HK40075734A

HK40075734A - Octahydropyrazinodiazanaphthyridine dione chemical organism

Info

Publication number: HK40075734A
Application number: HK62022065151.2A
Authority: HK
Inventors: 陈曙辉; 陈新海; 周凯; 江文; 胡国平; 黎健
Original assignee: 南京明德新药研发有限公司
Priority date: 2020-04-03
Filing date: 2021-04-06
Publication date: 2023-01-27

Description

Octahydropyrazinonanaphthyridinediones

The following priority is claimed in the present application:

cn202010260612.X, filing date 2020, 04/03;

CN202010568775.4, filing date 2020, 06 month 19;

CN202011616152.6, filed 2020, 12.30.

Technical Field

The invention relates to a novel octahydropyrazino naphthyridine dione compound, in particular to a compound shown as a formula (III) and a pharmaceutically acceptable salt thereof.

Background

The first RAS oncogene is found in rat sarcoma (rat sarcoma) and is hence the name. RAS proteins are products expressed by RAS genes, and refer to a closely related class of monomeric globulins consisting of 189 amino acids, which have a molecular weight of 21KDa. It may be bound to Guanine Trinucleotide Phosphate (GTP) or Guanine Dinucleotide Phosphate (GDP). The active state of RAS proteins has an influence on the growth, differentiation, cytoskeleton, protein transport and secretion, etc. of cells, and its activity is regulated by binding to GTP or GDP. When the RAS protein binds to GDP, it is in a dormant, i.e., "inactivated," state; upon stimulation by specific upstream cytokines, RAS proteins are induced to exchange GDP and bind GTP, which is referred to as the "activated" state. RAS proteins bound to GTP are able to activate downstream proteins for signaling. The RAS protein itself has weak GTP hydrolyzing activity, and is able to hydrolyze GTP to GDP. This allows the transition from the activated state to the deactivated state to be achieved. GAP (GTPase activating proteins) is also required to participate in this hydrolysis process. It can interact with RAS protein to greatly promote its ability to hydrolyze GTP to GDP. Mutations in the RAS protein affect its interaction with GAP and thus its ability to hydrolyze GTP to GDP, leaving it in an activated state. The activated RAS protein continuously gives downstream protein growth signals, eventually leading to the ceaseless growth and differentiation of cells, eventually leading to tumor development. The members of the RAS gene family are numerous, among the subfamilies closely related to various cancers are the Kresting rat sarcoma virus oncogene homolog (KRAS), the Harvey rat sarcoma virus oncogene Homolog (HRAS), and the neuroblastoma rat sarcoma virus oncogene homolog (NRAS). It was found that approximately 30% of human tumors carry certain mutated RAS genes, with KRAS mutations being the most prominent, accounting for 86% of all RAS mutations. For KRAS mutations, the most common mutations occur at glycine 12 (G12), glycine 13 (G13) and glutamine 61 (Q61) residues, with the G12 mutation accounting for 83%.

The G12C mutation is a common one in KRAS gene mutation, and refers to the mutation of No. 12 glycine to cysteine. KRAS G12C mutation is most common in lung cancer, and calculated according to data reported in literature, KRAS G12C mutation accounts for about 10% of all lung cancer patients.

Disclosure of Invention

The invention provides a compound shown in a formula (III) or a pharmaceutically acceptable salt thereof,

wherein, the first and the second end of the pipe are connected with each other,

m is selected from 0, 1 and 2;

n is selected from 0, 1, 2, 3 and 4;

x is selected from NH and O;

y is selected from CH and N;

z is selected from CH and N;

R ₁ are respectively H, F, cl, br, I, OH and NH independently ₂ And C _1-3 Alkyl radical, said C _1-3 Alkyl is optionally substituted by 1, 2 or 3R _a Substitution;

R ₂ selected from H, F, cl, br, I and C _1-3 Alkyl radical, said C _1-3 Alkyl is optionally substituted by 1, 2 or 3R _b Substitution;

R ₃ selected from H, F, cl, br, I and C _1-6 Alkyl radical, said C _1-3 Alkyl is optionally substituted by 1, 2 or 3R _c Substitution;

R ₄ selected from H, F, cl, br, I and C _1-6 Alkyl radical, said C _1-6 Alkyl is optionally substituted by 1, 2 or 3R _c Substitution;

R ₅ selected from H, F, cl, br and I;

R _a 、R _b and R _c Each independently selected from H, F, cl, br and I.

In some embodiments of the invention, the compound has the structure of formula (II):

R ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ x, Y, m and n are as defined herein.

In some embodiments of the invention, R is as defined above ₁ Each independently selected from F, cl, NH ₂ And OH, the other variables being as defined herein.

In some embodiments of the invention, R is as defined above ₂ Is selected from CH ₃ Said CH ₃ Optionally substituted by 1, 2 or 3R _b And, the other variables are as defined herein.

In some embodiments of the invention, R is as defined above ₂ Selected from CF ₃ And the other variables are as defined herein.

In some embodiments of the invention, R is as defined above ₃ Is selected from CH ₃ 、CH ₂ CH ₃ Andother variables are as defined herein.

In some embodiments of the invention, R is as defined above ₄ Is selected from CH ₃ 、CH ₂ CH ₃ Andthe other variables are as defined herein.

In some embodiments of the invention, R is as defined above ₅ Selected from H and F, and the other variables are as defined herein.

In some embodiments of the invention, the structural unitIs selected fromOther variables are as defined herein.

In some embodiments of the invention, the compound is selected from

Wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ And R ₅ As defined herein.

In some embodiments of the invention, the compound or pharmaceutically acceptable salt thereof is selected from

Wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ And R ₅ As defined herein.

The present invention provides a compound of the formula or a pharmaceutically acceptable salt thereof, selected from

In some embodiments of the invention, the compound, or a pharmaceutically acceptable salt thereof, is selected from

Still other embodiments of the present invention are derived from any combination of the above variables.

The invention provides a pharmaceutical composition comprising as an active ingredient a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

The invention provides application of the compound or the pharmaceutically acceptable salt thereof or the composition in preparing KRAS G12C mutant protein inhibitors.

The invention provides application of the compound or the pharmaceutically acceptable salt thereof or the composition in preparing a medicament for treating cancer.

Definitions and explanations

As used herein, the following terms and phrases are intended to have the following meanings, unless otherwise indicated. A particular term or phrase, unless specifically defined, should not be considered as indefinite or unclear, but rather construed according to ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding commodity or its active ingredient. The term "pharmaceutically acceptable" as used herein is intended to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salts" refers to salts of the compounds of the present invention, prepared from the compounds of the present invention found to have particular substituents, with relatively nontoxic acids or bases. When compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of a base in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amines or magnesium salts or similar salts. When compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of an acid in neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include salts with inorganic acids including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, and the like; and salts of organic acids including acids such as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-toluenesulfonic, citric, tartaric, methanesulfonic, and the like; also included are salts of amino acids such as arginine and the like, and salts of organic acids such as glucuronic acid and the like. Certain specific compounds of the invention contain both basic and acidic functionalities and can thus be converted to any base or acid addition salt.

The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, which contains an acid or base, by conventional chemical methods. In general, such salts are prepared by the following method: prepared by reacting these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid, in water or an organic solvent or a mixture of the two.

In addition to salt forms, the compounds provided herein also exist in prodrug forms. Prodrugs of the compounds described herein readily undergo chemical changes under physiological conditions to convert to the compounds of the present invention. In addition, prodrugs can be converted to the compounds of the present invention in an in vivo environment by chemical or biochemical means.

Certain compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention.

The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-) -and (+) -enantiomers, (R) -and (S) -enantiomers, diastereomers, (D) -isomers, (L) -isomers, as well as racemic and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.

Unless otherwise indicated, the terms "enantiomer" or "optical isomer" refer to stereoisomers that are mirror images of each other.

Unless otherwise indicated, the term "cis-trans isomer" or "geometric isomer" results from the inability of a double bond or a single bond to rotate freely within a ring-forming carbon atom.

Unless otherwise indicated, the term "diastereomer" refers to stereoisomers whose molecules have two or more chiral centers and which are in a non-mirror image relationship between the molecules.

Unless otherwise indicated, "(D)" or "(+)" means dextrorotation, "(L)" or "(-) -means levorotation," (DL) "or" (+ -) "means racemization.

Unless otherwise indicated, with solid wedge-shaped keysAnd wedge dotted bondRepresenting the absolute center of a solidFor configuration, with straight solid keysAnd straight dotted line bondShowing the relative configuration of the centres of solids, by wavy linesRepresenting solid-line keys of wedge shapeOr wedge dotted bondOr by wavy linesIndicating straight solid-line keysAnd straight dotted line bond

The compounds of the invention may be present specifically. Unless otherwise indicated, the term "tautomer" or "tautomeric form" means that at room temperature, the isomers of different functional groups are in dynamic equilibrium and can be rapidly interconverted. If tautomers are possible (e.g., in solution), then the chemical equilibrium of the tautomers can be reached. For example, proton tautomers (prototropic tautomers), also known as proton transfer tautomers (prototropic tautomers), include interconversions by proton transfer, such as keto-enol isomerization and imine-enamine isomerization. Valence isomers (valencetatomer) include interconversion by recombination of some of the bonding electrons. A specific example of where keto-enol tautomerism is the interconversion between two tautomers of pentane-2,4-dione and 4-hydroxypent-3-en-2-one.

Unless otherwise indicated, the terms "enriched in one isomer", "isomer enriched", "enantiomer enriched" or "enantiomeric enrichment" refer to a content of one isomer or enantiomer of less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

Unless otherwise indicated, the term "isomeric excess" or "enantiomeric excess" refers to the difference between the relative percentages of two isomers or enantiomers. For example, if the content of one isomer or enantiomer is 90%, and the content of the other isomer or enantiomer is 10%, the isomer or enantiomeric excess (ee value) is 80%.

