CN116249683A

CN116249683A - Deuteromethyl substituted pyrazinopyrazinoquinolinone derivative, preparation method and application thereof in medicine

Info

Publication number: CN116249683A
Application number: CN202280006447.2A
Authority: CN
Inventors: 陈友喜; 程超英; 向清; 朱明江; 叶成; 钱文建; 陈磊; 戴露媚; 吴恩国
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: 2021-07-06
Filing date: 2022-07-05
Publication date: 2023-06-09
Anticipated expiration: 2042-07-05
Also published as: WO2023280136A1; CN116249683B; CN116249683A8

Abstract

The invention relates to a deuteromethyl substituted pyrazinopyrazinoquinolinone derivative, a preparation method and application thereof in medicines. Specifically, the invention relates to a deuteromethyl substituted pyrazinopyrazinoquinolinone derivative shown in a general formula (I), a preparation method and pharmaceutically acceptable salts thereof, and application of the derivative as a therapeutic agent, particularly as a KRAS GTPase inhibitor, wherein the definition of each substituent in the general formula (I) or the general formula (I) is the same as that in the specification.

Description

Deuteromethyl substituted pyrazinopyrazinoquinolinone derivative, preparation method and application thereof in medicine

Technical Field

The invention relates to a deuteromethyl substituted pyrazinopyrazinoquinolinone derivative, a preparation method thereof, a pharmaceutical composition containing the derivative and application of the derivative as a therapeutic agent, in particular to application of the derivative as a K-Ras GTPase inhibitor.

Background

RAS represents a closely related group of monomeric globular proteins (21 kDa molecular weight) with 189 amino acids and which are associated with the plasma membrane and bind GDP or GTP. Under normal developmental or physiological conditions, the RAS is activated by receiving 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, the on/off state of the RAS protein is determined by nucleotide binding, the active signaling conformation binds GTP, and the inactive conformation binds GDP. When the RAS contains bound GDP, it is in a dormant or quiescent or off state and is "inactive". When cells are exposed to certain growth promoting stimuli in response, the RAS is induced to convert the bound GDP to GTP. As GTP is bound, the RAS is "on" and is able to interact with and activate other proteins (its "downstream targets"). RAS proteins themselves have a very low inherent ability to hydrolyze GTP back to GDP and thereby turn themselves into an off state. Conversion of the RAS to shut down requires exogenous proteins called Gtpase Activating Proteins (GAPs) that interact with the RAS and greatly promote the conversion of GTP to GDP. Any mutation in the RAS that affects its ability to interact with GAP or convert GTP back to GDP will result in prolonged activation of the protein and thus produce a prolonged signal to the cell that signals it to continue growth and division. These signals may therefore cause cell growth and division, and overactivated RAS signaling may ultimately lead to cancer.

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

Among RAS family members, oncogenic mutations are most common in KRAS (85%), while NRAS (12%) and HRAS (3%) are less common. KRAS mutations are prevalent in three major deadly cancer types in the united states: pancreatic cancer (95%), colorectal cancer (45%) and lung cancer (25%), KRAS mutations are also found in other cancer types including multiple myeloma, uterine cancer, cholangiocarcinoma, gastric cancer, bladder cancer, diffuse large B-cell lymphoma, rhabdomyosarcoma, cutaneous squamous cell carcinoma, cervical cancer, testicular germ cell carcinoma, etc., whereas KRAS mutations are rarely found (< 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 the frequency of specific allelic mutations comes mostly from classical smoking-induced classical 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 driving oncogenic mutations in NSCLC, including EGFR, ALK, ROS, RET, and BRAF, indicating the uniqueness of KRAS mutations in lung cancer. While at the same time KRAS mutations often coincide with certain co-mutations, such as STK11, KEAP1 and TP53, which in cooperation with the mutated RAS transform the cells into highly malignant and invasive tumor cells.

Three RAS oncogenes constitute the most frequently mutated gene family in human cancers. It is disappointing that despite thirty years of research efforts, there is still no clinically effective anti-RAS therapy, and targeting the gene using small molecules is a challenge. Accordingly, there is an urgent need in the art for small molecules for targeting the RAS (e.g., K-RAS, H-RAS, and/or N-RAS) and using the same to treat a variety of diseases, such as cancer.

At present, the clinical development of KRAS inhibitor at home and abroad is very competitive, wherein KRAS enzyme inhibitor MRTX-849 developed by Mirati Therapeutics Inc company already enters clinical stage two for preventing and treating diseases such as advanced solid tumor, metastatic colorectal cancer, metastatic non-small cell lung cancer and the like. There are other KRAS inhibitors under investigation, 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, including WO2020047192, WO2019099524 and WO2018217651, and the like, and the research and use of KRAS inhibitors has advanced to some extent, but the room for improvement is still enormous, and there is still a need to continue to research and develop new KRAS inhibitors.

