Calibur, Becton-Dickinson, Franklin Lakes, NJ, USA). The data were analyzed using Flowjo
software (FlowJo Vx 10.0, Ashland, Covington, KY, USA).
3
.6. Cell Migration and Invasion Assay
Cell migration assays were evaluated in transwell chambers (Corning Incorporated, Corning,
NY, USA). Cell invasion assays were evaluated in Matrigel invasion chambers (Becton-Dickinson).
5
First, 0.5
×
10 tumor cells were plated in the top chamber with RPMI-1640 medium without
FBS. Test compound was added to the bottom chamber with 500
µL medium containing 20% FBS.
◦
After incubation for 24 h at 37 C, the cells were fixed in 100% methanol and stained with 0.1% crystal
violet, then washed with PBS; the cells that had not migrated from the top surface of the filters were
removed with cotton. Migrated cells were quantified by counting them in three randomly selected
fields on the diameter under a microscope at 200× magnification
3
.7. Molecular Docking Study
X-ray protein structure of PAK4 obtained from the Protein Data Bank (ID: 2X4Z) was prepared
with AutoDockTools (AutoDock 4.2, The Scripps Research Institute, La Jolla, CA, USA). Both the
native ligand and the indicated compounds were built using CORINA Classic (CORINA Version 4.1.0,
Altamira, LLC, Nürnberg, Germany). The size of the box was set to 60
points, and grid spacing = 0.375 Å centered in native ligand using AutoGrid4. Docking was performed
using the Lamarckian genetic algorithm in AutoDock4. Each docking experiment was performed
×
60
×
60 units in number of grid
1
0 times, yielding 10 docked conformations, and the most favorable pose of 8d was displayed.

Molecules 2018, 23, 417
11 of 12
4
. Conclusions
In conclusion, a series of novel 2,4-diaminoquinazoline derivatives was designed and synthesized,
and preliminary inhibitory activity on PAK4 was evaluated. Among 19 derivatives, MTT and flow
cytometry studies suggested that 8d and 9c showed strong antiproliferative activity by inhibiting the
transition of cells from the G1 phase to the S phase, and transwell assay demonstrated that 8d strongly
inhibited migration and invasion of A549 cell line. Finally, the molecular docking study demonstrated
possible binding modes for the interactions between compound 8d and PAK4. These suggest that
compound 8d could act as a potential PAK4 inhibitor to be used for further optimization.
Supplementary Materials: The following are available online.
Acknowledgments: We gratefully acknowledge the financial support from the National Natural Science
Foundation of China (grant 81230077), the Program for Innovative Research Team of the Ministry of Education,
and the Program for Liaoning Innovative Research Team in University.
Author Contributions: Tianxiao Wu designed and carried out the experiment and wrote the paper; Yu Pang,
Jing Guo, Wenbo Yin, Mingyue Zhu, Chenzhou Hao, Kai Wang, and Jian Wang assisted with the experiment;
Dongmei Zhao and Maosheng Cheng supervised the whole experiment and provided technical guidance.
All authors read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1
2
3
4
5
.
.
.
.
.
Kumar, R.; Gururaj Anupama, E.; Barnes Christopher, J. p21-activated kinases in cancer. Nat. Rev. Cancer
006, 6, 459–471. [CrossRef] [PubMed]
Jin, D.; Durgan, J.; Hall, A. Function cross-talk between Cdc42 and two downstream targets, Par6B and
PAK4. Biochem. J. 2015, 467, 293–302. [CrossRef] [PubMed]
Baskaran, Y.; Ng, Y.W.; Selamat, W.; Ling, F.T.P.; Manser, E. Group I and II mammalian PAKs have different
modes of activation by Cdc42. EMBO Rep. 2012, 13, 653–659. [CrossRef] [PubMed]
Vega, F.M.; Ridley, A.J. Rho GTPases in cancer cell biology. FEBS Lett. 2008, 582, 2093–2101. [CrossRef]
2
[PubMed]
Eswaran, J.; Lee, W.H.; Debreczeni, J.E.; Filippakopoulos, P.; Turnbull, A.; Fedorov, O.; Deacon, S.W.;
Peterson, J.R.; Knapp, S. Crystal structures of the p21-Activated Kinases PAK4, PAK5 and PAK6 Reveal
Catalytic Domain Plasticity of Active Group II PAKs. Structure 2007, 15, 201–213. [CrossRef] [PubMed]
Wells, C.M.; Jones, G.E. The emerging importance of group II PAKs. Biochem. J. 2010, 425, 465–473. [CrossRef]
6
.
.
[PubMed]
7
Song, S.; Li, X.; Guo, J.; Hao, C.; Feng, Y.; Liu, B.G.T.; Zhang, Q.; Zhang, Z.; Li, R.; Wang, J.; et al.
