CN115385912A

CN115385912A - Pyrazinopyrazinoquinolinone derivatives, preparation method and medical application thereof

Info

Publication number: CN115385912A
Application number: CN202210568555.0A
Authority: CN
Inventors: 陈友喜; 程超英; 向清; 叶成; 钱文建; 陈磊
Original assignee: Zhejiang Hisun Pharmaceutical Co Ltd; Shanghai Aryl Pharmtech Co Ltd
Current assignee: Zhejiang Hisun Pharmaceutical Co Ltd; Shanghai Aryl Pharmtech Co Ltd
Priority date: 2021-05-25
Filing date: 2022-05-24
Publication date: 2022-11-25

Abstract

The invention relates to pyrazino-quinolinone derivatives, a preparation method thereof and application thereof in medicines. In particular, the invention relates to pyrazino-quinolinone derivatives, a preparation method thereof, pharmaceutically acceptable salts thereof, and applications thereof as therapeutic agents, particularly as KRAS GTP enzyme inhibitors.

Description

Pyrazinopyrazinoquinolinone derivatives, preparation method and medical application thereof

Technical Field

The invention relates to a pyrazino-quinolinone derivative, a preparation method thereof, a pharmaceutical composition containing the derivative and application of the derivative as a therapeutic agent, in particular as a K-Ras GTP enzyme inhibitor.

Background

RAS represents a group of closely related monomeric globular proteins (21 kDa molecular weight) of 189 amino acids that are associated with the plasma membrane and bind GDP or GTP. Under normal developmental or physiological conditions, RAS is activated upon receipt of growth factors and various other extracellular signals, and is responsible for regulating functions such as cell growth, survival, migration and differentiation. RAS functions as a molecular switch, with the on/off state of the RAS protein determined by nucleotide binding, the active signaling conformation binding GTP, and the inactive conformation binding GDP. When the RAS comprises bound GDP, it is in a dormant or quiescent or off state and is "inactive". RAS is induced to convert bound GDP to GTP when cells are exposed to certain growth-promoting stimuli to respond. As GTP is bound, RAS is "on" and is able to interact with and activate other proteins (their "downstream targets"). The RAS protein itself has a very low intrinsic ability to hydrolyze GTP back to GDP and thereby turn itself off. The conversion of RAS to shutdown requires an exogenous protein called Gtpase Activating Proteins (GAPs), which interact with RAS and greatly facilitate the conversion of GTP to GDP. Any mutation in the RAS that affects its ability to interact with GAPs or convert GTP back to GDP will result in prolonged activation of the protein and thus produce a prolonged signal to the cell that tells it to continue growth and division. These signals can therefore allow cells to grow and divide, and overactivated RAS signal transduction may ultimately lead to cancer.

Structurally, RAS proteins contain a G domain responsible for the enzymatic activity of RAS-guanine nucleotide binding and hydrolysis (gtpase reaction). It also includes a C-terminal extension containing a so-called CAAX box, which can be post-translationally modified and targets the protein to the membrane. The G domain is approximately 21-25kDa in size and contains a phosphate binding loop (P-loop). The P-loop represents the capsular bag of bound nucleotides in the protein, and this is a rigid part of the domain with conserved amino acid residues that are essential for nucleotide binding and hydrolysis (glycine 12, threonine 26 and lysine 16). The G domain also contains the so-called switch I (residues 30-40) and switch II (residues 60-76), which are both dynamic parts of the protein, often denoted as "spring-loaded" mechanisms due to the ability of the dynamic part to switch between resting and loaded states. The major interaction is the hydrogen bond formed by threonine-35 and glycine-60 with the gamma-phosphate of GTP, which maintains the switch I and switch II regions in their active conformations, respectively. After hydrolysis of GTP and release of phosphate, both relax into the inactive GDP conformation.

Among RAS family members, oncogenic mutations are most common in KRAS (85%), whereas NRAS (12%) and HRAS (3%) are less common. KRAS mutations are prevalent in three major cancer types in the united states: pancreatic (95%), colorectal (45%) and lung (25%), KRAS mutations were also found in other cancer types including multiple myeloma, uterine, cholangiocarcinoma, gastric, bladder, diffuse large B-cell lymphoma, rhabdomyosarcoma, squamous cell carcinoma of the skin, cervical, testicular germ cell carcinoma, etc., while rarely (< 2%) in breast, ovarian and brain cancers. In non-small cell lung cancer (NSCLC), KRAS G12C is the most common mutation, accounting for nearly half of all KRAS mutations, followed by G12V and G12D. In non-small cell lung cancer, the increase in frequency of specific allelic mutations is mostly due to classical smoking-induced canonical mutations (G: C to T: A substitutions), resulting in KRAS G12C (GGT to TGT) and G12V (GGT to GTT) mutations.

Large genomics studies indicate that lung cancer KRAS mutations, including G12C, are mutually exclusive from other known driver oncogenic mutations in NSCLC, including EGFR, ALK, ROS1, RET, and BRAF, indicating the uniqueness of KRAS mutations in lung cancer. While, KRAS mutations often occur simultaneously with certain co-mutations, such as STK11, KEAP1 and TP53, which cooperate with the mutated RAS to transform cells into highly malignant and aggressive tumor cells.

The three RAS oncogenes constitute the most frequently mutated gene family in human cancers. Disappointingly, despite over thirty years of research efforts, there is still no clinically effective anti-RAS therapy, and the use of small molecules to target this gene is a challenge. Thus, there is an urgent need in the art for small molecules for targeting and utilizing RAS (e.g., K-RAS, H-RAS and/or N-RAS) to treat various diseases, such as cancer.

At present, the competition of clinical development of KRAS inhibitors is intense at home and abroad, wherein the KRAS enzyme inhibitor MRTX-849 developed by Mirati Therapeutics Inc. enters the second stage of clinic and is used for preventing and treating diseases such as advanced solid tumors, metastatic colorectal cancer, metastatic non-small cell lung cancer and the like. There are also other KRAS inhibitors in the research including AMG-510 (Amgen Inc, phase 3). Early clinical studies showed that KRAS inhibitors significantly controlled and alleviated disease progression in non-small cell lung cancer patients and significantly reduced tumor size in patients with advanced lung cancer and colorectal cancer. A series of KRAS inhibitor patent applications have been published so far, including WO2020047192, WO2019099524 and WO2018217651, etc., and research and application of KRAS inhibitors have made some progress, but the increased space is still huge, and there is still a need to continue research and development of new KRAS inhibitors.

Disclosure of Invention

The invention aims to provide a pyrazino-quinolinone derivative shown in the following structure:

or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof.

Note: if there is a difference between a drawn structure and a given name for that structure, the drawn structure will be given more weight.

In another aspect, the present invention provides a pharmaceutical composition comprising an effective amount of a compound of the present invention, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or combination thereof.