Optically active (R) -and (S) -isomers as well as D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one of the enantiomers of a compound of the invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, where the molecule contains a basic functional group (e.g., amino) or an acidic functional group (e.g., carboxyl), diastereomeric salts are formed with an appropriate optically active acid or base, followed by diastereomeric resolution by conventional methods known in the art, and the pure enantiomers are recovered. Furthermore, enantiomersSeparation of the isomers and diastereomers is typically accomplished by using chromatography using a chiral stationary phase, optionally in combination with chemical derivatization (e.g., carbamate formation from amines). The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compound may be labeled with a radioisotope, such as tritium ( ³ H) Iodine-125 ( ¹²⁵ I) Or C-14 ( ¹⁴ C) In that respect For example, deuterium can be used to replace hydrogen to form a deuterated drug, the bond formed by deuterium and carbon is stronger than the bond formed by common hydrogen and carbon, and compared with an undeuterated drug, the deuterated drug has the advantages of reducing toxic and side effects, increasing the stability of the drug, enhancing the curative effect, prolonging the biological half-life period of the drug and the like. All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention. "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

The term "substituted" means that any one or more hydrogen atoms on the specified atom is replaced with a substituent that may include variations of deuterium and hydrogen, so long as the valence of the specified atom is normal and the substituted compound is stable. When the substituent is oxygen (i.e = O), it means that two hydrogen atoms are substituted. Substitution by oxygen does not occur on aromatic groups. The term "optionally substituted" means that it may or may not be substituted, and unless otherwise specified, the kind and number of substituents may be arbitrary on the basis of chemical realizability.

When any variable (e.g., R) occurs more than one time in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 0-2R, the group may optionally be substituted with up to two R, and there are separate options for R in each case. Furthermore, combinations of substituents and/or variants thereof are permissible only if such combinations result in stable compounds.

When the number of one linking group is 0, e.g., - (CRR) ₀ -, represents that the linking group is a single bond.

When one of the variables is selected from a single bond, it means that the two groups to which it is attached are directly connected, for example, where L represents a single bond in A-L-Z means that the structure is actually A-Z.

When a substituent is absent, it indicates that the substituent is absent, e.g., when X is absent in A-X, it indicates that the structure is actually A. When no atom through which a substituent is attached to a substituted group is indicated in the listed substituents, such substituents may be bonded through any atom thereof, for example, a pyridyl group as a substituent may be attached to a substituted group through any one of carbon atoms on the pyridine ring.

When the listed linking groups do not indicate their direction of attachment, the direction of attachment is arbitrary, for example,wherein the linking group L is-M-W-, in which case-M-W-can be formed by connecting the ring A and the ring B in the same direction as the reading sequence from left to rightThe ring A and the ring B may be connected in the reverse direction of the reading sequence from left to rightCombinations of the linking groups, substituents, and/or variants thereof are permissible only if such combinations result in stable compounds.

Unless otherwise specified, the term "C _1-6 Alkyl "is used to denote a straight or branched saturated hydrocarbon group consisting of 1 to 6 carbon atoms. Said C is _1-6 The alkyl group comprising C _1-5 、C _1-4 、C _1-3 、C _1-2 、C _2-6 、C _2-4 、C ₆ And C ₅ Alkyl groups and the like; it may be monovalent (e.g., methyl), divalent (e.g., methylene), or multivalent (e.g., methine). C _1-6 Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, s-butyl and t-butyl), pentyl (including n-pentyl, isopentyl and neopentyl), hexyl, and the like.

Unless otherwise specified, the term "C _1-3 Alkyl "is used to denote a straight or branched saturated hydrocarbon group consisting of 1 to 3 carbon atoms. Said C is _1-3 The alkyl group comprising C _1-2 And C _2-3 Alkyl, etc.; it may be monovalent (e.g. methyl), divalent (e.g. methylene) or polyvalent (e.g. methine). C _1-3 Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

Unless otherwise specified, C _n-n+m Or C _n -C _n+m Including any one particular case of n to n + m carbons, e.g. C _1-12 Comprising C ₁ 、C ₂ 、C ₃ 、C ₄ 、C ₅ 、C ₆ 、C ₇ 、C ₈ 、C ₉ 、C ₁₀ 、C ₁₁ And C ₁₂ Also included are any ranges of n to n + m, e.g. C _1- ₁₂ Comprising C _1-3 、C _1-6 、C _1-9 、C _3-6 、C _3-9 、C _3-12 、C _6-9 、C _6-12 And C _9-12 Etc.; similarly, n-to n + m-members represent n to n + m ring atoms, and for example, 3-12 membered rings include 3-membered rings, 4-membered rings, 5-membered rings, 6-membered rings, 7-membered rings, 8-membered rings, 9-membered rings, 10-membered rings, 11-membered rings, and 12-membered rings, and any range of n to n + m, for example, 3-12 membered rings include 3-6 membered rings, 3-9 membered rings, 5-6 membered rings, 5-7 membered rings, 6-7 membered ringsA 6-to 8-membered ring, a 6-to 10-membered ring, and the like.

The term "leaving group" refers to a functional group or atom that can be substituted by another functional group or atom through a substitution reaction (e.g., an affinity substitution reaction). For example, representative leaving groups include triflate; chlorine, bromine, iodine; sulfonate groups such as methanesulfonate, toluenesulfonate, p-bromobenzenesulfonate, p-toluenesulfonate and the like; acyloxy groups such as acetoxy, trifluoroacetyloxy, and the like.

The term "protecting group" includes, but is not limited to, "amino protecting group," hydroxyl protecting group, "or" thiol protecting group. The term "amino protecting group" refers to a protecting group suitable for use in preventing side reactions at the amino nitrogen position. Representative amino protecting groups include, but are not limited to: a formyl group; acyl, for example alkanoyl (such as acetyl, trichloroacetyl or trifluoroacetyl); alkoxycarbonyl such as tert-butoxycarbonyl (Boc); arylmethoxycarbonyl groups such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl groups such as benzyl (Bn), trityl (Tr), 1,1-bis- (4' -methoxyphenyl) methyl; silyl groups, such as Trimethylsilyl (TMS) and t-butyldimethylsilyl (TBS), and the like. The term "hydroxy protecting group" refers to a protecting group suitable for use in preventing side reactions of a hydroxy group. Representative hydroxy protecting groups include, but are not limited to: alkyl groups such as methyl, ethyl and tert-butyl; acyl groups, such as alkanoyl (e.g., acetyl); arylmethyl groups such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (benzhydryl, DPM); silyl groups, such as Trimethylsilyl (TMS) and t-butyldimethylsilyl (TBS), and the like.

The compounds of the present invention may be structurally confirmed by conventional methods well known to those skilled in the art, and if the present invention relates to the absolute configuration of a compound, the absolute configuration may be confirmed by means of conventional techniques in the art. For example, single crystal X-ray diffraction (SXRD), diffraction intensity data of the cultured single crystal is collected by Bruker D8 vision diffractometer, the light source is CuK α radiation, and the scanning mode:after scanning and collecting relevant data, the crystal structure is further analyzed by a direct method (Shelxs 97), so that the absolute configuration can be confirmed.

The compounds of the present invention may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations thereof with other chemical synthetic methods, and equivalents thereof known to those skilled in the art, with preferred embodiments including, but not limited to, examples of the present invention.

The solvent used in the present invention can be commercially available.

The compounds are named according to the conventional nomenclature in the art or usedThe software names, and the commercial compounds are under the supplier catalog name.

Technical effects

The compounds of the present invention are potent inhibitors of KRAS G12C muteins.

Drawings

FIGS. 1 and 2 show the protein expression of ERK and p-ERK in the tumor tissue of human colon cancer CO-04-0070 after administration.

Detailed Description

The present invention is described in detail below by way of examples, but is not meant to be limited to any of the disadvantages of the present invention. The compounds of the present invention may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations thereof with other chemical synthetic methods, and equivalents thereof known to those skilled in the art, with preferred embodiments including, but not limited to, examples of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made in the specific embodiments of the invention without departing from the spirit and scope of the invention.

Example 1

1-3 Synthesis: under nitrogen, brettPhos-Pd-G3 (2.96G, 3.27 mmol) and cesium carbonate (30.44G, 93.41 mmol) were added in one portion to a solution of compound 1-1 (14.82G, 46.71 mmol) and compound 1-2 (10G, 46.71 mmol) in t-amyl alcohol (100 mL) and stirred at 105 ℃ for 16 hours. The reaction mixture was concentrated and then separated by silica gel column chromatography (petroleum ether: ethyl acetate 10: 1 to 5: 1 (v/v)) to obtain compound 1-3.LCMS (ESI) m/z:451 (M + 1).

1-4 Synthesis: nitrogen bromosuccinimide (4.90 g, 27.53 mmol) was added in portions to a dichloromethane (120 ml) solution of compounds 1-3 (12.4 g, 27.53 mmol) at 0 ℃ under nitrogen blanket, and stirred at 0 ℃ for 0.5 h. The reaction was quenched with saturated sodium sulfite solution (30 ml) and extracted with dichloromethane (30 ml,1 time). The combined organic phases were washed with saturated brine (10 ml,1 time), dried over sodium sulfate, filtered and concentrated to give the crude product. The crude product was slurried with (petroleum ether: ethyl acetate 10: 1 (v/v), 50 mL) to provide compounds 1-4.

1-6 Synthesis: to a mixed solvent of compounds 1-4 (2.90 g, 5.48 mmol) and compounds 1-5 (784.37 mg, 6.03 mmol) in toluene (40 ml) was added chlorine [ (tri-tert-butylphosphine) -2- (2-aminobiphenyl) ] palladium (II) (561.50 mg, 1.10 mmol) and N, N-dicyclohexylmethylamine (1.18 g, 6.03 mmol) under nitrogen. The reaction was stirred at 100 ℃ for 16 h. The reaction mixture was concentrated and then separated by silica gel column chromatography (petroleum ether: ethyl acetate 5: 1 to 1: 1 (v/v)) to obtain compounds 1 to 6.

1-7 Synthesis: under nitrogen, compounds 1-6 (8 g, 15.03 mmol) were added to aqueous hydrochloric acid (12 mol per liter, 150 ml). The reaction was stirred at 100 ℃ for 16 hours. After completion of the reaction, pH =4 was adjusted with sodium bicarbonate, followed by extraction with dichloromethane (150 ml, 2 times), and the organic phase was dried over sodium sulfate, filtered, and concentrated to give compounds 1-7.LCMS (ESI) m/z:519 (M + 1).

