Identification of Novel Piperazinylquinoxaline Derivatives as Potent Phosphoinositide 3-Kinase (PI3K) Inhibitors

Background Development of small-molecule inhibitors targeting phosphoinositide 3-kinase (PI3K) has been an appealing strategy for the treatment of various types of cancers. Methodology/Principal Finding Our approach was to perform structural modification and optimization based on previously identified morpholinoquinoxaline derivative WR1 and piperidinylquinoxaline derivative WR23 with a total of forty-five novel piperazinylquinoxaline derivatives synthesized. Most target compounds showed low micromolar to nanomolar antiproliferative potency against five human cancer cell lines using MTT method. Selected compounds showed potent PI3Kα inhibitory activity in a competitive fluorescent polarization assay, such as compound 22 (IC50 40 nM) and 41 (IC50: 24 nM), which induced apoptosis in PC3 cells. Molecular docking analysis was performed to explore possible binding modes between target compounds and PI3K. Conclusions/Significance The identified novel piperazinylquinoxaline derivatives that showed potent PI3Kα inhibitory activity and cellular antiproliferative potency may be promising agents for potential applications in cancer treatment.


Introduction
The phosphoinositide 3-kinase (PI3K) family includes lipid kinases that catalyze the phosphorylation of the 39-hydroxyl group of phosphatidylinositols to generate second messengers, such as phosphatidylinositol-3,4,5-triphosphate (PIP 3 ) [1,2]. PIP 3 recruits downstream effectors along the PI3K/protein kinase B (PKB orAkt)/mammalian target of rapamycin (mTOR) signaling cascade that is of crucial importance for the regulation of cellular growth, survival, and proliferation [3]. Based on sequence homology and substrate preference, PI3Ks are divided into three classes. Class I PI3Ks are subdivided into four isoforms, PI3Ka, PI3Kb, PI3Kd, and PI3Kc, according to different activation mechanism and varied catalytic and regulatory subunits [4]. Many studies have demonstrated that gain-of-function mutations in the gene encoding the catalytic subunit of PI3Ka, PIK3CA, amplification of PIK3CA, and loss-of-function mutations in PTEN, a lipid phosphatase that dephosphorylates PIP 3 result in constitutive activation of the PI3K signaling cascade, which contributes to tumor growth and progression [5,6,7]. These observations make targeting PI3Ks, especially PI3Ka, with small-molecule inhibitors a promising strategy for cancer therapy [8,9,10,11].
Considerable efforts have been devoted toward the development of small-molecule inhibitors targeting PI3K with more than twenty promising molecules have been progressed into various stages of clinical trials [11,12].
In our efforts to identify novel inhibitors of PI3K [13], we established a pharmacophore model based on reported PI3K inhibitors and identified the morpholinoquinoxaline derivative WR1 (1) as an initial hit with good potency against PI3Ka (IC 50 : 0.44 mM) [14], which is equivalent to that of the extensively studied tool compound LY294002 (2, PI3Ka, IC 50 : 0.63 mM) ( Fig. 1) [15,16]. Following modification based on WR1 led to the discovery of a series of piperidinylquinoxaline derivatives with good to potent PI3Ka inhibitory activity and cellular antiproliferative activity, such as WR23 (3, PI3Ka, IC 50 : 0.025 mM) (Fig. 1) [17]. In this paper, we describe our ongoing efforts in this field that led to the identification of this series of novel piperazinylquinoxaline derivatives as potent PI3Ka inhibitors.
Among compounds synthesized based on modifying the 4morpholino group at the 2-position of the quinoxaline scaffold of WR1, compounds 4-8 with a 4-carbamoylpiperidin-1-yl group at the 2-position of the quinoxaline were identified as interesting leads for further study due to their potent in vitro antiproliferative activity that was equivalent to that of WR23.
Thus, compounds 4-8 were chosen for further optimization. Reversion of the carboxamide group at the 4-position of the piperidinyl ring of 4-8 led to compounds 9-13 with a 4acetylpiperazin-1-yl group. To fully assess the impact of different piperidinyl substituents on cellular and enzymatic potency, modification in the following facets were made. Firstly, replacement of the 4-acetyl group on the piperazinyl ring with a smaller group, i.e. methyl, led to compounds 14-18. Removing the 4-methyl group and relocating the 4-methyl group as 3methyl group on the piperazinyl ring led to compounds 19-23 and 24-28, respectively. Secondly, replacement of the 4-acetyl group of 9-13 with a benzoyl or 4-chlorobenzoyl group afforded compounds 29-33 and 34-38, respectively, with a larger substituted piperazinyl group than that of 9-13. Thirdly, replacement of the 4-acetyl group of 9-13 with a methylsulfonyl or 4-methylphenylsulfonyl group led to compounds 39-43 and 44-48, respectively. Lastly, different from above rigid substituted piperazinyl group, a flexible 4-(3-morpholinopropyl)piperazin-1-yl group was introduced to the 2-position of the quinoxaline scaffold to afford compounds 49-53 (Fig. 2). This work led to the identification of a series of piperazinylquinoxaline derivatives, whose synthesis, in vitro evaluation, apoptosis inductive effort, and docking analysis are described herein.
Fifty new derivatives including forty-five piperazinylquinoxalines were synthesized. Their purities were above 95% indicated by HPLC.