In another aspect, the present invention provides a method of inhibiting KRAS gtpase, wherein the method comprises administering to a patient a pharmaceutical composition comprising an effective amount of a compound of the present invention, or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein the KRAS gtpase is preferably KRAS G12C enzyme, and a pharmaceutically acceptable carrier, excipient or combination thereof.

The invention also provides the use of a compound of the invention, or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for the manufacture of a medicament for the treatment of a disease mediated by KRAS mutation, wherein the disease mediated by KRAS mutation is selected from cancer, wherein the cancer is selected from pancreatic cancer, colorectal cancer, lung cancer, multiple myeloma, uterine cancer, cholangiocarcinoma, gastric cancer, bladder cancer, diffuse large B-cell lymphoma, rhabdomyosarcoma, cutaneous squamous cell carcinoma, cervical cancer, testicular germ cell cancer, preferably pancreatic cancer, colorectal cancer and lung cancer; wherein said lung cancer is preferably non-small cell lung cancer, and wherein said KRAS mutation is preferably a KRAS G12C mutation.

In another aspect, the present invention provides a use of a compound of the present invention, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for the preparation of a KRAS gtpase inhibitor, preferably a KRAS G12C inhibitor.

Another aspect of the present invention relates to a method for preventing and/or treating a KRAS mutation-mediated disease, comprising administering to a patient a therapeutically effective amount of a compound of the present invention, or a tautomer, mesomer, racemate, enantiomer, or diastereomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, wherein the KRAS mutation is preferably a KRAS G12C mutation.

The invention also provides an application of the compound or the stereoisomer, the tautomer or the pharmaceutically acceptable salt thereof or the pharmaceutical composition thereof in preparing a medicament for treating cancers, wherein the cancers are selected from pancreatic cancer, colorectal cancer, lung cancer, multiple myeloma, uterine cancer, bile duct cancer, gastric cancer, bladder cancer, diffuse large B-cell lymphoma, rhabdomyosarcoma, squamous cell carcinoma of skin, cervical cancer and testicular germ cell cancer, and preferably pancreatic cancer, colorectal cancer and lung cancer; wherein the lung cancer is preferably non-small cell lung cancer.

The pharmaceutical formulations of the present invention may be administered topically, orally, transdermally, rectally, vaginally, parenterally, intranasally, intrapulmonary, intraocularly, intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intradermally, intraperitoneally, subcutaneously, subcortically, or by inhalation. The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, dragees, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.

The formulations of the present invention are suitably presented in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form generally refers to the amount of compound that produces a therapeutic effect.

Dosage forms for topical or transdermal administration of the compounds of the present invention may include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

When the compounds of the present invention are administered to humans and animals as pharmaceuticals, the compounds can be provided alone or in pharmaceutical compositions containing the active ingredient in combination with a pharmaceutically acceptable carrier, e.g., from 0.1% to 99.5% (more preferably, from 0.5% to 90%) of the active ingredient.

Examples of pharmaceutically acceptable carriers include, but are not limited to: (1) sugars such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) Cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered gum tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) Oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) Polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solution; (21) Cyclodextrins, e.g., targeting ligands attached to nanoparticles, e.g., accurins tm; and (22) other non-toxic compatible materials used in pharmaceutical formulations, such as polymer-based compositions.

Examples of pharmaceutically acceptable antioxidants include, but are not limited to: (1) Water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) Oil-soluble antioxidants, such as ascorbyl palmitate, butylated Hydroxyanisole (BHA), butylated Hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Solid dosage forms (e.g., capsules, dragee pills, dragees, powders, granules, and the like) can include one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) Fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) Binding agents, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) Disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) dissolution retarders, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) Humectants, such as cetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) Lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof; and (10) a colorant. Liquid dosage forms may include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents; solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum oxyhydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Ointments, pastes, creams and gels may also contain, in addition to the active compound, excipients, for example animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can also contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder or mixtures of these substances. The spray may contain other conventional propellants such as chlorofluorohydrocarbons, and volatile unsubstituted hydrocarbons such as butane and propane.

Detailed description of the invention

Unless stated to the contrary, some of the terms used in the specification and claims of the present invention are defined as follows:

the compounds of the invention may contain asymmetric or chiral centers and thus exist in different stereoisomeric forms. It is contemplated that all stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers and atropisomers (atropisomers) and geometric (conformational) isomers and mixtures thereof, such as racemic mixtures, are within the scope of the present invention.

Unless otherwise indicated, all isomers (e.g., diastereomers, enantiomers, and atropisomers and geometric (conformational) isomeric forms; e.g., R and S configurations at each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers) of the structures are also encompassed by the structures described herein.

"Boc" refers to tert-butoxycarbonyl.

"pharmaceutically acceptable salts" refers to certain salts of the above compounds which retain their biological activity and which are suitable for pharmaceutical use. Pharmaceutically acceptable salts of the compounds may be metal salts, amine salts with suitable acids.

"pharmaceutical composition" means a mixture containing one or more compounds described herein, or a physiologically acceptable salt or prodrug thereof, in admixture with other chemical components, as well as other components such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient, and exert biological activity.

Detailed Description

The present invention will be further described with reference to the following examples, which are not intended to limit the scope of the present invention.

Examples

The examples show the preparation of representative compounds represented by formula (I) and the associated structural identification data. It must be noted that the following examples are intended to illustrate the invention and not to limit it. ¹ HNMR spectra were obtained using a Bruker instrument (400 MHz) and chemical shifts are expressed in ppm. Tetramethylsilane internal standard (0.00 ppm) was used. ¹ Representation method of HNMR: s = singlet, d = doublet, t = triplet, m = multiplet, br = broadened, dd = doublet of doublet, dt = doublet of triplet. If a coupling constant is provided, it is in Hz.

The mass spectrum is measured by an LC/MS instrument, and the ionization mode can be ESI or APCI.

The thin layer chromatography silica gel plate adopts HSGF254 of tobacco yellow sea or GF254 of Qingdao, the specification of the silica gel plate used by Thin Layer Chromatography (TLC) is 0.15 mm-0.2 mm, and the specification of the thin layer chromatography separation and purification product is 0.4 mm-0.5 mm.

The column chromatography generally uses 200-300 mesh silica gel of the Tibet Huanghai silica gel as a carrier.

In the following examples, unless otherwise indicated, all temperatures are in degrees celsius and unless otherwise indicated, the starting materials and reagents are commercially available or synthesized according to known methods, and are used without further purification, unless otherwise indicated, commercially available manufacturers include, but are not limited to, shanghai haohnhong biomedical science and technology limited, shanghai shaoshimo reagents limited, shanghai beide medical science and technology limited, saen chemical technology (shanghai) limited, shanghai ling medical science and technology limited, and the like.

CD ₃ OD: deuterated methanol.

CDCl ₃ : deuterated chloroform.

DMSO-d ₆ : deuterated dimethyl sulfoxide.

In the examples, the solution in the reaction is an aqueous solution unless otherwise specified.

