Synthesis And Pharmacological Activity Of Protein Tyrosine Kinase Inhibitor AW1

Reports from WHO indicate that the number of people suffering from malignant tumor is getting larger along with the situation tougher and tougher. Tumor incidence is estimated to increase by 50% by the year 2020 with number of newly increased cancer sufferers rising from 10,000,000 to 15,000,000 per year. Epidemiologic survey in China revealed that cancer mortality was higher than that of any other disease causing death, both in country and city,104.01/10,000 for the country and 94.71/10,000for city. All data indicated that tumor therapy had become a global project to promote human health and raise life quality and level.Drug therapy has developed quickly in recent years for tumor suffers and already played a critical role in the area of chemotherapy. Regular chemical drugs used in chemotherapy including alkylating agents, antimetabolites, antitumor antibiotics, hormones, etc. did ease cancer syndrome, decrease death rate, enhance patients' quality life and prolong life-expectancy. However, as a result of the popular and frequent use of them, traditional anti-tumor drugs inevitably show disadvantages: low-selectivity, intensive toxicity, and drug resistance.Along with the rapid progress on life science research, human beings gained rich knowledge about malignant tumor, especially cellular signal transduction, regulation of cell cycle, induction of cell apoptosis, angiogenesis and basic interaction mechanism intra and outra cellularlly. As a result, targeting therapy advocated in recent years by targeting specific molecule within correlative signal channel in tumors, is a new therapy method and proved available.1. Protein tyrosine kianseProtein tyrosine kianse are enzymes that catalyze the transfer of the yphosphate group from adenosine triphosphate to target proteins. They play an important role in diverse normal cellular regulatory processes. Disturbance of PTK may induce a series disease. It's reported that over 50% of oncogene and oncogenic product display PTK activity. Dysfunction of them will cause disorders during cell proliferation, thus tumor occurs. People all over the world invest a lot of money on the research and development of PTK. Tyrosine kinases can be classified as receptor protein kinases (RPK) and nonreceptor protein kinases (NRPK) according to whether they locate on cell membrane or not.1) nonreceptor protein tyrosine kinasesNonreceptor protein tyrosine kinases can be ascribed to 11 families:SRC, ABL, JAK, FAK, etc. NRPK plays an important role in cell signal transduction and correlates with a lot of human cancers.1.1 SRCWithin SRC members include Fgr,Fyn,Sre,Yes,BIK,Hck,Lck,Lyn. Overexpression of SRC protein indicates tumor diseases such as breast carcinoma, liver cancer and colon cancer. SRC play an important role in proliferation, survival, adhesion and metastasis by showing oncogene activity and coaccomandate growth of tumor cells. High similarity is observed within Sre family, including acylating position in amino terminal needed for membrane allocation, SH2 domain, SH3 domain, catalyting domain and inhibitory position Tyr529 in carboxyl domain.1.2 ABLThere are 2 members in ABL family:Abl and Arg. Abl protein structure includes SH3,SH2,PTK,DNA binding domain, actin binding domain, etc, exerting cell inhibitory activity.1.3 JAKJAK is highly correlated with several kind of human leukemia and plays a critical role in initial process of cellular signal transduction.4 members are included in JAK: TYK2, JAK1T, JAK2, JAK3.TYK2, JAK1T, JAK2 are expressed commonly while JAK3 is limitedly expressed in hematopoietic cell.7 conserved sequence exit in amino sequence of JAK.JH1,JH2 domain in carboxyl terminal show kinase activity. No homogeneity is observed in domain JH7-JH3 in amino terminal compared with other NRPK families members.1.4 FAKFAK family includes FAK, Pyk2, CAKβ, RAFTK, cadTK, FAK2. Different cancer express different amount of FAK. FAK is expressed more during the process of prostatic cancer, breast cancer, colon cancer, ovarian cancer, oral carcinoma and thyroid cancer. FAK is a special cytoplasm kinase, without SH2 and SH3.2) Receptor protein tyrosine kinasesReceptor tyrosine kinases are