CN112480109A

CN112480109A - Pyrido [2,3-b ] pyrazine-3 (4H) -ketone derivatives and application thereof

Info

Publication number: CN112480109A
Application number: CN202011278827.0A
Authority: CN
Inventors: 陈建忠; 李刚剑; 潘有禄
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2020-11-16
Filing date: 2020-11-16
Publication date: 2021-03-12
Anticipated expiration: 2040-11-16
Also published as: CN112480109B

Abstract

本发明提供吡啶并[2,3‑b]吡嗪‑3(4H)‑酮类衍生物及其用途，所述吡啶并[2,3‑b]吡嗪‑3(4H)‑酮类衍生物的结构通式为式Ⅴ，包括其药学上可接受的盐、溶剂化物、水合物或晶型。本发明提供的化合物是成纤维细胞生长因子受体(FGFR)的活性配体，研究表明结构Ⅴ所示化合物对KATO Ⅲ胃癌细胞(FGFR2扩增)和Huh‑7肝癌细胞(FGFR4过表达)具有较好的抗增殖活性，作为FGFR抑制剂在制备治疗由FGFR异常激活导致肿瘤相关疾病的药物中应用。结构通式Ⅴ如下：

The present invention provides pyrido[2,3-b]pyrazine-3(4H)-one derivatives and uses thereof, the pyrido[2,3-b]pyrazine-3(4H)-one derivatives The general structural formula of the compound is formula V, including its pharmaceutically acceptable salts, solvates, hydrates or crystal forms. The compound provided by the present invention is the active ligand of fibroblast growth factor receptor (FGFR). Research shows that the compound represented by structure V has the effect on KATO III gastric cancer cells (FGFR2 amplification) and Huh-7 liver cancer cells (FGFR4 overexpression). The better anti-proliferative activity is used as an FGFR inhibitor in the preparation of a drug for treating tumor-related diseases caused by abnormal activation of FGFR. The general structural formula V is as follows:

Description

Pyrido [2,3-b ] pyrazine-3 (4H) -ketone derivatives and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a compound taking pyrido [2,3-b ] pyrazine-3 (4H) -ketone as a parent nucleus, pharmaceutically acceptable salt, solvate, hydrate or crystal form thereof, and application of the compound in preparation of medicines for treating tumor-related diseases caused by abnormal activation of FGFR.

Background

At present, malignant tumors are one of the important factors that pose serious threats to human life. Despite the advances in tumor detection and treatment methods and techniques in recent years, patient survival rates for many types of tumors remain low. Therefore, anti-tumor research remains a very challenging and significant area in life sciences. Receptor Tyrosine Kinases (RTKs) are a branch of Protein Tyrosine Kinases (PTKs) and generally comprise a domain that binds a specific ligand extracellularly, a transmembrane domain, and an intracellular kinase domain. RTKs bind to their corresponding ligands through their extracellular domains, which, upon ligand binding, cause kinase oligomerization and phosphorylation of tyrosine residues in the intracellular domain, which in turn stimulate a series of intracellular signaling cascades that cause numerous biochemical reactions within the cell to regulate cellular behavior, such as proliferation, differentiation, apoptosis, and the like. The receptor tyrosine kinases include Epidermal Growth Factor Receptor (EGFR), platelet growth factor receptor (PDGFR), Vascular Endothelial Growth Factor Receptor (VEGFR), insulin receptor (instr), Fibroblast Growth Factor Receptor (FGFR), and the like. Wherein, FGFR is closely related to the generation and development of tumors and becomes an important target protein for the research and development of antitumor drugs.

The corresponding ligand of FGFR in vivo is Fibroblast Growth Factor (FGF), and 22 kinds of ligands are used, and FGF used as ligand interacts with FGFR and conducts cell signals. Fibroblast Growth Factor Receptors (FGFRs) are a class of highly conserved transmembrane receptor tyrosine kinases consisting essentially of 5 FGFRs, 4 highly conserved single transmembrane tyrosine kinase receptors FGFR1, FGFR2, FGFR3, FGFR4, and FGFR5 that have no intracellular kinase domain although they bind FGF. FGFR structures are composed of three important parts: (1) an extracellular domain consisting of 3 immunoglobulins, IgI, IgII, Ig III, wherein Ig II and Ig III constitute a FGF-conjugated serine-rich region; (2) a single-channel transmembrane region; (3) two intracellular kinase domains.

FGFs are usually present in the extracellular matrix as secreted glycoproteins or on the cell surface via heparan sulfate proteoglycans (HPSGs). When signaling is required, FGF is released from the cell matrix by heparinase, proteases and proteins that specifically bind FGF, unbound FGF ligands bind to FGFR with the aid of the cofactor HPSG, forming the ternary complex FGF-FGFR-HPSG, directing dimerization of FGFR, followed by phosphorylation of tyrosine residues in the kinase domain of FGFR intracellular close to the cell membrane. Activated FGFRs achieve downstream transduction of signals mainly through two pathways: (a) activated FGFR phosphorylates FGFR substrate 2(FRS2), recruits growth factor receptor binding protein (GRB2), and then recruits guanine nucleotide exchange factors, resulting in activation of the mitogen-activated protein kinase (MAPK) signaling pathway; (b) growth factor receptor binding protein (GRB2) activates a dependent signaling pathway. In addition, phospholipase C-gamma (PLC-gamma) can bind to phosphorylated tyrosine in FGFR kinase domain independently of rat sarcoma protein (Ras), and then hydrolyze phosphatidylinositol diphosphate (PIP2) into second messenger inositol triphosphate (IP3) and Diacylglycerol (DAG), IP3 can cause intracellular calcium ion release to activate downstream signaling pathway, DAG activates protein kinase C (pkc), and finally the signal is imported into MAPK pathway to achieve cellular response. It is noted that activated FGFRs can also signal through signaling and activator of transcription (STAT) signaling pathways. FGF/FGFRs signaling pathways play important roles in many physiological and biochemical processes in the body, such as embryonic development, metabolic homeostasis in the body, tissue repair and wound healing, angiogenesis, and a range of cellular functions including proliferation, differentiation, apoptosis and migration.

However, studies have shown that abnormalities in the FGF/FGFR signaling pathway are closely associated with tumor development and resistance to the therapeutic course of antitumor drugs in many types of cancer. During the development of tumors, the possible mechanisms by which ligand-dependent and ligand-independent FGF/FGFR signaling abnormalities lead to tumors are genetic alterations (receptor amplification, mutations, and chromosomal ectopy), autocrine and paracrine signaling, angiogenesis, epithelial mesenchymal transition, and acquired drug resistance. Amplification, mutation and chromosomal translocation of FGFR1-4 were detected in various types of tumors, such as breast cancer, lung cancer, bladder cancer, stomach cancer, prostate cancer and the like.

Since the abnormalities in FGF/FGFR signaling pathway are closely related to tumors, scientists have studied many FGF/FGFR targeting therapeutic drugs, such as small molecule kinase inhibitors, monoclonal antibodies, FGF ligand traps, etc., many of which have entered clinical trials. Small molecule kinase inhibitors are the most studied class at present and can be divided into multi-kinase target FGFR inhibitors, FGFR1/2/3 inhibitors, FGFR4 inhibitors and pan-FGFR inhibitors. Multi-kinase target FGFR inhibitors generally have a good inhibitory effect on multiple types of tyrosine kinases, which may increase the risk of side effects of such inhibitors, such as hypertension, hyperphosphatemia, and the like. Here we only describe representative selective FGFR inhibitors. AZD4547 and Infigrtinib (BGJ398) are FGFR1/2/3 inhibitors, BLU9931 and H3B-6527 are FGFR4 inhibitors, LY2874455, Erdafitinib (JNJ-42756493) and PRN1371 are pan-FGFR inhibitors. Such inhibitors typically contain a nitrogen-containing heterocyclic core structure that hydrogen bonds to Adenosine Triphosphate (ATP) binding pocket hinge regions, such as pyrazole, pyrimidine, benzopyrimidine, benzopyridine, and the like, hydrophobic groups occupying hydrophobic cavities in the pocket, such as dimethoxyphenyl, 2, 6-dichloro-3, 5-dimethoxyphenyl, and hydrophilic groups extending into the solvent regions at the outer ends of the pocket, such as piperazine, hydroxyl, and the like. If irreversible binding is to be achieved, it is generally desirable to introduce the propenyl group towards the cysteine in order to achieve covalent binding.

