The Pteridine Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Design, Synthesis And Anticancer Activity

The protein tyrosine kinases (PTKs) play crucial roles in many of the signal transduction pathways regulating lots of cellular functions, such as cell growth, proliferation and apoptosis. Over-expression of the PTKs is thought to be related to cell proliferation, invasion, metastasis, resistance to chemoradiotherapy and poor prognosis of various human cancers. Targeted interference with PTKs activation and/or with intracellular PTK-activated signal transduction pathways represents a promising strategy for the development of novel and selective anticancer therapies.Epidermal growth factor receptor tyrosine kinase (EGFR) is the first PTK to be examined seriously as a drug target. EGFR has its own ATP binding site in the intracellular domain. EGFR inhibitors can bind competitively to the ATP binding site and subsequently inhibit the phosphorylation of the EGFR as well as the downstream signaling pathways, then depress the growth, proliferation and metastasis of tumor. Two drugs, Gefitinib and Erlotinib, inhibiting the EGFR, have been launched for clinical use. Thus, EGFR has been identified as a promising target of tumor therapy.During the research for drugs targeting the EGFR, anilinoquinazolines were found to inhibit EGFR potently and selectively by binding to the ATP binding site reversibly. PD 0153035, a quinazoline derivative, played an important role in establishing the structure-activity relationship(SAR) and many of the quinazoline derivatives were designed on the basis of PD 0153035. However, the research found that the quinazoline ring is not necessary for inhibiting EGFR. Many of the aza quinazolines have showed potent activity, such as pyrido[2,3-d]pyrimidine, pyrido [3,2-d]pyrimidine, pyrido[4,3-d]pyrimidine, pyrido[3,4-d]pyrimidine, and pyrimido [5,4-d]pyrimidine. Moreover, the compounds that the benzene in quinazoline ring was substituted by five-member rings, such as pyrrolo[2,3-d]pyrimidine, pyrazolo[3,4-d]pyrimidine, can also inhibit the EGFR and show bright prospects.The studies stated above reminded us of the pteridine ring, as pteridine ring could be regarded as quinazoline ring that 5-C and 8-C are substituted by nitrogen atoms. Meanwhile the 6-substituted pteridines are the common forms in natural pteridine derivatives. In view of this, according to the SAR, we designed 2 series of pteridine derivatives : 6-alkoxy-4-arylaminopteridinesâ… and 6,7-dialkoxy-4- arylaminopteridinesâ…¢, using the bioisoster theory and PD 0153035 as lead compound, the structures of the compounds we designed are shown as follow:Pteridine-like compounds exhibit a wide range of physiological activity in human body and provide a novel template for new drugs, such as anticancer agents, adenosine kinase inhibitors and nitric oxide synthase inhibitors. At present, the study of pteridine derivatives is extensive abroad, but less in our country. Moreover, the preparation has seldom been reported for the alkoxy-substituted pteridines.As no related reports about the synthesis of the designed compounds, we by ourselves designed the synthetic routes by means of retro-synthetic analysis. Through modifying and justifying the routes repeatedly, we eventually found the practical routes.During the preparing of compoundsâ…¢, we discovered unexpectedly that the cyclization of N⁴-aryl-4,5,6-triaminopyrirnidine with oxalic acid is regioselective, which led to the discovery of a series of novel compounds: N⁸-aryl-6-alkoxy (alkylamino)-4-amino-7(8H)pteridinonesâ…¡(As shown above).The flexible molecular docking method was applied to investigate the binding of the designed compounds to EGFR. The binding modes and the interaction mechanism between the designed compounds and EGFR were understood. The results, which are helpful for new drugs design, showed the designed compounds can be able to dock into the ATP binding domain by hydrophobic and electrostatic interactions.Compoundsâ… started from 3-aminopyrazine-2-formic acid, from which 20 6-alkoxy-4-arylaminopteridine derivatives were obtained by esterification, amidation, bromination, cyclization, alkoxylation, chlorination and aromatic amination in a 7-step procedure with an overall yield of 39%. Compoundsâ…¡commenced from diethyl malonate, from which 13 new pteridine derivatives were prepared in an overall yield of 22% in a 9-step procedure including cyclization, nitration, chlorination and arylamine substitution, amination, reduction, cyclization with oxalic acid, chlorination, alkoxylation or alkylamination. For compoundsâ…¢, 3-aminopyrazine-2-formic acid was used as starting material, which underwent esterification, chlorination, alkoxylation, ester hydrolysis, lactonization, amidation and cyclization, alkoxylation, chlorination and aromatic amination to produce 5 new compounds in a total yield of 27% in a 9 steps procedure.The structure of all compounds had been confirmed by ¹H NMR, ¹³C NMR, ESI-MS and elemental analysis. More than 50 new intermediates were prepared through the experimental process, and elucidated from the corresponding spectra according to their structural characteristics.Two possible isomers may occur in the cyclization of N⁴-aryl-4,5,6-tri aminopyrimidine with oxalic acid. Their structural similarity makes it difficult to be confirmed by ¹H NMR, ¹³C NMR, ESI-MS and elemental analysis. A single crystal of its chlorinated compound revealed its structure, and the cyclization pattern and regioselectivity were understood. However, the regioselectivity disappeared when aliphatic amino group replaced the arylamino group. Thus, the method can only be used to the cyclization of N⁴-aryl-4,5,6-triaminopyrimidine with oxalic acid.The toxicity of the compoundsâ…¡against human tumor cell proliferation was evaluated using MTT assay. Three kinds of cancer cell lines, including human promyelocytic leukemia HL-60 cells, human gastric adenocarcinoma SGC-7901 cells and human colon cancer cells SW-480, were selected to test the target compoundsâ…¡a-â…¡m, using 5-FU as the contrast. The results suggested that the designed compounds II showed a certain inhibitory effect on SGC-7901 cells, some of them on HL-60 cells. At the similar concentration, the individual compounds showed equivalent inhibition against SGC-7901 and HL-60 cells to that of 5-FU. The designed compoundsâ…¡exhibited little effects on SW-480 cells.The pharmacological experiments of compoundsâ… andâ…¢are in progress.