Disclosure of Invention

The invention aims to provide a tetracyclic derivative shown in a general formula (I), or a stereoisomer, a tautomer or pharmaceutically acceptable salts thereof:

wherein:

ring a is selected from aryl, heteroaryl or fused ring;

R ¹ the same OR different are each independently selected from hydrogen atom, alkyl, halogen, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, =o, -OR ² 、-C(O)R ² 、-C(O)OR ² 、-NHC(O)R ² 、-NHC(O)OR ² 、-NR ³ R ⁴ 、-C(O)NR ³ R ⁴ 、-CH ₂ NHC(O)OR ² 、-CH ₂ NR ³ R ⁴ or-S (O) _r R ² Is substituted by a substituent of (2); wherein said alkyl, cycloalkyl, heterocyclyl, aryl OR heteroaryl is optionally further substituted with one OR more substituents selected from alkyl, halo, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, =o, -OR ² 、-C(O)R ² 、-C(O)OR ² 、-NHC(O)R ² 、- NHC(O)OR ² 、-NR ³ R ⁴ 、-C(O)NR ³ R ⁴ 、-CH ₂ NHC(O)OR ² 、-CH ₂ NR ³ R ⁴ or-S (O) _r R ² Is substituted by a substituent of (2);

R ² selected from a hydrogen atom, an alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group, wherein the alkyl group, the cycloalkyl group, the heterocyclic group, the aryl group or the heteroaryl group is optionally further substituted with one or more groups selected from a hydroxyl group, a halogen group, a nitro group, a cyano group, an alkyl group, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyl group, a heterocyclic group, an aryl group, a heteroaryl group, =o, -C (O) R ⁵ 、-C(O)OR ⁵ 、-OC(O)R ⁵ 、-NR ⁶ R ⁷ 、-C(O)NR ⁶ R ⁷ 、-SO ₂ NR ⁶ R ⁷ or-NR ⁶ C(O)R ⁷ Is substituted by a substituent of (2);

R ³ and R is ⁴ Each independently selected from a hydrogen atom, hydroxy, halogen, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein said alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further substituted with one or more groups selected from hydroxy, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, =o, -C (O) R ⁵ 、-C(O)OR ⁵ 、-OC(O)R ⁵ 、-NR ⁶ R ⁷ 、-C(O)NR ⁶ R ⁷ 、-SO ₂ NR ⁶ R ⁷ or-NR ⁶ C(O)R ⁷ Is substituted by a substituent of (2);

alternatively, R ³ And R is ⁴ Together with the atoms to which they are attached form a 4-8 membered heterocyclic group, wherein the 4-8 membered heterocyclic group contains one or more of N, O or S (O) _r And said 4-8 membered heterocyclyl is optionally further substituted with one or more substituents selected from hydroxy, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, =o, -C (O) R ⁵ 、-C(O)OR ⁵ 、-OC(O)R ⁵ 、-NR ⁶ R ⁷ 、-C(O)NR ⁶ R ⁷ 、-SO ₂ NR ⁶ R ⁷ or-NR ⁶ C(O)R ⁷ Is substituted by a substituent of (2);

R ⁵ 、R ⁶ and R is ⁷ Each independently selected from the group consisting of hydrogen, alkyl, amino, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein said alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally further substituted with one or more substituents selected from the group consisting of hydroxy, halogen, nitro, amino, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxyl, or carboxylate;

m is selected from 0, 1 or 2;

r is selected from 0, 1 or 2.

In a preferred embodiment of the invention, the compound of formula (I) or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein ring a is selected from phenyl, naphthyl, pyridinyl, benzothiazolyl or benzothienyl, preferably benzothiazolyl or benzothienyl.

In a preferred embodiment of the invention, the compounds of formula (I) or stereoisomers, tautomers or pharmaceutically acceptable salts thereof, wherein

Selected from:

typical compounds of the present invention include, but are not limited to:

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

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

Further, the present invention provides a process for preparing a compound of formula (I), or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, comprising:

carrying out Suzuki coupling reaction on a compound shown in a general formula (IA) and a compound shown in a general formula (IB) under the action of a palladium catalyst and an alkaline reagent, further removing a protecting group to obtain a compound shown in a general formula (IC), and reacting the compound shown in the general formula (IC) with acryloyl chloride under an alkaline condition, and optionally further removing the protecting group to obtain a compound shown in the general formula (I);

wherein:

X ¹ a leaving group, preferably bromine;

m is selected from-B (OH) ₂ 、-BF ₃ K or

PG is a protecting group, preferably t-butoxycarbonyl;

ring A, R ¹ And m is as defined in formula (I).

Still further, the present invention provides a compound of the general formula (IC) or a stereoisomer, tautomer thereof, pharmaceutically acceptable salt thereof,

Wherein: ring A, R ¹ And m is as defined in formula (I).

Typical compounds of formula (IC) include, but are not limited to:

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

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

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

The present invention also provides the use of a compound of formula (I) or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for the manufacture of a medicament for the treatment of a disease mediated by KRAS mutations, wherein the disease mediated by KRAS mutations is preferably selected from cancer, wherein the cancer is preferably 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 carcinoma, more preferably pancreatic cancer, colorectal cancer and lung cancer; wherein the lung cancer is preferably non-small cell lung cancer, and wherein the KRAS mutation is preferably a KRAS G12C mutation.

In another aspect, the invention provides the use of a compound of formula (I), or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the preparation of a K-Ras GTPase inhibitor, wherein the K-Ras GTPase inhibitor is 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 formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, wherein said KRAS mutation is preferably a KRAS G12C mutation.