Many selective FGFR inhibitors are currently in clinical trials. AZD4547 has completed phase I clinical trials in patients with squamous cell lung carcinoma containing FGFR1 expansion, and in patients with advanced HER containing FGFR1 expansion^-Clinical phase ii trials of breast cancer and gastroesophageal cancer patients with FGFR2 amplification have also been completed, and in addition, many clinical trials are underway (clinical trials. gov, NCT01213160, NCT 01824901). Infiratib (BGJ398) has also completed clinical phase i experiments against advanced solid tumors containing FGFR alterations and clinical phase ii experiments against advanced or metastatic cholangiocarcinoma with FGFR2 alterations, and in addition, there are many clinical experiments in progress (clinical trials. gov, NCT01004224, NCT01928459, NCT01975701, NCT02150967, NCT02160041, NCT 02706691). H3B-6527 is currently undergoing clinical trials for treatment of advanced hepatocellular carcinoma and intrahepatic bile duct cancer (clinical trials. gov, NCT02834780), and is approved by the U.S. FDA as an orphan drug for hepatocellular carcinoma in 2017. LY2874455 has also completed phase i clinical trials on advanced solid tumors (clinical trials. gov, NCT01212107) and has good therapeutic activity and tolerability in patients with solid tumors, LY2874455 in combination with MeresClinical phase i trials of tinib on relapsed or refractory acute myeloid leukemia are ongoing (clinical trials. gov, NCT 03125239). PRN1371 is currently undergoing phase i clinical trials for the treatment of solid tumors (clinical trials. gov, NCT 02608125). It is particularly noteworthy that Erdafitinib was approved by the FDA in the united states as the first FGFR selective inhibitor for the treatment of adult patients with locally advanced or metastatic urothelial cancer in month 4 2019, and that there are also many clinical trials on Erdafitinib ongoing (clinical trials. gov, NCT02952573, NCT03155620, NCT03210714, NCT02699606, NCT03238196, NCT02421185, NCT 02365597).

In the clinical course of treating cancer, tumor cells can develop drug resistance in a variety of ways, such as changing drug targets, expelling drugs, repairing DNA damage, inhibiting cell death, and the like. Numerous lines of evidence have suggested that abnormalities in the FGF/FGFR signaling pathway are closely associated with resistance during treatment with many antineoplastic agents. With the clinical experiments of the FGFR inhibitor and the subsequent marketing of related drugs, the drug resistance of tumors to selective FGFR inhibitors is inevitable. Therefore, there is an urgent need to continuously develop new selective FGFR inhibitors to meet the current clinical needs.

Disclosure of Invention

The invention provides a pyrido [2,3-b ] pyrazine-3 (4H) -ketone derivative and a pharmaceutically acceptable salt thereof, wherein the structural general formula V of the derivative is as follows:

wherein:

R₁selected from 2-acetoxy, 2- (4-acryloylpiperazin-1-yl) -2-oxoethyl;

R₂selected from 3, 5-dimethoxyphenyl, 2, 6-dichlorophenyl, 3,4, 5-trimethoxyphenyl, 2, 6-dichloro-3, 5-dimethoxyphenyl;

R₂selected from the group consisting of chloro, phenylamino, 4- (4-methylpiperazine) anilino, 2, 6-dichloro-3, 5-dimethoxyphenyl;

the derivatives include pharmaceutically acceptable salts, solvates, hydrates or crystal forms thereof.

Preferred specific compounds are as follows:

(1)4- (2- (4-acryloylpiperazin-1-yl) -2-oxoethyl) -6-phenylamino-2- (3,4, 5-trimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one;

(2)4- (2- (4-acryloylpiperazin-1-yl) -2-oxoethyl) -6-chloro-2- (3,4, 5-trimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one;

(3)4- (2- (4-acryloylpiperazin-1-yl) -2-oxoethyl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -2- (3,4, 5-trimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one;

(4)4- (2- (4-acryloylpiperazin-1-yl) -2-oxoethyl) -2- (3, 5-dimethoxyphenylamino) -6- (4- (4-methylpiperazin-1-yl) phenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one;

(5)4- (2- (4-acryloylpiperazin-1-yl) -2-oxoethyl) -6-chloro-2- (2, 6-dichlorophenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one;

(6)4- (2- (4-acryloylpiperazin-1-yl) -2-oxoethyl) -6-chloro-2- (3, 5-dimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one;

(7)4- (2- (4-acryloylpiperazin-1-yl) -2-oxoethyl) -6-phenylamino-2- (3, 5-dimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one;

(8)4- (2- (4-acryloylpiperazin-1-yl) -2-oxoethyl) -6-chloro-2- (2, 6-dichloro-3, 5-dimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one;

(9)4- (2-acetoacetoxy) -6-chloro-2- (3, 5-dimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one;

(10)4- (2-acetoacetoxy) -6-chloro-2- (2, 6-dichlorophenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one;

(11)4- (2-acetoacetoxy) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -2- (3,4, 5-trimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one;

(12)4- (2-acetoacetoxy) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -2- (3, 5-dimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one;

(13)4- (2-acetoacetoxy) -6-chloro-2- (2, 6-dichloro-3, 5-dimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one;

(14)4- (2-acetoacetoxy) -6-phenylamino-2- (3, 5-dimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one;

(15)4- (2-acetoacetoxy) -6-phenylamino-2- (2, 6-dichloro-3, 5-dimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one;

(16)4- (2-acetoacetoxy) -6-phenylamino-2- (2, 6-dichlorophenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one;

(17)4- (2-Acetocarbethoxy) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -2- (2, 6-dichloro-3, 5-dimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one.

And pharmaceutically acceptable salts, solvates, hydrates or crystal forms of the above specific compounds.

Specific examples of the "pharmaceutically acceptable salt" in the present specification include salts of the compounds provided by the present invention with organic acids such as propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, and citric acid; or forming salt with inorganic acid such as hydrochloric acid, phosphoric acid, sulfuric acid, hydrofluoric acid, hydrobromic acid, etc.; or a quaternary ammonium salt formed with a haloalkane, said haloalkane being a fluoro, chloro, bromo or iodo alkane.

The second purpose of the invention is to provide a preparation method of pyrido [2,3-b ] pyrazine-3 (4H) -ketone derivatives and pharmaceutically acceptable salts, solvates, hydrates or crystal forms thereof, and the following is a preferred embodiment of the invention and comprises the following technical scheme: processes for the preparation of route 1, route 2, route 3, route 4 and salts, solvates, hydrates or crystalline forms thereof.

(1) Scheme 1, a process for the preparation of compounds of formula i:

reaction conditions are as follows: a)1, ammonia gas and ethanol for 3 hours; reacting overnight at room temperature; b) ammonium chloride, iron powder, ethyl acetate/water, room temperature; c) oxalic acid, concentrated hydrochloric acid/water, 100 ℃; d) thionyl chloride, N, N-dimethylformamide, Dichloromethane (DCM), at 45 ℃ for 6 hours; e) lithium hydroxide, tetrahydrofuran/water (5:2), 65 ℃.

(2) Scheme 2, preparation of compound G:

reaction conditions are as follows: a) acryloyl chloride, dichloromethane and triethylamine are added at the temperature of 0-room temperature; b) trifluoroacetic acid/dichloromethane (1:1), room temperature, 30 min.

(3) Scheme 3, preparation of compound J:

reaction conditions are as follows: a) acetic anhydride, toluene, 25 ℃; b) SO (SO)₂Cl₂Acetonitrile, 0 ℃; c)2N potassium hydroxide, ethanol.

(4) Scheme 4, a process for the preparation of compounds of formula v:

reaction conditions are as follows: a) ethyl bromoacetate, potassium carbonate, N-dimethylformamide, room temperature; b) r₂-NH₂Palladium acetate, 4, 5-bis (diphenylphosphino) -9, 9-dimethyl xanthene, potassium carbonate, argon, 1, 4-dioxane, 90 ℃; c) r₃-NH₂Palladium acetate, 4, 5-bis-diphenylphosphine-9, 9-dimethyl xanthene, potassium tert-butoxide, argon, toluene, 110 ℃; d) lithium hydroxide, tetrahydrofuran/water, room temperature; 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate, N, N-diisopropylethylamine, N, N-dimethylformamide, at room temperature.

Wherein R is₂、R₃The same as defined above for formula V.