Biological Evaluation and Structure-Activity Relationships (SAR)
Antiproliferative activity against human cancer cell lines. All synthesized target compounds were firstly tested for their antiproliferative activity against five human cancer cell lines, PC3, A549, HCT116, HL60, and KB, using MTT assay. Compounds WR1 and LY294002 were used as positive controls. As shown in Table 1, 2, 3, both pieridinylquinoxalines 4-8 and piperazinylquinoxalines 9-53 exhibited significantly improved antiproliferative activity against most tested cell lines than that of WR1 and LY294002, for example, compounds 4-8 showed IC 50 ranging from 1.17 to 4.36 mM against PC3 cell, compounds 14-18 showed IC 50 ranging from 0.84 to 3.09 mM against PC3 cell, while the corresponding IC 50 values for WR1 and LY294002 were 18.88 and 61.35 mM, respectively. Some of the most potent compounds showed nanomolar antiproliferative activity against certain cancer cell lines, such as compound 22 and 25, which showed IC 50 values of 100 and 90 nM against HL60, respectively.
Inhibition of PI3Ka. Selected compounds were then tested for their enzymatic inhibitory activity against PI3Ka using a competitive fluorescence polarization (FP) assay to determine the molecular target of synthesized compounds [21]. As shown in Table 4, compound 4 with a 4-carbamoylpiperidin-1-yl group did not show significant inhibitory activity against PI3Ka (IC 50 value .10 mM). Most tested piperazinylquinoxaline derivatives showed comparable PI3Ka inhibitory activity with that of WR1 and LY294002. The most potent compounds 2-(piperazin-1-yl)-3-(4-bromophenylsulfonyl)quinoxaline 22 (IC 50 : 40 nM) and 2-(4-(methylsulfonyl)piperazin-1-yl)-3-(4-methoxyphenylsulfonyl)quinoxaline 41 (IC 50 : 24 nM) showed nanomolar inhibitory activity against PI3Ka. Consistent with the result of antiproliferative test, compound 29 with a 4-benzoylpiperazin-1-yl group (IC 50 : .10 mM) and compound 44 with a 4-(4-methylphenylsulfonyl)piperazin-1-yl group (IC 50 : .10 mM) showed less potent PI3Ka inhibitory activity than that of compound 24 with a 3methylpiperazin-1-yl group (IC 50 : 0.059 mM) and compound 39 with a 4-(methylsulfonyl)piperazin-1-yl group (IC 50 : 1.34 mM). The values of binding efficiency index (BEI), a modified ligand efficiency index based on a molecular weight (MW) scale [22], were calculated for target compounds that exhibited good to potent PI3Ka inhibitory activity to evaluate binding efficiency of these compounds. As shown in Table 5, although most compounds showed BEI values comparable to that of WR1, LY294002 or WR23, no significant improvement in ligand binding efficiency was observed. This analysis based on BEI indicated that further modification with an aim to improve ligand binding efficiency might expedite the optimization process for this series of compounds.
Apoptosis assay. Piperazinylquinoxaline derivative 41 was further tested for its ability to induce apoptosis in PC3 cells. GDC0941, one of the most advanced PI3K inhibitors revealed so far, was used as the positive control [23] (Fig. 5). With an apoptotic percent of 1.71% of the control, the percent of apoptotic PC3 cells induced by compound 41 and GDC0941 in 5 mM after treatment of 24 h were 4.48% and 3.12%, respectively. The fact that compound 41 showed an apoptotic percent of 32.83% in 10 mM, in comparison with that of 5.85 for GDC0941, indicated the potent apoptosis inductive activity of compound 41.
Cell cycle arrest. Moreover, flow cytometric analysis was performed to determine whether target compounds could induce cell cycle arrest in PC3 cells. GDC0941 was used as the positive control. PC3 cells were treated with compound 41 and GDC0941 in two different concentrations (2 and 4 mM) for 24 h, the results are presented as Figure 6. GDC0941 induced cell cycle arrest in G1 phase with a simultaneous decrease of cells in S phase. Compound 41 showed similar trend while the percent of cell in G1 phase was smaller.