The invention also provides the use of a compound of formula (I) or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for the manufacture of a medicament for the treatment of cancer, wherein the cancer is preferably selected from pancreatic cancer, colorectal cancer, lung cancer, multiple myeloma, uterine cancer, cholangiocarcinoma, gastric cancer, bladder cancer, diffuse large B-cell gonorrhoeae, rhabdomyosarcoma, cutaneous squamous cell carcinoma, cervical cancer, testicular germ cell carcinoma, more 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, intraocular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intradermal, intraperitoneal, subcutaneous, subcuticular or by inhalation. Pharmaceutical compositions containing the active ingredient may be in a form suitable for oral administration, for example, as tablets, troches, 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-dose form and may be prepared by any method well known in the pharmaceutical arts. The amount of active ingredient that can be combined with the carrier material to produce a single dosage form can 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 is capable of producing 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 admixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers or propellants which may be required.

When the compounds of the invention are administered to humans and animals in the form of a medicament, the compounds may be provided alone or in the form of a pharmaceutical composition containing the active ingredient in combination with a pharmaceutically acceptable carrier, for example 0.1% to 99.5% (more preferably 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 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., accursinTM; and (22) other non-toxic compatible substances 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, cysteamine 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 chelators such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like. Solid dosage forms (e.g., capsules, dragees, powders, granules and the like) may 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) Binders, such as carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerin; (4) Disintegrants, for example agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) dissolution retarders, such as paraffin; (6) an absorption accelerator, such as a quaternary ammonium compound; (7) Humectants, such as cetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite; (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 form may contain inert diluents commonly used in the art, such as water or other solvents; solubilizing agents and emulsifiers, for example, ethanol, isopropanol, 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 also contain suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum hydroxide oxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

In addition to the active compounds, ointments, pastes, creams and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

In addition to the active compounds, the powders and sprays can also contain 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:

"alkyl" when taken as a group or part of a group is meant to include C ₁ -C ₂₀ Straight chain or branched aliphatic hydrocarbon groups. Preferably C ₁ -C ₁₀ Alkyl, more preferably C ₁ -C ₆ An alkyl group. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylpropylButyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl, and the like. Alkyl groups may be substituted or unsubstituted.

"alkenyl" refers to an alkyl group as defined above consisting of at least two carbon atoms and at least one carbon-carbon double bond, representative examples include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-, 2-or 3-butenyl, and the like. Alkenyl groups may be optionally substituted or unsubstituted.

"alkynyl" refers to an aliphatic hydrocarbon group containing one carbon-carbon triple bond, which may be straight or branched. Preferably is C ₂ -C ₁₀ More preferably C ₂ -C ₆ Alkynyl, most preferably C ₂ -C ₄ Alkynyl groups. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-, 2-, or 3-butynyl, and the like. Alkynyl groups may be substituted or unsubstituted.

"alkylene" is a divalent alkyl group. Preferably C ₁ -C ₁₀ Alkylene, more preferably C1-C6 alkylene, particularly preferably C ₁ -C ₄ An alkylene group. Examples of alkylene groups include, but are not limited to, methylene, ethylene, -CH (CH) ₃ ) ₂ N-propylene group, and the like. The alkylene group may be substituted or unsubstituted.

"cycloalkyl" refers to saturated or partially saturated monocyclic, fused, bridged, and spiro carbocycles. Preferably C ₃ -C ₁₂ Cycloalkyl, more preferably C ₃ -C ₈ Cycloalkyl, most preferably C ₃ -C ₆ Cycloalkyl groups. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like, with cyclopropyl, cyclohexenyl being preferred. Cycloalkyl groups may be optionally substituted or unsubstituted.

"spirocycloalkyl" refers to a 5 to 18 membered, two or more cyclic structure, and monocyclic polycyclic groups sharing one carbon atom (called spiro atom) with each other, containing 1 or more double bonds within the ring, but no ring has a completely conjugated pi-electron aromatic system. Preferably 6 to 14 membered, more preferably 7 to 10 membered. The spirocycloalkyl group is classified into a single spiro group, a double spiro group or a multiple spirocycloalkyl group according to the number of common spiro atoms between rings, preferably single spiro group and double spirocycloalkyl group, preferably 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered. Non-limiting examples of "spirocycloalkyl" include, but are not limited to: spiro [4.5] decyl, spiro [4.4] nonyl, spiro [3.5] nonyl, spiro [2.4] heptyl.

"fused ring alkyl" refers to an all-carbon polycyclic group having 5 to 18 members, two or more cyclic structures sharing a pair of carbon atoms with each other, one or more of the rings may contain one or more double bonds, but none of the rings has a fully conjugated pi-electron aromatic system, preferably 6 to 12 members, more preferably 7 to 10 members. The number of constituent rings may be classified as a bicyclic, tricyclic, tetracyclic or polycyclic fused ring alkyl group, preferably a bicyclic or tricyclic, more preferably a 5-membered/5-membered or 5-membered/6-membered bicycloalkyl group. Non-limiting examples of "fused ring alkyl" include, but are not limited to: bicyclo [3.1.0] hexyl, bicyclo [3.2.0] hept-1-enyl, bicyclo [3.2.0] heptyl, decalinyl, or tetradecahydrophenanthryl.