Preparation of pharmaceutically acceptable salts, solvates, hydrates or crystal forms:

1. dissolving the compound V in anhydrous methanol, adding a proper amount of organic acid such as propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid and the like or inorganic acid such as hydrochloric acid, phosphoric acid, sulfuric acid, hydrofluoric acid, hydrobromic acid and the like under ice bath, and spin-drying the solvent to obtain a pharmaceutically acceptable acid addition salt; or dissolving the compound V in anhydrous ethanol, adding equivalent sodium hydroxide, potassium iodide and halogenated hydrocarbon such as methyl iodide, heating and refluxing overnight, and recrystallizing and purifying the crude product with acetone to obtain pharmaceutically acceptable quaternary ammonium salt of the compound V.

2. Dissolving the compound V in an aqueous solution of acid, adding a non-acidic organic solvent into the system, and obtaining a solvate or hydrate of the compound V by a crystallization method. Wherein the suitable acid is selected from hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, acetic acid, hydrobromic acid, nitric acid, formic acid, tartaric acid, benzoic acid, phenylacetic acid, maleic acid, oxalic acid, trifluoroacetic acid; the non-acidic organic solvent is selected from ethanol, methanol, acetonitrile, ethyl acetate, tetrahydrofuran, diethyl ether, petroleum ether, isopropanol, N-butanol, and N, N-dimethylformamide.

The third purpose of the invention is to provide an application of pyrido [2,3-b ] pyrazine-3 (4H) -ketone derivatives and pharmaceutically acceptable salts, solvates, hydrates or crystal forms thereof in preparing medicines for treating tumor-related diseases caused by abnormal activation of FGFR. The tumor-related diseases such as gastric cancer and liver cancer.

The fourth purpose of the invention is to provide a pharmaceutical composition of pyrido [2,3-b ] pyrazine-3 (4H) -ketone derivatives and pharmaceutically acceptable salts, solvates, hydrates or crystal forms thereof, which can further comprise excipients, diluents and carriers. The compounds of the present invention may exist in unsolvated forms as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, polyethylene glycol, propylene glycol, and the like. In general, the solvated forms are considered equivalent to unsolvated forms for the purposes of the present invention. The pharmaceutical compositions of the invention may include one or more compounds of the invention, typically formulated by mixing a compound of the invention, and a pharmaceutically acceptable salt, solvate or hydrate thereof, with a carrier, excipient or diluent. Suitable carriers, excipients or diluents are well known to those skilled in the art and include materials such as carbohydrates, waxes, water soluble and/or swellable polymers, hydrophilic or hydrophobic materials, gelatin, solvents, water and the like. The dosage form of the medicine is solid preparation or liquid preparation, and specifically is tablet, capsule, powder, solution, syrup, suspension or aerosol.

A fifth object of the invention is to provide a method for testing the proliferative activity of FGFR2 and FGFR4, which method comprises the antiproliferative activity of a compound represented by structure V, typically an FGFR inhibitor, on KATO iii gastric cancer cells (FGFR2 amplification) and Huh-7 liver cancer cells (FGFR4 overexpression).

The compound provided by the invention is an FGFR inhibitor, and the compound comprises a compound with a structure shown in a general formula V and pharmaceutically acceptable salt, solvate, hydrate or crystal form thereof. The compound is an FGFR inhibitor, can be used for preparing medicaments for treating tumor-related diseases caused by abnormal activation of FGFR, and has good medicament development prospect.

Detailed Description

The invention is further illustrated by reference to examples, which are intended to further illustrate the invention but are not intended to be limiting.

The starting materials, reaction reagents and the like used in the examples of the present invention are commercially available products. Example 30 illustrates a method for preparing a hydrochloride, a quaternary ammonium salt, or a monohydrate of compound v-17, to which other compounds may be referred, or other salts may be formed by a method generally used in the art.

EXAMPLE 16 Synthesis of chloro-3-nitropyridin-2-amine (B)

9.2g of the raw material 2, 6-dichloro-3-nitropyridine (A) is taken to be put into a bottle, 200mL of absolute ethyl alcohol is added, ammonia gas is introduced for 3 hours at the temperature of 0 ℃, and the reaction system turns turbid from clear and has yellow brown color. After aeration, the reaction was allowed to proceed overnight at room temperature. The next day, the reaction solution was poured into 200mL of water, filtered with suction, and the filter cake was washed with 200mL of ice water and 200mL of petroleum ether to give B (15.2g) as a yellow solid with a yield of 88.0%. The melting point is measured: 186 ℃ and 187 ℃, and the literature reports that: 190 ℃ and 192 ℃.

EXAMPLE 26 Synthesis of chloropyridine-2, 3-diamine (C)

Intermediate B (1g, 1.0equiv.) and ammonium chloride (620.6mg, 2.0equiv.) were weighed out into a bottle, ethyl acetate (10mL) and water (6mL) were added, followed by iron powder (972.7mg, 3.0equiv.) and reacted at room temperature for 24 hours, followed by Thin Layer Chromatography (TLC) detection and reaction was complete. Filtering with diatomaceous earth, washing filter cake with water and ethyl acetate, collecting organic layer, extracting water layer with ethyl acetate for 2 times, combining organic layers, drying, filtering, evaporating to dryness, and purifying with column eluting agent of dichloromethane: methanol 50:1 to 30:1 gave 780.0mg of a brown solid in 93.7% yield. The melting point is measured: 117 ℃ and 119 ℃, and the literature reports that: 120-122 ℃. Wherein the volume of the ethyl acetate is 8-12 times of the mass of the raw materials, and the volume of the water is 4-8 times of the mass of the raw materials.

Example 36 Synthesis of chloro-1, 4-dihydropyrido [2,3-b ] pyrazine-2, 3-dione (D)

Intermediate C (776.4mg, 1.0equiv.) and oxalic acid dihydrate (1.0g, 1.5equiv.) were weighed into a bottle, and 8mL of water and 0.8mL of concentrated hydrochloric acid were added, and the reaction was stopped after 6 hours at 100 ℃. After cooling, 968.0mg of intermediate D were obtained by suction filtration, the yield being 90.6%.¹H NMR(500MHz,DMSO-d₆):δ12.53(s,1H),12.07(s,1H),7.47(d,J＝8.1Hz,1H),7.23–7.17(m,1H)。

Example 42 Synthesis of 3, 6-trichloropyrido [2,3-b ] pyrazine (E)

Intermediate D (1.3g, 1.0equiv.) was added to the bottle, the reaction solvent dichloromethane (7mL) was added followed by thionyl chloride (0.95mL, 2.0equiv.) and N, N-dimethylformamide was added slowly under nitrogen. Then, the temperature was increased to 45 ℃ and the reaction was stopped after 6 hours. After quenching the reaction by adding ice water, dichloromethane was used for 3 times, the organic layer was washed with saturated brine for 3 times, dried, filtered and evaporated to dryness, and the intermediate E was obtained as a slightly pink solid in a yield of 90.7% after passing through a column.¹H NMR (500MHz, chloroform-d): delta 8.37(d, J ═ 8.7Hz,1H),7.79(d,J＝8.7Hz,1H)。

EXAMPLE 5 Synthesis of N-acryloyl-N-Boc-piperazine (F)

The starting material, 1-Boc-piperazine (200.0mg,1equiv.) was weighed into a bottle, 2mL of anhydrous dichloromethane was added followed by triethylamine (297.5 μ L,2equiv.), the reaction bottle was cooled in an ice bath, followed by the slow addition of acryloyl chloride (130.2 μ L,1.5 equiv.). After the addition was completed, the reaction system was allowed to stand at room temperature to continue the reaction overnight. The next day, after quenching with water, dichloromethane was extracted three times, dried, filtered, evaporated to dryness, and column-passed to obtain 206.1mg of white solid, 80.2% yield, melting point: 75-76 ℃.¹H NMR (500MHz, chloroform-d) δ 6.56(dd, J ═ 16.8,10.6Hz,1H),6.32(dd, J ═ 16.8,1.8Hz,1H),5.73(dd, J ═ 10.6,1.8Hz,1H),3.66(s,2H),3.54(s,2H), 3.48-3.42 (m,4H),1.48(s, 10H).