Purifying the compound using an eluent system selected from the group consisting of column chromatography and thin layer chromatography, wherein the system is selected from the group consisting of: a: petroleum ether and ethyl acetate systems; b: dichloromethane and methanol systems; c: dichloromethane and ethyl acetate system, D: dichloromethane and ethanol system, E: ethyl acetate and tetrahydrofuran, wherein the volume ratio of the solvent is different according to the polarity of the compound, or a small amount of acidic or basic reagent such as acetic acid or triethylamine can be added for carrying out the conditions.

Room temperature: 20-30 ℃.

Example 1

(2R, 4aR) -10- (2-amino-5, 7-difluorobenzo [ d ] thiazol-4-yl) -11-chloro-9-fluoro-3- (2-fluoroacryloyl) -2,6-dimethyl-2,3, 4a-tetrahydro-1H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinolin-5 (6H) -one (2R, 4aR) -10- (2-amino-5, 7-difluorobenzo [ d ] thiazol-4-yl) -11-chloro-9-fluoro-3- (2-fluoroacryloyl) -2,6-dimethyl-2,3,4,4a-tetrahydro-1H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinolinone (6H) -5H-quinolinone (2R, 4aR) -10- (2-amino-5, 7-difluorobenzo [ d ] thiazol-4-yl) -11-chloro-9-fluoro-3- (2-fluoroacryloyl) -2,6-dimethyl-2,3,4,4a-tetrahydro-1H-pyrazino [2, 5-c ] quinolinone (6H) -one

First step of

1-(tert-butyl)3-methyl

(3R, 6R) -4- (7-bromo-6-chloro-8-fluoro-3-nitroquinolin-4-yl) -6-methylpiperazine-1,3-dicarboxylate1- (tert-butyl) 3-methyl (3R, 6R) -4- (7-bromo-6-chloro-8-fluoro-3-nitroquinolin-4-yl) -6-methylpiperazine-1,3-dicarboxylate

1- (tert-butyl) 3-methyl (3R, 6R) -6-methylpiperazine-1,3-dicarboxylate 1b (911.87mg, 3.53mmol, prepared according to the published patent WO 2019110751), 7-bromo-4,6-dichloro-8-fluoro-3-nitroquinoline 1a (527.47mg, 1.55mmol, prepared according to the published patent WO 2019110751) was added to acetonitrile (5 mL) and allowed to warm to 80 ℃ for reaction for 3 hours. Ethyl acetate (20 mL) and water (20 mL) were added to the reaction system, a saturated sodium carbonate solution was added dropwise to adjust the pH to neutrality, the reaction mixture was separated, the organic phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and the obtained residue was separated and purified by silica gel column chromatography (eluent: system A) to give the product 1- (tert-butyl) 3-methyl (3R, 6R) -4- (7-bromo-6-chloro-8-fluoro-3-nitroquinolin-4-yl) -6-methylpiperazine-1,3-dicarboxylate 1c (1.6 g, 2.85mmol), yield: 96.82 percent.

MS m/z(ESI):562.1[M+1] ⁺

Second step of

tert-butyl

(2R,4aR)-10-bromo-11-chloro-9-fluoro-2-methyl-5-oxo-1,2,4,4a,5,6-hexahydro-3H-pyrazino[1',2':4,5]pyrazino[2,3-c]quinoline-3-carboxylate

(2R, 4aR) -10-bromo-11-chloro-9-fluoro-2-methyl-5-oxo-1, 2,4,4a,5, 6-hexahydro-3H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinoline-3-carboxylic acid tert-butyl ester

1- (tert-butyl) 3-methyl (3R, 6R) -4- (7-bromo-6-chloro-8-fluoro-3-nitroquinolin-4-yl) -6-methylpiperazine-1,3-dicarboxylate 1c (3.3g, 5.87mmol), ammonium chloride (1.57g, 29.37mmol) and iron powder (1.64g, 29.37mmol) were dissolved in a mixed solvent of methanol (20 mL) and water (5 mL), and heated to 80 ℃ for reaction for 3 hours. After the reaction was completed, the reaction was filtered while hot, and the filtrate was concentrated under reduced pressure to obtain a crude product of (2r, 4ar) -10-bromo-11-chloro-9-fluoro-2-methyl-5-oxo-1, 2,4,4a,5, 6-hexahydro-3H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinoline-3-carboxylic acid tert-butyl ester 1d (2.9g, 5.80mmol), yield: 98.79 percent.

MS m/z(ESI):499.0[M+1] ⁺

The third step

tert-butyl

(2R,4aR)-10-bromo-11-chloro-9-fluoro-2,6-dimethyl-5-oxo-1,2,4,4a,5,6-hexahydro-3H-pyrazino[1',2':4,5]pyrazino[2,3-c]quinoline-3-carboxylate

(2R, 4aR) -10-bromo-11-chloro-9-fluoro-2, 6-dimethyl-5-oxo-1, 2,4,4a,5, 6-hexahydro-3H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinoline-3-carboxylic acid tert-butyl ester

Tert-butyl (2R, 4aR) -10-bromo-11-chloro-9-fluoro-2-methyl-5-oxo-1, 2,4,4a,5, 6-hexahydro-3H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinoline-3-carboxylate 1d (2.9g, 5.80mmol) was added to N, N-dimethylformamide (10 mL), iodomethane (1.65g, 11.61mmol, 722.50. Mu.L), potassium carbonate (2.41g, 17.41mmol) were added in this order, and the reaction was allowed to proceed overnight at room temperature. The system was extracted with ethyl acetate (50 mL) and water (50 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the obtained residue was separated and purified by silica gel column chromatography (eluent: system a) to give the product (2r, 4 ar) -10-bromo-11-chloro-9-fluoro-2, 6-dimethyl-5-oxo-1, 2, 4a,5, 6-hexahydro-3H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinoline-3-carboxylic acid tert-butyl ester 1e (1.8g, 3.50mmol), yield: 60.37 percent.