Design, synthesis and biological evaluation of 1-phenanthryl-tetrahydroisoquinoline derivatives as novel
p21-Activated kinase (PAK4) inhibitors. Org. Biomol. Chem. 2015, 13, 3803–3818. [CrossRef] [PubMed]
Fulciniti, M.; Martinez-Lopez, J.; Senapedis, W.; Oliva, S.; Bandi, R.L.; Amodio, N.; Xu, Y.; Szalat, R.; Gulla, A.;
Samur, M.K.; et al. Functional role and therapeutic targeting of p21-activated kinase 4 in, multiple myeloma.
Haematologica. Blood 2017, 129, 2233–2245. [CrossRef] [PubMed]
8
.
.
9
Wang, Y.; Gratzke, C.; Tamalunas, A.; Wiemer, N.; Ciotkowska, A.; Rutz, B.; Waidelich, R.; Strittmatter, F.;
Liu, C.; Stief, C.G.; et al. p-21 Activated Kinase Inhibitors FRAX486 and IPA3: Inhibition of Prostate
Stromal Cell Growth and Effects on Smooth Muscule Contraction in the Human Prostate. PLoS ONE 2016
1, e0153312.
,
1
1
1
0. Shao, Y.-G.; Ning, K.; Li, F. Group II p21-activated kinases as therapeutic targets in gastrointestinal cancer.
World J. Gastroneterol. 2016, 22, 1224–1235. [CrossRef] [PubMed]
1. Rudolph, J.; Murray, L.J.; Ndubaku, C.O.; O’Brien, T.; Blackwood, E.; Wang, W.; Aliagas, I.; Gazzard, L.;
Crawford, J.J.; Drobnick, J.; et al. Chemically Diverse Group I p21-Activated Kinase (PAK) InhibitorsImpart
Acute Cardiovascular Toxicity with a Narrow Therapeutic Window. J. Med. Chem. 2016, 59, 5520–5541.
[CrossRef] [PubMed]
1
2. Kim, H.; Woo, D.J.; Kim, S.Y.; Yang, E.G. p21-activated kinase 4 regulates HIF-1a translation in cancer cells.
Biochem. Biophys. Res. Commun. 2017, 486, 270–276. [CrossRef] [PubMed]

Molecules 2018, 23, 417
12 of 12
1
1
3. Hoeflich, K.P.; Lyle, K.S.; Staben, S. p21-Activated Kinase 4(PAK4) Inhibitors as Potential Cancer Therapy.
ACS Med. Chem. Lett. 2015, 6, 17–18.
4. Ma, Y.; McCarty, S.K.; Kapuriya, N.P.; Brendel, V.J.; Wang, C.; Zhang, X.; Jarjoura, D.; Saji, M.; Chen, C.;
Ringel, M.D. Development of p21 Activated Kinase-Targeted Multikinase Inhibitors That Inhibit Thyroid
Cancer Cell Migration. J. Clin. Endocr. Metab. 2013, 98, E1314–E1322. [CrossRef] [PubMed]
5. Zhang, J.; Wang, J.; Guo, Q.; Wang, Y.; Zhou, Y.; Peng, H.; Cheng, M.; Zhao, D.; Li, F. LCH-7749944, a novel
and potent p21-activated kinase 4 inhibitor, suppresses proliferation and invasion in human gastric cancer
cells. Cancer Lett. 2012, 317, 24–32. [CrossRef] [PubMed]
1
1
1
1
6. Statsuk, A.V.; Maly, D.J.; Seeliger, M.A.; Fanian, M.A.; Biggs, W.H., III; Lockhart, D.J.; Zarrinkar, P.P.;
Kuriyan, J.; Shokat, K.M. Tuning a three-component reaction for trapping kinase substrate complexes. J. Am.
Chem. Soc. 2008, 130, 17568–17574. [CrossRef] [PubMed]
7. Zeng, Z.-S.; He, Q.-Q.; Liang, Y.-H.; Feng, X.-Q.; Chen, F.-E.; De Clercq, E.; Balzarini, J.; Pannecouque, C.
Hybrid diarylbenzopyrimidine non-nucleoside reverse transcriptase inhibitors as promising new leads for
improved anti-HIV-1 chemotherapy. Bioorg. Med. Chem. 2010, 18, 5039–5047. [CrossRef] [PubMed]
8. Cho, N.-C.; Cha, J.H.; Kim, H.; Kwak, J.; Kim, D.; Seo, S.-H.; Shin, J.; Kim, T.H.; Park, K.D.; Lee, J.;
No, K.T.; Kim, Y.K.; Lee, K.; Pae, A.N. Discovery of 2-aryloxy-4-amino-quinazoline derivatives as novel
protease-activated receptor 2 (PAR ) antagonists. Bioorg. Med. Chem. 2015, 23, 7717–7727. [CrossRef]
2
[PubMed]
Sample Availability: Samples of the compounds 6a,b, 7a–i, 8a–e, 9a–c are available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(