Design, Synthesis, and In Vitro Activity of Pyrazine Compounds

Despite the fact that there are several anticancer drugs available, cancer has evolved using different pathways inside the cell. The protein tyrosine phosphatases pathway is responsible for monitoring cell proliferation, diversity, migration, and metabolism. More specifically, the SHP2 protein, which is a member of the PTPs family, is closely related to cancer. In our efforts, with the aid of a structure-based drug design, we optimized the known inhibitor SHP099 by introducing 1-(methylsulfonyl)-4-prolylpiperazine as a linker. We designed and synthesized three pyrazine-based small molecules. We started with prolines as cyclic amines, confirming that our structures had the same interactions with those already existing in the literature, and, here, we report one new hydrogen bond. These studies concluded in the discovery of methyl (6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)prolylprolinate hydrochloride as one of the final compounds which is an active and acceptable cytotoxic agent.


Introduction
The definition of cancer is that it is a disease caused by an uncontrolled division of abnormal cells in a part of the body. According to the World Health Organization [1], 9.6 million people worldwide are estimated to have died from cancer in 2018, making cancer the second most deadly disease globally. The most common cancers are lung (2.09 million cases), breast (2.09 million cases), and colorectal (1.8 million cases). Thus, discovery of novel drugs for the treatment of cancer patients is vital.
The activation of the PTP's (Protein tyrosine phosphatases) pathway in human cancers has triggered the development of a variety of pharmacological inhibitors targeting catalytic sites of the SHP2 cascade. The SHP2 protein belongs to the protein tyrosine phosphatases group of enzymes. It is a non-receptor protein which is constituted from two SH2 (N-terminal Src homology 2) domains, a PTP domain, and a C-terminal tail. The activity of SHP2 is auto-inhibited, as the N-SH2 domain is binding with the PTP domain [2]. The upregulation expression of the PTPN11 gene that encodes SHP2 protein exists in melanoma [3,4], liver cancer [5,6], and lung cancer [7].
More specifically, the SHP2 oncogene activates the RAS-ERK signaling pathway, so it can regulate cancer cell survival and proliferation [8,9]. In 2016, Fortanet et al. [10] revealed a new allosteric binding site of SHP2 with X-ray analysis. After structure-based drug design, they synthesized a small molecule, SHP099. The same molecule was used by Chen's group [11], who reported that receptor tyrosine kinases-driven cancer cells depend on SHP2 survival. One year later, LaRochelle et al. [12] followed an alternative route to find allosteric inhibitors by making use of a partially active cancer mutant, SHP2F285S. In recent years, many different attempts have been performed for the discovery of new SHP2 inhibitors [13]. Cyanoacrylamides [14], 6-amino-3-methylpyrimidinones [15], substituted thiazoles [16], and fused bicyclic compounds [17] have shown interesting results in SHP2 inhibition ( Figure 1).
In order to evaluate and determine the utility of substituted pyrazine compounds, such as SHP2 inhibitors, we built a pharmacophore model using the highly selective SHP2 allosteric inhibitor SHP099-SHP2 complex. In the present work, we designed three small molecules using structure-based drug design. The substituted pyrazine compounds incorporated with novel 1-(methyl sulfonyl)-4-prolylpiperazine, as a linker to amides and sulphonamides, and the cytotoxic effect on different human cancer cell lines were examined.
Molecules 2019, 24, x 2 of 17 a small molecule, SHP099 . The same molecule was used by Chen's group [11], who reported that receptor tyrosine kinases-driven cancer cells depend on SHP2 survival. One year later, LaRochelle et al. [12] followed an alternative route to find allosteric inhibitors by making use of a partially active cancer mutant, SHP2F285S. In recent years, many different attempts have been performed for the discovery of new SHP2 inhibitors [13]. Cyanoacrylamides [14], 6-amino-3-methylpyrimidinones [15], substituted thiazoles [16], and fused bicyclic compounds [17] have shown interesting results in SHP2 inhibition (Figure 1). In order to evaluate and determine the utility of substituted pyrazine compounds, such as SHP2 inhibitors, we built a pharmacophore model using the highly selective SHP2 allosteric inhibitor SHP099-SHP2 complex. In the present work, we designed three small molecules using structurebased drug design. The substituted pyrazine compounds incorporated with novel 1-(methyl sulfonyl)-4-prolylpiperazine, as a linker to amides and sulphonamides, and the cytotoxic effect on different human cancer cell lines were examined.