MS m/z(ESI):513.0[M+1] ⁺

The fourth step

tert-butyl

(2R,4aR)-10-(2-((tert-butoxycarbonyl)amino)-5,7-difluorobenzo[d]thiazol-4-yl)-11-chloro-9-fluoro-2,6-dimethyl-5-oxo-1,2,4,4a,5,6-hexahydro-3H-pyrazino[1',2':4,5]pyrazino[2,3-c]quinoline-3-carboxylate

(2R, 4aR) -10- (2- ((tert-butoxycarbonyl) amino) -5,7-difluorobenzo [ d ] thiazol-4-yl) -11-chloro-9-fluoro-2, 6-dimethyl-5-oxo-1, 2,4,4a,5, 6-hexahydro-3H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinoline-3-carboxylic acid tert-butyl ester

(2- ((tert-butoxycarbonyl) amino) -5,7-difluorobenzo [ d ] thiazol-4-yl) boronic acid 1f (192.75mg,583.90. Mu. Mol, prepared according to published patent US 20200115375A 1), (2R,4aR) -10-bromo-11-chloro-9-fluoro-2,6-dimethyl-5-oxo-1,2,4,4a,5,6-hexahydro-3H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinoline-3-carboxylic acid tert-butyl ester 1e (200mg,389.27. Mu. Mol) was added to a mixed solvent of 1,4-dioxane (1 mL) and water (0.2 mL), tetrakis triphenylphosphine palladium (44.98mg,38.93. Mu. Mol), sodium carbonate (123.78mg,1.17mmol) was added, and the reaction was warmed to 110 ℃ for 3 hours. Ethyl acetate (10 mL) and water (10 mL) were added to the system, followed by liquid separation, ethyl acetate extraction (10 mL. Times.3), organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the obtained residue was separated and purified by preparative HPLC (separation column: AKZONOBEL Kromasil; 250X 21.2mm I.D.;5 μm; mobile phase A:0.05 TFA (+) H2O; mobile phase B: acetonitrile; flow rate: 20 mL/min), to obtain the product (2R, aR) -10- (2- ((t-butoxycarbonyl) amino) -5,7-difluorobenzo [ d ] thiazol-4-yl) -11-chloro-9-fluoro-2, 6-dimethyl-5-oxo-1, 2, 4a,5, 6-hexahydro-3H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinoline-3-carboxylic acid t-butyl ester 1g (69.52 mol), yield: 17.86 percent.

MS m/z(ESI):719.8[M+1] ⁺

The fifth step

(2R,4aR)-10-(2-amino-5,7-difluorobenzo[d]thiazol-4-yl)-11-chloro-9-fluoro-2,6-dimethyl-2,3,4,4a-tetrahydro-1H-pyrazino[1',2':4,5]pyrazino[2,3-c]quinolin-5(6H)-one

(2R, 4aR) -10- (2-amino-5, 7-difluorobenzo [ d ] thiazol-4-yl) -11-chloro-9-fluoro-2, 6-dimethyl-2,3,4,4a-tetrahydro-1H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinolin-5 (6H) -one

1g (50mg, 69.52. Mu. Mol) of (2R, 4aR) -10- (2- ((tert-butoxycarbonyl) amino) -5,7-difluorobenzo [ d ] thiazol-4-yl) -11-chloro-9-fluoro-2, 6-dimethyl-5-oxo-1, 2,4,4a,5, 6-hexahydro-3H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinoline-3-carboxylic acid tert-butyl ester was dissolved in dichloromethane (2 mL), and a solution of 1, 4-dioxane (4M, 1mL) of hydrochloric acid was added and reacted at room temperature overnight. After the reaction was completed, concentration was performed under reduced pressure to obtain a crude product (2r, 4ar) -10- (2-amino-5, 7-difluorobenzo [ d ] thiazol-4-yl) -11-chloro-9-fluoro-2, 6-dimethyl-2,3, 4a-tetrahydro-1H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinolin-5 (6H) -one 1H (30mg, 57.81 μmol), yield: 83.15 percent.

MS m/z(ESI):517.8[M+1] ⁺

The sixth step

(2R,4aR)-10-(2-amino-5,7-difluorobenzo[d]thiazol-4-yl)-11-chloro-9-fluoro-3-(2-fluoroacryloyl)-2,6-dimethyl-2,3,4,4a-tetrahydro-1H-pyrazino[1',2':4,5]pyrazino[2,3-c]quinolin-5(6H)-one

(2R, 4aR) -10- (2-amino-5, 7-difluorobenzo [ d ] thiazol-4-yl) -11-chloro-9-fluoro-3- (2-fluoropropenyl) -2,6-dimethyl-2,3,4,4a-tetrahydro-1H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinolin-5 (6H) -one

Reacting (2R, 4aR) -10- (2-amino-5, 7-difluorobenzo [ d ]]Thiazol-4-yl) -11-chloro-9-fluoro-2, 6-dimethyl-2,3, 4a-tetrahydro-1H-pyrazino [1',2':4,5]Pyrazino [2,3-c ] s]Quinolin-5 (6H) -one 1H (5mg, 9.64. Mu. Mol) was added to dichloromethane (2 mL)N, N-diisopropylethylamine (4.98mg, 38.54. Mu. Mol) was added, cooled to 0 ℃ and 2-fluoropropenylchloride (4.33mg, 39.92. Mu. Mol, prepared according to published patent WO 2020101736A 1) was added and stirred at room temperature for 0.5 hour. Ethyl acetate (10 mL) and water (10 mL) were added to the system, and the mixture was separated, extracted with ethyl acetate (10 mL. Times.3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the obtained residue was separated and purified by preparative HPLC (separation column: AKZONOBEL Kromasil; 250X 21.2mm I.D.;5 μm; mobile phase A:0.05% TFA + H2O, mobile phase B: acetonitrile; flow rate: 20 mL/min) to obtain the product (2R, 4aR) -10- (2-amino-5, 7-difluorobenzo [ d ] d]Thiazol-4-yl) -11-chloro-9-fluoro-3- (2-fluoroacryloyl) -2,6-dimethyl-2,3, 4a-tetrahydro-1H-pyrazino [1',2':4,5]Pyrazino [2,3-c ] s]Quinolin-5 (6H) -one 1 (1mg, 1.69. Mu. Mol), yield: 20 percent. MS m/z (ESI) 591.1[ deg. ] M +1 ]] ⁺

Examples 2,3 and 4

(2R,4aR)-10-(2-amino-5-fluorobenzo[d]thiazol-4-yl)-11-chloro-9-fluoro-3-(2-fluoroacryloyl)-2,6-dimethyl-2,3,4,4a-tetrahydro-1H-pyrazino[1',2':4,5]pyrazino[2,3-c]quinolin-5(6H)-one 2

(2R, 4aR) -10- (2-amino-5-fluorobenzo [ d ] thiazol-4-yl) -11-chloro-9-fluoro-3- (2-fluoropropenyl) -2,6-dimethyl-2,3,4,4a-tetrahydro-1H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinolin-5 (6H) -one 2

(2R,4aR,10R)-10-(2-amino-5-fluorobenzo[d]thiazol-4-yl)-11-chloro-9-fluoro-3-(2-fluoroacryloyl)-2,6-dimethyl-2,3,4,4a-tetrahydro-1H-pyrazino[1',2':4,5]pyrazino[2,3-c]quinolin-5(6H)-one 3

(2R, 4aR, 10R) -10- (2-amino-5-fluorobenzo [ d ] thiazol-4-yl) -11-chloro-9-fluoro-3- (2-fluoropropenyl) -2,6-dimethyl-2,3,4,4a-tetrahydro-1H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinolin-5 (6H) -one 3