1-8 Synthesis: compounds 1-7 (5 g, 9.64 mmol) were added to a mixed solution of acetic acid (30 ml) and fuming nitric acid (3.04 g, 48.22 mmol) at 0 degrees celsius, followed by addition of sodium nitrite (3.33 g, 48.22 mmol), and stirred at 20 degrees celsius for 0.5 hours. The reaction solution was diluted with ethyl acetate (150 ml), then washed with saturated brine (50 ml, 3 times), the resulting organic phase was adjusted to pH =10 with saturated aqueous sodium bicarbonate, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give compounds 1 to 8.LCMS (ESI) m/z:564 (M + 1).

1-9 Synthesis: to a solution of compounds 1-8 (5.02 g, 8.91 mmol) in acetonitrile (12 ml) was added phosphorus oxychloride (4.14 ml, 44.55 mmol) at room temperature under nitrogen, and stirred at 70 ℃ for 15 minutes. The reaction was concentrated, dissolved in acetonitrile (12 ml), concentrated once more, dried under reduced pressure from an oil pump for 15 minutes, dissolved in ethyl acetate (150 ml), washed with cold water (0-10 ℃,50 ml × 3), dried over saturated brine (50 ml), dried over sodium sulfate, filtered, and concentrated to give crude compounds 1-9.LCMS (ESI) m/z:582.0 (M + 1).

1-11 Synthesis: to compound 1-9 (4.00 g, 6.87 mmol) and compound 1-10 (4.75 g, 20.62 mmol) in acetonitrile (60 ml) under nitrogen protection was added 4A molecular sieve (10 g), and the reaction was stirred at room temperature for 14 hours. TLC showed the reaction was complete. The reaction mixture was concentrated and dissolved in ethyl acetate (300 ml), washed with water (50 ml. Times.2), the organic phase was concentrated, and the residue was separated by silica gel column chromatography (petroleum ether: ethyl acetate 3: 1 to 1: 1 (v/v)) to obtain compounds 1-11.

1-12 Synthesis: to a solution of compound 1-11 (2.0 g, 2.58 mmol) in dry DMF (20 mL) under nitrogen was added 1,8-diazabicyclo [5.4.0] undec-7-ene (0.79 g, 5.10 mmol). The reaction solution was stirred at 130 ℃ for 30 minutes. LCMS showed the reaction was complete. The reaction solution was diluted with ethyl acetate (350 ml), washed with water (50 ml. Times.3) and saturated brine (50 ml), concentrated, and the residue was subjected to silica gel column chromatography (petroleum ether: ethyl acetate 3: 1 to 1: 1 (v/v)) to isolate the compound 1-12.LCMS (ESI) m/z:729.26 (M + 1).

1-13 Synthesis: compounds 1 to 12 (1.4 g, 1.92 mmol) were added to a mixed solution of ethanol (30 ml) and water (10 ml), and then ammonium chloride (1.03 g, 19.2 mmol) and iron powder (1.07 g, 19.2 mmol) were added to the above mixed solution and stirred at 80 degrees celsius for 2 hours. The reaction was filtered, the filtrate was diluted with water (20 ml) and extracted with dichloromethane (50 ml,1 time), the organic phase was washed with saturated brine (10 ml,1 time) and dried over sodium sulfate, filtered, and the filtrate was concentrated to give compounds 1-13.LCMS (ESI) m/z:699.2 (M + 1).

1-14 Synthesis: compounds 1-13 (0.716 g, 1.02 mmol) were dissolved in acetic acid (10 ml) and acetonitrile (5 ml), then cooled to 0-5 ℃ and dichlorohydantoin (0.232 g, 1.18 mmol) was added to the above mixed solution and stirred for 25 minutes. The reaction solution was diluted with ethyl acetate (150 ml), washed with saturated sodium hydrogen sulfite (50 ml. Times.2) and saturated sodium hydrogen carbonate (50 ml), concentrated, and the residue was separated by silica gel column chromatography (petroleum ether: ethyl acetate 1: 1 to 0: 1 (v/v)) to give compounds 1 to 14.

1-15 Synthesis: compounds 1-14 (0.70 g, 0.95 mmol) were dissolved in acetic acid (7 ml) and acetonitrile (3.5 ml), then cooled to 0-5 ℃ and dichlorohydantoin (0.216 g, 1.02 mmol) was added to the above mixed solution and stirred for 30 minutes. The reaction was diluted with ethyl acetate (150 ml), washed with saturated sodium bisulfite (50 ml. Times.2) and saturated sodium bicarbonate (50 ml), concentrated and isolated by preparative HPLC (column: phenomenex luna C18. Times.50mm. Times.10 μm; mobile phase: [ water (0.225% formic acid) -acetonitrile ]; acetonitrile%: 35% -70%,25 min.) to give compounds 1-15.

1-16 Synthesis: compounds 1-15 (138 mg, 0.18 mmol) were dissolved in dichloromethane (2.0 ml), cooled to 0-5 degrees celsius trifluoroacetic acid (1.5 ml) was added and stirred for 40 minutes. The reaction solution was washed with ethyl acetate (100 ml), saturated sodium bicarbonate (40 ml. Times.2) and saturated brine (40 ml), dried over anhydrous sodium sulfate, filtered and concentrated to give compounds 1 to 16.

1A to 1D Synthesis: to a solution of compounds 1-16 (120 mg, 0.18 mmol) in dichloromethane (2 ml) was added triethylamine (36.38 mg, 0.36 mmol) and acryloyl chloride (17.00,0.188 mmol) under nitrogen and stirred at 0 degrees celsius for 30 minutes. The reaction solution was quenched with water (10 ml) and extracted with ethyl acetate (50 ml. Times.2). The combined organic phases were washed with saturated brine (10 ml), dried over sodium sulfate, filtered and concentrated to give the crude compound 1. The crude product was separated by SFC (column information: REGIS (R, R) WHELK-O1 (250mm × 25mm,10 μm); mobile phase: [0.1% ammonia-methanol ];% methanol: 40% -40%,3 min) to give 1ac (3.205-4.163 min), compound 1D (4.7 min), compound 1B (5.62 min). The 1ac was again SFC isolated (column information (s, s) WHELK-O1 (250mm. Times.50mm, 10 μm); mobile phase: [0.1% ammonia-isopropanol ];% isopropanol: 60% -60%,5.5min 90 min) to give compound 1C (6.175 min), compound 1A (7.905 min).

Compound 1A: LCMS (ESI) m/z:721.0 (M + 1). ¹ HNMR(400MHz，CDCl3)δ＝8.38(br，1H)，7.31(d，J＝6.8Hz，1H)，7.17(s，1H)，6.93(m，1H)，6.35(d，J＝16.8Hz，1H)，5.75(d，J＝10.4Hz，1H)，4.62(br，2H)，4.30-4.14(br，3H)，3.88(br，2H)，3.58-3.43(m，3H)，3.42-3.40(m，2H)，2.50(br，1H)，2.25(br，2H)，2.04(s，3H)，1.18-1.14(m，6H)。

Compound 1B: LCMS (ESI) m/z:721.2 (M + 1). ¹ HNMR(400MHz，CDCl3)δ＝8.48(d，J＝5.2Hz，1H)，7.32(d，J＝6.8Hz，1H)，7.17(s，1H)，6.93(m，1H)，6.32(d，J＝16.8Hz，1H)，5.76(dd，J＝10.4，1.6Hz，1H)，4.55-4.95(br，2H)，4.25-4.10(m，3H)，3.84(br，2H)，3.60-3.40(m，3H)，3.00-3.25(m，2H)，2.75(br，1H)，2.24(br，2H)，2.04(s，3H)，1.18-1.14(m，6H)。

Compound 1C: LCMS (ESI) m/z:721.0 (M + 1). ¹ HNMR(400MHz，CDCl3)δ＝8.48(d，J＝4.8Hz，1H)，7.31(d，J＝6.8Hz，1H)，7.14(s，1H)，7.02(d，J＝4.4Hz，1H)，6.52(br，1H)6.36(d，J＝16.8Hz，1H)，5.75(d，J＝10.4Hz，1H)，4.55-4.80(m，2H)，4.07-4.14(m，3H)，3.85(br，2H)，3.60(br，1H)，3.45(br，1H)，2.95-3.20(br，2H)，2.54(m，1H)，2.25(br，2H)，2.00(s，3H)，1.18-1.14(m，6H)。

Compound 1D: LCMS (ESI) m/z:721.1 (M + 1). ¹ HNMR(400MHz，CDCl3)δ＝8.48(d，J＝5.2Hz，1H)，7.31(d，J＝6.8Hz，1H)，7.14(s，2H)，6.50(m，1H)，6.36(d，J＝18.8Hz，1H)，5.75(d，J＝11.2Hz，1H)，4.62-4.80(br，2H)，4.07-4.14(m，2H)，3.50-3.85(m，3H)，3.20-3.60(m，4H)，2.50(br，1H)，2.25(br，2H)，2.04(s，3H)，1.18-1.14(m，6H)。

In the same way, chiral piperazine compounds 1-18 are used in the synthesis of steps 1-11, and compounds 1-25 are obtained through the same synthesis steps and methods. Compounds 1-25 were prepared by chiral SFC (column information: (s, s) WHELK-O1 (250mm. About.50mm, 10 μm), mobile phase A supercritical carbon dioxide, mobile phase B [0.1% ammonia-isopropanol ]; elution gradient: 40% -40%) to give compound 1D, followed by HPLC (column information: phenomenex Luna C8 250. About.50mm 10 μm, mobile phase A water containing 0.225% formic acid, mobile phase B acetonitrile; elution gradient: 20% -60. About.30min.) to give compound 1D.

Compound 1D: LCMS (ESI) m/z:721.2 (M + 1). ¹ HNMR(400MHz，CDCl3)δ＝8.52(d，J＝5.02Hz，1H)，7.39(d，J＝7.2Hz，1H)，7.23(s，1H)，7.09(br d，J＝4.0Hz，1H)，6.59(br s，1H)，6.43(dd，J＝16.8，1.6Hz，1H)，5.84(br d，J＝10.4Hz，1H)，4.96(br s，1H)，4.73(br s，1H)，4.21(br s，1H)，3.85-4.05(m，4H)，3.43- 3.73(m，2H)，3.04-3.34(m，2H)，2.58(m，1H)，2.32(br s，2H)，2.06(s，3H)，1.21(d，J＝6.8Hz，3H)1.15(d，J＝6.8Hz，3H)。

SFC: ee value 100%, retention time 2.951 minutes. (column information (S, S) Whelk-O1 100X 4.6mm I.D.,5.0 μm; mobile phase: mobile phase A is supercritical carbon dioxide, mobile phase B is [ acetonitrile (containing 0.05% dimethylamine) -isopropanol (V: V = 1: 2) ], elution gradient: 40% mobile phase B in supercritical carbon dioxide at a flow rate of 2.5mL/min, detector: PDA, column temperature 40 ℃ and back pressure 100Bar.

