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

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 μM). Among them, compounds 8d and 9c demonstrated the most potent inhibitory activity against PAK4 (IC50 = 0.060 μM and 0.068 μM, 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.


Introduction
The p21-activated kinases (PAKs), which are members of the serine/threonine protein kinases, 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][4][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][7][8][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-1α in hypoxia [12]. 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.
In our prior study [15], N 2 -(3-methoxyphenyl)-N 4 -((tetrahydrofuran-2-yl)methyl)quinazoline-2,4-diamine (LCH-7749944) was identified as a novel and potent inhibitor of PAK4 ( Figure 1A). 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 hingebinder fragment was also the focus of our investigation, by replacing the original group with a 3amino-5-cyclopropyl-pyrazole fragment, which can form the classic three-point donor-acceptordonor 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. 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.

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 4substituted 3-amino-5-cyclopropylpyrazole moiety and introduced piperazines or piperidines at the 2-position 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

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 3-amino-5-cyclopropylpyrazole moiety and introduced piperazines or piperidines at the 2-position 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 PAK4 potency, except for 7a, indicating that flat geometric configuration was beneficial. Phenyl rings containing substituents (compounds 7b-i) exhibited higher kinase inhibition than non-substituted derivatives (compound 7a), and the para-substituted phenyl analogs (7e,g) showed higher enzyme activity than the meta-substituted analogs 7f,h. Furthermore, we examined the effects of different substituents at the same position (para-substituted phenyl ring derivatives 7b-e,i). The activities were in the order 4-Br > 4-Cl > 4-F > 4-Me > 4-CF 3 . Analog 7c displayed strong inhibition against PAK4, 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,c,d) and pyrimidine (compounds 9a−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 (IC 50 = 0.060 µM) and pyrimidine 9c (IC 50 = 0.068 µM), like Br-substituted phenyl 7c, showed better potency than other analogs 8a,c and 9a,b.

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 (IC 50 = 4.685 µM, and 4.751 µM, respectively), and negligible activity was observed for HT1080 (Table 2).  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. in Figure 2C-F, 8d decreased the migration and invasion potential of the A549 cells in a dosedependent manner. These results demonstrate that 8d efficiently exhibits antimetastatic potential against cancer cells.

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).

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). in Figure 2C-F, 8d decreased the migration and invasion potential of the A549 cells in a dosedependent manner. These results demonstrate that 8d efficiently exhibits antimetastatic potential against cancer cells.

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).  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.

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 light. Proton nuclear magnetic resonance ( 1 H-NMR, 400 MHz) and carbon-13 nuclear magnetic resonance ( 13 C-NMR, 151 MHz) spectra were recorded using a Bruker 400 MHz NMR spectrometer (Bruker, Karlsruhe, Germany) in DMSO-d 6
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 µs for the delay and 500 µs integration time. Date were calculated using 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 50 .

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% CO 2.
The in vitro antiproliferative activity of some of the target compounds was determined by the MTT assay (Amresco, Seattle, WA, USA). A suspension of 100 µL human cancer cells (5 × 10 4 /mL) was plated in a 96-well cell culture plate and incubated with 5% CO 2 at 37 • C for 24 h. Then cells were 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 × 800 microplate reader (BioTek, Winooski, VT, USA). Dose-response curves were plotted to determine the IC 50 values using GraphPad Prism 5.0. Each IC 50 value was expressed as mean ± standard deviation.

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).

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). First, 0.5 × 10 5 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 × 60 × 60 units in number of grid 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 10 times, yielding 10 docked conformations, and the most favorable pose of 8d was displayed.

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.