(2R,4aR,10S)-10-(2-amino-5-fluorobenzo[d]thiazol-4-yl)-11-chloro-9-fluoro-3-(2-fluoroacryloyl)-2,6-dimethyl-2,3,4,4a-tetrahydro-1H-pyrazino[1',2':4,5]pyrazino[2,3-c]quinolin-5(6H)-one 4

(2R, 4aR, 10S) -10- (2-amino-5-fluorobenzo [ d ] thiazol-4-yl) -11-chloro-9-fluoro-3- (2-fluoropropoyl) -2,6-dimethyl-2,3,4,4a-tetrahydro-1H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinolin-5 (6H) -one 4

First step of

1-(3-fluoro-2-methoxyphenyl)thiourea

1- (3-fluoro-2-methoxyphenyl) thiourea

Adding 3-fluoro-2-methoxyaniline 2a (20g, 141.70mmol) into tetrahydrofuran (500 mL), slowly dropwise adding a tetrahydrofuran solution (100 mL) of benzoyl isothiocyanate 2b (23.13g, 141.70mmol), continuing to react for 3 hours at room temperature after dropwise adding, after monitoring that all raw materials are reacted into an intermediate by LCMS, adding water (80 mL) and sodium hydroxide (6.80g, 170.04mmol), and heating to 80 ℃ for reacting overnight. Cooled to room temperature, extracted with ethyl acetate (100 mL × 1), the organic phase washed with saturated brine (100 mL × 1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the obtained residue was separated and purified by silica gel column chromatography (eluent: system a) to obtain the product 1- (3-fluoro-2-methoxyphenyl) thiourea 2c (22g, 109.87mmol), yield: 77.55 percent. MS m/z (ESI) 201.1[ 2 ], [ M ] +1] ⁺

Second step of

5-fluoro-4-methoxybenzo[d]thiazol-2-amine

5-fluoro-4-methoxybenzo [ d ] thiazol-2-amine

1- (3-fluoro-2-methoxyphenyl) thiourea 2c (7.09g, 35.41mmol) was added to acetic acid (200 mL), lithium bromide (4.61g, 53.11mmol) was added, liquid bromine (5.77g, 36.12mmol) was slowly added dropwise, maintaining the temperature below 30 ℃. After the dropwise addition, the reaction solution was heated to 40 ℃ for overnight reaction. The reaction solution was cooled, poured into water (500 mL), the PH was adjusted to be alkaline with a saturated sodium carbonate solution, extracted with ethyl acetate (300 mL × 1), the organic phase was washed with saturated brine (100 mL × 1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the obtained residue was separated and purified by silica gel column chromatography (eluent: system a) to give the product 5-fluoro-4-methoxybenzo [ d ] thiazol-2-amine 2d (7g, 35.31mmol), yield: 99.73 percent.

MS m/z(ESI):199.1[M+1] ⁺

The third step

2-amino-5-fluorobenzo[d]thiazol-4-ol

2-amino-5-fluorobenzo [ d ] thiazol-4-ol

5-fluoro-4-methoxybenzo [ d ] thiazol-2-amine 2d (7g, 35.31mmol) was added to dichloromethane (70 mL), cooled to 0 deg.C, boron tribromide (22.12g, 88.29mmol, 8.51mL) was added dropwise, and the mixture was allowed to warm to room temperature for reaction overnight. The reaction mixture was poured into ice water (300 mL), the pH was adjusted to basic with a saturated sodium carbonate solution, extracted with ethyl acetate (500 mL. Times.1), the organic phase was washed with saturated brine (100 mL. Times.1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product 2-amino-5-fluorobenzo [ d ] thiazol-4-ol 2e (5.2g, 28.23mmol), yield: 79.94 percent.

MS m/z(ESI):185.1[M+1] ⁺

The fourth step

tert-butyl (4- ((tert-butyloxycarbonyl) oxy) -5-fluorobenzoxy [ d ] thiazol-2-yl) carbamate (4- ((tert-butoxycarbonyl) oxy) -5-fluorobenzo [ d ] thiazol-2-yl) carbamate

2-amino-5-fluorobenzo [ d ] thiazol-4-ol 2e (10g, 54.29mmol), dimethylaminopyridine (1.33g, 10.86mmol), triethylamine (10.99g, 108.58mmol, 15.13mL), di-tert-butyl dicarbonate (23.70g, 108.58mmol) were added to dichloromethane (80 mL) and reacted at room temperature for 4 hours. The reaction mixture was concentrated under reduced pressure, ethyl acetate (100 mL) and water (50 mL) were added, the mixture was separated, the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product, tert-butyl (4- ((tert-butoxycarbonyl) oxy) -5-fluorobenzo [ d ] thiazol-2-yl) carbamate 2f (20g, 52.03mmol), yield: 95.83 percent.

MS m/z(ESI):385.1[M+1] ⁺

The fifth step

tert-butyl(5-fluoro-4-hydroxybenzo[d]thiazol-2-yl)carbamate

(5-fluoro-4-hydroxybenzo [ d ] thiazol-2-yl) carbamic acid tert-butyl ester

Tert-butyl (4- ((tert-butoxycarbonyl) oxy) -5-fluorobenzo [ d ] thiazol-2-yl) carbamate 2f (20g, 52.03mmol) was added to a mixed solvent of tetrahydrofuran (80 mL) and water (20 mL), cooled to 0 ℃, added with lithium hydroxide monohydrate (10.92g, 260.13mmol), and allowed to react at room temperature overnight. The reaction mixture was added with ethyl acetate (100 mL) and water (50 mL), extracted with ethyl acetate (100 mL. Times.2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 2g (14.45g, 50.83mmol) of the crude product, tert-butyl (5-fluoro-4-hydroxybenzo [ d ] thiazol-2-yl) carbamate, yield: 97.69 percent.

MS m/z(ESI):228.9[M+1-56] ⁺

The sixth step

2-((tert-butoxycarbonyl)amino)-5-fluorobenzo[d]thiazol-4-yl trifluoromethanesulfonate

2- ((tert-Butoxycarbonyl) amino) -5-fluorobenzo [ d ] thiazol-4-yl trifluoromethanesulfonate

Under ice bath, (5-fluoro-4-hydroxybenzo [ d ]]Thiazol-2-yl) carbamic acid tert-butyl ester 2g (20g, 70.35mmol) was dissolved in methylene chloride (80 mL), and pyridine (11.13g, 140.69mmol, 11.36mL) and trifluoromethanesulfonic anhydride (23.82g, 84.42mmol, 14.26mL) were added in this order, followed by stirring for 30 minutes. Adding water (150 mL) into the reaction liquid, extracting with dichloromethane (150 mL multiplied by 3), combining organic phases, washing with citric acid monohydrate aqueous solution (50 mL) and saturated saline solution (50 mL), drying with anhydrous sodium sulfate, filtering, concentrating under reduced pressure, and separating and purifying the obtained residue by silica gel column chromatography (eluent: A system) to obtain the product 2- ((tert-butoxycarbonyl) amino) -5-fluorobenzo [ d]Thiazol-4-yl triflate 2h (13.6g, 32.66mmol), yield: 46.43 percent. MS m/z (ESI) 361.0[ m ] +1-56 ]] ⁺