Structure Retrieval and Validation
The three-dimensional structure of SHP2 was downloaded from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank and used for the structure-guided design of low molecular weight organic compounds [18]. The crystal structure (PDB ID: 5EHR) was solved through X-ray crystallography with a resolution of 1.7 Å and contained 526 amino acids which covered 87.8% of the canonical protein sequence (UniProtKB ID: Q06124) [11]. The quality of the crystal structure was validated using the SWISS-MODEL Structure Assessment tools [19]. Ramachandran plot analysis showed that 96.62% of the residues were within energetically favored regions, while only 0.85% (VAL505, LYS324, GLU313, GLY115) were Ramachandran outliers (Figure 2A) [20]. The outliers were found adjacent to the gaps in the protein structure. The Qualitative Model Energy Analysis 4 (QMEAN4) global score of 5EHR indicates that the overall quality of the structure was good when compared to experimental structures of similar size ( Figure 2B) [21]. Assessment of the local quality of the structure showed that only a few compounds were characterized by low quality ( Figure 2C).

Structure Retrieval and Validation
The three-dimensional structure of SHP2 was downloaded from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank and used for the structure-guided design of low molecular weight organic compounds [18]. The crystal structure (PDB ID: 5EHR) was solved through X-ray crystallography with a resolution of 1.7 Å and contained 526 amino acids which covered 87.8% of the canonical protein sequence (UniProtKB ID: Q06124) [11]. The quality of the crystal structure was validated using the SWISS-MODEL Structure Assessment tools [19]. Ramachandran plot analysis showed that 96.62% of the residues were within energetically favored regions, while only 0.85% (VAL505, LYS324, GLU313, GLY115) were Ramachandran outliers (Figure 2A) [20]. The outliers were found adjacent to the gaps in the protein structure. The Qualitative Model Energy Analysis 4 (QMEAN4) global score of 5EHR indicates that the overall quality of the structure was good when compared to experimental structures of similar size ( Figure 2B) [21]. Assessment of the local quality of the structure showed that only a few compounds were characterized by low quality ( Figure 2C). The allosteric inhibitor SHP099 was co-crystallized with the protein structure of SHP2. The SHP099 (Chemical name: 6-(4-azanyl-4-methyl-piperidin-1-Yl)-3-(2,3-bis(chloranyl)phenyl)pyrazin-2-amine) is a highly potent and selective allosteric inhibitor of SHP2 with an IC50 value of 0.071 µM. The inhibitor binds to the interface of the N-terminal SH2, C-terminal SH2, and PTP domains and stabilizes an auto-inhibited conformation of SHP2 [11]. We used the above protein structure for the structured-guided design of novel allosteric SHP2 inhibitors.

Small Molecule Docking
An in silico study based on molecular docking was implemented to explore the interactions between the newly synthesized small molecules and the allosteric binding site on SHP2. Docking was performed against the 3D structure of SHP2, and the docking scores and binding poses of the compounds were obtained. Based on a co-crystallized ligand, SHP099, several compounds were designed in order to improve the binding affinity. To enhance the affinity between the binding pocket and the compounds, the third ring was changed while the aminopyrazine moiety and the dichlorophenyl moiety were kept identical to the reference compound. That design path was followed, because the two last moieties had the strongest bonds in the binding pocket, while the third part could be improved. Compounds 10, 18, and 19 (Scheme 1) showed scores comparable to that of the cocrystallized ligand, SHP099. The models of the protein-ligand complexes reveal the interactions between compounds 10, 18, and 19 and the surrounding residues of the binding pocket ( Figure 3). All three compounds form a hydrogen bond with Glu250 of SHP2's PTP domain and a hydrogen bond with Thr218 that lies on the loop between domains C-SH2 and PTP. The allosteric inhibitor SHP099 was co-crystallized with the protein structure of SHP2. The SHP099 (Chemical name: 6-(4-azanyl-4-methyl-piperidin-1-Yl)-3-(2,3-bis(chloranyl)phenyl)pyrazin-2-amine) is a highly potent and selective allosteric inhibitor of SHP2 with an IC 50 value of 0.071 µM. The inhibitor binds to the interface of the N-terminal SH2, C-terminal SH2, and PTP domains and stabilizes an auto-inhibited conformation of SHP2 [11]. We used the above protein structure for the structured-guided design of novel allosteric SHP2 inhibitors.