"bridged cycloalkyl" means an aromatic system having 5 to 18 members, containing two or more cyclic structures, sharing two all-carbon polycyclic groups with one another that are not directly attached to a carbon atom, one or more of the rings may contain one or more double bonds, but none of the rings has a fully conjugated pi electron, preferably 6 to 12 members, more preferably 7 to 10 members. Preferably 6 to 14 membered, more preferably 7 to 10 membered. Cycloalkyl groups which may be classified as bicyclic, tricyclic, tetracyclic or polycyclic bridged according to the number of constituent rings are preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of "bridged cycloalkyl" include, but are not limited to: (1 s,4 s) -bicyclo [2.2.1] heptyl, bicyclo [3.2.1] octyl, (1 s,5 s) -bicyclo [3.3.1] nonyl, bicyclo [2.2.2] octyl, and (1 r,5 r) -bicyclo [3.3.2] decyl.

"heterocyclyl", "heterocycle" or "heterocyclic" are used interchangeably herein to refer to a non-aromatic heterocyclic group in which one or more of the ring-forming atoms are heteroatoms, such as oxygen, nitrogen, sulfur atoms, and the like, including monocyclic, fused, bridged and spiro rings. Preferably having a 5 to 7 membered single ring or a 7 to 10 membered bi-or tricyclic ring, which may contain 1,2 or 3 atoms selected from nitrogen, oxygen and/or sulfur. Examples of "heterocyclyl" include, but are not limited to, morpholinyl, oxetanyl, thiomorpholinyl, tetrahydropyranyl, 1-dioxothiomorpholinyl, piperidinyl, 2-oxopiperidinyl, pyrrolidinyl, 2-oxopyrrolidinyl, piperazin-2-one, 8-oxa-3-aza-bicyclo [3.2.1] octyl, and piperazinyl. The heterocyclic group may be substituted or unsubstituted.

"spiroheterocyclyl" refers to a 5-to 18-membered, two or more cyclic structure, polycyclic group having single rings sharing one atom with each other, containing 1 or more double bonds in the ring, but no ring having a completely conjugated pi-electron aromatic system in which one or more ring atoms are selected from nitrogen, oxygen or S (O) _r (wherein r is selected from 0, 1 or 2) and the remaining ring atoms are carbon. Preferably 6 to 14 membered, more preferably 7 to 10 membered. The spirocycloalkyl group is classified into a single spiro heterocyclic group, a double spiro heterocyclic group or a multiple spiro heterocyclic group according to the number of common spiro atoms between rings, and preferably a single spiro heterocyclic group and a double spiro heterocyclic group. More preferably a 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered single spiro heterocyclic group. Non-limiting examples of "spiroheterocyclyl" include, but are not limited to: 1, 7-dioxaspiro [4.5 ] ]Decyl, 2-oxa-7-azaspiro [4.4 ]]Nonyl, 7-oxaspiro [3.5 ]]Nonyl and 5-oxaspiro [2.4 ]]A heptyl group.

"fused heterocyclyl" refers to an all-carbon polycyclic group containing two or more cyclic structures sharing a pair of atoms with each other, one or more of the rings may contain one or more double bonds, but none of the rings has a fully conjugated pi-electron aromatic system in which one or more of the ring atoms is selected from nitrogen, oxygen, or S (O) _r (wherein r is selected from 0, 1 or 2) and the remaining ring atoms are carbon. Preferably 6 to 14 membered, more preferably 7 to 10 membered. The condensed heterocyclic groups which may be classified into a double ring, a triple ring, a tetra ring or a polycyclic ring according to the number of constituent rings are preferably double ring or triple ring, more preferably 5-membered/5-membered or 5-membered/6-membered double ringFused heterocyclic groups. Non-limiting examples of "fused heterocyclyl" include, but are not limited to: octahydropyrrolo [3,4-c ]]Pyrrolyl, octahydro-1H-isoindolyl, 3-azabicyclo [3.1.0 ]]Hexyl, octahydrobenzo [ b ]][1,4]Dioxin (dioxin).

"bridged heterocyclyl" means a 5 to 14 membered, 5 to 18 membered, polycyclic group containing two or more cyclic structures sharing two atoms not directly attached to each other, one or more of the rings may contain one or more double bonds, but none of the rings has a fully conjugated pi electron aromatic system in which one or more of the ring atoms is selected from nitrogen, oxygen or S (O) _r (wherein r is selected from 0, 1 or 2) and the remaining ring atoms are carbon. Preferably 6 to 14 membered, more preferably 7 to 10 membered. Heterocyclic groups which may be classified as bicyclic, tricyclic, tetracyclic or polycyclic bridged according to the number of constituent rings are preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of "bridged heterocyclyl" include, but are not limited to: 2-azabicyclo [2.2.1]Heptyl, 2-azabicyclo [2.2.2]Octyl and 2-azabicyclo [3.3.2]And (3) a decyl group.

"aryl" refers to a carbocyclic aromatic system containing one or two rings, wherein the rings may be linked together in a fused manner. The term "aryl" includes monocyclic or bicyclic aryl groups such as phenyl, naphthyl, tetrahydronaphthyl aromatic groups. Preferably aryl is C ₆ -C ₁₀ Aryl groups, more preferably aryl groups are phenyl and naphthyl. Aryl groups may be substituted or unsubstituted.

