Synthesis, and Biological Evaluations of Novel Pyrazinoic Acid Derivatives as Anticancer Agents

Pyrazinoic acid or pyrazine-2-carboxylic acid (PA), due to its nitrogenous heteroaromatic ring, can be explored as an anticancer agent. Here, a series of twenty novels PA derivatives have been synthesized and characterized using IR, NMR, and mass spectrums. Their cytotoxic activity was evaluated against three different cancer cell lines, including lung (A549), breast (MCF-7), and colon (HT-29). P16, the most potent compound, showed moderate cytotoxicity with IC 50 of 6.11 μM, 10.64 μM, and 14.92 μM, against the A549, MCF-7, and HT-29 cell lines, respectively. Furthermore, the effect of this compound against MRC5 as a non-tumoral lung cell line, exhibited a selectivity index of 9.02. The apoptotic induction activity of P16 was also performed on the A549 cell line. The results showed that as the concentration of the compound increases (from 3 to 6 μM), the percentage of induction of apoptosis increases from 8.54% to 72.4%. Electrophoretic gel mobility shift assays showed that P16 was also able to ROS induce DNA cleavage in the presents of H 2 O 2 (1.0 mM) in dose-dependent manner. Molecular docking was also applied to anticipate the binding locations and the binding of the synthesized compound with Bcl-2 apoptosis regulator and DNA as their proposed targets.


Introduction
The raising number of cancer patients throughout the world encourages medicinal chemists to seek novel anticancer agents.The global burden of cancer prevalence and death is steadily rising.In general, an estimated 28.4 million new cases of cancer are predicted in 2040, a 47 percent raise over the corresponding 19.3 million cases in 2020, assuming local rates remain stable in 2020 [1].DNA contains all genetic information, including cell replication and transcription [2].Thus, DNA is a signi cant target for anticancer therapies.Recently, studies have been directed at the interaction of chemicals with this target, causing DNA damage by stopping cancer cells from rapidly dividing.Small heterocyclic molecules interact with DNA through two grooves that run the length of the DNA double helix [3,4].The e ciency of DNA cleavage can be improved using promising coordination compounds, which usually include DNA.The disturbance in the apoptotic pathway has been identi ed as a characteristic of cancer.[5].Development of new anticancer agents for apoptosis reactivation methods, which can be used to treat a variety of cancers [6].Cell shrinkage, and nuclear DNA fragmentation are common biochemical markers of apoptosis [7].
The various nitrogen heterocyclic rings have recently exhibited extraordinarily complicated biological characteristics, making them one of the most in uential groups of medical chemistry molecules [8].Pyrazinoic acid or pyrazine-2carboxylic acid (PA) can be explored as an anticancer agent due to its heterocyclic pyrazine ring.Its derivatives have a wide range of biological activities, such as anti-tuberculosis [9], hepatitis C [10], antidepressant [11], antitumor [12], antifungal-antitumor [13], Antiviral [14].
The structure modi cation of PA may be utilized to convert its antibacterial to anticancer activity.Since then, derivatives of PA have been recognized as an alteration of DNA methylation and apoptosis.Recently, PA was used to treat colorectal cancer (CRC).Treatment with PA ameliorated the metabolic changes caused by CRC, indicating that it has an acceptable anti-proliferative effect [15].Bortezomib is a combination of PA, phenylalanine, and boronic acid that has been approved by the FDA for the treatment of multiple myeloma.One of the most important pathways of bortezomib associated with antitumor e cacy is a proteasome inhibitor that could improve signal transduction and apoptosis [16].
In this study, we designed and synthesized novel PA derivatives utilizing the Passerini multicomponent reaction.The compounds were identi ed by Fourier transform infrared spectroscopy (FTIR) and Nuclear Magnetic Resonance ( 1 H-NMR, 13 C-NMR), and Mass spectrometry (MS), as well as physical properties determination such as appearance, and melting point.Furthermore, their cytotoxicity was evaluated against three cancer cell lines, A549 (lung cancer), MCF7 (breast cancer), and HT29 (colon cancer), as well as non-tumorigenic lung cell line (MCR5).To experimentally evaluate the DNA-binding activity of the compound, the electrophoretic mobility shift assay was performed.The effects of the most cytotoxic compounds (P5 and P16) on the induction of apoptosis in the A549 cell line were also investigated.Using molecular docking, the binding energy of the synthesized compounds, as well as their types of interactions had been studied with Bcl-2 apoptosis regulator and DNA as their proposed targets.