Seventh step

(2-((tert-butoxycarbonyl)amino)-5-fluorobenzo[d]thiazol-4-yl)boronic acid

(2- ((tert-Butoxycarbonyl) amino) -5-fluorobenzo [ d ] thiazol-4-yl) boronic acid

2- ((tert-Butoxycarbonyl) amino) -5-fluorobenzo [ d ] thiazol-4-yl trifluoromethanesulfonate 2h (5g, 12.01mmol), pinacol diboron (24.40g, 96.07mmol) was mixed in 1, 4-dioxane (80 mL), potassium acetate (3.54g, 36.03mmol) was added, argon was substituted, stirring was carried out for 10 minutes, [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride (2.64g, 3.60mmol) was added, argon was used for protection, and stirring was carried out at 100 ℃ for 8 hours. And adding pinacol diboron (24.40g, 96.07mmol) and [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride (2.64g, 3.60mmol), stirring for 8 hours at 100 ℃ under the protection of argon. The reaction solution was concentrated under reduced pressure, and the obtained residue was separated and purified by silica gel column chromatography (eluent: system a) to obtain the product (2- ((tert-butoxycarbonyl) amino) -5-fluorobenzo [ d ] thiazol-4-yl) boronic acid 2i (2g, 6.41mmol), yield: 53.36 percent.

MS m/z(ESI):312.9[M+1] ⁺

Eighth step

tert-butyl

(2R,4aR)-10-(2-((tert-butoxycarbonyl)amino)-5-fluorobenzo[d]thiazol-4-yl)-11-chloro-9-fluoro-2,6-dimethyl-5-oxo-1,2,4,4a,5,6-hexahydro-3H-pyrazino[1',2':4,5]pyrazino[2,3-c]quinoline-3-carboxyl

ate

(2R, 4aR) -10- (2- ((tert-butoxycarbonyl) amino) -5-fluorobenzo [ d ] thiazol-4-yl) -11-chloro-9-fluoro-2, 6-dimethyl-5-oxo-1, 2,4,4a,5, 6-hexahydro-3H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinoline-3-carboxylic acid tert-butyl ester

(2- ((tert-Butoxycarbonyl) amino) -5-fluorobenzo [ d ] thiazol-4-yl) boronic acid 2i (182.25mg, 583.90. Mu. Mol), (2R, 4aR) -10-bromo-11-chloro-9-fluoro-2, 6-dimethyl-5-oxo-1, 2,4,4a,5, 6-hexahydro-3H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinoline-3-carboxylic acid tert-butyl ester 1e (200mg, 389.27. Mu. Mol) was added to a mixed solvent of 1, 4-dioxane (1 mL) and water (0.2 mL), tetrakis (triphenylphosphine) palladium (44.98mg, 38.93. Mu. Mol), sodium carbonate (123.78mg, 1.17mmol), argon-protected, and warmed to 110 ℃ for reaction overnight. Ethyl acetate (10 mL) and water (10 mL) were added to the system, and liquid separation, ethyl acetate extraction (10 mL. Times.3), organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the obtained residue was separated and purified by preparative HPLC (separation column: AKZONOBEL Kromasil; 250X 21.2mm I.D.;5 μm; mobile phase A:0.05 TFA (+) H2O, mobile phase B: acetonitrile; flow rate: 20 mL/min), to obtain the product (2R, aR) -10- (2- ((t-butoxycarbonyl) amino) -5-fluorobenzo [ d ] thiazol-4-yl) -11-chloro-9-fluoro-2, 6-dimethyl-5-oxo-1, 2, 4a,5, 6-hexahydro-3H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinoline-3-carboxylic acid t-butyl ester 2j (yield, 71. Mu. Mol, 5031 mol): 18.32 percent.

MS m/z(ESI):700.8[M+1] ⁺

The ninth step

(2R,4aR)-10-(2-amino-5-fluorobenzo[d]thiazol-4-yl)-11-chloro-9-fluoro-2,6-dimethyl-2,3,4,4a-tetrahydro-1H-pyrazino[1',2':4,5]pyrazino[2,3-c]quinolin-5(6H)-one

(2R, 4aR) -10- (2-amino-5-fluorobenzo [ d ] thiazol-4-yl) -11-chloro-9-fluoro-2, 6-dimethyl-2,3,4, 4a-tetrahydro-1H-pyrazino-and

[1',2':4,5] pyrazino [2,3-c ] quinolin-5 (6H) -one

(2R, 4aR) -10- (2- ((tert-butoxycarbonyl) amino) -5-fluorobenzo [ d ] thiazol-4-yl) -11-chloro-9-fluoro-2, 6-dimethyl-5-oxo-1, 2,4,4a,5, 6-hexahydro-3H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinoline-3-carboxylic acid tert-butyl ester 2j (50mg, 71.31. Mu. Mol) was dissolved in dichloromethane (5 mL), and a solution of 1, 4-dioxane (4M, 3mL) of hydrochloric acid was added and reacted at room temperature overnight. After the reaction was completed, concentration was performed under reduced pressure to obtain a crude product (2r, 4ar) -10- (2-amino-5-fluorobenzo [ d ] thiazol-4-yl) -11-chloro-9-fluoro-2, 6-dimethyl-2,3, 4a-tetrahydro-1H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinolin-5 (6H) -one 2k (30mg, 59.89 μmol), yield: 83.98 percent.

MS m/z(ESI):500.8[M+1] ⁺

The tenth step

(2R,4aR) -10- (2-amino-5-fluorobenzo [ d ] thiazol-4-yl) -11-chloro-9-fluoro-2,6-dimethyl-2,3,4,4a-tetrahydro-1H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinolin-5 (6H) -one 2k (20mg, 39.92. Mu. Mol) was added to methylene chloride (2 mL), triethylamine (12.12mg, 119.77. Mu. Mol) was added, cooled to 0 ℃ and 2-fluoropropenylchloride (4.33mg, 39.92. Mu. Mol, prepared according to the laid-open patent WO 2020106A 1731) was added and reacted for 0.5 hour. Ethyl acetate (10 mL) and water (10 mL) were added to the system, and the mixture was separated, extracted with ethyl acetate (10 mL. Times.3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give crude (2R, 4aR) -10- (2-amino-5-fluorobenzo [ d ] thiazol-4-yl) -11-chloro-9-fluoro-3- (2-fluoropropy-loyl) -2,6-dimethyl-2,3,4,4a-tetrahydro-1H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinolin-5 (6H) -one 2 (12mg, 20.94. Mu. Mol).