"heteroaryl" refers to an aromatic 5-to 6-membered monocyclic or 8-to 10-membered bicyclic ring, which may contain 1 to 4 atoms selected from nitrogen, oxygen and/or sulfur. Preferred bicyclic heteroaryl groups, examples of "heteroaryl" include, but are not limited to, furyl, pyridyl, 2-oxo-1, 2-dihydropyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, thienyl, isoxazolyl, oxazolyl, oxadiazolyl, imidazolyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, 1,2, 3-thiadiazolyl, benzodioxolyl, benzothienyl, benzimidazolyl, indolyl, isoindolyl, 1, 3-dioxo-isoindolyl, quinolinyl, indazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl, benzoisoxazolyl, benzothiophenyl, benzofuranyl, and the like,

Heteroaryl groups may be substituted or unsubstituted.

"fused ring" means a polycyclic group having two or more cyclic structures sharing a pair of atoms with each other, one or more of the rings may contain one or more double bonds, but at least one of the rings does not have a fully conjugated pi-electron aromatic system, wherein the ring atoms are selected from 0, one or more of the ring atoms are selected from nitrogen, oxygen, or S (O) _r (wherein r is selected from 0, 1 or 2) and the remaining ring atoms are carbon. The fused ring preferably includes a double-or triple-ring fused ring, wherein the double-ring fused ring is preferably a fused ring of an aryl or heteroaryl group and a monocyclic heterocyclic group or a monocyclic cycloalkyl group. Preferably 7 to 14 membered, more preferably 8 to 10 membered. Examples of "fused rings" include, but are not limited to:

"alkoxy" refers to a group of (alkyl-O-). Wherein alkyl is as defined herein. C (C) ₁ -C ₆ Is preferably selected. Examples include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy and the like.

"haloalkyl" refers to a group wherein the alkyl is optionally further substituted with one or more halogens, where alkyl is as defined herein.

"deuterated alkyl" refers to a group in which the alkyl group is optionally further substituted with one or more deuterium atoms, where alkyl is as defined herein. "deuterated alkyl" is preferably deuterated methyl, including: mono-, di-and tri-deuterated methyl groups are preferred as tri-deuterated methyl groups.

"hydroxyalkyl" refers to a group in which the alkyl group is optionally further substituted with one or more hydroxyl groups, where alkyl is as defined herein.

"haloalkoxy" refers to a group in which the alkyl group of (alkyl-O-) is optionally further substituted with one or more halogens, wherein alkoxy is as defined herein.

"hydroxy" refers to an-OH group.

"halogen" refers to fluorine, chlorine, bromine and iodine.

"amino" means-NH ₂ 。

"cyano" refers to-CN.

"nitro" means-NO ₂ 。

"benzyl" means-CH ₂ -phenyl.

"carboxy" means-C (O) OH.

"carboxylate" refers to-C (O) O-alkyl or-C (O) O-cycloalkyl, wherein alkyl, cycloalkyl are as defined above.

"DMSO" refers to dimethyl sulfoxide.

"BOC" refers to t-butoxycarbonyl.

"Ts" refers to p-toluenesulfonyl.

"T3P" refers to propyl phosphoric anhydride.

"DPPA" refers to diphenyl azide phosphate.

"DEA" refers to diethylamine.

"TFA" refers to trifluoroacetic acid.

"X-PHOS Pd G2" refers to chloro (2-dicyclohexylphosphino-2 ',4',6 '-triisopropyl-1, 1' -biphenyl) [2- (2 '-amino-1, 1' -biphenyl) ] palladium (II).

"substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are independently substituted with a corresponding number of substituents. It goes without saying that substituents are only in their possible chemical positions, and that the person skilled in the art is able to determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups having free hydrogen may be unstable when bound to carbon atoms having unsaturated (e.g., olefinic) bonds.

"substituted" or "substituted" as used herein, unless otherwise indicated, means that the group may be substituted with one or more groups selected from the group consisting of: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, alkenyl, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, amino, haloalkyl, hydroxyalkyl, carboxyl, carboxylate, =o, -OR ² 、-C(O)R ² 、-C(O)OR ² 、-NHC(O)R ² 、-NHC(O)OR ² 、-NR ³ R ⁴ 、-C(O)NR ³ R ⁴ 、-CH ₂ NHC(O)OR ² 、-CH ₂ NR ³ R ⁴ or-S (O) _r R ² Is substituted by a substituent of (2);

r is 0, 1 or 2.

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 (attopiomers) and geometric (conformational) isomers and mixtures thereof, such as racemic mixtures, are within the scope of the present invention.

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

"pharmaceutically acceptable salts" refers to certain salts of the above compounds which retain the original biological activity and are suitable for pharmaceutical use. The pharmaceutically acceptable salts of the compounds represented by formula (I) may be metal salts, amine salts with suitable acids.

"pharmaceutical composition" means a mixture comprising one or more of the compounds described herein or a physiologically acceptable salt or prodrug thereof, and other chemical components, such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.

Synthesis method of compound of the invention

In order to achieve the purpose of the invention, the invention adopts the following technical scheme:

the preparation method of the compound shown in the general formula (I) or the stereoisomer, the tautomer or the pharmaceutically acceptable salt thereof comprises the following steps:

wherein:

X ¹ a leaving group, preferably bromine;

m is selected from-B (OH) ₂ 、-BF ₃ K or

PG is a protecting group, preferably t-butoxycarbonyl;

ring A, R ¹ And m is as defined in formula (I).