Small Molecule Docking
An in silico study based on molecular docking was implemented to explore the interactions between the newly synthesized small molecules and the allosteric binding site on SHP2. Docking was performed against the 3D structure of SHP2, and the docking scores and binding poses of the compounds were obtained. Based on a co-crystallized ligand, SHP099, several compounds were designed in order to improve the binding affinity. To enhance the affinity between the binding pocket and the compounds, the third ring was changed while the aminopyrazine moiety and the dichlorophenyl moiety were kept identical to the reference compound. That design path was followed, because the two last moieties had the strongest bonds in the binding pocket, while the third part could be improved. Compounds 10, 18, and 19 (Scheme 1) showed scores comparable to that of the co-crystallized ligand, SHP099. The models of the protein-ligand complexes reveal the interactions between compounds 10, 18, and 19 and the surrounding residues of the binding pocket ( Figure 3). All three compounds form a hydrogen bond with Glu250 of SHP2 s PTP domain and a hydrogen bond with Thr218 that lies on the loop between domains C-SH2 and PTP. Hydrophobic interactions between the dichlorophenyl ring and residues of the PTP domain are shared with Thr218 which lies on the loop between domains C-SH2 and PTP. Hydrophobic interactions between the dichlorophenyl ring and the surrounding residues further aid the protein-ligand interactions. between the dichlorophenyl ring and residues of the PTP domain are shared with Thr218 which lies on the loop between domains C-SH2 and PTP. Hydrophobic interactions between the dichlorophenyl ring and the surrounding residues further aid the protein-ligand interactions.

Synthetic Strategy
The synthesis of compound 10 (Scheme 2) started with the Suzuki-Miyaura coupling between (2,3-dichlorophenyl) boronic acid (2) and 3-bromo-6-chloropyrazin-2-amine (1), affording intermediate (3) in 74% yield, and then protection of amine on pyrazine with di-tert-butyl dicarbonate gave the di-protected intermediate (4)  between the dichlorophenyl ring and residues of the PTP domain are shared with Thr218 which lies on the loop between domains C-SH2 and PTP. Hydrophobic interactions between the dichlorophenyl ring and the surrounding residues further aid the protein-ligand interactions.
Molecules 2019, 24, x 5 of 17 drolysis with base in homolytic mixture of water and THF (tetrahydrofuran) ended up with acid intermediate (7) in 54% yield. Amide coupling with L-proline methyl ester (5) resulted in proline amide intermediate (8) with 49% yield. Final deprotection using 4 M HCl in MeOH gave a 36% yield of compound 9, which, upon reduction with LAH (lithium aluminum hydride) in THF, afforded compound 10 as free base after acid/base extraction with very low yield (13%). The synthesis of compounds 18 and 19 (Scheme 3) started with the sulphonamide synthesis on tert-butyl piperazine-1-carboxylate (11) with methane sulfonyl chloride and trifluoromethane sulfonic anhydride and ended up with intermediates 12 and 13. Deprotection with 4 M methanolic HCl resulted in intermediates 14 and 15 with 50% and 58% yields, respectively. Amide coupling between (6-((ditert-butoxycarbonyl)amino)-5-(2,3-dichlorophenyl)pyrazin-2-yl)proline (7) and intermediates 14 and 15 resulted in intermediates 16 and 17 in 40% and 45% yields, accordingly. Final deprotection of amine on pyrazine gave the final compound 18 with a 40% yield and compound 19 with a 35% yield. The synthesis of compounds 18 and 19 (Scheme 3) started with the sulphonamide synthesis on tert-butyl piperazine-1-carboxylate (11) with methane sulfonyl chloride and trifluoromethane sulfonic anhydride and ended up with intermediates 12 and 13. Deprotection with 4 M methanolic HCl resulted in intermediates 14 and 15 with 50% and 58% yields, respectively. Amide coupling between (6-((ditert-butoxycarbonyl)amino)-5-(2,3-dichlorophenyl)pyrazin-2-yl)proline (7) and intermediates 14 and 15 resulted in intermediates 16 and 17 in 40% and 45% yields, accordingly. Final deprotection of amine on pyrazine gave the final compound 18 with a 40% yield and compound 19 with a 35% yield. The synthesis of compound 10 was performed by another synthetic route in order to increase the final yield. We succeeded to synthesize two different intermediates which were tested for their biological activity. However, we did not receive the final product (compound 10) with this synthetic route. The synthesis of 5 and 6 (Scheme 4) started with an SNAr reaction on the Boc protected biphenyl intermediate (4) with L-prolinol (17) and which provided the pyrazine ortho prolinol compound 5 with a 60% yield. Compound 5 on deprotection with 4 M methanolic in MeOH gave compound 6 as yellow colored hydrochloride salt with a 45% yield. The synthesis of compound 10 was performed by another synthetic route in order to increase the final yield. We succeeded to synthesize two different intermediates which were tested for their biological activity. However, we did not receive the final product (compound 10) with this synthetic route. The synthesis of 5 and 6 (Scheme 4) started with an S N Ar reaction on the Boc protected biphenyl intermediate (4) with l-prolinol (17) and which provided the pyrazine ortho prolinol compound 5 with a 60% yield. Compound 5 on deprotection with 4 M methanolic in MeOH gave compound 6 as yellow colored hydrochloride salt with a 45% yield. The synthesis of compound 10 was performed by another synthetic route in order to increase the final yield. We succeeded to synthesize two different intermediates which were tested for their biological activity. However, we did not receive the final product (compound 10) with this synthetic route. The synthesis of 5 and 6 (Scheme 4) started with an SNAr reaction on the Boc protected biphenyl intermediate (4) with L-prolinol (17) and which provided the pyrazine ortho prolinol compound 5 with a 60% yield. Compound 5 on deprotection with 4 M methanolic in MeOH gave compound 6 as yellow colored hydrochloride salt with a 45% yield.     (Table 3) to have a significant effect on MCF7 cell viability at the same time point in two of the three assays were at 48 h with 0.001 µM and at 72 h with 1 µM of intermediate 9. The percentages of untreated cells were 88.48% for living cells, 0.41% for early apoptotic, 1.84% for late apoptotic, and 1.3% for dead cells. After treatment with 0.001 µM of intermediate 9, the percentages of cells were 86.53% for living cells, 0.38% for early apoptotic, 1.38% for late apoptotic, and 2.34% for dead cells. Generally, an increasing dose of intermediate 9 led to an increasing percentage of apoptosis and necrosis. At 72 h, the percentages of untreated cells were 78.21% for living cells, 1.62% for early apoptotic, 6.45% for late apoptotic, and 1.52% for dead cells. After treatment with 1 µM of intermediate 9, the percentages of cells were 78.21% for living cells, 1.51% for early apoptotic, 5.91% for late apoptotic, and 2.34% for dead cells.  On the other hand, final molecule 10 appeared (Table 5) to have significant cytotoxic activity against MDA-MB-231 at 0.1 µM with MTT assay. At 24 h, the percentages of untreated cells were 69.6% for living cells, 3.02% for early apoptotic, 14.24% for late apoptotic, and 2.39% for dead cells. After treatment with 0.1 µM of final compound 10, the percentages of cells were 56.79% for living cells, 3.16% for early apoptotic, 27.72% for late apoptotic, and 2.52% for dead cells.