MS m/z(ESI):573.1[M+1] ⁺

Eleventh step (2R, 4aR, 10R) -10- (2-amino-5-fluorobenzol [ d ] thiazol-4-yl) -11-chloro-9-fluoro-3- (2-fluoroacylyl) -2

,6-dimethyl-2,3,4,4a-tetrahydro-1H-pyrazino[1',2':4,5]pyrazino[2,3-c]quinolin-5(6H)-one 3

(2R, 4aR, 10R) -10- (2-amino-5-fluorobenzo [ d ] thiazol-4-yl) -11-chloro-9-fluoro-3- (2-fluoropropy l) -2,6-dimethyl-2,3,4,4a-tetrahydro-1H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinolin-5 (6H) -one 3

(2R, 4aR, 10S) -10- (2-amino-5-fluorobenzo [ d ] thiazol-4-yl) -11-chloro-9-fluoro-3- (2-fluoropropenyl) -2,6-dimethyl-2,3,4,4a-tetrahydro-1H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinolin-5 (6H) -one 4

The crude product (2R, 4aR) -10- (2-amino-5-fluorobenzo [ d ] thiazol-4-yl) -11-chloro-9-fluoro-3- (2-fluoropropenyl) -2,6-dimethyl-2,3,4,4a-tetrahydro-1H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinolin-5 (6H) -one 2 was purified by preparative HPLC separation (separation column: AKZONOBEL Kromasil; 250X 21.2mm I.D.;5 μm; mobile phase A:0.05 TFA + 2HO, mobile phase B: acetonitrile; flow rate: 20 mL/min) to give products 3 and 4.

Product 3 (2R, 4aR, 10R) -10- (2-amino-5-fluorobenzo [ d ] thiazol-4-yl) -11-chloro-9-fluoro-3- (2-fluoropropenyl) -2,6-dimethyl-2,3,4,4a-tetrahydro-1H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinolin-5 (6H) -one 3 (5mg, 5.85. Mu. Mol), yield: at the beginning of the production of 14.64%,

MS m/z(ESI):573.1[M+1] ⁺ ；

product 4 (2R, 4aR, 10S) -10- (2-amino-5-fluorobenzo [ d ] thiazol-4-yl) -11-chloro-9-fluoro-3- (2-fluoropropenyl) -2,6-dimethyl-2,3,4,4a-tetrahydro-1H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinolin-5 (6H) -one (5mg, 8.64. Mu. Mol), yield: 21.64 percent of the total weight of the steel,

MS m/z(ESI):573.1[M+1] ⁺

biological evaluation

Test example 1 determination of the ability of the Compounds of the invention to covalently bind to KRAS G12C protein

The following method was used to determine the ability of the compounds of the invention to covalently bind to recombinant human KRAS G12C protein under in vitro conditions.

The experimental procedure is briefly described as follows: reaction buffers (20mM HEPES,150mM NaCl,1mM MgCl) were used ₂ 1mM DTT) was prepared at a concentration of 4. Mu.M for use as recombinant human KRAS G12C protein (aa 1-169). Test compounds were prepared as 10mM stock solutions dissolved in DMSO and subsequently diluted with reaction buffer for use. First, 1.5. Mu.L of the test compound diluted with the reaction buffer (final concentration of the reaction system was 3. Mu.M) was added to the well, 23.5. Mu.L of the reaction buffer was added thereto, followed by mixing, 25. Mu.L of 4. Mu.M recombinant human KRAS G12C protein was added thereto, the reaction was terminated by adding 5. Mu.L of acetic acid after incubation at room temperature for 5 minutes, and the sample was transferred to a sample bottle. The covalent binding rate of the test compound to the KRAS G12C Protein was measured using an Agilent 1290/6530 instrument, and the sample was purified on a liquid chromatography column (XBridge Protein BEH C4,

3.5 μm,2.1mm × 50 mm), mobile phase a is 0.1% formic acid in water, mobile phase B is acetonitrile, mobile phase elution procedure is: 0-0.5 min, keeping mobile phase A: at 95%, at 2.5 minutes, mobile phase a became 30% and held for 0.5 minutes, 3.1 minutes, mobile phase a became 95% and held for 1.9 minutes; flow rate: 0.5mL/min; finally, the data are analyzed by using MassHunter Workstation Software bioconjugate Version B.08.00 Software, the concentration of the tested compound is 3 mu M, and the tested compound and KRAS G12C protein are incubated for 5minBinding Rate (Binding Rate).

The covalent binding rate of the compounds of the invention to KRAS G12C protein is shown in the table below.

Compound numbering	Binding Rate(％)
		4	40.23

The conclusion is that the compound has better covalent binding rate with KRAS G12C protein under the conditions of 3 mu M and 5 min.

Test example 2 assay for inhibition of NCI-H358 cell proliferation by Compounds of the present invention

The following method was used to determine the effect of the compounds of the invention on NCI-H358 cell proliferation. NCI-H358 cells (containing KRAS G12C mutation) were purchased from the cell resource center of Shanghai Life sciences institute of Chinese academy of sciences, and cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100U penicillin, 100. Mu.g/mL streptomycin and 1mM Sodium Pyruvate. Cell viability by

The Luminescent Cell Viability Assay kit (Promega, cat # G7573) was used for the Assay.

The experimental method is operated according to the steps of the kit specification, and is briefly as follows: test compounds were first prepared as 10mM stock solutions dissolved in DMSO and then diluted in culture medium to prepare test samples with compound concentrations ranging from 1000nM to 0.015nM. Cells in logarithmic growth phase were seeded at a density of 800 cells per well in 96-well cell culture plates and at 37 ℃,5% CO ₂ Incubated overnight in an incubator, followed by addition of test compoundThe culture was continued for 120 hours. After the incubation was completed, 50. Mu.L of CellTiter-Glo detection solution was added to each well, shaken for 5 minutes and then allowed to stand for 10 minutes, and then Luminescence values of each well of the sample were read on a microplate reader using a Luminescence mode. The percentage inhibition of the compounds at each concentration point was calculated by comparison with the values of the control (0.3% DMSO), after which non-linear regression analysis was performed in GraphPad Prism 5 software at the log-inhibition of the compound concentration, to obtain the IC of the compounds for inhibition of cell proliferation ₅₀ The value is obtained.

The compounds of the present invention inhibit NCI-H358 (human non-small cell lung carcinoma) cell proliferation IC ₅₀ Values, see the following Table

Compound numbering	IC ₅₀ (nM)
		4	48.42

The conclusion is that the compound of the invention has better proliferation inhibition effect on NCI-H358 (human non-small cell lung cancer) cells, and the IC of the compound is preferred ₅₀ <500nM, more preferably IC of the compound ₅₀ <200nM。

Test example 3 determination of p-ERK1/2 inhibitory Activity of Compounds of the present invention in NCI-H358 cells

The following method was used to determine the p-ERK1/2 inhibitory activity of the compounds of the present invention in NCI-H358 cells. The method uses Advanced phosphor-ERK 1/2 (Thr 202/tyr 204) kit (cat. 64 AERPEH) of Cisbio company, and the detailed experimental operation can refer to the kit instruction. NCI-H358 cells (containing KRAS G12C mutation) were purchased from the Shanghai Life sciences research institute cell resource center, chinese academy of sciences.