Chemistry
The general synthetic strategy employed to synthesized the PA derivatives was based on the passerine multicomponent reaction [17] under normal state, using isocyanide, different aldehyde derivatives, and PA (Scheme 1).To purify the product, the recrystallization process, using a combination of methanol and water, or plate chromatography using 230-400 mesh silica gel, and a solution of 50% ethyl acetate and 50% n-hexane, was applied.The obtained compounds were characterized using (IR, 1 HNMR, 13 CNMR, and Mass) spectrums.The existence of a chloro pattern (1:2) was also convincingly demonstrated by mass spectrometry (MS).

In vitro cytotoxic activity
In this study, novel PA derivatives were designed, synthesized and their cytotoxic activity against three human cancer cell lines such asA549 (lung), MCF7 (breast), and HT-29 (colon) was evaluated.To demonstrate their selectivity among normal and cancerous cell lines, the cytotoxicity of these compounds was also assessed against a normal lung cell line (MRC5).Almost all of the synthesized compounds had shown higher anti-proliferative activity than PA.Of twenty synthesized compounds, P16 has shown promising anticancer activity with IC 50 of 6.11 µM, 10.64 µM, and 14.92 µM against the A549, MCF-7, and HT-29 cell lines, respectively.Also, P5 showed moderate cytotoxicity with IC 50 of 14.09 µM, 8.90 µM, and 16.38 µM, against A549, MCF7-7, and HT-29 cell lines, respectively.P5 exhibited higher cytotoxicity than P16 against the MCF-7 cell line (Table 1).One-way ANOVA statistical analysis showed that the differences between the IC 50 of cisplatin, and P16 are statistically signi cant in all the studied cell lines.Thus, P16 showed higher anti-proliferative activity than cisplatin in all the studied cell lines.On the other hand, P5 was also shown higher cytotoxicity in comparison to cisplatin in MCF-7, and HT-29 cell lines.
MRC5, a normal epithelial lung cell line, is utilized to evaluate the toxicity of the synthesized compounds on normal cells and determine the selectivity index between cancerous and normal cells.All the synthesized compounds, particularly P1, P2, P5, P6, P16, and P17 with a selectivity index of 2.23, 1.62, 5.18, 2.14, 9.02, and 2.35, respectively, demonstrated higher selectivity for human lung cancer cells while causing less harm to normal epithelial lung cells.
Obviously, this series of compounds showed a better selectivity index and therapeutic window than cisplatin.

Structure-activity relationship
The activity of the synthesized PA derivatives illustrates that substituted by both electron-withdrawing groups like (F, Cl, Br, and NO 2 ) and electron-donating groups like (OCH 3 , Me) in the B-ring of the structure have a promising impact on the cytotoxic activity against all the studied cancer cell lines.For example, P16, which has a chloro group in the B ring, has higher cytotoxic activity on the A549, MCF-7, and HT-29 cell lines than the other halo groups.However, the movement of the chloro group in ring B from para to meta, and ortho reduced the inhibitory effect on the studied cancer cell lines.The presence of one Cl group on the B ring showed good to moderate cytotoxic activity (IC 50 < 40 M in MCF-7 and HT-29 cell lines).Besides, by inserting the chloro group in positions 2 and 4, such as P17, a suitable compound in terms of in vitro cytotoxicity activity is produced, although weaker than P16.In all the studied cancer cell lines, substituting withdrawing groups like halogens in the B-ring in the order of Cl > Br > F, can affect the antitumor activity of compounds.P10, with NO 2 group in 2, and 4 positions, showed weak anticancer activity.Also, the placement of other electron-withdrawing groups such as nitro did not show effective activity on the studied cancer cell lines.On the other hand, when an electron donor groups such as methoxy (OCH 3 ), is embedded in the B ring as illustrated in the (P1-P6) compounds, it shows signi cant cytotoxic activity against studied cancer cell lines.
As evident in P5, which encompasses OCH 3 in 2 and 5 positions, the highest antitumor activity is observed in the MCF-7 cell line.The P4 with the OCH 3 group in 3 and 4 positions in the B ring has the most potent anticancer activity against the HT-29 cell line.