Detailed Description

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

Examples

The preparation of representative compounds represented by formula (I) and related structural identification data are presented in the examples. It must be noted that the following examples are given by way of illustration and not by way of limitation. ¹ HNMR spectra were determined using a Bruker instrument (400 MHz) and chemical shifts were expressed in ppm. Tetramethylsilane internal standard (0.00 ppm) was used. ¹ HNMR representation method: s=singlet, d=doublet, t=triplet, m=multiplet, br=broadened, dd=doublet of doublet, dt=doublet of triplet. If coupling constants are provided, they are 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 uses a smoke table yellow sea HSGF254 or Qingdao GF254 silica gel plate, the specification of the silica gel plate used by the 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.

Column chromatography generally uses tobacco stand yellow sea silica gel 200-300 mesh silica gel as a carrier.

In the following examples, unless otherwise indicated, all temperatures are in degrees celsius and unless otherwise indicated, various starting materials and reagents are either commercially available or synthesized according to known methods, all of which are used without further purification and unless otherwise indicated, commercially available manufacturers include, but are not limited to, shanghai Haohong biological medicine technologies, shanghai Shaoshao reagent, shanghai Pico medicine, saen chemical technologies (Shanghai) and Shanghai Ling Kai medicine technologies, and the like.

CD ₃ OD: deuterated methanol.

CDCl ₃ : deuterated chloroform.

DMSO-d ₆ : deuterated dimethyl sulfoxide.

The examples are not particularly described, and the solution in the reaction is an aqueous solution.

Purifying the compound using an eluent system 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: methylene chloride and methanol systems; c: dichloromethane and ethyl acetate system, D: dichloromethane and ethanol system, E: tetrahydrofuran/petroleum ether system, F: tetrahydrofuran and methanol systems, in which the volume ratio of the solvents varies according to the polarity of the compound, may also be subjected to conditions in which a small amount of an acidic or basic reagent such as acetic acid or triethylamine is added.

Example 1 and example 2

(2R,4aR,10S)-3-acryloyl-10-(2-amino-5,7-difluorobenzo[d]thiazol-4-yl)-11-chloro-9-fluoro-2-methyl-6-(methyl-d3)-2,3,4,4a-tetrahydro-1H-pyrazino[1',2':4,5]pyrazino[2,3-c]quinolin-5(6H)-one

(2R,4aR,10R)-3-acryloyl-10-(2-amino-5,7-difluorobenzo[d]thiazol-4-yl)-11-chloro-9-fluoro-2-methyl-6-(methyl-d3)-2,3,4,4a-tetrahydro-1H-pyrazino[1',2':4,5]pyrazino[2,3-c]quinolin-5(6H)-one

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

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

First step

1-(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) -4- (7-bromo-6-chloro-8-fluoro-3-nitroquinolin-4-yl) -6-methylpiperazine-1, 3-dicarboxylic acid ester

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

MS m/z(ESI):562.1[M+1] ⁺

Second step

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, 4 aR) -10-bromo-11-chloro-9-fluoro-2-methyl-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

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

MS m/z(ESI):499.0[M+1] ⁺

Third step

tert-butyl(2R,4aR)-10-bromo-11-chloro-9-fluoro-2-methyl-6-(methyl-d3)-5-oxo-1,2,4,4a,5,6-hexahydro-3H-pyrazino[1',2':4,5]pyrazino[2,3-c]quinoline-3-carboxylate

(2R, 4 aR) -10-bromo-11-chloro-9-fluoro-2-methyl-6- (methyl-d 3) -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

(2R, 4 aR) -10-bromo-11-chloro-9-fluoro-2-methyl-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 1d (350 mg, 700.34. Mu. Mol) was added to N, N-dimethylformamide (2 mL), deuterated iodomethane (203.04 mg,1.40 mmol) and potassium carbonate (290.38 mg,2.10 mmol) were sequentially added, and the reaction was carried out at room temperature overnight. The system was extracted with ethyl acetate (20 mL) and water (20 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: A system) to give the product (2R, 4 aR) -10-bromo-11-chloro-9-fluoro-2-methyl-6- (methyl-d 3) -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 (200 mg, 386.99. Mu. Mol), yield: 55.26%.

MS m/z(ESI):517.1[M+1] ⁺

Fourth step

(2- ((tert-Butoxycarbonyl) amino) -5, 7-difluorobenzo [ d ]]Thiazol-4-yl) boronic acid 1f (191.63 mg,580.49 μmol, prepared according to published patent US20200115375 A1), (2 r,4 ar) -10-bromo-11-chloro-9-fluoro-2-methyl-6- (methyl-d 3) -5-oxo-1, 2, 4a,5, 6-hexahydro-3H-pyrazine And [1',2':4,5]Pyrazino [2,3-c ]]Tert-butyl quinoline-3-carboxylate 1e (200 mg, 386.99. Mu. Mol) was added to a mixed solvent of 1, 4-dioxane (5 mL) and water (1 mL), tetrakis (triphenylphosphine) palladium (223.60 mg, 193.50. Mu. Mol), sodium carbonate (123.05 mg,1.16 mmol) and argon shield were added, and the mixture was heated to 110℃for 3 hours. Ethyl acetate (10 mL) and water (10 mL) were added to the system, the mixture was separated, the ethyl acetate extract (10 mL. Times.3) was taken up, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the resulting residue was purified by preparative HPLC (separation column: AKZONOBEL Kromasil; 250X 21.2mm I.D.;5 μm; mobile phase A:0.05% TFA+H) ₂ O, mobile phase B: acetonitrile; flow rate: 20 mL/min) to give 1g of compound and 1h of compound belonging to one of the single configuration compound (shorter retention time) and the single configuration compound (longer retention time), respectively.