The experimental procedure is briefly described as follows:NCI-H358 cells were cultured in RPMI 1640 complete medium containing 10% fetal bovine serum, 100U penicillin, 100. Mu.g/mL streptomycin, and 1mM Sodium Pyruvate. 30000 cells per well of NCI-H358 were plated in 96-well plates in complete medium at 37 ℃ 5% CO ₂ The culture was carried out overnight in an incubator. Test compounds were dissolved in DMSO to prepare a 10mM stock solution, which was then diluted with RPMI 1640 basic medium, to which 90. Mu.L of RPMI 1640 basic medium containing the test compound at the corresponding concentration was added per well, the final concentration of the test compound in the reaction system ranged from 1000nM to 0.015nM, and the cells were cultured in a cell culture chamber for 3 hours and 40 minutes. Then 10. Mu.L of hEGF (purchased from Roche under the trademark 11376454001) prepared in RPMI 1640 basic medium was added to a final concentration of 5nM and incubated in an incubator for 20 minutes. Cell supernatants were discarded and cells were washed with ice-cooled PBS, after which 45. Mu.L of 1 xcell phosphate/total protein lysis buffer (Advanced phosphate-ERK 1/2 kit component) was added per well for lysis, 96-well plates were placed on ice for half an hour for lysis, and lysates were then detected with reference to the Advanced phosphate-ERK 1/2 (Thr 202/tyr 204) kit instructions. Finally, the fluorescence intensity of each well with the emission wavelength of 620nM and 665nM under the excitation wavelength of 304nM is measured in a microplate reader in TF-FRET mode, and the ratio of the fluorescence intensity of 665/620 of each well is calculated. The percent inhibition of the test compound at each concentration was calculated by comparison with the fluorescence intensity ratio of the control group (0.1% DMSO), and the IC of the compound was obtained by nonlinear regression analysis of the value-inhibition at the test compound concentration using GraphPad Prism 5 software ₅₀ The value is obtained.

The conclusion is that the compound has better proliferation inhibition effect on p-ERK1/2 in NCI-H358 cells, and the IC of the compound is optimized ₅₀ <500nM, more preferably IC of the compound ₅₀ <200nM。

Test example 4 Metabolic stability Studies of Compounds of the invention in human and rat liver microsomes

1. Purpose of experiment

The purpose of this experimental study was to study the metabolic stability of compound 4 of the present invention in human and rat liver microsomes.

2. Reagent information

Name (R)	Suppliers of goods
		Rat liver microsome	Corning Inc. of USA
Rat liver microsome	Corning Inc. of USA
		Midazolam maleate	China Institute for food and drug control
NADPH	Roche Switzerland
		Potassium dihydrogen phosphate	SINOPHARM CHEMICAL REAGENT Co.,Ltd.
Dipotassium hydrogen phosphate	SINOPHARM CHEMICAL REAGENT Co.,Ltd.
		Magnesium chloride (MgCl) ₂ )	SINOPHARM CHEMICAL REAGENT Co.,Ltd.
Verapamil hydrochloride	China Institute for food and drug control
		Glibenclamide	China Institute for food and drug control
DMSO	Amresco Inc. of USA
		Methanol	Honeywell Corp, USA
Acetonitrile	Honeywell corporation, USA
		Formic acid	SHANGHAI ALADDIN BIOCHEMICAL TECHNOLOGY Co.,Ltd.

3. Experimental protocol

The test compound is incubated with human or rat liver microsomes and the reaction is initiated by addition of the coenzyme NADPH. The reaction was stopped at 0, 5, 15, 30 and 60 minutes by removing 20. Mu.L of the incubation and transferring to 200. Mu.L of acetonitrile containing an internal standard. After protein precipitation, the supernatant was centrifuged at 3,700rpm for 10 minutes. The supernatant was diluted with water 1 and analyzed by LC-MS/MS method. Intrinsic clearance in vitro was calculated from the clearance half-life of the test compound in the incubation system. Midazolam was used as an internal reference compound, and 2 portions were incubated in parallel. The incubation conditions are summarized in the following table:

4. data analysis

Area ratio of analyte/internal standard peaks (A) _analyte /A _IS ) Will be obtained from the instrument, the percentage remaining (% Control) is determined from A in the non-zero time point sample and the zero time sample _analyte /A _IS The ratio of the two is calculated. Ln (% Control) was plotted against incubation time and a linear fit was made. Subject to testCompound clearance constant (k, min) ^-1 ) And elimination half-life (T) _1/2 Min) is calculated from the following equation.

k＝-slope

T _1/2 ＝0.693/k

5. Results of the experiment

The relevant parameters for the stability of human and rat liver microsomes for compound 4 of the invention are shown in the following table:

compound numbering	half-life/(T) _1/2 Min, human)	Half life/(T) _1/2 Min, rat)
			4	291.27	226.65

And (4) conclusion: the compound 4 of the invention has long half-life period and high stability of liver microsomes of rats and human beings.

Claims

1. A compound represented by the structure or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein said compound comprises:

2. a compound according to claim 1, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein said compound comprises:

3. a pharmaceutical composition comprising an effective amount of a compound according to claim 1 or 2, or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or combination thereof.

4. Use of a compound according to claim 1 or 2, or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 3, for the preparation of a KRAS gtpase inhibitor, preferably a KRAS G12C inhibitor.

5. Use of a compound according to claim 1 or 2 or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 3 for the manufacture of a medicament for the treatment of a disease mediated by a KRAS mutation, wherein the disease mediated by a KRAS mutation is selected from the group consisting of cancer, wherein the cancer is selected from the group consisting of pancreatic cancer, colorectal cancer, lung cancer, multiple myeloma, uterine cancer, cholangiocarcinoma, gastric cancer, bladder cancer, diffuse large B-cell lymphoma, rhabdomyosarcoma, squamous cell carcinoma of the skin, cervical cancer, testicular germ cell cancer, preferably pancreatic cancer, colorectal cancer and lung cancer, wherein the KRAS mutation is preferably a KRAS G12C mutation.

6. Use of a compound according to claim 1 or 2, or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 3, for the manufacture of a medicament for the treatment of a cancer selected from the group consisting of pancreatic cancer, colorectal cancer, lung cancer, multiple myeloma, uterine cancer, bile duct cancer, gastric cancer, bladder cancer, diffuse large B-cell lymphoma, rhabdomyosarcoma, cutaneous squamous cell carcinoma, cervical cancer, testicular germ cell carcinoma, preferably pancreatic cancer, colorectal cancer and lung cancer.

7. The use of claim 5 or 6, wherein the lung cancer is non-small cell lung cancer.