membrane-spanning cell surface proteins that play critical roles in the transduction of extracellular signals to the cytoplasm. They are characterized by immunoglobulin-like sequences in their amino-terminal extracellular domains, alipophilic transmembrane segment, and an intracellular carboxyl-terminal domain that includes the tyrosine kinase catalytic site. Ligand binding induces dimerization of these receptor tyrosine kinases, resulting in autophosphorylation of their cytoplasmic domains and activation of tyrosine kinase activity. Multiple cytoplasmic signaling pathways, including the Ras/Raf mitogen-activated protein kinase pathway, the phosphoinositol3-kinase/Akt pathway, the signal transducer and activator of transcription 3 pathway, the protein kinase C pathway, and scaffolding proteins may then be activated. Intracellular mediators in these pathways transduce signals from membrane receptors through the cytosol and into the nucleus, culminating in altered DNA synthesis and cell division as well as effects on a variety of biological processes, including cell growth, migration, differentiation, and death 2.1 EGFR (Epidermal growth factory receptor)The EGFR family comprises four transmembrane tyrosine kinase growth factor receptors:EGFR itself (ErbB1) (EGFR/HER1), ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4(HER4). Binding of a specific set of ligands to the receptor promotes EGFR dimerization and the autophosphorylation of the receptors on tyrosine residues. Upon autophosphorylation of the receptor, several signal transduction pathways downstream of EGFR become activated. The Ras/Raf mitogen-activated protein kinase pathway and the phosphoinositol3-kinase/Akt pathway are two major signaling routes for the HER family. The EGFR signal transduction pathways have been implicated in the regulation of various neoplastic processes, includingcell cycle progression, and inhibition of apoptosis, tumor cell motility, invasion, and metastasis. EGFR activation also stimulates vascular endothelial growth factor, which is the primary inducer of angiogenesis2.2 PDGFR (platelet-derived growth factor receptor)Platelet-derived growth factor (PDGF) signals through a cell surface tyrosine kinase receptor (PDGFR) to stimulate various cellular functions, including growth, proliferation, and differentiation. Two distinct PDGFR types have been identified:a andβ. Intracellular activation of this receptor can lead to cell transformation and generation of a mitotic signal. Both receptor types are over expressedin several solid tumors as well as in the surrounding stroma.2.3 VEGFR (vascular endothelial growth factor receptor)Angiogenesis is a complex process that occurs in a variety of physiologic and pathophysiologic states and is a remodeling of an established primitive network of blood vessels. VEGF is secreted by all almost all solid tumors and tumor-associated stroma in response to hypoxia. It is highly specific for vascular endothelium and regulates both vascular proliferation and permeability. Excessive expression of VEGF levels correlate with increased microvascular density, cancer recurrence, and decreased survival. There are six different ligands for the VEGFR, VEGF-A through -E and placenta growth factor. Ligands bind to specific receptors on endothelial cells, mostly VEGFR-2 (FLK-1/KDR), but it will also bind to VEGFR-1 (Flt-1) and-3. The binding of VEGF-A to VEGFR-1 induces endothelial cell migration. VEGFR-2 induces endothelial cell proliferation, permeability, and survival. VEGFR-3 is thought to mediate lymphangiogenesis. Binding of VEGF to VEGFR-2 receptors results in activation and autophosphorylation of intracellular tyrosine kinase domains, with triggering of intracellular signaling cascade.Reviewing data and the crystal structure of compound formed by VEGFR-2 and its inhibitors, we designed 38 chemicals using Sybyl 7. Also we calculated the dock score and found that AW1, with a furan ring binding to I molindole at the same time a benzene ring binding to the molindole get a high score. After the synthesis of it, we do the researches including in vitro enzyme activity, cell growth inhibitory activity and in vivo activity.The research methods and results are below:1. Synthesis of protein tyrosine kinase inhibitor AW 1 and its structure identification6-bromine-2-indolinone was synthesized from 2-Broaniline via condensation, cyclization, hydrolysis and reduction, after which AW1 was synthesized from 6-bromine-2-indolinone and 2-chloro benzoic acid.'H-NMR, IR, MS spectrum proved the compound to be AW1.1H-NMR (500 MHz)57.04 (s,1H),7.27(d,1H, J=8.0 Hz),7.38(s,1H),7.47 (dd,2H, J,=4.0Hz),7.72 (d,1H, J=8.5Hz),8.01(1d,1H, J=8.5 Hz),8.36 (d,2H, J=8.5 Hz),10.71 (s,1H); IR (KBr) cm -1 3037.1,1690.8,1592.6,1543.1,1398.6,1231.9,1304.5,684.4; MS (m/z),ESI-1,444(M-H)。