Single configuration compounds (shorter retention time):

MS m/z(ESI):723.9[M+1] ⁺

10mg; HPLC retention time 12.727 mins.

Single configuration compounds (longer retention time):

MS m/z(ESI):723.9[M+1] ⁺

10mg; HPLC retention time 12.828 mins.

Fifth step

1g (10 mg, 13.85. Mu. Mol) was dissolved in methylene chloride (1 mL), and a 1, 4-dioxane solution (4M, 1 mL) of hydrochloric acid was added thereto and reacted overnight at room temperature. After the reaction was completed, it was concentrated under reduced pressure to give crude product 1i (7 mg, 13.41. Mu. Mol), yield: 96.85%.

MS m/z(ESI):523.8[M+1] ⁺

Sixth step

Crude 1i (7 mg, 13.41. Mu. Mol) was added to dichloromethane (3 mL), triethylamine (4.07 mg, 40.23. Mu. Mol) was added, cooled to 0℃and acryloyl chloride (2.43 mg, 26.82. Mu. Mol) was added and stirred at room temperature for 0.5 hours. Ethyl acetate (10 mL) and water (10 mL) were added to the system, 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 resulting residue was 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 give product 1 (2 mg, 3.30. Mu. Mol), yield: 24.60%.

MS m/z(ESI):577.1[M+1] ⁺

Seventh step

1h (10 mg, 13.85. Mu. Mol) was dissolved in methylene chloride (1 mL), and a 1, 4-dioxane solution (4M, 1 mL) of hydrochloric acid was added thereto and reacted overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure to give crude product 1j (7.3 mg, 13.85. Mu. Mol), yield: 100%.

MS m/z(ESI):523.8[M+1] ⁺

Eighth step

The crude 1j (7.3 mg, 13.85. Mu. Mol) was added to dichloromethane (3 mL), triethylamine (4.07 mg, 40.23. Mu. Mol) was added, cooled to 0deg.C, acryloyl chloride (2.43 mg, 26.82. Mu. Mol) was added, and stirred at room temperature for 0.5 hours. Ethyl acetate (10 mL) and water (10 mL) were added to the system, the mixture was separated, ethyl acetate extracted (10 mL. Times.3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the resulting residue was 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 give product 2 (0.5 mg, 0.87. Mu. Mol), yield: 6.26%.

MS m/z(ESI):577.1[M+1] ⁺

Example 3

4-((2R,4aR)-3-acryloyl-11-chloro-9-fluoro-2-methyl-6-(methyl-d3)-5-oxo-2,3,4,4a,5,6-hexahydro-1H-pyrazino[1',2':4,5]pyrazino[2,3-c]quinolin-10-yl)-2-amino-7-fluorobenzo[b]thiophene-3-carbonitrile

4- ((2 r,4 ar) -3-propenoyl-11-chloro-9-fluoro-2-methyl-6- (methyl-d 3) -5-oxo-2, 3, 4a,5, 6-hexahydro-1H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinolin-10-yl) -2-amino-7-fluorobenzo [ b ] thiophene-3-carbonitrile

First step

tert-butyl(3-cyano-7-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[b]thiophen-2-yl)carbamate

(3-cyano-7-fluoro-4- (4, 5-tetramethyl l-1,3, 2-dioxaborolan-2-yl) benzo [ b ] thiophen-2-yl) carbamic acid tert-butyl ester

Tert-butyl (4-bromo-3-cyano-7-fluorobenzo [ b ] thiophen-2-yl) carbamate 3a (300 mg, 808.14. Mu. Mol, prepared according to WO2021118877A 1), 4', 5',5 '-octamethyl-2, 2' -bis (1, 3, 2-dioxaborolan) (779.82 mg,3.07 mmol), potassium acetate (237.93 mg,2.42 mmol) and bis (diphenylphosphinophenyl ether) palladium (II) dichloride (57.85 mg, 80.81. Mu. Mol) were added to 1, 4-dioxabicyclo (10 mL), and the mixture was heated to 95℃under argon atmosphere to react for 4 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: A system) and preparative high performance liquid chromatography (preparative conditions: separation column AKZONOBEL Kromasil; 250X 21.2mm I.D.;5 μm,20mL/min; mobile phase A:0.05% TFA+H2O; mobile phase B: CH3 CN) in this order to give tert-butyl 3B (200 mg, 478.14. Mu. Mol) carbamate as a product (3-cyano-7-fluoro-4- (4, 5-tetramethyl l-1,3, 2-dioxaborolan-2-yl) benzo [ B ] thiophen-2-yl), yield: 59.17%. MS m/z (ESI): 416.9[ M-1] + with a high-pressure gas

Second step

tert-butyl(2R,4aR)-10-(2-((tert-butoxycarbonyl)amino)-3-cyano-7-fluorobenzo[b]thiophen-4-yl)-11-chloro-9-fluoro-2-methyl-6-(methyl-d3)-5-oxo-1,2,4,4a,5,6-hexahydro-3H-pyrazino[1',2':4,5]pyrazino[2,3-c]quinoline-3-carboxylate