Example 2

Example 3

Example 4

4-5-C Synthesis:

after slowly dropwise adding malonyl chloride (51.20g, 374.97mmol,40.00mL, 1.17eq) to a suspension of the compound 4-5-A (50g, 320.28mmol, 1eq), potassium carbonate (55.00g, 397.93mmol, 1.24eq) and acetonitrile (500 mL) at 0 ℃,16 ℃ was stirred at 20 ℃ to complete the dropwise additionAnd (4) hours. The reaction solution was filtered through celite, the filter cake was washed with ethyl acetate (100ml × 2), the crude product obtained by concentrating the filtrate was slurried with petroleum ether/ethyl acetate (V/V = 3/1/150ml/50 mL) for 16 hours and then filtered, the filter cake was washed with the above slurried solvent (50ml × 2), and the solid was collected and dried to obtain compound 4-5-C. The relevant characterization data are as follows: LCMS m/z:256.9[ deg. ] M +1] ⁺ ； ¹ H NMR(400MHz，CDCl ₃ )δ＝9.92(br s，1H)，7.84(td，J＝1.2，8.4Hz，1H)，7.49-7.41(m，1H)，7.40-7.32(m，1H)，3.85(s，3H)，3.57(s，2H)。

4-5-E Synthesis:

to a solution of the compound 4-5-C (60g, 234.20mmol, 1eq) in acetonitrile (600 mL) was added cesium carbonate (78g, 239.40mmol, 1.02eq), and then the compound (E) -4-ethoxy-1,1,1-trifluorobut-3-en-2-one (40.12g, 238.65mmol,34mL, 1.02eq) was added dropwise to the reaction solution. The reaction solution was stirred at 20 ℃ for 1 hour, after the reaction was completed, the reaction solution was filtered, and the filter cake was washed with ethyl acetate (100ml × 2), and then the organic phases were combined and concentrated to obtain a crude product. The crude product was slurried with petroleum ether/ethyl acetate (V/V =10/1, 600mL/60 mL) for 1 hour and filtered, the resulting filter cake was washed with this solvent (50ml × 3), the filter cake was collected and dried in vacuo to give compound 4-5-E. The relevant characterization data are as follows: LCMS m/z:379.0[ M ] +1] ⁺ ；1H NMR(400MHz，DMSO-d ₆ )δ＝11.40(br s，1H)，8.67-8.31(m，1H)，7.81-7.56(m，2H)，7.34(dt，J＝5.5，8.3Hz，1H)，7.01-6.02(m，1H)，3.83-3.63(m，3H)。

4-5-F Synthesis:

to compound 4-5-E (129g, 341.06mmol, 1eq) in 2,2,2-trifluoroethanol (650 mL) was added triethylamine (50.89g, 502.92mmol,70mL, 1.47eq). The reaction mixture was reacted at 80 ℃ for 16 hours. After the reaction, the reaction mixture was concentrated to obtain a residue. To the residue was added 500mL of ethyl acetate and stirred for 1 hour, then filtered, the filter cake was washed with ethyl acetate (50ml × 2), the filter cake was collected and dried in vacuo to give compound 4-5-F. The relevant characterization data are as follows: LCMS m/z:346.9[ 2 ] M +1] ⁺ ；1H NMR(400MHz，DMSO-d ₆ )δ＝8.25(d，J＝8.3Hz，1H)，8.07-7.85(m，2H)，7.37(d，J＝8.0Hz，1H)，6.99(d，J＝8.0Hz，1H)。

4-5-K Synthesis:

to a suspension of compound 4-5-F (90G) in isopropyl acetate (810 mL) was added a solution of 4-5-G (15.75g, 0.5 eq) in isopropyl acetate (90 mL) at 25 deg.C to give a clear solution. Seed crystals (30mg, ee 91%) were added and the mixture was stirred at 25 ℃ for 16 h to precipitate a large amount of solid, which was filtered and the mother liquor washed with 2N hydrochloric acid. When 4-5-G was completely removed, the mother liquor was concentrated to give a residue. The residue was then taken up in acetonitrile (540 mL) and a solution of 4-5-H (29.1g, 0.8eq.) in acetonitrile (20 mL) to give a clear solution, and after 5 minutes a solid precipitated. The solid was dissolved at 80 ℃ to give a clear solution, cooled to 60-65 ℃, seeded (60mg, 100% ee) and stirred for 1 hour. Cooled to 25 ℃ and stirred for 16 hours, and the solid is filtered to obtain the chiral pure 4-5-K salt. Dissolving with 2N sodium hydroxide solution, extracting with methyl tert-butyl ether, drying, and vacuum distilling to obtain 4-5-K. EE value 99.5%; retention time 3.976 min, SFC analytical method: column type: chiralpak IC-3I.d.,3 μm, mobile phase: a: CO 2 ₂ B: ethanol (0.05% diethylamine, v/v) elution gradient: 5 minutes B from 5% to 40% and maintain B40% for 2.5 minutes, then maintain B5% 2.5 minute flow rate: 2.5mL/min column temperature: column pressure at 35 ℃:1500psi;

4-5 Synthesis:

4-5-K (20 g) in tetrahydrofuran at 80 deg.CPyridine (18.62g, 4.07eq) was added to the solution (160 mL), followed by dropwise addition of a solution of liquid bromine (18.6 g, 2.01eq) in tetrahydrofuran (40 mL). The mixture was stirred at 80 ℃ for 16 hours. LC-MS detects the product formation, organic phase is vacuum distilled to give a residue, the residue is diluted with ethyl acetate (120 mL), filtered, the filtrate is washed with 1M HCl solution (30 mL), water (30 mL), the organic phase is washed with anhydrous sodium sulfate, and concentrated to give a crude product of 4-5, which is slurried with methyl tert-butyl ether (6 mL) and petroleum ether (12 mL) to give an intermediate of 4-5.LCMS m/z:380.8[ mu ] M +1] ⁺ . EE value 99.5%; retention time 1.206 min, SFC analytical method: column type: chiralcel OJ-3, 100 × 4.6mm i.d.,3 μm. Mobile phase: a: CO 2 ₂ B: isopropanol (0.05% isopropylamine, v/v). Elution gradient: 0.0 min a 95% B5%, 0.5 min; a95% and B5% for 2.0 min; a60% and B40% for 3.0 min; a60% and B40% for 3.6 min; a 95% B5%, 4.0 min, a 95% B5% flow: column temperature of 3.4 mL/min: column pressure at 35 ℃:1800psi.

4-3 Synthesis:

to a mixed solution of dioxane (20 ml) and water (5 ml) of compound 4-1 (2.00 g, 12.20 mmol) and compound 4-2 (8.20 g, 48.78 mmol) was added 1,1-bis (diphenylphosphino) ferrocene palladium chloride (892.36 mg, 1.22 mmol) and potassium carbonate (3.37 g, 24.39 mmol) in one portion under nitrogen and stirred at 85 ℃ for 12 hours. The reaction mixture was diluted with water (50 ml), extracted with ethyl acetate (50 ml. Times.2), washed with saturated brine (50 ml), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and then separated by silica gel column chromatography (petroleum ether: ethyl acetate 1: 0 to 3: 1 (v/v)) to give compound 4-3.

4-4 Synthesis:

compound 4-3 (1.90 g, 10.84 mmol) was dissolved in methanol (20 ml) solution, wet palladium on carbon (190 mg, 10% pure) was added to the solution, and hydrogen (15 Psi) was passed through and stirred at 25 ℃ for 12 hours. The reaction mixture was filtered through celite, and concentrated to give compound 4-4.LCMS (ESI) m/z:180.2 (M + 1).

4-6 Synthesis:

to a mixed solvent of compound 4-4 (1.30 g, 7.25 mmol) and compound 4-5 (2.76 g, 7.25 mmol) in dioxane (30 ml) was added tris (dibenzylideneacetone) dipalladium (664.08 mg, 0.725 mmol), 4,5-bis (diphenylphosphino) -9,9-dimethylxanthene (839.23 mg, 1.45 mmol) and cesium carbonate (4.73 g, 14.50 mmol) under nitrogen. The reaction was stirred at 100 ℃ for 12 hours. The reaction solution was diluted with water (100 ml), extracted with ethyl acetate (50 ml. Times.2), then washed with saturated brine (100 ml), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and then separated by silica gel column chromatography (petroleum ether: ethyl acetate 10: 1 to 1: 1 (v/v)) to give compound 4-6.LCMS (ESI) m/z:480.1 (M + 1).

4-7 Synthesis:

to a solution of compound 4-6 (2.5 g, 5.21 mmol) in acetic acid (30 ml) was added N-bromosuccinimide (928.11 mg, 5.21 mmol). The reaction was stirred at 25 ℃ for 0.5 h. After the reaction was completed, the reaction mixture was extracted with saturated sodium sulfite solution (10 ml), the solution was adjusted to pH 8 with saturated sodium bicarbonate solution, and then extracted with ethyl acetate (50 ml), followed by washing with saturated brine (50 ml), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compounds 4 to 7.LCMS (ESI) m/z:558.0 (M + 1). 4-9 Synthesis:

to a mixed solvent of compounds 4-7 (1.90 g, 3.40 mmol) and compounds 1-5 (434.66 mg, 3.74 mmol) in toluene (20 ml) was added chloro [ (tri-tert-butylphosphino) -2- (2-aminobiphenyl) ] palladium (II) (384.75 mg, 0.68 mmol) and N, N-dicyclohexylmethylamine (731.24 mg, 3.74 mmol) under nitrogen protection. The reaction was stirred at 110 ℃ for 16 h. The reaction mixture was concentrated, diluted with water (100 ml), extracted with ethyl acetate (100 ml. Times.2), washed with saturated brine (100 ml), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and separated by silica gel column chromatography (petroleum ether: ethyl acetate 3: 1 to 1: 1 (v/v)) to give compound 4-9.LCMS (ESI) m/z:562.1 (M + 1).