2. Establishment of homogeneous time-resolved fluorescence immunoassay and activity evaluation of compound AWIThe most popular methods for screening tyrosine kinase inhibitors are in-plate binding assays and radiometric assays. The in-plate binding assays include enzyme- catalyzed colorimetric and luminescent read-outs and time-resolved fluorescence4. These methods require plate coating and multiple wash and incubation steps, which limits throughput. The radiometric assays using 33P or 32P, although extremely sensitive, can require filtration, which is labor intensive and relatively slow. A higher throughput radioisotopic method is SPA (scintillation proximity assay, Amersham International) as it does not require separation steps. However, because the amount of radioactivity used in the assay is kept to a minimum for cost and safety reasons, the measurement times can be the rate-limiting step in throughput.Specific fluorescence signals atλ=670 nm andλ,=612 nm are measured by multifunctional microplate reader when fluorescence is emitted through resonance energy transfer between fluorescent materials (EuK and XL-665). The inhibitory activity of AW Ion VEGFR-2 (Vascular Endothelia Growth Factor Receptor 2) kinase activity was investigated. In this system, the concentrations of VEGFR-2, adenosine triphosphate (ATP) and poly-peptide substrate were 5 ng/μl,100μM and 1μM, respectively. AW1 significantly inhibited VEGFR-2 kinase activity.3. Inhibitory proliferation of AW1 on human umbilical vein endothelial cellsHUVECs were collected from umbilical cord of infants. After the identification of HUVECs, the inhibitory activity of AW1 on HUVECs was assayed. The result showed that AW1 significantly inhibited the growth of HUVECs in a dose-dependent manner.4. Inhibitory effect of AW1 on GTL-16 cells xenograft tumor growth in nude mouseTumor cells GTL-16 were implanted in nude mouse. The mice with tumor size over 100 mm3 were selected for the study. Four dose groups were set:Omg/kg, 5mg/kg,15mg/kg,45mg/kg, bid, weight and tumor size were recorded every three days in order to observe the inhibitory activity of AW1.We observed that AW1 inhibited the growth GTL-16 in nude mouse. Significant difference (P<0.05) was showed between the drug-giving groups and the blank group, in a dose-dependent manner. Conclusion1.1H-NMR, IR, MS spectrum proved the compound synthesized to be AW1: 1H-NMR (500 MHz)δ7.04 (s,1H),7.27(d,1H, J=8.0 Hz),7.38(s,1H),7.47 (dd,2H, J1=4.0Hz),7.72 (d,1H, J=8.5Hz),8.01(1d,1H, J=8.5 Hz),8.36 (d,2H, J=8.5 Hz), 10.71 (s,1H);IR(KBr) cm-1 3037.1,1690.8,1592.6,1543.1,1398.6,1231.9,1304.5,684.4; MS (m/z), ESI-1,444 (M-H)。2. Homogeneous time-resolved fluorescence for high throughput screening of receptor protein tyrosine kinase was established by our lab, which leads all over the country. Three contents including VEGFR-2, adenosine triphosphate (ATP) and poly-peptide substrate. of the reaction were optimized to the most appropriate concentration,5 ng/μl,100μM and 1μM, respectively. Inhibitory activity of AW1 on VEGFR-2 was identified through homogeneous time-resolved fluorescence.3. HUVEC grows in a single layer, shaped short fusiform or pebble, with applanatus, polygon, clear boundary and abundant cytoplasm. After correlative antigen immunohistochemistry staining, the cells were shaped spherical, fusiform or polygon, with buffy particles, expecially intensive around the nucleus, while no staining was observed in the control group. AW1 significantly inhibited the proliferation of HUVEC.4. AW1 inhibited the growth GTL-16 in nude mouse. Significant difference was showed between the drug-giving groups and the blank group, in a dose-dependent manner.