SYNTHESIS OF AMINOPYRAZINE AND PYRAZINAMIDE DERIVATIVES AS TYROSINE KINASE INHIBITORS

• Main Author: Agustina E S, Widiastuti
• Format: Dissertations
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/72852
• Institution: Institut Teknologi Bandung

Several pyrazine derivatives have been reported as tyrosine kinase inhibitors (TKIs), one of the target proteins in cancer treatment. These pyrazine derivatives contain amine groups (aminopyrazine) and amides groups (pyrazinamide) which are reported to be active as TKIs against several families of kinases, namely IGF1R (insulin-like growth factor-1 receptor), ACK1 (activated Cdc42-associated kinase 1), JAK3 (janus kinase 3) and SYK (spleen tyrosine kinase). TKIs as an anticancer have advantages over cytotoxic drugs because they work more specifically on protein molecule targets that play a role in cancer cell proliferation, so they cause fewer side effects on patients. However, the study about this is still relatively limited. In this research, various aminopyrazine and pyrazinamide derivatives have been synthesized and their biological activity as TKIs also have been evaluated, against receptor tyrosine kinases (RTKs) and non-receptor tyrosine kinases (NRTKs). The syntheses of aminopyrazine and pyrazinamide were carried out using the starting materials 3-hydroxypyrazine-2-carboxamide (1) and 3-aminopyrazine-2- carboxylic acid (2). The syntheses were carried out by chemical transformations at the C-6 position in pyrazine 1, and transformations of the carboxylic acid group and amine group in pyrazine 2. The molecular structures of the synthesized compounds were determined using 1D and 2D NMR spectroscopy and high- resolution mass spectrum data, ESI-TOF (electrospray ionization-time of flight). The compounds were then tested in vitro for their biological activity as TKIs against eleven RTKs, including EGFR (epidermal growth factor receptor), HER2 (human epidermal growth factor receptor 2), HER4 (human epidermal growth factor receptor 4), IGF1R (insulin-like growth factor-1 receptor), InsR (insulin receptor), KDR (kinase domain receptor), PDGFR? (platelet-derived growth factor receptor alpha), PDGFR? (platelet-derived growth factor receptor beta), AXL1 (anexelekto receptor tyrosine kinase 1), EPHA1 (erythropoietin-producing human hepatocellular type-A receptor 1) and TRKA (tyrosine kinase receptor A), as well as against five NRTKs, including FAK (focal adhesion kinase), ITK (interleukin-2- inducible T-cell kinase), JAK3 (janus kinase 3), PYK2 (protein tyrosine kinase 2 beta) and SYK (spleen tyrosine kinase). The tests were carried out at 10 µM with the bioluminescence method. Molecular docking studies were carried out using AutoDock Vina. The syntheses of pyrazinamide between carboxylic groups attached to the pyrazine ring and various primary amines (aliphatic and aromatic) and between amine groups attached to the pyrazine ring and benzoyl chloride were developed in this study. CDI was used as a coupling agent in the one-pot syntheses. The use of CDI in the synthesis of pyrazinamide from the amine group attached to the pyrazine ring with benzoyl chloride leads to a significant synthesis yield. Nineteen aminopyrazine and pyrazinamide derivatives have been synthesized in this study. Two compounds were synthesized from pyrazine 1, namely 3-hydroxy-6- nitropyrazine-2-carboxamide (3) and 3-hydroxy-6-fluoropyrazine-2-carboxamide (4). In addition, seventeen compounds were synthesized from pyrazine 2, namely methyl-3-aminopyrazine-2-carboxylate (5), 3-amino-N-methylpyrazine-2- carboxamide (6), methyl 3-amino-6-bromopyrazine-2-carboxylate (7), methyl 3- benzamidopyrazine-2-carboxylate (8), 3-amino-N-butylpyrazine-2-carboxamide (9), 3-amino-N-isobutylpyrazine-2-carboxamide (10), 3-amino -N-phenylpyrazine- 2-carboxamide (11), 3-amino-N-(4-methoxyphenyl)pyrazine-2-carboxamide (12), 3-amino-N-(4-bromo-2-methoxyphenyl)pyrazine-2 -carboxamide (13), 3-amino-N- (3-aminophenyl)pyrazine-2-carboxamide (14), 3-amino-N-(3- chlorophenyl)pyrazine-2-carboxamide (15), 3-amino-N-benzylpyrazine-2- carboxamide (16), 3-amino-N-(4-methylbenzyl)pyrazine-2-carboxamide (17), 3- amino-N-(2-methylbenzyl)pyrazine-2-carboxamide (18), 3-amino-N-(4- methoxybenzyl)pyrazine-2-carboxamide (19), N,N'-(hexane-1,6-diyl)bis(3- aminopyrazine-2-carboxamide) (20), and 3-benzamido-N-phenylprazine-2- carboxamide (21). Pyrazinamide 13 - 15, bipyrazine 20 and benzamide phenylpyrazine 21 are new compounds that have never been isolated from natural sources or synthesized before. The results of the biological activity test showed that pyrazinamide 11 and 15 had the best inhibitory activity. Pyrazinamide 11 inhibited 41% of AXL1 activity and 34% of TRKA activity, while pyrazinamide 15 inhibited 35% of EGFR activity. Pyrazines 1 and 2, nitropyrazine 3, fluoropyrazine 4, benzamide esterpyrazine 8, and benzamide phenylpyrazine 21 showed monotarget inhibitory activity. Pyrazinamide 11, 12, 14, 15, 17, and bipyrazine 20 exhibit multitarget inhibitory activity. Meanwhile, pyrazinamides 13, 16, 17, 18, and 19 are very weak or inactive as tyrosine kinase inhibitors. The relationship between structure and activity shows that pyrazine compounds with aromatic chains on the amide group provide better inhibitory activity (5–41%) than pyrazine compounds with aliphatic chains (1– 16%). In aromatic-substituted pyrazinamides, a phenyl ring has higher inhibitory activity (35–14%) than a benzyl ring (14%). Molecular docking studies on pyrazinamide 11 and pyrazinamide 15 show that the interaction of the compounds (ligands) with tyrosine kinases occurs through hydrogen bonds and hydrophobic interactions. The oxygen atom of the amide group at the C-2 position plays a role in the formation of hydrogen bonds with the amino acid residues of AXL1, TRKA and EGFR. Other hydrogen bonds are also shown by amine (AXL1), amide (TRKA), and nitrogen heterocyclic (EGFR). Meanwhile, the hydrophobic interaction is shown by the aromatic hydrocarbons from the amide group at the C-2 position. Pyrazinamide 11 shows hydrogen bonding with Lys619 of AXL1. Pyrazinamide 11 shows hydrophobic interactions with Phe589 and Tyr591 of TRKA. Pyrazinamide 15 exhibits hydrogen bonding with Thr766 and hydrophobic interactions with Met769 of the EGFR. These interactions indicate that the position of the ligand is in the catalytic site of the kinase domain. Pyrazinamide 11 and 15 can be further developed by molecular docking approach to obtain compounds with higher ligand-enzyme affinity. The compounds that were developed were 3-((6,7-dimethoxyquinazolin-4-yl)amino-N-phenylpyrazine-2- carboxamide) (22), N-(3-chlorophenyl)-3-((6,7-dimethoxyquinazoline-4- yl)amino)pyrazine-2-carboxamide (23), 3-cinamido-N-phenylpyrazine-2- carboxamide (24) and N-(3-chlorophenyl)-3- cinamidopyrazine-2-carboxamide (25). Based on the results obtained, it can be concluded that 3-hydroxypyrazine-2- carboxamide (1) and 3-aminopyrazine-2-carboxylic acid (2) can be chemically transformed into nineteen aminopyrazine and pyrazinamide derivatives (3 – 21). Pyrazinamide 11 provided the best inhibition activity against AXL1 and TRKA, while pyrazinamide 15 provided the best inhibition activity against EGFR. Both compounds contain an aromatic amide group at the C-2 position and an amine group at the C-3 position. The results of this study make a scientific contribution in several aspects. Syntheses methods, especially for novel compounds, including the obtained syntheses yields, can be used as a reference in providing access to aminopyrazine and pyrazinamide derivatives. Furthermore, information on the activity of aminopyrazine and pyrazinamide derivatives as tyrosine kinase inhibitors, as well as the results of molecular docking analysis, also contributed to the development of active compounds as new anticancer candidates.