Example 6 Synthesis of N-acryloylpiperazine (G)

Weighing intermediate F (56.1mg,0.23mmol) in a bottle, adding dried dichloromethane (0.5mL) for dissolving, then adding trifluoroacetic acid (0.5mL) with the same volume as dichloromethane, reacting at room temperature for 30 minutes, evaporating most of the trifluoroacetic acid, washing with saturated saline solution for 3 times after dichloromethane is dissolved, evaporating to dryness and passing through a column to obtain solid intermediate G25.1 mg, the yield is 77.8%, and the melting point is: 54-56 ℃. The subsequent reaction with large amount can also be directly rotated to dry trifluoroacetic acid, and the product is stored at-20 ℃ and taken out when in use.¹H NMR (500MHz, chloroform-d): δ 10.01(s,1H),6.52(dd, J ═ 16.7,10.5Hz,1H),6.35(dd, J ═ 16.7,1.7Hz,1H),5.81(dd, J ═ 10.5,1.7Hz,1H),3.91(s,4H), 3.23-3.17 (m,4H).¹³C NMR (126MHz, chloroform-d). delta. 165.55,129.98,126.13, 43.32.

EXAMPLE 7 Synthesis of intermediate 2, 6-dichloro-3, 5-dimethoxyaniline (J)

3, 5-dimethoxyaniline (3.5g,1equiv.) was weighed into a bottle, 20ml of toluene was added as a solvent, and 2.5ml (1.05equiv.) of acetic anhydride was slowly dropped to maintain the reaction system at 35-45 ℃. After the completion of the addition of acetic anhydride, the reaction was carried out at room temperature for 20 hours. After the reaction was completed, an appropriate amount of petroleum ether was added, suction filtration was performed, the filter cake was washed with an appropriate amount of petroleum ether, and the filter cake was dried to obtain 4.4g of crude N- (3, 5-dimethoxyphenyl) acetamide (H), yield 91.5%, and measured as melting point: 155 ℃ and 156 ℃, literature values: 157 ℃.

Thionyl chloride (2.0equiv.) was added dropwise to a solution of N- (3, 5-dimethoxyphenyl) acetamide (4.4g) in acetonitrile (60ml) under nitrogen protection at 0 ℃. After the addition was completed, the reaction was continued in the ice bath for 1 hour, and the reaction was stopped. After quenching the reaction by slow addition of saturated sodium bicarbonate solution, suction filtration was carried out and the filter cake was washed with 40ml of water. After 40ml of saturated sodium bicarbonate solution was added to the filtrate, it was extracted with ethyl acetate, checked by TLC, containing a small amount of product and discarded. The filter cake was dried to give 2.3g of crude N- (2, 6-dichloro-3, 5-dimethoxyphenyl) (I) in 39.3% yield.

To a solution of N- (2, 6-dichloro-3, 5-dimethoxyphenyl) (I) (2.3g, 1equiv.) in ethanol (45ml) was added 2N potassium hydroxide (24.5ml), and the mixture was heated to 90 ℃ to react for 44 hours. After the reaction is finished and the temperature is reduced to room temperature, the reaction product is cooled for 1 hour at 0 ℃ to precipitate more solid, the more solid is filtered, the more solid is washed by cold ethanol/water (1:1) firstly, then washed by cold water until the water is neutral, and the 1.7g of 2, 6-dichloro-3, 5-dimethoxyaniline is obtained after drying, the yield is 86.8 percent, and the white solid can be directly used for the next reaction.

EXAMPLE 82 Synthesis of 6, 6-dichloropyrido [2,3-b ] pyrazin-3 (4H) -one (I)

Adding the intermediate E (1.2g, 1.0equiv.) into a bottle, adding reaction solvents tetrahydrofuran and water in a ratio of 5:2, adding lithium hydroxide (245.2mg, 2.0equiv.), then heating to 65 ℃, reacting for 9 hours, detecting by Thin Layer Chromatography (TLC), adding tetrahydrofuran and saturated saline water after the reaction is finished, layering to obtain a tetrahydrofuran organic layer, extracting the water layer with tetrahydrofuran for multiple times, combining the organic layers, drying, filtering and evaporating to dryness to obtain 1.0g of the intermediate I, wherein the yield is 90.4%.¹H NMR (500MHz, dimethylsulfoxide-d)₆):δ13.51(s,1H),8.21(d,J＝8.4Hz,1H),7.47(d,J＝8.3Hz,1H).

EXAMPLE 92 Synthesis of 6-dichloro-4- (2-acetoacetoxy) -3-carbonyl-4 (3H) -pyrido [2,3-b ] pyrazine (II)

Intermediate i (1.2g,5.6mmol, 1.0equiv.) was weighed into a bottle, solvent N, N-dimethylformamide (15mL) was added, potassium carbonate (921.9mg,6.7mmol,1.2equiv.) was added, followed by ethyl bromoacetate (74.74 equiv.)0.0 mu L,0.9mmol,6.7equiv.), reacting at room temperature for 3 hours, detecting by Thin Layer Chromatography (TLC) that the reaction is complete, adding saturated sodium chloride solution to quench the reaction, extracting by ethyl acetate for 3 times, washing an organic layer by saturated saline solution for 3 times, drying, filtering, evaporating to dryness, passing through a column, and obtaining petroleum ether and ethyl acetate by a washing and dehydrating machine to obtain 900.0mg of an intermediate II, wherein the yield is 53.6 percent.¹H NMR (500MHz, chloroform-d): δ 8.00(d, J ═ 8.3Hz,1H),7.28(d, J ═ 8.3Hz,1H),5.12(s,2H),4.20(q, J ═ 7.1Hz,2H),1.25(t, J ═ 7.1Hz,3H).

EXAMPLE 10 Synthesis of amino-substituted ethyl-2- (6-chloro-2-amino-3-oxopyridin [2,3, b ] pyrazin-4 (3H) -yl) -ethyl ester (III)

Weighing the intermediate II (1equiv.), amines (1.02equiv.), palladium acetate (5 mol%), 4, 5-bis (diphenylphosphine) -9, 9-dimethyl xanthene (10 mol%) and potassium carbonate (1.5equiv.) in a Schlenk reaction tube, plugging the mouth of the reaction tube with a rubber plug, vacuumizing and protecting with argon for 4 times, adding a reaction solvent dioxane, reacting for 1 hour at 90 ℃, stopping the reaction, cooling, detecting by thin-layer chromatography (TLC), adding a proper amount of ethyl acetate into a reaction solution, washing for 1 time with saturated saline solution, extracting a water phase with ethyl acetate for 2 times, combining organic layers, drying, filtering, evaporating to dryness, and passing through a column to obtain an important intermediate III.

EXAMPLE 11 Synthesis of amino-and aryl-substituted ethyl-2- (6-chloro-2-amino-3-oxopyridin [2,3, b ] pyrazin-4 (3H) -yl) -ethyl ester (IV)

Taking the intermediate III (1equiv.), amines (1.5equiv.), palladium acetate (5 mol%), 4, 5-bis-diphenylphosphine-9, 9-dimethyl xanthene (10 mol%) and potassium tert-butoxide (1.5equiv.) in a Schlenk reaction tube, plugging the mouth of the reaction tube with a rubber plug, vacuumizing and protecting with argon for 4 times, then adding a reaction solvent dioxane, reacting for 1 hour at 90 ℃, stopping the reaction and cooling, detecting by thin-layer chromatography (TLC), adding a proper amount of ethyl acetate into the reaction solution, washing for 1 time with saturated saline, extracting the aqueous phase for 2 times with ethyl acetate, combining organic layers, drying, filtering, evaporating to dryness, and passing through a column to obtain an important intermediate IV.

EXAMPLE 12 Synthesis of substituted 4- (2- (4-propenylpiperazin-1-yl) -2-ethylcarbonyl) -2-aminopyridine [2,3, b ] pyrazin-3 (4H) -one (V)

Important intermediate IV (1.0equiv.) was weighed into a bottle and tetrahydrofuran and water were added in a ratio of 4: 1. Subsequently, lithium hydroxide (1.5equiv.) was weighed and added to the reaction solution, and reacted at room temperature, followed by Thin Layer Chromatography (TLC) for about 6 h. Then, the tetrahydrofuran is removed by rotary evaporation, a proper amount of water is added, the pH value is adjusted to 2-3 by using 1N hydrochloric acid, and the intermediate acid compound is obtained by extracting with ethyl acetate, drying, filtering and evaporating to dryness. The reaction can be directly carried out in the next step without column purification. If purified, an ethyl acetate petroleum ether system can be used, with additional acetic acid added as eluent.