Molecular Docking Analysis
Compounds 41 and 22 that showed the most potent inhibitory activity against PI3Ka were subjected to molecular docking analysis to investigate possible binding mode between target compounds and PI3Ks. Co-crystal structures of mutant PI3Ka with small-molecule inhibitor (PDB ID: 3HHM) was utilized as the template to perform docking analysis [24]. Based on the docking results as shown in Figure 7, compound 41 might form three hydrogen bond interactions with PI3Ka, the methoxy oxygen with the NH of Val851 (distance: 2.1 Å), one of the methylsulfonyl oxygen with the OH of Ser774 (distance: 1.9 Å), and one of the quinoxaline nitrogen with the NH 2 of Lys802 (distance 2.4 Å) (Fig. 7A); the hydrogen bond interaction with Val882 is probably retained in the PI3Ka-22 docking complex (Fig. 7B). Although both 41 and 22 have the potential to interact with Val851 through the formation of a hydrogen bond interaction that is believed to be of significant importance for PI3K inhibition [25], 41 and 22 tend to bind with PI3Ka in different modes, the quinoxaline moiety of 41 might bind with an affinity pocket close to Lys802 and its methylsulfonylpiperazinyl moiety extends to the solvent front ( Fig. 7C), while the quinoxaline moiety of 22 might extend to the solvent front with its bromophenylsulfonyl moiety binds with the affinity pocket (Fig. 7D).

Experimental Methods
Reagents and apparatus. Melting points were determined with a B-540 Büchi apparatus and are uncorrected. NMR spectra were recorded on a Brüker 500 (500 MHz) spectrometer at room temperature (chemical shifts are reported in ppm (d) using TMS as  internal standard, coupling constants (J) are in hertz (Hz), and signals are designated as follows: s, singlet; d, doublet; t, triplet; m, multiplet; brs, broad singlet, etc.) Mass spectra (MS), ESI (positive) were recorded on an Esquire-LC-00075 spectrometer. Thin layer chromatography was carried out using plate silica gel F254 (Merck, Damstadt, Germany). All commercially obtained reagents were used as received unless otherwise noticed. All yields are unoptimized and generally represent the result of a single experiment.
General procedure for the synthesis of 4-8 (procedure A). To a microwave vial (2-5 mL) were added 2-chloro-3-(arylsulfonyl)quinoxaline 55-59 (0.1 mmol), N-carbamoylpiperidine (0.5 mmol), and isopropyl alcohol (2 mL). The sealed vial was heated at 80uC for 10 min by microwave irradiation in a Biotage TM Initiator Synthesizer using a fixed hold time. The mixture was then cooled to room temperature and the residue obtained after evaporating under vacuum was subjected to purification over silica gel chromatography eluting with PE: EtOAc (4:1, v/v) to afford target compounds as yellow solid.  General procedure for the synthesis of 9-13. Similar with procedure A, but N-carbamoylpiperazine was replaced by Nacetylpiperazine. Target compounds were obtained as yellow solid.    General procedure for the synthesis of 14-18. Similar with procedure A, but N-carbamoylpiperazine was replaced by Nmethylpiperazine. Target compounds were obtained as yellow solid.