apoptosis assay using ow cytometry
Here, we applied the AnnexinV/PI kit to evaluate the effect of P5, and P16, as the most potent antitumor compounds, on the apoptosis induction against A549 cell lines.As shown in Fig. 1, with the rise in the concentration of P5 from 7.0 to 14.0 µM, the percentage of cells in the apoptotic process increases signi cantly from 0.6 percent in untreated cells to 4.27%, and 42.7% in treated cells.On the other hand, p16 at concentrations of 3 µM, and 6 µM was used to induce apoptosis and the percentage of apoptotic cells is signi cantly increased from 8.28% at 3 µM to 72.4% at 6 µM.Although both compounds cause apoptotic cell death, P16 is more potent than P5.These ndings showed that, P16, and P5 could induce programmed cell death in a concentration-dependent manner in A549.
Hence, parts of their cytotoxic effects go through apoptosis induction.

Gel electrophoresis study
Gel electrophoresis has been carried out to monitor the cleavage of DNA by selective synthesized compounds.The experiment followed a technique that had already been reported in the literature [18].It's well de ned that there has been large accumulation of H 2 O 2 in several tumor tissues [19] and it was reported earlier that some complexes like Cu, and Pt containing compounds [20] induced DNA cleavage activity in the presence of reducing agents like H 2 O 2 , we also explored the possible nuclease activity of P5, and P16 in the presence of H 2 O 2 .
As shown in Fig. 2A, B, P5 was failed to induce DNA cleavage independently.It was unable to ROS induce DNA cleavage in the presents of H 2 O 2 (1.0 mM) and it was unable to displace with safe stain in DNA supercoil.On the other hand, Fig. 2C, D shows that, P16 was unable to induce DNA cleavage independently.However, it was able to ROS induce DNA cleavage in the presents of H 2 O 2 (1.0 mM) in dose dependent manner (Fig. 2D).It should be noted that P16 was unable to displace with safe stain in DNA supercoil.