Weighing the intermediate acid compound (1.0equiv.) obtained in the previous step, the intermediate G (1.5equiv.), and 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate (1.5equiv.) into a bottle, adding a reaction solvent N, N-dimethylformamide (1mL), then adding N, N-diisopropylethylamine (1.5equiv.), reacting overnight at room temperature, adding water for quenching, extracting with ethyl acetate for 3 times, washing an organic layer with saturated saline solution for 3 times, drying, filtering, evaporating to dryness, and passing through a column to obtain a target product V.

Example 13

The compounds 4- (2- (4-acryloylpiperazin-1-yl) -2-oxoethyl) -6-chloro-2- (3,4, 5-trimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one (V-2), 4- (2- (4-acryloylpiperazin-1-yl) -2-oxoethyl) -6-chloro-2- (2, 6-dichlorophenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one (V-5), 4- (2- (4-acryloylpiperazin-1-yl) -2-oxoethyl) -6-chloro-2- (3, synthesis of 5-dimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one (V-6), 4- (2- (4-acryloylpiperazin-1-yl) -2-oxoethyl) -6-chloro-2- (2, 6-dichloro-3, 5-dimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one (V-8)

After synthesis of intermediate iii, part iii (1.0equiv.) was weighed into a bottle and tetrahydrofuran and water were added in a ratio of 4: 1. Subsequently, lithium hydroxide (1.5equiv.) was weighed and added to the reaction solution, and reacted at room temperature, followed by Thin Layer Chromatography (TLC) for about 6 h. Then, the tetrahydrofuran is removed by rotary evaporation, a proper amount of water is added, the pH value is adjusted to 2-3 by using 1N hydrochloric acid, and the intermediate carboxyl-containing compound is obtained by extracting with ethyl acetate, drying, filtering and evaporating to dryness. The reaction can be directly carried out in the next step without column purification. If purified, an ethyl acetate petroleum ether system can be used, with additional acetic acid added as eluent.

The carboxyl group-containing compound (1.0equiv.) obtained in the previous step, the intermediate G (1.2equiv.), and 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate (1.2equiv.) were weighed out in a bottle, and N, N-dimethylformamide (1mL) as a reaction solvent was added, followed by N, N-diisopropylethylamine (1.2equiv.) and reacted overnight at room temperature, and the reaction solution was diluted with ethyl acetate, washed once with saturated saline, and the aqueous layer was extracted with ethyl acetate several times, and if the solubility of ethyl acetate was not good, dichloromethane was used. And combining the organic layers, drying, filtering, evaporating to dryness, and passing through a column to obtain a target product.

Example 144- (2- (4-Acryloylpiperazin-1-yl) -2-oxoethyl) -6-phenylamino-2- (3,4, 5-trimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one (V-1)

Compound V-1 was prepared according to the method of example 10. Silica gel column chromatography solvent ratio ethyl acetate/petroleum ether 12/7 to 12/4, yellow solid (13.2mg, 39.0%), melting point: 168 ℃ and 169 ℃. Purity: 96 percent.¹H NMR (500MHz, chloroform-d): δ 8.04(s,1H),7.60(d, J ═ 8.5Hz,1H),7.28(d, J ═ 7.8Hz,2H),7.22(t, J ═ 7.8Hz,2H),7.14(s,2H),6.98(t, J ═ 7.2Hz,1H), 6.63-6.57 (m,2H),6.51(dd, J ═ 16.8,10.5Hz,1H),6.31(dd, J ═ 16.7,1.9Hz,1H), 5.75-5.69 (m,1H),5.24(s,2H),3.83(s,6H),3.77(s,3H), 3.77-3.47 (m, 8H).

Example 154 Synthesis of- (2- (4-acryloylpiperazin-1-yl) -2-oxoethyl) -6-chloro-2- (3,4, 5-trimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one (V-2)

Compound V-2 was prepared according to the method of example 13. Silica gel column chromatography solvent ratio ethyl acetate/petroleum ether 12/7 to 12/4, yellow solid (120.7mg, 58.5%), melting point: 175 ℃ and 177 ℃. Purity: 96 percent.¹H NMR (500MHz, chloroform-d): δ 8.37(s,1H),7.79(d, J ═ 8.2Hz,1H),7.24 to 7.18(m,3H),6.60(dd, J ═ 16.8,10.5Hz,1H),6.37(dd, J ═ 16.8,1.9Hz,1H),5.79(dd, J ═ 10.4,1.9Hz,1H),5.31(s,2H),3.98 to 3.61(m, 17H).

Example 164 Synthesis of- (2- (4-acryloylpiperazin-1-yl) -2-oxoethyl) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -2- (3,4, 5-trimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one (V-3).

Compound V-3 was prepared according to the method of example 12. Silica gel column chromatography solvent ratio dichloromethane/methanol 100/1 to 25/1, yellow solid (35.9mg, 52.9%), melting point: 185 ℃ and 187 ℃. Purity: 96 percent.¹H NMR(500MHz,DMSO-d₆):δ9.21(s,1H),9.10(s,1H),7.74(d,J＝8.5Hz,1H),7.63(s,2H),7.43(d,J＝8.5Hz,2H),6.87(d,J＝9.0Hz,3H),6.73(d,J＝8.6Hz,1H),6.21(d,J＝16.3Hz,1H),5.82–5.74(m,1H),5.26(s,2H),3.84–2.78(m,25H),2.51(s,3H)。

Example 174- (2- (4-Acryloylpiperazin-1-yl) -2-oxoethyl) -2- (3, 5-dimethoxyphenylamino) -6- (4- (4-methylpiperazin-1-yl) phenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one (V-4)

Compound V-4 was prepared according to the method of example 12. Silica gel column chromatography solvent ratio dichloromethane/methanol 40/1 to 20/1, yellow solid (26.0mg, 53.9%), melting point: 178 ℃ and 180 ℃. Purity: 98 percent.¹H NMR (500MHz, chloroform-d): δ 8.08(s,1H),7.62(d, J ═ 8.5Hz,1H),7.21(d, J ═ 8.7Hz,2H),7.11(d, J ═ 2.2Hz,2H),6.84(d, J ═ 8.9Hz,2H),6.74(s,1H), 6.61-6.51 (m,2H),6.35(dd, J ═ 16.8,2.0Hz,1H),6.19(t, J ═ 2.2Hz,1H),5.77(dd, J ═ 10.5,2.0Hz,1H),5.23(s,2H), 3.86-3.53 (m,14H),3.18(t, J ═ 4.9, 4H), 2.81-3.42H), 3.42H (m, 3H).

Example 184- (2- (4-Acryloylpiperazin-1-yl) -2-oxoethyl) -6-chloro-2- (2, 6-dichlorophenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one (V-5)

Compound V-5 was prepared according to the method of example 13. Silica gel column chromatography solvent ratio petroleum ether/ethyl acetate 12/10 to 12/8, white solid (18.0mg, 85.8%), melting point: 160 ℃ and 162 ℃. Purity: 98 percent.¹H NMR (500MHz, chloroform-d): δ 7.91(s,1H),7.72(d, J ═ 8.3Hz,1H),7.44(d, J ═ 8.1Hz,2H), 7.27-7.22 (m,1H),7.15(d, J ═ 8.2Hz,1H),6.59(dd, J ═ 16.8,10.5Hz,1H),6.37(dd, J ═ 16.8,1.8Hz,1H),5.78(dd, J ═ 10.5,1.9Hz,1H),5.33(s,2H), 3.97-3.62 (m, 8H).

Example 194 Synthesis of- (2- (4-acryloylpiperazin-1-yl) -2-oxoethyl) -6-chloro-2- (3, 5-dimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one (V-6)

Compound V-6 was prepared according to the method of example 13. Silica gel column chromatography solvent ratio petroleum ether/ethyl acetate 12/10 to 12/8, white solid (26.7mg, 62.7%), melting point: 168 ℃ and 170 ℃. Purity: 96 percent.¹H NMR (500MHz, chloroform-d): δ 8.39(s,1H),7.83(d, J ═ 8.2Hz,1H),7.19(d, J ═ 8.2Hz,1H),7.13(d, J ═ 2.2Hz,2H),6.59(dd, J ═ 16.8,10.5Hz,1H),6.37(dd, J ═ 16.8,1.9Hz,1H),6.26(t, J ═ 2.2Hz,1H), 5.81-5.75 (m,1H),5.28(s,2H), 3.96-3.61 (m, 14H).