MTT Assay -Inhibition of Human Cancer Cell Lines
Human prostate cancer PC3 cells, lung adenocarcinoma epithelial A549 cells, colon cancer HCT116 cells, promyelocytic leukemia HL60 cells and nasopharyngeal carcinoma KB cells were obtained from the cell bank of the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The inhibitory activity of target compounds against tested cancer cell lines was measured using the MTT assay. PC3, A549, HCT116, HL60, and KB cancer cell lines were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) with heatinactivated 10% fetal bovine serum, penicillin (100 units/mL) and streptomycin (100 mg/mL) and incubated in an atmosphere with 20% oxygen and 5% carbon dioxide at 37uC. All tested compounds were dissolved in DMSO at concentrations of 10.0 mg/mL and diluted to appropriate concentrations. Cells were plated in 96-well plates for 24 h and subsequently treated with different concentrations of all tested compounds for 72 h. Viable cells were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay kit (MTT, Sigma) according to operation instructions provided by the manufacturer. The concentration of drug causing 50% inhibition in absorbance compared with control cells (IC 50 ) was calculated using the software of dose-effect analysis with microcomputers.

FP Assay -Inhibition of PI3Ka
The PI3Ka inhibitory test was determined using a competitive fluorescence polarization kinase activity assay. PI3K fluorescence polarization assay kit (catalog No. K-1100) and recombinant human PI3Ka (catalog No. E-2000) were purchased from Echelon Biosciences (Salt Lake City, UT, USA). PI3K reactions were performed in 5 mM HEPES, Ph 7, 2.5 mM MgCl 2 , 10 mM DTT and 50 mM ATP, using diC 8 -phosphatidylinositol-4, 5-bisphosphate (PIP 2 ) as the substrate, and the final reaction volumes were 10 mL. For evaluation of the PI3Ka inhibitory activity of target compounds, 50 ng enzyme and 10 mM substate were used per 10 mL reaction volume with the concentrations of inhibitors ranging from 3.2 nM to 10 mM. After incubating for 3 h at room temperature, reactions were quenched by adding chelators. A mixture of phosphoinositide binding protein was added and mixed, followed by the addition of a fluorophore-labeled phosphoinositide tracer. Samples were then mixed in 384-well black Corning nonbinding plates (Corning, NY, USA) and incubated in a dark environment for 1 h to equilibrate. Finally, polarization values were measure using red fluorophores with appropriate filters to determine the extent of enzyme activity in the reaction.

Molecular Docking
The X-ray co-crystal structure of mutant PI3Ka-wortmannin complex was downloaded from the RCSB Protein Data Bank (ID: 3HHM). The C-Dock protocol within DiscoveryStudio 2.1 was utilized to perform molecular docking analysis for compounds 22 and 41. For ligand preparation, the 3D structures of 22 and 41 were generated and minimized using DiscoveryStudio 2.1. For protein preparation, the hydrogen atoms were added, water was removed, and the CHARMm force field was employed. The whole PI3Ka enzyme was defined as a receptor and the site sphere was selected based on the ligand binding location of wortmannin. Compound 22 or 41 were placed in the binding site during the docking procedure. Docking parameters were set as follows: top hits, 25; random conformations, 25; random conformations dynamics steps, 1000; grid extension, 8.0; random dynamics time step, 0.002. All other parameters were set as default values. Types of interactions of the docked enzyme with ligand were analyzed upon the finish of molecular docking. All graphical pictures were made using PyMol.