4-10 Synthesis:

compound 4-9 (1.30 g, 2.32 mmol) was added to aqueous hydrochloric acid (12 mol per liter, 30 ml). The reaction was stirred at 115 ℃ for 36 hours. After completion of the reaction, the reaction mixture was concentrated, diluted with water (100 ml), extracted with ethyl acetate (150 ml. Times.2), washed with saturated brine (100 ml), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and separated by silica gel column chromatography (petroleum ether: ethyl acetate 2: 1 to 0: 1 (v/v)) to give compound 4-10.LCMS (ESI) m/z:548.0 (M + 1).

4-11 Synthesis:

compounds 4-10 (0.7 g, 1.28 mmol) were added to a mixed solution of acetic acid (10 ml) and fuming nitric acid (1.26 g, 20.00 mmol) at 0 degrees celsius, followed by sodium nitrite (441.10 mg, 6.39 mmol) and stirred at 25 degrees celsius for 0.5 hours. The reaction solution was diluted with ice water (50 ml), extracted with ethyl acetate (50 ml × 2), and washed with saturated brine (50 ml), and the resulting organic phase was adjusted to pH =6 with saturated aqueous sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 4-11.LCMS (ESI) m/z:593.0 (M + 1).

4-12 Synthesis:

to a solution of compounds 4-11 (0.70 g, 1.18 mmol) in acetonitrile (10 ml) was added phosphorus oxychloride (985.83 mg, 5.95 mmol) and N, N-dimethylformamide (863.62 μ g, 11.82 μmol) at room temperature and stirred at 45 ℃ for 1 hour. The reaction mixture was diluted with water (50 ml), extracted with ethyl acetate (50 ml), and washed with saturated brine (50 ml), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give compound 4-12.LCMS (ESI) m/z:611.0 (M + 1).

4-14 Synthesis:

to compound 4-12 (0.60 g, 0.982 mmol) and compound 4-13 (452.39 mg, 1.96 mmol) in acetonitrile (10 ml) was added 4A molecular sieve (1 g), and the reaction was stirred at 45 ℃ for 12 hours. The reaction solution was filtered through celite and concentrated, diluted with water (20 ml), extracted with ethyl acetate (20 ml. Times.2), and then washed with saturated brine (20 ml), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated by silica gel column chromatography (petroleum ether: ethyl acetate 2: 1 to 1: 1 (v/v)) to give compound 4-14.LCMS (ESI) m/z:805.1 (M + 1).

4-15 Synthesis:

to a solution of compound 4-14 (0.4 g, 0.497 mmol) in dry DMF (8 ml) was added 1,8-diazabicyclo [5.4.0] undec-7-ene (151.34 mg, 0.994 mmol). The reaction solution was stirred at 130 ℃ for 30 minutes. The reaction solution was diluted with ethyl acetate (50 ml), washed with water (30 ml. Times.3) and saturated brine (20X 2 ml), and the organic phase was dried over anhydrous sodium sulfate to obtain compounds 4-15.LCMS (ESI) m/z:758.2 (M + 1).

4-16 Synthesis:

compound 4-15 (0.35 g, 0.461 mmol) was added to a mixed solution of ethanol (5 ml) and water (3 ml), and then ammonium chloride (247.08 mg, 4.62 mmol) and iron powder (257.95 mg, 4.62 mmol) were added to the above mixed solution and stirred at 80 degrees celsius for 2 hours. The reaction solution was filtered, the filtrate was diluted with water (20 ml) and extracted with ethyl acetate (30 ml × 2), the organic phase was washed with saturated brine (20 ml) and dried over sodium sulfate, filtered, and the filtrate was concentrated to give compound 4-16.LCMS (ESI) m/z:728.2 (M + 1).

4-17 Synthesis:

compounds 4-16 (0.18 g, 0.247 mmol) were dissolved in DMF (10 ml) and THF (5 ml), then dichlorohydantoin (82.84 mg, 0.42 mmol) was added to the above mixed solution with cooling to 0 ℃ and stirring for 30 minutes. The reaction solution was diluted with water (20 ml), extracted with ethyl acetate (20 ml × 2), and the organic phase was washed with saturated brine (20 ml), dried over sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude compound 4-17.LCMS (ESI) m/z:796.1 (M + 1).

4-18 Synthesis:

compounds 4-17 (200 mg, 0.25 mmol) were dissolved in dichloromethane (3.0 ml), trifluoroacetic acid (1 ml) was added at 25 ℃ and stirred for 20 minutes. Concentrating the reaction solution to obtain crude compounds 4-18.

Synthesis of Compound 4:

compound 4-18 (200 mg, 0.287 mmol) was dissolved in a mixed solution of THF (4 ml) and water (2 ml), the pH of the above solution was adjusted to 8 with potassium carbonate, and acryloyl chloride (38.98 mg, 0.43 mmol) was added dropwise to the reaction solution and stirred for 5 minutes. The reaction mixture was diluted with water (20 ml), extracted with ethyl acetate (20 ml. Times.2), the organic phase was washed with saturated brine (20 ml), dried over sodium sulfate, filtered, the filtrate was concentrated and subjected to preparative HPLC (column: phenomenex luna C18 150. Times.25mm. Times.10 μm; mobile phase: [ water (0.225% formic acid) -acetonitrile](ii) a Acetonitrile%: 49% -69%,10 min) to obtain compound 4.LCMS (ESI) m/z:750.1 (M + 1). ¹ HNMR(400MHz，DMSO-d6)δ＝8.98(s，1H)，7.63(d，J＝7.6Hz，1H)，7.2(s，1H)，6.99-6.74(m，1H)，6.25-6.07(m，3H)，5.81-5.72(m，1H)，4.71-4.35(m，2H)，4.18(dd，J＝4.8，8.4Hz，2H)，4.02-3.70(m，2H)，3.66-3.43(m，3H)，2.92-2.74(m，1H)，2.14(s，2H)，1.13-0.83(m，13H)

Example 5

5-3 Synthesis: 1,1-bis (diphenylphosphino) ferrocene palladium chloride (2.5 g, 3.41 mmol) and potassium carbonate (35.40 g, 256.11 mmol) were added in one portion to a mixed solution of dioxane (150 ml) and water (30 ml) of compound 5-1 (14 g, 85.37 mmol) and compound 5-2 (52.59 g, 341.48 mmol) under nitrogen protection, and stirred at 85 degrees Celsius for 12 hours. The reaction mixture was concentrated, diluted with water (500 ml), extracted with ethyl acetate (500 ml. Times.2), washed with saturated brine (200 ml), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and separated by silica gel column chromatography (petroleum ether: ethyl acetate 1: 0 to 1: 1 (v/v)) to give compound 5-3.

5-4 Synthesis: compound 5-3 (5.7 g, 38.73 mmol) was dissolved in methanol (50 ml) solution, wet palladium on carbon (500 mg, 10% pure) was added to the solution, and hydrogen (15 Psi) was passed through and stirred at 25 ℃ for 12 hours. The reaction solution was filtered through celite and concentrated to give compound 5-4.LCMS (ESI) m/z:152.2 (M + 1).

5-6 Synthesis:

to a mixed solvent of compound 5-4 (3.7 g, 24.47 mmol) and compound 4-5 (10.26 g, 26.92 mmol) in dioxane (30 ml) was added tris (dibenzylideneacetone) dipalladium (2.24 g, 2.45 mmol), 4,5-bis (diphenylphosphino) -9,9-dimethylxanthene (2.83 g, 4.89 mmol) and cesium carbonate (15.95 g, 48.94 mmol) under nitrogen. The reaction was stirred at 100 ℃ for 12 hours. The reaction solution was diluted with water (100 ml), extracted with ethyl acetate (50 ml. Times.2), then washed with saturated brine (100 ml), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and then separated by silica gel column chromatography (petroleum ether: ethyl acetate 5: 1 to 1: 1 (v/v)) to give compound 5-6.LCMS (ESI) m/z:452.1 (M + 1).

5-7 Synthesis:

to a solution of compound 5-6 (5.4 g, 11.96 mmol) in acetic acid (50 ml) was added N-bromosuccinimide (3.19 g, 17.95 mmol). The reaction was stirred at 25 ℃ for 0.5 h. After the reaction was completed, the reaction mixture was quenched with a saturated sodium sulfite solution (50 ml), the solution was adjusted to pH 8 with sodium hydroxide, and then extracted with ethyl acetate (200 ml), followed by washing with a saturated brine (100 ml), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and then separated by silica gel column chromatography (petroleum ether: ethyl acetate 4: 1 to 2: 1 (v/v)) to obtain compounds 5 to 7.LCMS (ESI) m/z:529.9 (M + 1).

5-9 Synthesis:

to a mixed solvent of compounds 5-7 (1.5 g, 2.83 mmol) and compounds 1-5 (492.69 mg, 4.24 mmol) in toluene (15 ml) was added chloro [ (tri-tert-butylphosphino) -2- (2-aminobiphenyl) ] palladium (II) (289.89 mg, 0.565 mmol) and N, N-dicyclohexylmethylamine (607.84 mg, 3.11 mmol) under nitrogen protection. The reaction was stirred at 110 ℃ for 16 hours. The reaction mixture was concentrated, diluted with water (100 ml), extracted with ethyl acetate (100 ml. Times.2), washed with saturated brine (100 ml), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and separated by silica gel column chromatography (petroleum ether: ethyl acetate 3: 1 to 1: 1 (v/v)) to give compound 5-9.LCMS (ESI) m/z:534.1 (M + 1).

5-10 Synthesis:

compound 5-9 (0.5 g, 0.937 mmol) was added to aqueous hydrochloric acid (12 mol per liter, 20 ml). The reaction was stirred at 115 ℃ for 36 hours. After completion of the reaction, the reaction mixture was concentrated, diluted with water (50 ml), extracted with ethyl acetate (50 ml × 2), and washed with saturated brine (50 ml), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 5-10.LCMS (ESI) m/z:520.0 (M + 1).

5-11 Synthesis:

compound 5-10 (0.40 g, 0.77 mmol) was added to a mixed solution of acetic acid (5 ml) and fuming nitric acid (0.96 g, 15.23 mmol) at 0 deg.c, followed by sodium nitrite (265.67 mg, 3.85 mmol) and stirred at 25 deg.c for 0.5 h. The reaction solution was diluted with ice water (50 ml), extracted with ethyl acetate (20 ml × 2), and washed with saturated brine (20 ml × 2), and the resulting organic phase was adjusted to pH =6 with saturated aqueous sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 5-11.LCMS (ESI) m/z:565.0 (M + 1).

