Design And Synthesis Of Novel VEGF Receptor Inhibitors

Malignant tumor is currently the major disease threatening human's health and life. So anti-cancer is then taken as an important task by WHO and Health Department of countries. Most of the current anti-cancer drugs in clinic are cytotoxic which bears properties of nonselective, severer side-effect and drug resistance. Therefore, new targets with high efficiency and low toxicity is the urgent requirement for developing anti-cancer drugs. Recently, many targets based on mechanics of cancer developing are identified, such as: protein tyrosine kinase (PTK) involving cellular signal transduction, protein kinase C (PKC), farnesyl transferase (Ftase), cyclin-dependent protein kinases (CDK), Bcl-2 protein, vascular endothelial growth factor (VEGF), matrix metallo-proteinase (MMP), etc. Discovering novel potent anti-cancer drugs based on these new targets are main task in medicinal chemistry.Angiogenesis refers to the formation of new capillaries sprouting or splitting from pre-existing vessels. physiological angiogenesis in the adult is associated with the maintenance of the vasculature, female reproductive cycling, and wound healing. Pathological angiogenesis is upregulated in a wide range of diseases such as retinopathies, arthritis, endometriosis and cancer. Starting from Folkman's studies in the early 70s, it has been demonstrated that the growth of primary tumors and their subsequent metastasis are angiogenesis-dependent processes. Today, there are 10 anti-angiogenesis agents as anti-cancer drugs approved by US FDA and more than 1.2 million people taking anti-angiogenesis therapy.VEGF receptors are critical targets of anti-angiogenesis. In the recent years, various kind of small molecule inhibitors targeting VEGFR have been reported such as 2-indolinones, Quinazolines and Phthalazines. Some inhibitors are under clinical trials value and show promising prospect. Otherwise, some shortages still remain. For example, as a ATP competitor, it's IC50 should be below nanomolar level to show efficacy. On the other hand, VEGFR belongs to receptor tyrosine kinase super family, of which members play broadly roles in signal transduction. Their ATP binding site is highly conserved, so selectivity of inhibitors among those RTKs are also important. With development of computer-aided drug design technology, elucidating of structure and function of VEGFR and binding mode of receptor and inhibitor, discovering new VEGFR inhibitors is turning to rational drug design from random screen.Our aim is to design and synthesis a novel VEGFR inhibitor lead compound based on exploring properties of VEGFR active site and identifying interaction model between VEGFR and inhibitors by using CADD methods. For this sake, we have achieved following works:1. Study of properties of VEGFR active siteThe properties of active site and critical residues involving ligands binding is the base of rational drug design. Unfortunately, there is no reports about comprehensive study of active site of VEGFR-2 yet. So, we identified the shape and size of the active site of VEGFR-2 using Binding Site Analysis method. Then Multiple Copy Simultaneous Search (MCSS) method was applied to study the properties of the active site. The results show that the active site can be divided into three hydrophobic sections and one polar section by the characters bound with ligand: Sectionâ… is a hydrophobic pocket formed by the hydrophobic side chain of Val846, Ala864, Val897, Val914, Phe916, Leu1033; Section II is a relatively larger hydrophobic lumen formed by the hydrophobic side chain of Ile886, Leu887, Ile890, Val896, Val897, Leu1017, Ile1042 and Phe1047. A ring-like polar section formed by the side chain of Lys866, Glu883 and the carbonyl oxygen on the skeleton of Asp1044 between Sectionâ… andâ…¡, is very important to ligand binding; Sectionâ…¢lies at the active site open to solvent, and is a small hydrophobic area formed by the side chains of Leu838 and Phe916; Sectionâ…£is a polar section formed by the hydrophobic residues of Asn921, Leul033, Cys1043 and hydrophilic residues of Arg1030, Asn1031.In order to study the differences of conformation of active site between before and after ligand's binding, we compared free state crystal structure with 8 published complex crystal structures of VEGFR-2. These 9 structures were superimposed by Cαof peptide around the active site while RMSD values were calculated and the differences of conformation were examined. The results show that the skeleton of peptide and side chains of residues in sectionâ… ,â…¢andâ…£are comparatively similar but the conformation of DFG motif at the beginning of the activation loop in section II is changed greatly. These changes result in different shape and size of section II. It implicates that the flexibility of section II must be considered when carrying out molecular docking.2. Molding of binding mode of VEGFR-2 and inhibitorsStudying of various binding mode of different inhibitors and the receptor can provide much useful information for understanding the mechanism of the interaction between ligand and receptor. So various kind of inhibitors were docked into the active site of VEGFR-2 by flexible docking method and the critical residues involving ligand's binding were identified. In section I, there are two critical action sites for hydrogen bonding: C=O on Glu917 skeleton as hydrogen bond receptor; N-H of Cys919 skeleton as hydrogen bond donor which are used by most inhibitors. The side chain of Lys868, Glu885 and the carbonyl oxygen on the skeleton of Asp1046 between Sectionâ… andâ…¡can provide additional action site for ligand binding to improve affinity. Hydrophobic pocketâ…¢and polar areaâ…£can also contribute to improve affinity. N-H on the side chain of Asn923 in sectionâ…£is the critical hydrogen bond donor site, too.3. de novo design of VEGFR-2 inhibitorBase on above work, small molecular segments from reported inhibitors were put into the active site valued their affinity by MCSS. The favorable segments in each section of the active site were linked by LUDI program to construct new ligand molecues. Several scoring functions such as empirical binding free energy function, CScore, were applying to value the affinity of these constructed molecules. Human's knowledge of organic synthesis was also introduced in to value the possibility of synthesis. At last, we picked out two kinds of molecules 3,4-dihydroisoquinolines and 1-isoquinolinones as our target molecules to synthesis.4. Synthesis and inhibition test of novel VEGFR-2 inhibitorsThe general synthesis method of 3,4-dihydroisoquinolines is: 3,4,5-trimethoxy-benzaldehyde and nitromethane condensed to give 1,2,3-trimethoxy- 5-(2-nitrovinyl) benzene, then treated by LiAlH4 to give 3,4,5-trimethoxy- phenylethanamine, then treated by various substituted benzoyl chloride to give amide, then cyclized via Bischler-Napieralski reaction to give target productions. The productions can further dehydroed by Pd/C or reduced by NaBH₄ to give corresponding isoquinoline or tetrahydroisoquinoline.The general synthesis method of 1-isoquinoliones is: Homophthalic acid and aqua ammonia mixed to give ammonium, then dehydrolysised in high temperature to give homophtalimide, then condensed with various Benaldehyde via Knoevenagel reaction, then reduced by NaBH₄, hydrolysised by dilute HC1 solution to give target productions . 24 products have been synthesized and 22 among them are not reported in literature. The target products show anit-angiogenesis activity by HUVEC assay and CAM method. Compound 6 shows IC50 of inhibition to HUVEC up to 3.14μmol/L and compound 10 inhibits angiogenesis of CAM greatly at the concentration of 1μg/ml.