Example 204- (2- (4-Acryloylpiperazin-1-yl) -2-oxoethyl) -6-phenylamino-2- (3, 5-dimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one (V-7)

Compound V-7 was prepared according to the method of example 12. Silica gel column chromatography solvent ratio dichloromethane/methanol 150/1 to 80/1, yellow solid (26.5mg, 72.4%), melting point: 176 ℃ and 178 ℃. Purity: 97 percent.¹H NMR (500MHz, chloroform-d): δ 8.14(s,1H),7.74(d, J ═ 8.0Hz,1H),7.37 to 7.27(m,4H),7.13(s,2H),7.05(q, J ═ 7.5,6.9Hz,1H),6.70 to 6.53(m,3H),6.37(dd, J ═ 16.8,4.2Hz,1H),6.22(s,1H),5.83 to 5.76(m,1H),5.30(s,2H),3.90 to 3.57(m, 14H).

Example 214 Synthesis of- (2- (4-acryloylpiperazin-1-yl) -2-oxoethyl) -6-chloro-2- (2, 6-dichloro-3, 5-dimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one (V-8)

Compound V-8 was prepared according to the method of example 13. Silica gel column chromatography solvent ratio ethyl acetate/petroleum ether 12/8, white solid (31.4mg, 74.1%), melting point: 156 and 158 ℃. Purity: 99 percent.¹H NMR (500MHz, chloroform-d): δ 7.92(s,1H),7.72(d, J ═ 8.3Hz,1H),7.14(d, J ═ 8.2Hz,1H), 6.64-6.55 (m,2H),6.36(dd, J ═ 16.8,1.8Hz,1H),5.78(dd, J ═ 10.5,1.9Hz,1H),5.32(s,2H),3.96(s,6H), 3.95-3.63 (m, 8H).

Example 224- (2-Acetocarbethoxy) -6-chloro-2- (3, 5-dimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one (V-9)

Compound V-9 was prepared according to the method of example 10. Silica gel column chromatography solvent ratio petroleum ether/ethyl acetate 15/1 to 6/1, white solid (76.8mg, 61.6%), melting point: 163 ℃ and 165 ℃. Purity: 96 percent.¹H NMR (500MHz, chloroform-d): δ 8.38(s,1H),7.87(d, J ═ 8.2Hz,1H),7.23(d, J ═ 8.2Hz,1H),7.15(d, J ═ 2.2Hz,2H),6.27(t, J ═ 2.2Hz,1H),5.21(s,2H),4.27(q, J ═ 7.1Hz,2H),3.84(s,6H),1.32(t, J ═ 7.1Hz,3H).

Example 234- (2-Acetocarbethoxy) -6-chloro-2- (2, 6-dichlorophenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one (V-10)

Compound V-10 was prepared according to the method of example 10. Silica gel column chromatography solvent ratio petroleum ether/ethyl acetate 15/1 to 6/1, white solid (15.6mg, 9.2%), melting point: 156 ℃ and 157 ℃. Purity: 97 percent.¹H NMR (500MHz, chloroform-d): δ 7.90(s,1H),7.72(d, J ═ 8.2Hz,1H),7.44(d, J ═ 8.1Hz,2H),7.25(t, J ═ 8.1Hz,1H),7.17(d, J ═ 8.3Hz,1H),5.23(s,2H),4.29(q, J ═ 7.1Hz,2H),1.33(t, J ═ 7.1Hz,3H).

Example 244- (2-Acetocarbethoxy) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -2- (3,4, 5-trimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one (V-11) Synthesis method

Compound V-11 was prepared according to the method of example 11. Silica gel column chromatography solvent ratio petroleum ether/ethyl acetate/triethylamine 10/10/1, yellow solid (39.8mg, 65.9%), melting point: 181 ℃ and 183 ℃. Purity: 98 percent.¹H NMR (500MHz, chloroform-d): δ 8.11(s,1H),7.66(d, J ═ 8.6Hz,1H), 7.29-7.27 (m,2H),7.23(s,2H), 6.97-6.90 (m,2H),6.62(d, J ═ 8.6Hz,1H),6.49(s,1H),5.20(s,2H),4.24(q, J ═ 7.1Hz,2H),3.91(s,6H),3.84(s,3H), 3.26-3.20 (t, J ═ 5.0Hz,4H),2.66(t, J ═ 5.0Hz,4H),2.41(s,3H),1.28(t, J ═ 7.1Hz,3H).

EXAMPLE 254- (2-Acetocarbethoxy) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -2- (3, 5-dimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one (V-12) synthesis method

Compound V-12 was prepared according to the method of example 11. Silica gel column chromatography solvent ratio petroleum ether/ethyl acetate/triethylamine 10/10/1, yellow solid (47.7mg, 69.6%), melting point:177 and 179 ℃. Purity: 96 percent.¹H NMR (500MHz, chloroform-d): δ 8.14(s,1H),7.71(d, J ═ 8.6Hz,1H), 7.29-7.25 (m,2H),7.15(d, J ═ 2.2Hz,2H), 6.96-6.90 (m,2H),6.60(d, J ═ 8.6Hz,1H),6.45(s,1H),6.21(t, J ═ 2.2Hz,1H),5.19(s,2H),4.23(q, J ═ 7.1Hz,2H),3.83(s,6H),3.21(t, J ═ 5.0Hz,4H), 2.64-2.59 (t, J ═ 5.0, 4H),2.37(s,3H),1.28(t, J ═ 7, 3H), 1.7, 3H.

Example 264- (2-Acetocarbethoxy) -6-chloro-2- (2, 6-dichloro-3, 5-dimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one (V-13)

Compound V-13 was prepared according to the method of example 10. Silica gel column chromatography solvent ratio petroleum ether/ethyl acetate 8/1 to 2/1, white solid (10.0mg, 7.0%), melting point: 161-163 ℃. Purity: 97 percent.¹H NMR (500MHz, chloroform-d): δ 7.84(s,1H),7.65(d, J ═ 8.3Hz,1H),7.08(d, J ═ 8.2Hz,1H),6.53(s,1H),5.15(s,2H),4.21(q, J ═ 7.1Hz,2H),3.90(s,6H),1.25(t, J ═ 7.1Hz,3H).

Example 274- (2-Acetocarbethoxy) -6-phenylamino-2- (3, 5-dimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one (V-14)

Compound V-14 was prepared according to the method of example 11. Silica gel column chromatography solvent ratio petroleum ether/ethyl acetate 8/1 to 3/1, white solid (26.0mg, 57.3%), melting point: 174 ℃ and 176 ℃. Purity: 96 percent.¹H NMR (500MHz, chloroform-d): δ 8.10(s,1H),7.69(d, J ═ 8.6Hz,1H), 7.35-7.30 (m,2H), 7.30-7.23 (m,2H),7.08(d, J ═ 2.2Hz,2H),6.99(tt, J ═ 7.3,1.2Hz,1H),6.64(d, J ═ 8.6Hz,1H),6.55(s,1H),6.15(t, J ═ 2.2Hz,1H),5.14(s,2H),4.16(q, J ═ 7.1Hz,2H),3.76(s,6H),1.25(t, J ═ 7.1Hz,3H).

EXAMPLE 284- (2-Acetocarbethoxy) -6-phenylamino-2- (2, 6-dichloro-3, 5-dimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one (V-15) synthesis method

Compound V-15 was prepared according to the method of example 11. Silica gel column chromatography solvent ratio petroleum ether/ethyl acetate 3/1 to 2/1, yellow solid (25.8mg, 77.1%), melting point: 168 ℃ and 170 ℃. Purity: 99 percent.¹H NMR (400MHz, chloroform-d): delta 7.69(s,1H),7.60(d, J ═ 8.6Hz,1H), 7.41-7.36 (m,2H), 7.36-7.26 (m,2H),7.04(t ═ tt,J＝7.2,1.3Hz,1H),6.72(s,1H),6.60(d,J＝8.7Hz,1H),6.52(s,1H),5.23(s,2H),4.24(q,J＝7.1Hz,2H),3.91(s,6H),1.28(t,J＝7.2Hz,3H)。

Example 294- (2-Acetocarbethoxy) -6-phenylamino-2- (2, 6-dichlorophenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one (V-16)

Compound V-16 was prepared according to the method of example 11. Silica gel column chromatography solvent ratio petroleum ether/ethyl acetate 8/1 to 5/1, yellow solid (16.8mg, 49.5%), melting point: 161-162 ℃. Purity: 96 percent.¹H NMR (400MHz, chloroform-d): δ 7.66(s,1H),7.62(d, J ═ 8.6Hz,1H), 7.43-7.29 (m,6H), 7.24-7.14 (m,1H),7.06(tt, J ═ 7.2,1.3Hz,1H),6.64(d, J ═ 8.6Hz,1H),6.60(s,1H),5.23(s,2H),4.24(q, J ═ 7.1Hz,2H),1.28(t, J ═ 7.1Hz,3H).