5-12 Synthesis:

to a solution of compound 5-11 (0.30 g, 0.53 mmol) in acetonitrile (3 ml) was added phosphorus oxychloride (407.51 mg, 2.66 mmol) and N, N-dimethylformamide (388.52 μ g, 5.32 mmol) at room temperature and stirred at 45 ℃ for 1 hour. The reaction mixture was diluted with water (10 ml), extracted with ethyl acetate (10 ml), washed with saturated brine (10 ml), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give compounds 5 to 12.LCMS (ESI) m/z:583.0 (M + 1).

5-14 Synthesis:

to compound 5-12 (0.28 g, 0.48 mmol) and compound 4-13 (221.28 mg, 0.96 mmol) in acetonitrile (3 ml) was added 4A molecular sieve (0.5 g), and the reaction was stirred at 45 ℃ for 12 hours. The reaction solution was filtered through celite and concentrated, diluted with water (20 ml), extracted with ethyl acetate (20 ml. Times.2), and then washed with saturated brine (20 ml), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated by silica gel column chromatography (petroleum ether: ethyl acetate 2: 1 to 0: 1 (v/v)) to give compound 5-14.LCMS (ESI) m/z:777.1 (M + 1).

5-15 Synthesis:

to a solution of compound 5-14 (0.15 g, 0.19 mmol) in dry DMF (2 mL) was added 1,8-diazabicyclo [5.4.0] undec-7-ene (44.10 mg, 0.29 mmol). The reaction solution was stirred at 130 ℃ for 30 minutes. The reaction solution was diluted with ethyl acetate (20 ml), washed with water (10 ml. Times.3) and saturated brine (10X 2 ml), and the organic phase was dried over anhydrous sodium sulfate to give compounds 5 to 15.LCMS (ESI) m/z:730.2 (M + 1).

5-16 Synthesis:

compounds 5-15 (0.11 g, 0.15 mmol) were added to a mixed solution of ethanol (1 ml) and water (1 ml), and then ammonium chloride (80.64 mg, 1.51 mmol) and iron powder (84.19 mg, 1.51 mmol) were added to the above mixed solution and stirred at 80 degrees celsius for 12 hours. The reaction solution was filtered, the filtrate was diluted with water (10 ml) and extracted with ethyl acetate (10 ml), the organic phase was washed with saturated brine (10 ml) and dried over sodium sulfate, filtered, and the filtrate was concentrated to give compound 5-16.LCMS (ESI) m/z:700.2 (M + 1).

5-17 Synthesis:

compound 5-16 (0.1 g, 0.14 mmol) was dissolved in DMF (2 ml), then cooled to 0 ℃ and dichlorohydantoin (47.87 mg, 0.24 mmol) was added to the above mixed solution and stirred for 30 minutes. The reaction solution was diluted with water (20 ml), extracted with ethyl acetate (20 ml × 2), and the organic phase was washed with saturated brine (20 ml), dried over sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude compound 5-17.LCMS (ESI) m/z:768.2 (M + 1).

5-18 Synthesis:

compounds 5-17 (140 mg, 0.18 mmol) were dissolved in dichloromethane (2 ml), trifluoroacetic acid (0.5 ml) was added at 25 ℃ and stirred for 20 min. Concentrating the reaction solution to obtain a crude product, namely a compound 5-18.

Synthesis of Compound 5:

compounds 5 to 18 (120 mg, 0.18 mmol) were dissolved in a mixed solution of THF (2 ml) and water (1 ml), the pH of the above solution was adjusted to 8 with potassium carbonate, and acryloyl chloride (24.37 mg, 0.27 mmol) was added dropwise to the reaction solution and stirred for 5 minutes. The reaction was diluted with water (10 ml), extracted with ethyl acetate (10 ml), the organic phase was washed with saturated brine (10 ml), dried over sodium sulfate, filtered, and the filtrate was concentrated and separated by preparative HPLC (column: phenomenex luna C18 150 × 25mm × 10 μm; mobile phase: [ water (0.1% trifluoroacetic acid) -acetonitrile ];% acetonitrile: 50% -80%,10 min) to give compound 5.LCMS (ESI) m/z:722.1 (M + 1).

Example 6

6-3 Synthesis: to a solution of compound 6-1 (10 g, 37.70 mmol) and compound 5-2 (24.46 g, 158.79 mmol) dioxane (80 ml) and water (20 ml) was added Pd (dppf) Cl in one portion under nitrogen protection ₂ (2.90 g, 3.97 mmol) and potassium carbonate (16.46 g, 119.09 mmol) were stirred at 100 degrees celsius for 12 hours. The reaction mixture was concentrated and then separated by silica gel column chromatography (petroleum ether: ethyl acetate 0: 1 (v/v)) to obtain compound 6-3.LCMS (ESI) m/z:147.2 (M + 1).

6-4 Synthesis: to a solution of compound 6-3 (5.2 g, 35.57 mmol) in methanol (20 ml) at 20 deg.c was added palladium on carbon (0.5 g, 17.10 mmol) under nitrogen and stirred at 20 deg.c under hydrogen (15 PSI) for 10 minutes. Filtering the reaction solution through diatomite, and spin-drying to obtain a crude product 6-4.LCMS (ESI) m/z:151.2 (M + 1)

6-6 Synthesis:

to a mixed solution of compounds 6-4 (5 g, 33.28 mmol) and compounds 4-5 (12.68 g, 32.28 mmol) in dioxane (150 ml) was added tris (dibenzylideneacetone) dipalladium (1.52 g, 1.66 mmol), 4,5-bis (diphenylphosphino) -9,9-dimethylxanthene (1.93 g, 3.33 mmol) and cesium carbonate (21.69 g, 66.57 mmol) under nitrogen. The reaction was stirred at 100 ℃ for 3 hours. The reaction mixture was concentrated and then separated by silica gel column chromatography (petroleum ether: ethyl acetate 1: 1 (v/v)) to obtain compound 6-6.LCMS (ESI) m/z:451.1 (M + 1)

6-7 Synthesis:

under nitrogen, compound 6-6 (6 g, 13.323 mmol) was dissolved in ethyl acetate (100 ml) and NBS (2.37 g, 13.32 mmol) was added in portions. The reaction was carried out at 20 ℃ for 0.5 hour. After the reaction is finished, the mixture is extracted and concentrated and then is separated by silica gel column chromatography (petroleum ether: ethyl acetate 1: 1 (v/v)) to obtain the compound 6-7.LCMS (ESI) m/z:530.9 (M + 1). ¹ H NMR(400MHz，CDCl ₃ )δ＝8.25(s，2H)，8.07(d，J＝8.4Hz，1H)，7.66(dt，J＝5.6，8.4Hz，1H)，7.54(dt，J＝1.2，8.4Hz，1H)，6.88(s，2H)，2.63-2.36(m，4H)，1.18-1.04(m，6H)。

6-9 Synthesis:

to dioxane (50 ml) under nitrogen protection at 20 degrees celsius were added compounds 6-7 (5 g, 9.45 mmol) and compounds 1-5 (4.39 g, 37.79 mmol) dicyclohexylmethylamine (2.77 g, 14.17 mmol), lithium chloride (1.6 g, 37.79 mmol) and tri-tert-butylphosphine palladium (724.17 mg, 1.42 mmol) in that order. The reaction was carried out at 105 ℃ under nitrogen for 12 hours. The reaction mixture was diluted with ethyl acetate (150 ml), washed with saturated brine (50 ml, 3 times), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and then separated by silica gel column chromatography (petroleum ether: ethyl acetate 0: 1 (v/v)) to obtain compound 6-9.

6-10 Synthesis:

under nitrogen, compound 6-9 (2 g, 3.76 mmol) was added to aqueous hydrochloric acid (12 mol per liter, 20 ml). The reaction was stirred at 110 ℃ for 2 hours. After the reaction was completed, the mixture was directly concentrated under reduced pressure, and then extracted with ethyl acetate (25 ml, 2 times), and the organic phase was dried over sodium sulfate, filtered, and concentrated to obtain compound 6-10.LCMS (ESI) m/z:519.0 (M + 1).

6-11 Synthesis:

compound 6-10 (2 g, 3.86 mmol) was added to a mixed solution of acetic acid (15 ml) and fuming nitric acid (1.22 g, 19.29 mmol) at 0 deg.c, followed by addition of sodium nitrite (1.33 g, 19.29 mmol), and stirred at 15 deg.c for 0.5 h. The reaction was diluted with ethyl acetate (50 ml), then washed with saturated sodium bicarbonate (40 ml), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give compound 6-11.LCMS (ESI) m/z:564.2 (M + 1)

6-12 Synthesis:

to a solution of compounds 6-11 (1.85 g, 3.28 mmol) and DMF (24 mg, 0.33 mmol) in acetonitrile (15 ml) was added phosphorus oxychloride (2.52 g, 16.42 mmol) at room temperature under nitrogen and stirred at 40 ℃ for 0.5 h. After the reaction is finished, the compound 6-12 is obtained by extraction and concentration and then separation by silica gel column chromatography (petroleum ether: ethyl acetate 2: 1 (v/v)). LCMS (ESI) m/z:582.0 (M + 1).

6-14 Synthesis:

to acetonitrile (15 ml) of compounds 6-12 (0.5 g, 0.859 mmol) and 4-13 (435.39 mg, 1.89 mmol) was added 4A molecular sieve (2 g) under nitrogen and the reaction was stirred at 45 ℃ for 12 hours. The reaction was filtered through celite, extracted with ethyl acetate (20 ml. Times.2) and water (20 ml), concentrated and separated by silica gel column chromatography (petroleum ether: dichloromethane: ethyl acetate 6: 7: 8 (v/v)) to give compound 6-14.LCMS (ESI) m/z:776.5 (M + 1).