Molecular docking studies
Recently, pyrazine derivatives have been studied due to their heteroaromatic nature in medicinal chemistry.The interaction of pyrazine was evaluated using different targets.The results revealed that the nitrogen atoms of pyrazine have the prominent roles in the binding primarily through acceptor hydrogen bonding [21].In this study, all the synthesized compounds were docked conducted to molecular docking studies to understand their interactions and acquire further information on their molecular binding mechanism with Bcl-2 apoptosis regulator (PDB ID: 6QGK) and DNA (PDB ID: 1LEX).
The interactions of the synthesized PA derivatives with Bcl-2 were explored, and it was found that the most signi cant amino acids in binding pyrazine to Bcl-2 were Phe104, Ala149, Met115, and Phe112 (Fig. 3).The main interaction site of pyrazine was through arene-H, donor hydrogen bonding, and hydrophobic interactions with Phe104, Met115, and Ala149, respectively.For example, P10 interacts with Bcl-2 through three different interactions, including hydrophobic interactions, donor hydrogen bonding, and π-Stacking.As seen in Fig. 3A, the pyrazine ring in P10 via hydrophobic interaction bind to the Leu137 and Ala149, as well as binds to Phe104 through π-Stacking.
There exists a hydrogen bonding between the nitrogen of amid group and Asp111.A hydrophobic interaction between the cyclohexane ring and Met115 is also observed.There is a hydrogen bonding between the nitrogen of amid group and Asp111, and a hydrophobic interaction between cyclohexane and Met115.According to Fig. 3A, π-Stacking binding has also existed between pyrazine with Phe112, and benzene with Phe104.The hydrophobic interactions between cyclohexane and Phe153, Ala149, and Val133 were also observed.
According to Fig. 3B, P16 has shown hydrophobic interactions between its cyclohexane ring and Phe153, Ala149, and Val133.There are also existed two stacking binding between pyrazine with Phe112, and benzene with Phe104.
Furthermore, the molecular binding of the synthesized compounds with DNA was also studied.Thymine and cytosine base was found to have the highest interaction in binding with PA derivatives.Both P10, and P16 compounds are located in the minor groove of DNA.There exists a hydrogen bonding between the carbonyl of amide group and T8.Also, pyrazine has a donor hydrogen bonding with the C9 base and the nitro group has a hydrophobic interaction with C6 (Fig. 3C).The main interaction of P16 with DNA was also explored (Fig. 3D).It interacts through the nitrogen on the pyrazine ring with the T7 base via a donor hydrogen bonding and a carbonyl ester group via an acceptor hydrogen bonding with the same thymine base (T7).Also, this group has a hydrophobic interaction with another T8 base.There is also a donor hydrogen bonding between the nitrogen of amide group and the C9 base (Fig. 3D).
Apart from experimental tests, the validation of the docking protocol was also explored using RMSD.According to Fig. 4, the RMSD of the co-crystal ligand has been reported as 0.95 Å, and 0.79 Å with Bcl-2 apoptosis regulator, and DNA, respectively.The ΔGbind values for Bcl-2, ranged from − 7.59 to-6.02kcal/mol, whereas the ΔGbind values for DNA ranged from − 9.65 to 4.43 kcal/mol (Table 2).
The docking binding energies of all the synthesized compounds on Bcl-2, and DNA receptors as well as some ADME descriptors, especially drug-likeness rules (Lipinski's rule), which were calculated by DruLiTo are shown in Table 2.
The Lipinsky rule of ve (RO5) descriptors such as Molecular Weight (MW), logP, AlogP, H-bond Acceptor (HBA), Hbond Donor (HBD), Total Polar Surface Area (TPSA), Active Metabolic Rate (AMR), and the number of Rotatable Bonds (nRB), intrinsic solubility (log S), and gastrointestinal absorption (GA) were calculated for all the synthesized compounds.All compounds pass the drug-likeness lter, which is based on Lipinski's rule of ve (Table 2).

Conclusion
In general, PA derivatives were successfully synthesized using the Passerini multicomponent reaction.The compounds were entirely characterized by (IR, 1 HNMR, 13 CNMR, Mass) spectrums.These PA derivatives were evaluated for cytotoxic activity against three different cell lines, including lung (A549), breast (MCF-7), and colon (HT-29) and one normal human lung cell line (MRC5).The ndings of this study indicated satisfying selectivity of the compounds between the cancerous cell and non-cancerous cell line (MRC5).P5, and P16 have high e cacy in suppressing cell growth and considerably more potent in vitro cytotoxic activity than cisplatin and the others.Electrophoresis mobility shift assay revealed that P16 was able to ROS induce DNA cleavage in the presents of H 2 O 2 (1.0 mM) in dose-dependent manner.P5, and P16 can signi cantly cause apoptosis in A549 cells via a dosedependent manner.The molecular docking studies of the synthesized compounds on Bcl-2 apoptosis regulator and DNA were also conducted and their interactions and further information on their molecular binding mechanism were acquired.These compounds (especially P5, and P16) have the ability to play an essential role in the future treatment of cancer diseases after further clinical studies.

General
All chemical reagents and solvents were acquired from Merck Chemical or Sigma Aldrich.The passerine multicomponent reaction was used to synthesize the novel PA derivatives.To purify the product, the recrystallization process, which uses a combination of methanol and water, or chromatography plate, using 230-400 mesh silica gel and solution of 50% ethyl acetate and 50% n-hexane, was applied.The advance of the reaction was monitored by TLC.It was conducted on silica gel polygrams SIL G/UV 254 plates.The analysis of the compound was recorded by IR spectra were run on a (Bruker's VERTEX 70 Series FTIR Spectrometers) and the 1 H-NMR and 13 C-NMR spectra were run on a Bruker Avance DPX 300, 500 FT-NMR spectrometers.Chemical shifts were reported in parts per million (ppm), down led from tetramethylsilane coupling constant (J) values in Hz, and spin multiplicities were denoted by the letters s (singlet), d (doublet), t (triplet), and m (multiple) (multiplet).Mass spectrum recorded by Manufacturer Company Agilent Technology -(HP)-MS Model: 5973).

