CN103301133A - Anti-tumor treatment application of amino quinazoline epidermal growth factor receptor antagonist - Google Patents

Abstract

The present invention relates to an epidermal growth factor receptor antagonist of formula (I) or a pharmaceutically acceptable salt thereof. Such compounds can antagonize the activity of epidermal growth factor receptors, and can be developed as molecularly targeted antitumor drugs.

Description

Anti-tumor therapeutic application of a class of aminoquinazoline epidermal growth factor receptor antagonists

technical field

The invention relates to an anti-tumor therapeutic application of a class of aminoquinazoline epidermal growth factor receptor antagonists. the

Background technique

Epidermal growth factor receptor (EGFR) is a multifunctional glycoprotein widely distributed on the cell membrane of various tissues in the human body. One of the members, it is also known as HER1 or ErbB-1. EGFR belongs to the tyrosine kinase type I receptor family and is the expression product of the proto-oncogene Cerb-1. EGFR can activate receptor tyrosine kinase after binding with ligands epidermal growth factor and transforming growth factor, leading to phosphorylation of receptor itself and intracellular tyrosine residues, thereby causing cell division and proliferation. EGFR plays an important role in regulating cell growth and tissue repair, and the cellular effects caused by its signaling system include cell proliferation, migration, adhesion and other links. Related studies have also shown that overexpression of EGFR can promote tumor cell proliferation and tumor angiogenesis, promote cell migration, and cause tumor metastasis. More than 70% of the growing tumor cells have this receptor and its ligands, and its overexpression or regulation disorder will change the intracellular signaling pathway and affect the survival and apoptosis of tumor cells in different ways. Therefore, this receptor can be used as a target for tumor treatment. Target, 8 small molecule EGFR inhibitors have been approved for marketing as molecularly targeted anti-tumor drugs in foreign countries. In this experiment, homogeneous time-resolved fluorescence technology was used to conduct high-throughput screening of the anti-epidermal growth factor receptor activity of the tested samples, and the lead compounds with better activity were selected for further development of molecularly targeted anti-tumor drugs. The tested samples were purchased from the United States. ChemDiv high-throughput screening of compound libraries. the

Gefitinib, the first antineoplastic drug targeting the epidermal growth factor receptor, has potent efficacy in patients with non-small cell lung cancer who do not respond to one or more chemotherapy regimens, including platinum-based and docetaxel. In May 2003, the U.S. Food and Drug Administration (FDA) approved gefitinib as an antitumor drug because of its antitumor activity. Erlotinib, another therapeutic drug acting on this target, was approved for marketing by the FDA and the European Drug Evaluation Agency in November 2004 and October 2005, respectively. Studies have found that epidermal growth-dependent tumor cells change their affinity to drugs through genetic mutation, and can activate downstream intracellular signaling effectors through other transforming growth factor receptor pathways, which can escape growth inhibition caused by drug treatment and produce drug resistance Therefore, the development of a new generation of targeted EGFR antagonists has important application value. the

Contents of the invention

In order to develop epidermal growth factor receptor antagonists so as to find new candidate drugs for anti-tumor treatment, the present invention uses a high-throughput screening model of epidermal growth factor receptor antagonists to find lead compounds through functional verification, and discovers a class of Epidermal growth factor receptor antagonists provide lead compounds for the development of new anti-tumor molecular targeted drugs. The invention can provide lead compounds for the development of such clinical anti-tumor therapeutic drugs, and provide clues and experimental basis for the development of novel anti-tumor molecular targeted drugs. the

The technical scheme of the present invention is: adopting the homogeneous time-resolved fluorescence method to establish a high-throughput screening model of the epidermal growth factor receptor antagonist in vitro, and discovering a class of candidate drugs with anti-tumor activity through preliminary screening and secondary screening. Specific steps are as follows:

Step 1: Establish a high-throughput screening model for EGFR antagonists. the

Step 2: Positive drug testing and model stability verification. the

Step 3: Establish a high-throughput screening protocol and carry out activity screening of relevant samples to be tested. the

Description of drawings:

Figure 1: EGFR kinase concentration gradient optimization experiment. n=3,

Figure 2: EGFR kinase reaction time optimization experiment. n=3,

Figure 3: EGFR kinase substrate concentration optimization experiment. n=3,

Figure 4: EGFR kinase ATP concentration optimization experiment. n=3,

Figure 5: Positive drug gefitinib dose-effect curve. n=3,

Figure 6: Activity list of lead compounds

Detailed ways

The specific embodiment of the present invention is described below in conjunction with accompanying drawing:

1 Detection method

In this experiment, the homogeneous time-resolved fluorescence method was used to detect the reaction system. This method is divided into two steps: one is the enzymatic reaction, that is, when the reaction is started after adding ATP, the kinase causes the phosphorylation reaction of the substrate to link the phosphate On the biotin-labeled substrate; the second is the termination and detection process, in which EDTA terminates the reaction, there is europium element labeled anti-phosphotyrosine antibody close to the phosphate of the substrate, and the isotope of XL665 is labeled Phycocyanin is bound to the biotin label of the substrate, and the two fluorescent groups undergo energy resonance transfer and generate fluorescence at 665nm when they approach each other, and the free TK antibody (labeled europium ion) generates fluorescence at 620nm, This signal can be used as a background signal, while free SA-XL665 only produces transient fluorescence, which can be ignored by delaying the detection time (detection 1 hour after adding the terminator). In the optimization experiment of the high-throughput screening model of epidermal growth factor, the results of the optimization experiment of enzyme concentration are shown in Figure 1, the results of optimization experiment of enzyme reaction time are shown in Figure 2, the results of optimization time of substrate concentration are shown in Figure 3, and the results of optimization experiment of ATP concentration are shown in Figure 4 , the results of quality control experiments for positive compounds are shown in Figure 5. the

2 Model establishment and compound screening

Using 384 low-volume white plate as the reaction vessel, add 0.8 μL enzyme solution, 0.8 μL substrate solution, 1.6 μL buffer solution (or 1.6 μL compound solution), 0.8 μL ATP solution to each well of the reaction system, a total of 4 μL reaction system, in Reaction times were specified at room temperature. When the reaction was terminated, 2 μL of antibody solution and 2 μL of isophycocyanin solution were added, and incubated at room temperature for 60 min. Then use Envision to detect. The reagent solution in the experimental reaction was added using Biomek NXP automatic sampling instrument and Multidrop automatic dispenser. After the model was established, the small molecular compounds to be tested were screened first, and those with better activity were re-screened. The IC ₅₀ of the active compound was calculated using Prism5.0 (Graphpad Software, USA) statistical analysis software. For the activity results of the lead compound, see Figure 6.

The results of the screening experiment of epidermal growth factor receptor antagonist activity

The general structural formula and _IC50 of the screened EGFR receptor antagonist are as follows:

I: 2-carbamoyl-4-aminoquinazoline

Claims (2)

1. The chemical compound of the claimed formula (I) of claim 1 or its pharmaceutically acceptable salt in the purposes of epidermal growth factor receptor antagonists.

   

   Wherein:

   R ₁Represent C ₁-C ₆Alkyl, cycloalkyl or aryl

   R ₂Represent phenyl or substituted-phenyl.
2. 2. according to the purposes of claim 1, as epidermal growth factor receptor antagonists, be used for developing the purposes of molecular targeted antitumor drug.

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