Discussion
The aim of our study was to determine the utility of substituted pyrazine compounds such as SHP2 inhibitors. We built a pharmacophore model using the highly selective SHP2 allosteric inhibitor SHP099-SHP2 complex. A set of three novel compounds was discovered using a structure-based drug design. Compared with the existing inhibitors, the new compounds not only had a similar ability to be bound in the allosteric binding site of SHP2, but also a new hydrogen bond was determined during this binding mode. The modulation of existing inhibitors and designing new inhibitors of SHP2 are really crucial if we consider that SHP2 is a therapeutic target due to the fact of its importance in known oncogenic pathways and emerging role in immuno-oncology. According to the literature, there are amino pyrazines, such as kinase inhibitors [11,16,[22][23][24][25], which have different biological data depending on their structures. Hence, in this work, we have focused on allosteric inhibition of SHP2-PTP catalytic sites through small molecules, particularly in amino pyrazine compounds with different novel cyclic amine rings with substitutions. Initially, we started with condensed ring systems. We started with prolines as cyclic amines; through in silico modelling, we came to know that the interactions are as reported in the literature with one new hydrogen bond. Also, we noticed the space available with the ortho position of proline for functionalization. Then, we designed structures with different substitutions at the 2 and 3 positions of the prolines followed by a chain of amides with substitutions.
As result of our efforts, we synthesized three novel pyrazine small molecular prolinamides and sulphonamides as allosteric inhibitors of SHP2 which were determined by structure-based drug design. The new compounds contained the 1-(methylsulfonyl)-4-prolylpiperazine group as a linker. The final compound (10) and some of its intermediates (compounds 9, 21, 22) were evaluated for their cytotoxic effect on different human cancer cell lines, and they were examined by four different biological assays. Generally, an increasing dose of the intermediates led to an increasing percentage of apoptosis and necrosis. Concerning the final compound 10, after treatment with 0.1 µM, the percentages of MDA-MB-231 human breast adenocarcinoma cells were 56.79% for living cells, 3.16% for early apoptotic, 27.72% for late apoptotic, and 2.52% for dead cells. The methodology described to identify allosteric inhibitors also avoided the drug discovery challenges related to the polarity and homology of the orthosteric binding pocket of SHP2. Finally, the compounds 18 and 19 will be evaluated for their cytotoxic effect in different cancer cell lines, and the compound 10 will be modified in order to achieve a better effect, as the results we presented in this work encourage us to further modify our structures.