Example Synthesis of 304- (2-Acetocarbethoxy) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -2- (2, 6-dichloro-3, 5-dimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one (V-17)

Compound V-17 was prepared according to the method of example 11. Silica gel column chromatography solvent ratio dichloromethane/methanol 80/1 to 20/1, yellow solid (20.8mg, 48.6%), melting point: 173 ℃ and 175 ℃. Purity: 98 percent.¹H NMR (500MHz, chloroform-d): δ 7.66(s,1H),7.56(d, J ═ 8.6Hz,1H),7.26 to 7.23(m,2H),6.93 to 6.87(m,2H),6.55 to 6.48(m,3H),5.21(s,2H),4.24(q, J ═ 7.1Hz,2H),3.93(s,6H),3.22 to 3.17(m,4H),2.62(t, J ═ 5.0Hz,4H),2.37(s,3H),1.28(t, J ═ 7.1Hz,3H).

EXAMPLE 31 hydrochloride salt of the Compound V-17 4- (2-Acetocarbethoxy) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -2- (2, 6-dichloro-3, 5-dimethoxyphenylamino) pyrido [2,3-B ] pyrazin-3 (4H) -one hydrochloride (V-17A), 4- (2-Acetocarbethoxy) -6- (4- (4-methylpiperazin-1-yl) phenylamino) -2- (2, 6-dichloro-3, 5-dimethoxyphenylamino) pyrido [2,3-B ] pyrazin-3 (4H) -one Quaternary ammonium salt (V-17B) and 4- (2-Acetocarbethoxy) -6- (4- (4-methylpiperazin-1-yl) pyrazin-3 (4H) -one Quaternary ammonium salt (V-17B) Preparation of monohydrate of phenylpiperazin-1-yl) phenylamino) -2- (2, 6-dichloro-3, 5-dimethoxyphenylamino) pyrido [2,3-b ] pyrazin-3 (4H) -one (V-17C)

a) Preparation of hydrochloride salt V-17A of Compound V-17

At room temperatureThen, compound v-17 (64.3mg, 0.1mmol) was dissolved in anhydrous methanol (2mL), and a saturated methanolic hydrogen chloride solution (2mL) was slowly added dropwise under ice bath, after the solvent was evaporated under reduced pressure, ether was added with stirring to precipitate a yellow solid, which was filtered and washed with ether to obtain compound v-17 hydrochloride v-17A as a yellow solid 61.8mg, yield: 91 percent. ESI-MS M/z 676.17[ M ]⁺]。

b) Preparation of Quaternary ammonium salt V-17B of Compound V-17

At room temperature, compound v-17 (64.3mg, 0.1mmol) was dissolved in absolute ethanol (2mL), and equal equivalents of sodium hydroxide, potassium iodide and methyl iodide were added, after heating under reflux overnight, the solvent was dried under reduced pressure, and the crude product was purified by recrystallization from acetone to give compound v-17 quaternary ammonium salt v-17B as a yellow solid 31.4mg, yield: 40.1 percent. ESI-MS M/z 782.12[ M ]⁺]。

c) Preparation of monohydrate V-17C of Compound V-17

At room temperature, dissolving the compound V-17 (64.3mg, 0.1mmol) in 1N hydrochloric acid solution, slowly dropwise adding petroleum ether under stirring until the solution becomes turbid, standing at room temperature until no crystal is separated out, and filtering to obtain the monohydrate V-17C of the compound V-17. Elemental analysis (elem. anal.) C, 54.55%; h, 5.34%; cl, 10.73; n, 14.84%; o,14.53 percent.

According to analogous procedures to those described in this example, pharmaceutically acceptable salts, solvates, hydrates, or crystal forms of compound v of the present invention, including salts with organic acids such as propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, and the like; or forming salt with inorganic acid such as hydrochloric acid, phosphoric acid, sulfuric acid, hydrofluoric acid, hydrobromic acid, etc.; or a quaternary ammonium salt formed with a haloalkane, said haloalkane being a fluoro, chloro, bromo or iodo alkane.

Example 32: pharmaceutical composition

Compound V-17A 20g

140g of starch

Microcrystalline cellulose 60g

According to the conventional operation method, the 3 substances are physically and uniformly mixed and then are filled into a gelatin capsule to obtain 1000 capsules.

Example 33: pharmaceutical composition

50g of Compound V-17A

Starch 400g

Microcrystalline cellulose 200g

Example 34: experimental example for antiproliferative Activity

Experimental materials: KATO III type human gastric cancer cells (FGFR2 amplified) are semi-suspension cells and purchased from cell resource center of Shanghai Life science research institute of Chinese academy of sciences, and the culture solution conditions are RPMI1640+ 10% FBS + 1% double antibody. Huh-7 type human hepatoma cells (FGFR4 overexpression) are adherent cells and purchased from cell resource center of Shanghai Life science research institute of Chinese academy of sciences, and the culture medium conditions are DMEM + 10% FBS + 1% double antibody. The cells were cultured in a medium containing 5% CO₂The culture was carried out routinely in a 37 ℃ saturated humidity incubator. The cell counting reagent (CCK-8) is Japanese colleague, and is stored at 4 deg.C in a refrigerator in dark place, and is packaged.

The experimental method comprises the following steps: a) 5000 KATO III human gastric cancer cells in logarithmic growth phase are inoculated in a 96-well plate, each well of culture solution is 100 mu L, after the cells are stabilized for one day, 50 mu L of drug-containing culture solution is added, each concentration is provided with three multiple wells, and dimethyl sulfoxide solution with corresponding concentration is set as a control group. After the drug is added, the 96-well plate is put into an incubator to be cultured for 4 days. After 4 days, the 96-well plate was removed, 10. mu.L of CCK-8 solution was added to each well, the incubation was continued in the incubator for 1-4 hours, and the absorbance of each well was measured in a microplate reader at a wavelength of 450 nM.

b) 6000 Huh-7 human hepatoma cells in logarithmic growth phase are inoculated in a 96-well plate, each well is 100 mu L of culture solution, then the cells are cultured in an incubator for one day until the cells are stable, 50 mu L of drug-containing culture solution is added, three multiple wells are arranged for each concentration, and dimethyl sulfoxide solution with corresponding concentration is set as a control group. After the drug is added, the 96-well plate is put into an incubator to be cultured for 3 days. After 3 days, the 96-well plate was removed, 10. mu.L of CCK-8 solution was added to each well, the incubation was continued in the incubator for 1-4 hours, and the absorbance of each well was measured in a microplate reader at a wavelength of 450 nM.

The inhibition rate of the compound on the proliferation of tumor cells is calculated by the following formula:

inhibition (%) was (OD control well-OD administration well)/(OD control well-OD blank well) × 100%

OD control wells: culture solution, cells, dimethyl sulfoxide with corresponding concentration, and CCK-8

OD administration well: culture solution, cells, dimethyl sulfoxide and compound with corresponding concentration, and CCK-8

OD blank wells: culture solution, dimethyl sulfoxide with corresponding concentration, and CCK-8

IC₅₀The values were estimated by a four parameter method. All experimental results were from two independent experiments and the results are presented as mean ± standard deviation.

Results of antiproliferative activity test:

TABLE 1 inhibition of KATO III human gastric carcinoma cells by Compounds V-1 to V-17 (10. mu.M)

Numbering	Inhibition rate	Numbering	Inhibition rate	Numbering	Inhibition rate
						Ⅴ-1	90％	Ⅴ-7	89％	Ⅴ-13	43％
Ⅴ-2	52％	Ⅴ-8	44％	Ⅴ-14	9％
						Ⅴ-3	5％	Ⅴ-9	14％	Ⅴ-15	49％
Ⅴ-4	92％	Ⅴ-10	13％	Ⅴ-16	22％
						Ⅴ-5	53％	Ⅴ-11	93％	Ⅴ-17	95％
Ⅴ-6	36％	Ⅴ-12	95％

TABLE 2 partial compound on KATO III human gastric carcinoma cell IC₅₀Value of (. mu.M)

Numbering	IC₅₀(μM)	Numbering	IC₅₀(μM)	Numbering	IC₅₀(nM)
						Ⅴ-1	1.87±0.21	Ⅴ-11	0.55±0.09	FIIN2	0.62±0.14
Ⅴ-4	1.05±0.17	Ⅴ-12	0.33±0.12
						Ⅴ-7	1.31±0.30	Ⅴ-17	0.15±0.07

Note: n- [4- [ [3- (3, 5-dimethoxyphenyl) -3, 4-dihydro-7- [ [4- (4-methyl-1-piperazinyl) phenyl]Amino group]-2-oxopyrimidino [4,5-D]Pyrimidin-1 (2H) -yl]Methyl radical]Phenyl radical]-2-acrylamide; FGFR inhibitor (FIIN2) IC₅₀(literature value) 1.9 nM.