Synthesis of Pyrazinoic acid analogs
Firstly, 1 mmol of pyrazinoic acid was dissolved in 5 ml of water by heating.Then 1 mmol of aldehyde derivatives and 1.2 mmol isocyanide were added to the reaction mixture, and stirred at room temperature for 4 to 48 hours.The solid obtained was washed with water and ltered and dried in a desiccator.For puri cation, recrystallization (methanol and water) or plate chromatography (silica gel with 50% ethyl acetate and 50% n-hexane) was applied.
Below are the physical and spectral data of the synthesized compounds:

Apoptosis assay
The apoptotic activity of compounds P5, and P16 was further investigated utilizing the eBioscience TM Annexin V apoptosis detection kit and AnnexinV/PI labeling (Invitrogen) [22].The A549 cell lines are pre-cultured for 16 hours before being exposed to compounds over the next 24 hours.In sum, 0.5×10 The apoptosis rates were then determined by BD FACS CaliburTM ow cytometry (BD Biosciences, San Jose, CA, USA).This assay can show apoptosis rates were calculated as the sum of early apoptosis and late apoptosis.

Gel electrophoresis
Gel electrophoresis analysis was performed using 500 ng plasmid, pBlu2KSM, DNA in 10 mM Tris-HCl buffer, pH 7.4 with the nal concentrations of 20-150 µM complex in a total volume of 10 µl.The samples were then incubated for 60 min at 37°C, followed by electrophoresis on 1% agarose gel in TAE buffer containing 1.0 µg ml − 1 safe stain at 100 V for 30 min and nally, photographed under UV light.To investigate the DNA-ligand ability of each compound to unwind the supercoiled conformation of pBlu2KSM was determined by electrophoretic mobility on the native plasmid.As expected, three different pBlu2KSM conformations were observed on the agarose gels.These were the negative open circular form (NSC) as well as single break nicked supercoil (SSNSC) and a strong bond of the positive twisted supercoiled (PSC) [23].The Double-strand break of the circular supercoil transform circular DNA to Linear (L) format with natural length of the plasmid is shown as these conformations resolve on agarose gels as distinct bands, as shown in Fig. 2.

Molecular docking
The molecular docking study of the synthesis compounds was carried out using conventional methodology and techniques, as detailed in the report [24].The crystal arrangements of three-dimensional crystals Bcl-2 (PDB ID: 6qgk), and DNA (PDB ID: 1LEX) is downloaded from the protein data bank (www.rcsb.org/pdb).The protein was prepared by removing water molecules and ligands that were not implicated in the active site and co-crystal ligands

Table 1
In vitro cytotoxic activity of the synthesized compounds against three cancerous cell lines: A549, MCF-7, and HT-29, as well as a normal lung cell line (MRC5).

Table 2
The docking binding energies as well as some ADME descriptors of all the synthesized compounds.
Molecular Weight (MW), H-Bond Acceptor (HBA), H-Bond Donor (HBD), Total Polar Surface Area (TPSA), Active Metabolic Rate (AMR), number of Rotatable Bonds (nRB), intrinsic solubility (log S), and gastrointestinal absorption (GA) Hz, 3H)H3',H 2' ), in 96-well microplates with a density of 0.8 × 10 4 cells per well and kept for 24h to recover.The cells were then treated in a triplicate manner with compounds (P1-P20) at varying concentrations ranging from 1 to 100 µM and incubated in a humidi ed CO 2 incubator for at least 72 hours at 37°C.The media was discarded entirely after incubation and replaced with 150 µl of RPMI 1640 containing 0.5 mg/mL MTT solution and incubated for 3h at room temperature.The media containing MTT was discarded again to dissolve the formazan crystals, and 150 µl of DMSO was applied to each well and incubated in the dark for at least 30 min at 37 ° C. The absorbance of the individual well was then read by ELISA reader at 490 nm.CurveExpert 1.4 was used to measure the 50% inhibitory concentration of each compound, re ecting IC 50 .The data are given as mean ± SD.