Small Molecule Docking
The molecular docking studies on SHP2 were performed using ICM version 3.8-7c (Molsoft LLC, San Diego, CA, USA) on a 2.0 GHz Intel Xeon Gold 6138 processor. The X-ray structure of SHP2 (PDB ID: 5EHR) included two copies of the protein, and chain A was chosen as the receptor for docking. The protein was prepared for docking through ICM's automated protocol. This process includes: the global optimization of hydrogens, the optimization of the orientation of His, Pro, Asn, Gln, and Cys residues, and the deletion of water molecules that are not tightly bound to the protein structure. The binding pocket was defined around the co-crystallized ligand, SHP099, by creating a box around the ligand with a margin of 3. The box defines the potential energy maps and the selected margin value was enough, in this case, to encompass the whole binding site. The co-crystallized ligand, SHP099, was removed before docking. The chosen docking approach involved a rigid receptor and fully flexible ligands. A table of compounds was imported into ICM and docked against the allosteric binding pocket of SHP2 with an effort of 2. The effort value (ranges from 1 to 10) determines the "thoroughness" which relates to the length of the simulation's sampling time. Conformational sampling uses the Monte Carlo minimization procedure, and the scoring is according to the ICM scoring function [26][27][28]. A docking score was assigned to each docked compound and used to assess the predicted interaction.

General Information
One milligram of synthesized compound was dissolved in 1 mL of a methanol/water mixture (90/10), filtered through a 0.45 µM filter, and inserted in an amber glass vial. The LC/MS analysis (1260 Infinity Series HPLC, 6120 Quadrupole MSD, Agilent Technologies Inc., Richardson, TX, USA) was carried out with the use of a reverse phase column, Zorbax RX-C8 (5 µM, 250 mm × 4.6 mm, Agilent Technologies, Santa Clara, CA, USA) maintained at 40 • C. Chromatograms were integrated and analyzed using OpenLAB Chemstation (version M8301AA, Revision C.01.07 Agilent Technologies Inc., Richardson, TX, USA). The solvent system used was: (A) aqueous solution of 0.1% ammonium acetate and (B) solution of 0.1% ammonium acetate in methanol. The separation was carried out at a 0.4 mL/min flow rate with isocratic elution of 10% solvent A and 90% solvent B for 20 min. The injection volume was 10 µL and ultraviolet (UV) detection was carried out at 278, 280, and 325 nm. Mass spectrometry analysis was performed with a 6120 Series Quadrupole System (Agilent Technologies, Santa Clara, CA, USA) equipped with an electrospray ionization source (ESI) operating in negative mode by scan analysis with a 100-1000 m/z scan range. Proton and carbon nuclear magnetic resonance ( 1 H and 13 C-NMR) spectra were recorded with dimethylsulfoxide-d 6 (in DMSO-d 6 ) on the following instrument: Bruker ( 1 H at 400 MHz and 13 C-NMR at 100 MHz, Milano, Italy).