TABLE 3 inhibition of Huh-7 hepatoma cells by Compounds V-1 to V-17 (10. mu.M)

Numbering	Inhibition rate	Numbering	Inhibition rate	Numbering	Inhibition rate
						Ⅴ-1	84％	Ⅴ-7	56％	Ⅴ-13	5％
Ⅴ-2	45％	Ⅴ-8	7％	Ⅴ-14	×
						Ⅴ-3	×	Ⅴ-9	6％	Ⅴ-15	×
Ⅴ-4	55％	Ⅴ-10	3％	Ⅴ-16	43％
						Ⅴ-5	×	Ⅴ-11	91％	Ⅴ-17	93％
Ⅴ-6	10％	Ⅴ-12	85％

Note: and x represents no inhibition.

TABLE 4 partial compound on Huh-7 hepatoma cell IC₅₀Value of (. mu.M)

Numbering	IC₅₀(μM)	Numbering	IC₅₀(μM)	Numbering	IC₅₀(nM)
						Ⅴ-1	1.49±0.46	Ⅴ-7	5.23±1.20	Ⅴ-17	1.07±0.70
Ⅴ-2	6.45±0.97	Ⅴ-11	3.82±0.63	AZD4547	0.88±0.12
						Ⅴ-4	9.61±1.05	Ⅴ-12	2.05±0.59

Note: REL-N- [5- [2- (3, 5-Dimethoxyphenyl) ethyl]-1H-pyrazol-3-yl]-4- [ (3R,5S) -3, 5-dimethyl-1-piperazinyl]Benzamide (AZD4547) IC₅₀(literature value) ═ 0.28. mu.M.

Experimental results show that the compound has better antiproliferative activity on KATO III type human gastric cancer cells (FGFR2 amplification) and Huh-7 liver cancer cells (FGFR4 overexpression), and particularly the compound V-17 has IC (integrated Circuit) of the two cells₅₀The values were 0.15. mu.M and 1.07. mu.M, respectively. Also, R in the compounds of the present invention₁ R₂And R₃Have an important effect on the antiproliferative activity of the compounds. In general, the compound is an inhibitor of FGFR2 and FGFR4, and has better drug development prospect.

Claims

1. A pyrido[2,3-b]pyrazin-3(4H)-one derivative, wherein the derivative has the following general structural formula V:

in,

R ₁ is selected from 2-ethyl acetate, 2-(4-acryloylpiperazin-1-yl)-2-oxoethyl;

R ₂ is selected from 3,5-dimethoxyphenyl, 2,6-dichlorophenyl, 3,4,5-trimethoxyphenyl, 2,6-dichloro-3,5-dimethoxyphenyl phenyl;

R ₂ is selected from chlorine, aniline, 4-(4-methylpiperazine)anilino, 2,6-dichloro-3,5-dimethoxyphenyl,

The derivatives include pharmaceutically acceptable salts, solvates, hydrates or crystalline forms thereof.

2. A pyrido[2,3-b]pyrazin-3(4H)-one derivative according to claim 1, characterized in that it is selected from the following compounds:

(1) 4-(2-(4-Acryloylpiperazin-1-yl)-2-oxoethyl)-6-anilino-2-(3,4,5-trimethoxyanilino)pyridine iso[2,3-b]pyrazin-3(4H)-one;

(2) 4-(2-(4-Acryloylpiperazin-1-yl)-2-oxoethyl)-6-chloro-2-(3,4,5-trimethoxyanilino)pyrido [2,3-b]pyrazin-3(4H)-one;

(3) 4-(2-(4-Acryloylpiperazin-1-yl)-2-oxoethyl)-6-(4-(4-methylpiperazin-1-yl)anilino)- 2-(3,4,5-Trimethoxyanilino)pyrido[2,3-b]pyrazin-3(4H)-one;

(4) 4-(2-(4-Acryloylpiperazin-1-yl)-2-oxoethyl)-2-(3,5-dimethoxyphenylamino)-6-(4-( 4-Methylpiperazin-1-yl)anilino)pyrido[2,3-b]pyrazin-3(4H)-one;

(5) 4-(2-(4-Acryloylpiperazin-1-yl)-2-oxoethyl)-6-chloro-2-(2,6-dichlorophenylamino)pyrido[2, 3-b]pyrazin-3(4H)-one;

(6) 4-(2-(4-Acryloylpiperazin-1-yl)-2-oxoethyl)-6-chloro-2-(3,5-dimethoxyanilino)pyrido[ 2,3-b]pyrazin-3(4H)-one;

(7) 4-(2-(4-Acryloylpiperazin-1-yl)-2-oxoethyl)-6-phenylamino-2-(3,5-dimethoxyphenylamino)pyrido [2,3-b]pyrazin-3(4H)-one;

(8) 4-(2-(4-Acryloylpiperazin-1-yl)-2-oxoethyl)-6-chloro-2-(2,6-dichloro-3,5-dimethoxy phenylamino)pyrido[2,3-b]pyrazin-3(4H)-one;

(9) 4-(2-acetoethyl)-6-chloro-2-(3,5-dimethoxyanilino)pyrido[2,3-b]pyrazin-3(4H)-one ;

(10) 4-(2-acetoethyl)-6-chloro-2-(2,6-dichlorophenylamino)pyrido[2,3-b]pyrazin-3(4H)-one;

(11) 4-(2-acetoethyl ester)-6-(4-(4-methylpiperazin-1-yl)anilino)-2-(3,4,5-trimethoxyanilino) pyrido[2,3-b]pyrazin-3(4H)-one;

(12) 4-(2-acetoethyl)-6-(4-(4-methylpiperazin-1-yl)anilino)-2-(3,5-dimethoxyanilino)pyridine iso[2,3-b]pyrazin-3(4H)-one;

(13) 4-(2-acetoethyl)-6-chloro-2-(2,6-dichloro-3,5-dimethoxyanilino)pyrido[2,3-b]pyrazine -3(4H)-one;

(14) 4-(2-acetoethyl)-6-anilino-2-(3,5-dimethoxyanilino)pyrido[2,3-b]pyrazine-3(4H)- ketone;

(15) 4-(2-acetoethyl)-6-phenylamino-2-(2,6-dichloro-3,5-dimethoxyphenylamino)pyrido[2,3-b]pyridine oxazin-3(4H)-one;

(16) 4-(2-acetoethyl)-6-phenylamino-2-(2,6-dichlorophenylamino)pyrido[2,3-b]pyrazin-3(4H)-one;

(17) 4-(2-acetoethyl ester)-6-(4-(4-methylpiperazin-1-yl)phenylamino)-2-(2,6-dichloro-3,5-di Methoxyanilino)pyrido[2,3-b]pyrazin-3(4H)-one;

and pharmaceutically acceptable salts, solvates, hydrates or crystalline forms thereof of the above specific compounds.

3. A pyrido[2,3-b]pyrazin-3(4H)-one derivative according to claim 1, wherein the pharmaceutically acceptable salt is of general formula V The represented compound forms the salt with organic acid or inorganic acid, or the quaternary ammonium salt formed with haloalkane, and described organic acid is selected from propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, Malic acid, tartaric acid, citric acid, hydrochloric acid, phosphoric acid, sulfuric acid, hydrofluoric acid, hydrobromic acid are selected as the inorganic acid, and the haloalkane is fluorine, chlorine, bromine or iodoalkane.

4. A pyrido[2,3-b]pyrazin-3(4H)-one derivative according to claim 1 and a pharmaceutically acceptable salt, solvate, hydrate or crystal form thereof in The invention relates to an application in preparing a medicine for treating tumor-related diseases caused by abnormal activation of FGFR.

The application according to claim 4, wherein the tumor-related diseases are gastric cancer and liver cancer.