6-15 Synthesis:

to a solution of compound 6-14 (300 mg, 386.75 micromoles) in dry DMF (3 ml) was added 1,8-diazabicyclo [5.4.0] undec-7-ene (88.32 mg, 580.12 micromoles) under nitrogen. The reaction solution was stirred at 130 ℃ for 0.5 hour. LCMS showed the reaction was complete. The reaction solution was diluted with ethyl acetate (20 ml), washed with water (20 ml), concentrated, and the residue was subjected to silica gel column chromatography (petroleum ether: ethyl acetate 0: 1 (v/v)) to isolate compound 6-15.LCMS (ESI) m/z:729.1 (M + 1).

6-16 Synthesis:

compounds 6-15 (0.12 g 164.68 micromoles) were added to a mixed solution of ethanol (4 ml) and water (2 ml), and then ammonium chloride (44.04 mg, 823.40 micromoles) and iron powder (45.99 mg, 823.40 micromoles) were added to the above mixed solution and stirred at 80 degrees celsius for 0.5 hour. The reaction solution was filtered and spun-dried, and the residue was separated by silica gel column chromatography (petroleum ether: ethyl acetate 0: 1 (v/v)) to obtain compound 6-16.LCMS (ESI) m/z:699.2 (M + 1).

6-17 Synthesis:

compounds 6-16 (50 mg, 71.56 micromoles) were dissolved in DMF (2 ml) and ethyl acetate (2 ml), then dichlorohydantoin (23.97 mg, 121.65 micromoles) was added to the above mixed solution with stirring to 0 ℃. The reaction was diluted with ethyl acetate (20 ml), washed with saturated sodium bisulfite (10 ml × 2) and saturated sodium bicarbonate (10 ml), and concentrated to give crude compound 6-17.LCMS (ESI) m/z:767.1 (M + 1).

6-18 Synthesis:

compound 6-17 (50 mg, 65.14 mmol) was dissolved in dichloromethane (3.0 ml), cooled to 0-5 degrees celsius trifluoroacetic acid (1.54 g, 13.51 mmol) was added and stirred at 15 degrees celsius for 15 minutes. The reaction solution is directly decompressed and concentrated to obtain the compounds 6-18.LCMS (ESI) m/z:667.4 (M + 1).

Synthesis of Compound 6:

to a solution of compounds 6-18 (50 mg, 65.14 micromol) in tetrahydrofuran (5 ml) and water (2 ml) was added potassium carbonate (20.71 mg, 149.82 micromol) and acryloyl chloride (8.14 mg, 89.89 micromol) under nitrogen with stirring at 25 ℃ for 30 minutes. The reaction solution was extracted with water (10 ml), ethyl acetate (50 ml. Times.2). The combined organic phases were dried over sodium sulfate, filtered and concentrated to give the crude compound 6. The crude product was purified by prep-HPLC (column information: phenomenex Gemini-NX C18 75 x 30mm x 3 μm; mobile phase: [ water (0.225% formic acid) -acetonitrile ]; acetonitrile%: 32% -42%,7 min) to give compound 6.LCMS (ESI) m/z:721.1 (M + 1).

¹ H NMR(400MHz，CDCl ₃ )δ＝8.45(br d，J＝8.5Hz，2H)，7.42(d，J＝7.0Hz，1H)，7.27-7.16(m，1H)，6.73-6.35(m，2H)，5.89-5.78(m，1H)，5.07-4.59(m，1H)，4.30-3.83(m，4H)，3.75-2.93(m，3H)，2.51-2.17(m，7H)，1.22-0.88(m，8H)。

Experimental example 1: anti-proliferation assay for H358 cells

Experimental materials:

RPMI-1640 medium, penicillin/streptomycin antibiotics from Verben, fetal bovine serum from Biosera. CellTiter-Glo (cell viability chemiluminescence detection reagent) reagent was purchased from Promega. The NCI-H358 cell line was purchased from the cell bank of Chinese academy of sciences. Nivo multi-label analyzer (PerkinElmer).

The experimental method comprises the following steps:

NCI-H358 cells were seeded in white 96-well plates, 80. Mu.L of cell suspension per well, containing 4000 NCI-H358 cells. The cell plates were placed in a carbon dioxide incubator overnight.

The test compounds were diluted 3-fold with a rifling to the 9 th concentration, i.e. from 2mM to 304nM, setting up a duplicate well experiment. Add 78. Mu.L of medium to the intermediate plate, transfer 2. Mu.L of each well of the gradient dilution compound to the intermediate plate according to the corresponding position, mix well and transfer 20. Mu.L of each well to the cell plate. The concentration of compound transferred to the cell plate ranged from 10. Mu.M to 1.52nM. The cell plates were placed in a carbon dioxide incubator for 5 days. A separate cell plate was prepared, and the signal values were read on the day of drug addition as maximum values (Max values in the following equation) for data analysis. To each well of this cell plate, 25. Mu.L of a cell viability chemiluminescence detection reagent was added, and the plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal. Reading with a multi-label analyzer.

Add 25. Mu.L of cell viability chemiluminescence detection reagent to the cell plate and incubate for 10 min at room temperature to stabilize the luminescence signal. Reading with a multi-label analyzer.

And (3) data analysis:

the raw data was converted to inhibition rate, IC, using the equation (Sample-Min)/(Max-Min) × 100% ₅₀ The values of (A) can be obtained by curve fitting of four parameters (obtained in the GraphPad Prism "log (inhibitor) vs. response- -Variable slope" model). Table 1 provides the inhibitory activity of the compounds of the present invention on NCI-H358 cell proliferation.

TABLE 1

Sample (I)	H358 IC 50 (nanomole per liter)
Compound 1A	5.27
Compound 1B	16.2
Compound 1C	0.462
Compound 1D	0.917
Compound 4	0.9
Compound 5	4
Compound 6	2

And (4) conclusion:

the compound has strong inhibition effect on H358 cell proliferation.

Experimental example 2: MIA-PA-CA-2 cell antiproliferation assay

Experimental materials:

DMEM medium, fetal bovine serum from Biosera and horse serum from Gibco. CellTiter-Glo (cell viability chemiluminescence detection reagent) reagent was purchased from Promega. MIA-PA-CA-2 cell line was purchased from Nanjing Kebai Biotech Co. EnVision multi-label analyzer (PerkinElmer).

The experimental method comprises the following steps:

MIA-PA-CA-2 cells were seeded in white 96-well plates, 80. Mu.L of cell suspension per well, containing 1000 MIA-PA-CA-2 cells. The cell plates were placed in a carbon dioxide incubator overnight.

The test compounds were diluted 5-fold with a calandria to the 8 th concentration, i.e. from 2mM to 26nM, setting up a double-well experiment. Add 78. Mu.L of medium to the intermediate plate, transfer 2. Mu.L of each well of the gradient dilution compound to the intermediate plate according to the corresponding position, mix well and transfer 20. Mu.L of each well to the cell plate. The concentration of compound transferred to the cell plate ranged from 10. Mu.M to 0.13nM. The cell plates were incubated in a carbon dioxide incubator for 3 days. A separate cell plate was prepared, and the signal values were read on the day of drug addition as maximum values (Max values in the following equation) for data analysis. To each well of this cell plate, 25. Mu.L of a cell viability chemiluminescence detection reagent was added, and the plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal. Reading with a multi-label analyzer.

And (3) data analysis:

the raw data was converted to inhibition, IC, using the equation (sample-min)/(max-min) × 100% ₅₀ Values of (d) are then obtained by curve fitting of four parameters (from the "log (inhibitor)/response-variable domain of action" model in GraphPad Prism software). Table 2 provides the inhibitory activity of the compounds of the invention on MIA-PA-CA-2 cell proliferation.

TABLE 2

Sample (I)	MIA-PA-CA-2 IC 50 (nanomole per liter)
Compound 1A	46
Compound 1B	52
Compound 1C	7
Compound 1D	8
Compound 4	1
Compound 5	5
Compound 6	4

And (4) conclusion: the compound of the invention shows better inhibitory activity on MIA-PA-CA-2 cell proliferation.

Experimental example 3: antitumor Activity test on in vivo xenograft tumor model

Purpose of the experiment:

the human non-small cell lung cancer H358 is used for examining the single tumor inhibition effect of the compound to be tested on an in vivo tumor model.

The experimental method comprises the following steps:

female Balb/c nude mice were subcutaneously inoculated with H358 human non-small cell lung cancer cell lines, randomly grouped on day 6 after inoculation according to tumor size and animal body weight, and administered as described below.

Solvent control group: dosing was started in the afternoon of the day, once a day, and vehicle (10% DMSO/60% PEG400/30% deionized water) was gavaged at a dose of 0.1mL/10g body weight for 28 consecutive days.

Administration group: administration was started in the afternoon of the day on a daily basis, and 3 groups were gavaged with compound 1D (10% dmso/60% peg400/30% deionized water) at doses of 1mg/kg, 3mg/kg, and 10mg/kg body weight, respectively, for 28 consecutive days.

Mice were weighed twice a week during the experiment and tumor volume was measured (length x width) ² The equation of/2 calculates tumor volume). Tumor inhibition rate TGI (%) = [ (1-mean tumor volume at the end of treatment group administration-mean tumor volume at the time of treatment group administration)/(mean tumor volume at the end of solvent control group treatment-mean tumor volume at the time of solvent control group administration)]×100％。

The formula for relative tumor volume calculation was RTV = TVn/TV1 × 100%

Where TV1 is the tumor volume on the day of group administration and TVn is the tumor volume on the day of measurement.

Relative tumor proliferation rate (T/C%) = RTVt/RTVc × 100%

Wherein RTVt is the mean relative tumor volume of the treatment group, and RTVc is the mean relative tumor volume of the vehicle control group.

The experimental results are as follows:

the mean tumor volume of the solvent control group reached 669.16mm 28 days after administration ³ Mean tumor volumes of 341.36mm for compound 1D at 1, 3 and 10mg/kg doses, respectively ³ 、199.4mm ³ And 37.5mm ³ The TGI was 58.78%, 84.17% and 113.19%, respectively, and the T/C was 51.35%, 27.59% and 5.76%, respectively, which significantly inhibited tumor growth compared to the solvent control group (P values of 0.0629,0.0029 and P < 0.0001, respectively).

And (4) experimental conclusion:

the compound has good dose dependence trend, can obviously inhibit the growth of tumors, does not obviously reduce the body weight of mice, and has better tolerance.