Experimental Procedures and Analytical Data
All reactions were carried out under N 2 atmosphere unless specified. Methyl (6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)prolylprolinate hydrochloride (10): To a clean dry round-bottom flask, which was purged with nitrogen to remove water traces and other particles, 3-bromo-6-chloropyrazin-2-amine (1.0 eq) was added and dissolved in dry THF (10 volumes). Then, (2,3-dichlorophenyl) boronic acid (1.3 to 1.5 eq) was added and stirred for 10 min under inert atmosphere. The reaction mass was degassed using a nitrogen inlet and vacuum outlet for 15 min. Tetrakis (triphenylphosphine) palladium (0) (0.1 eq) was added and stirred for 15 min. After the addition of a solution of 2 M Na 2 CO 3 , the reaction mixture was slowly heated to reflux. The process of the reaction was evaluated by TLC and LCMS. After the solution was stirred for 4 h, it was cooled to RT. Ethyl acetate and water were added, and the reaction mixture was stirred for another 10 min. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate. The combined organic layer was washed with brine and dried over sodium sulfate. After evaporation of the solvent, the crude product was purified by flash chromatography (0% to 50% gradient of ethyl acetate/n-hexane) to get 6-chloro-3-(2,3-dichlorophenyl)pyrazin-2-amine (3)  To a cooled solution (0 • C) of 6-chloro-3-(2,3-dichlorophenyl)pyrazin-2-amine (3) (1.0 eq) in DCM (10 volumes), triethyl amine (2.5 eq) was added and stirred for 10 min. Then, Boc anhydride (1.5 eq) and DMAP (0.1 eq) were slowly added and stirred for 2 h at room temperature. The reaction mixture was diluted with DCM and washed with water and brine solution. The organic layer was dried over sodium sulphate, filtered, and concentrated to get crude compound as a solid. The crude solid was purified by flash chromatography (0% to 15% ethyl acetate/n-hexane) to get di-tert-butyl-(6-chloro-3-(2,3-dichlorophenyl)pyrazin-2-yl)carbamate (4) as a white amorphous solid. MS m/z 374.1 (ES-, -100 PEAK).

Cell Viability Analysis
Viability was measured with MTT for cell-metabolism activity, with SRB and CVE for cellular protein content. The viable cells were seeded at a density of 2 × 10 4 (200 µL/well) in 96 well plates and incubated at 37 • C and 5% CO 2 for 24 h to form a cell monolayer. After 24 h of incubation, supernatant on the monolayer was aspirated and 200 µL of medium and varying concentrations of the natural substances were added and incubated for 24, 48, and 72 h time points.
After the specific time points, 20 µL of 5 mg/mL MTT Catalog #M2128 (Sigma-Aldrich, Darmstadt, Germany) in PBS Catalog #P3813 (Sigma-Aldrich, Darmstadt, Germany) was added to each well and incubated for 3 h at 37 • C and 5% CO 2 . Supernatants were discarded and 100 µL of DMSO Catalog #445103 (Carlo Erbo Reagents, Barcelona, Spain) was added and the plates were incubated for 5 min at 37 • C and 5% CO 2 to solubilize the formazan crystals. The absorbance was measured at 560 nm, and the reference wavelength was at 605 nm.
For the SRB assay, at the specific time points cells were fixed by adding 100 µL trichloroacetic acid Catalog #91228 (Sigma-Aldrich, Darmstadt, Germany) and with incubation at 4 • C for 1 h. Next, 100 µL of SRB solution Catalog #341738 (Sigma-Aldrich, Darmstadt, Germany) was added in the wells for 30 min at room temperature. After that, supernatant was discarded, cells were rinsed three times with 1% glacial acetic acid Catalog #1.00063.1011 (Merck, Darmstadt, Germany) and were left to air dry. Finally, the dye was solubilized with 200 µL of 10 mM Tris-base pH 10.5 Catalog #T6791 (Sigma-Aldrich, Darmstadt, Germany) and absorbance was measured at 570 nm and the reference wavelength was at 605 nm.
For the CVE assay, at the specific time points, supernatant was removed and cells were fixed with 100 µL 10% formalin for 20 min. Formalin was discarded and cells were left to air dry. Next, cells were dyed with 100 µL of 0.25% aqueous crystal violet solution Catalog #HT901 (Sigma-Aldrich, Darmstadt, Germany) and left for 10 min at room temperature. Then, the supernatant was discarded, and cells were rinsed twice with 100 µL WFI (water for injection) and were left to air dry. The dye was solubilized with 100 µL of 33% glacial acetic acid Catalog #1.00063.1011 (Merck, Darmstadt, Germany). Absorbance was measured at 570 nm and the reference wavelength was at 605 nm.

Apoptosis Assessment
Apoptosis detection was conducted with flow cytometry to determine the membrane phospholipid phosphatidylserine exposed apoptotic cells by Annexin V-PE and 7-AAD double staining. The PE Annexin V Apoptosis Detection Kit I Catalog #559763 (BD Biosciences, San Jose, CA, USA) was used for carrying out the experiments by following the manufacturer's instructions. Samples were analyzed on FACScan Calibur (Becton Dickinson, San Jose, CA, USA) and FSC 6 Express software.

Conclusions
In this work we established that, with the effectiveness of structure-based drug design, we were able to accomplish the novel 1-(methylsulfonyl)-4-prolylpiperazine group as a linker in pyrazine amine compounds. These structures have shown a receivable anti-cancerous activity.