Experimental example 4: pharmacokinetic study of female BALB/c nude mice

This experiment is a pharmacokinetic study of female BALB/c nude mice after oral administration of compound 1D, respectively. Three groups were tested in total, and 1, 3 and 10mg/kg of compound 1D were gavaged separately. Each group of animals was divided into two batches, and plasma and tumor tissue samples were collected at different time points after dosing. The collection time points for each group of animal plasma samples were: 0.083, 0.25, 0.5, 1, 2,4, 6 and 8 hours, tumor tissue samples were taken at time points of 1, 4 and 8 hours, respectively, and plasma was collected at the time points of respective sample collection after administration, and tumor tissue was exfoliated and weighed. Storing the plasma at-80 deg.C, placing the tumor tissue in a freezing tube, and quick-freezing in liquid nitrogen. Cross-plasma sample collection the mean concentration values for each group at the corresponding time points were calculated for calculation of PK parameters.

The experimental results are as follows:

TABLE 3 plasma and tumor pharmacokinetic parameters of female BALB/c nude mice after intragastric administration of Compound 1D

"NR" lacks a terminal elimination phase or is too low exposed;

"ND" cannot be determined;

AUC ratio = tumor AUC _0-last Plasma AUC _0-last 。

TABLE 4 drug concentrations in BALB/c nude mice mean plasma and tumor at various times in females following gavage administration of Compound 1D

"ND" cannot be determined.

And (4) conclusion: the compound has moderate metabolism speed in mice, high drug concentration in tumor tissues and good pharmacokinetic property.

Experimental example 5: in vivo pharmacodynamic study of compound on human colon cancer CO-04-0070 subcutaneous xenograft tumor model

Purpose of the experiment:

in vivo efficacy of the compounds was evaluated in a nude mouse model of human colon cancer CO-04-0070 subcutaneous xenograft tumor BALB/c.

Tumor tissue inoculation and grouping:

each animal was inoculated with a volume of about 30mm at the right dorsal position ³ The average tumor volume of the tumor mass of the CO-04-0070FP7 generation tumor mass reaches 151mm ³ At that time, the administration was started using random grouping.

TABLE 5 Experimental animal groups and dosing regimens

Group of	Medicine	Dosage (mg/kg)	Administration volume (. Mu.L/g) < 2 >	Route of administration	Frequency of administration
1	Solvent control	--	10	PO	QD x 3 weeks
2	Compound 1D	3	10	PO	QD x 3 weeks
3	Compound 1D	10	10	PO	QD x 3 weeks
4	Compound 1D	30	10	PO	QD x 3 weeks

Tumor measurements and experimental indices:

tumor diameters were measured twice weekly using a vernier caliper. The formula for tumor volume is: v =0.5a × b ² And a and b represent the major and minor diameters of the tumor, respectively.

The tumor suppressor therapeutic effect of the compound was evaluated as TGI (%) or relative tumor proliferation rate T/C (%). Relative tumor proliferation rate T/C (%) = T _RTV /C _RTV ×100％(T _RTV : treatment group mean RTV; c _RTV : negative control group mean RTV). Calculating Relative Tumor Volume (RTV) according to the tumor measurement result, wherein the calculation formula is RTV = V _t /V ₀ In which V is ₀ The resulting tumor volume, V, was measured at the time of group administration (i.e., D0) _t Tumor volume at a certain measurement, T _RTV And C _RTV The same day data was taken. TGI (%), reflecting the rate of tumor growth inhibition.

TGI (%) = [ (1- (average tumor volume at the end of administration of a certain treatment group-average tumor volume at the start of administration of the treatment group))/(average tumor volume at the end of treatment of the solvent control group-average tumor volume at the start of treatment of the solvent control group) ] × 100%.

After the experiment is finished, the tumor weight is detected, and T/C is calculated _weight Percent, T _weight And C _weight Tumor weights of the administration group and vehicle control group are shown, respectively.

The experimental results are as follows:

TABLE 6 antitumor Effect of Compounds on human Colon cancer CO-04-0070 xenograft tumor model

Note: a. mean ± SEM, n =8.

TABLE 7 tumor weights of the groups

Note: a. mean ± SEM, n =8.

b. Tumor growth inhibition by T/C _weight ＝TW _treatment /TW _vehicle And (4) calculating.

c.p values were obtained by analyzing tumor weights using one-way ANOVA and vehicle treated groups, and F values were significantly different (p < 0.01) and analyzed by Games-Howell method.

The change of body weight: all the administered groups did not significantly decrease in weight average in this model

And (4) conclusion: the compound of the invention has obvious effect of inhibiting tumor growth and shows certain dose dependence. .

Experimental example 6: protein level assay of ERK and p-ERK in tumor tissue

This experiment analyzed the protein levels of ERK and p-ERK in tumor tissues of different groups of administration 6 hours after administration of compound 1D (30 mg/kg) in a CO-04-0070 subcutaneous xenograft tumor model of human colon cancer by immunoblotting.

Experimental materials:

1) Reagent

2) Instrument for measuring the position of a moving object

3) Antibodies

The experimental method comprises the following steps:

1) Protein extraction quantification

And taking out the quick-frozen tissue sample from a refrigerator at the temperature of minus 80 ℃. The procedure was performed on dry ice, and a portion of the tissue (about 50-100 mg) was excised and placed in a 2mL centrifuge tube containing a steel ball, and 500. Mu.L of cell lysate RIPA (1% protease inhibitor and phosphatase inhibitor had been freshly added). Tissue was disrupted using the highest frequency for 5 minutes by Tissuelyser LT. The tissue lysate was lysed on ice for 30 minutes. Centrifuge at 12,000rpm at 4 ℃ for 10 minutes and take the supernatant into a new 1.5mL centrifuge tube. Protein quantification was performed using BCA quantification kit. According to the quantitative result, protein samples for sample loading are prepared, the protein concentration of the samples is unified to 2 mug/mu L, LDS sample loading buffer solution (4 times) and sample reducing agent (10 times) are added, and the samples are heated at constant temperature of 100 ℃ for 10 minutes. Western blot, or the denatured sample was stored in a-80 ℃ freezer.

2) Immunoblotting

And (4) unfreezing the loaded sample. Sampling: in SDS-PAGE gels, 10. Mu.L of each well was loaded. Electrophoresis: 80 volts, 30 minutes, then 120 volts, 90 minutes. Film transfer: the film is rotated by an iBlot2 film rotating set and a film rotating instrument, and the P3 program is run for 7 minutes. And after the membrane is switched, cutting the membrane according to the molecular weight of the protein to be detected. And (3) sealing: the membrane was blocked in blocking solution (5% skim milk prepared with 1-fold TBST), shaken at room temperature for 1 hour. Incubating the primary antibody: primary antibody (1: 1000) was added at the appropriate dilution (diluted with 5% skim milk or bovine serum albumin in 1-fold TBST) overnight at 4 ℃ with slow shaking. Membranes were washed with 1-fold TBST 3 times for 10 min each time at room temperature with shaking. Incubation of secondary antibody: add a suitable dilution of secondary antibody (1: 10000) at room temperature with slow shaking for 1 hour. Membranes were washed with 1-fold TBST 3 times for 10 min each time at room temperature with shaking. Chemiluminescence: HRP substrate in West Femto super-sensitive chemiluminescence kit was added to the membrane. Chemiluminescence was detected on a Tanon 5200Multi machine and photographed.

3) Quantification of expression

The intensity of the density of the immunoblot chemiluminescence bands was relatively quantified using software such as ImageJ.

The experimental results are as follows: see fig. 1 and 2.

In fig. 2 ". Star" indicates p value < 0.01 two-tailed T test. "normalized fold change" is the normalized fold change.

And (4) conclusion: the p-ERK protein expression level in the tumor tissues of the compound treatment group is obviously reduced.

Claims

A compound represented by the formula (III) or a pharmaceutically acceptable salt thereof,

wherein, the first and the second end of the pipe are connected with each other,

m is selected from 0, 1 and 2;

n is selected from 0, 1, 2, 3 and 4;

x is selected from NH and O;

y is selected from CH and N;

z is selected from CH and N;

R ₁ are respectively H, F, cl, br, I, OH and NH independently ₂ And C _1-3 Alkyl radical, said C _1-3 Alkyl is optionally substituted by 1, 2 or 3R _a Substitution;

R ₂ selected from H, F, cl, br, I and C _1-3 Alkyl radical, said C _1-3 Alkyl is optionally substituted by 1, 2 or 3R _b Substitution;

R ₃ selected from H, F, cl, br, I and C _1-6 Alkyl radical, said C _1-6 Alkyl is optionally substituted by 1, 2 or 3R _c Substitution;

R ₄ selected from H, F, cl, br, I and C _1-6 Alkyl radical, said C _1-6 Alkyl is optionally substituted by 1, 2 or 3R _c Substitution;

R ₅ selected from H, F, cl, br and I;

R _a 、R _b and R _c Each independently selected from H, F, cl, br and I.
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound has the structure of formula (II):

R ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ 、X、Y、m and n are as defined in claim 1.
A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R ₁ Each independently selected from F, cl, NH ₂ And OH.
A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R ₂ Is selected from CH ₃ Said CH ₃ Optionally substituted by 1, 2 or 3R _b And (4) substitution.
A compound or pharmaceutically acceptable salt thereof according to claim 4, wherein R ₂ Selected from CF ₃ 。
A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R ₃ Is selected from CH ₃ 、CH ₂ CH ₃ And
a compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R ₄ Is selected from CH ₃ 、CH ₂ CH ₃ And
a compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R ₅ Selected from H and F.
According to claim 1 or 2The compound or a pharmaceutically acceptable salt thereof, wherein the structural unitIs selected from
A compound or pharmaceutically acceptable salt thereof according to claim 9, wherein the building blockIs selected from
A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the building blockIs selected from
A compound according to claim 2, or a pharmaceutically acceptable salt thereof, wherein the building blockIs selected from
The compound according to any one of claims 1 to 9, wherein the compound is selected from the group consisting of

Wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ And R ₅ As defined in any one of claims 1 to 9.
A compound of the formula selected from
The compound according to claim 14, or a pharmaceutically acceptable salt thereof, selected from
A pharmaceutical composition comprising as an active ingredient a therapeutically effective amount of a compound according to any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
Use of a compound according to any one of claims 1 to 15 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 16 for the preparation of a KRAS G12C mutein inhibitor.
Use of a compound according to any one of claims 1 to 15 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 16 for the manufacture of a medicament for the treatment of cancer.