(2R, 4 aR) -10- (2- ((tert-Butoxycarbonyl) amino) -3-cyano-7-fluorobenzo [ b ] thiophen-4-yl) -11-chloro-9-fluoro-2-methyl-6- (methyl-d 3) -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

Tert-butyl (3-cyano-7-fluoro-4- (4, 5-tetramethyl l-1,3, 2-dioxaborolan-2-yl) benzo [ b ] thiophen-2-yl) carbamate 3b (1.26G, 3.02 mmol) and (2 r,4 ar) -10-bromo-11-chloro-9-fluoro-2-methyl-6- (methyl-d 3) -5-oxo-1, 2, 4a,5, 6-hexahydro-3H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinoline-3-carboxylate 1e (1.3G, 2.52 mmol) were added to tetrahydrofuran (20 mL), potassium phosphate (1.60G, 7.55 mmol) and X-PHOS Pd G2 (988.31 mg,1.26 mmol) were added in sequence, and the mixture was reacted at 50 ℃ for 3 hours under argon atmosphere. The reaction solution was cooled to room temperature, ethyl acetate (30 mL) and water (30 mL) were added to the system, the solution was separated, ethyl acetate extraction (30 mL. Times.3) was performed, 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 silica gel column chromatography (eluent: A system) to give the product (2R, 4 aR) -10- (2- ((t-butoxycarbonyl) amino) -3-cyano-7-fluorobenzo [ b ] thiophen-4-yl) -11-chloro-9-fluoro-2-methyl-6- (methyl-d 3) -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 3c (200 mg, 274.64. Mu. Mol), yield: 10.92%.

MS m/z(ESI):729.9[M+1] ⁺

Third step

2-amino-4-((2R,4aR)-11-chloro-9-fluoro-2-methyl-6-(methyl-d3)-5-oxo-2,3,4,4a,5,6-hexahydro-1H-pyrazino[1',2':4,5]pyrazino[2,3-c]quinolin-10-yl)-7-fluorobenzo[b]thiophene-3-carbonitrile

2-amino-4- ((2 r,4 ar) -11-chloro-9-fluoro-2-methyl-6- (methyl-d 3) -5-oxo-2, 3, 4a,5, 6-hexahydro-1H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinolin-10-yl) -7-fluorobenzo [ b ] thiophene-3-carbonitrile

(2R, 4 aR) -10- (2- ((tert-Butoxycarbonyl) amino) -3-cyano-7-fluorobenzo [ b ] thiophen-4-yl) -11-chloro-9-fluoro-2-methyl-6- (methyl-d 3) -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 3c (300 mg, 411.96. Mu. Mol) was added to dichloromethane (5 mL), 1, 4-dioxane solution (4M, 8 mL) of hydrochloric acid was added dropwise, and the reaction was carried out overnight at room temperature. After the reaction was completed, the crude product 2-amino-4- ((2 r,4 ar) -11-chloro-9-fluoro-2-methyl-6- (methyl-d 3) -5-oxo-2, 3, 4a,5, 6-hexahydro-1H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinolin-10-yl) -7-fluorobenzo [ b ] thiophene-3-carbonitrile 3d (200 mg,378.79 μmol) was obtained by concentration under reduced pressure, yield: 91.95%.

MS m/z(ESI):529.9[M+1] ⁺

Fourth step

Crude 2-amino-4- ((2R, 4 aR) -11-chloro-9-fluoro-2-methyl-6- (methyl-d 3) -5-oxo-2, 3, 4a,5, 6-hexahydro-1H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinolin-10-yl) -7-fluorobenzo [ b ] thiophene-3-carbonitrile 3d (200 mg, 378.79. Mu. Mol) was added to dichloromethane (3 mL), triethylamine (114.99 mg,1.14mmol, 158.39. Mu. L) was added, cooled to 0 ℃, and acryloyl chloride (37.71 mg, 416.67. Mu. Mol) was added dropwise and reacted at room temperature for 1 hour. Dichloromethane (10 mL) and water (10 mL) were added to the system, extracted with dichloromethane (10 ml×3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the resulting residue was separated and purified by silica gel column chromatography (eluent: a system) to give the product 4- ((2 r,4 ar) -3-propenoyl-11-chloro-9-fluoro-2-methyl-6- (methyl-d 3) -5-oxo-2, 3, 4a,5, 6-hexahydro-1H-pyrazino [1',2':4,5] pyrazino [2,3-c ] quinolin-10-yl) -2-amino-7-fluorobenzo [ b ] thiophene-3-carbonitrile 3 (33 mg,52.73 μmol), yield: 13.92%.

MS m/z(ESI):582.9[M+1] ⁺

Example 4 and example 5

4- ((2R, 4 aR) -3-propenoyl-11-chloro-9-fluoro-2-methyl-6- (methyl-d 3) -5-oxo-2, 3, 4a,5, 6-hexahydro-1H-pyrazino [1',2':4, 5)]Pyrazino-ring[2,3-c]Quinolin-10-yl) -2-amino-7-fluorobenzo [ b]Thiophene-3-carbonitrile 3 (5 mg, 8.59. Mu. Mol) was purified by preparative high performance liquid chromatography (conditions for preparation: separation column AKZONOBEL Kromasil; 250X 21.2mm I.D.;5 μm,20mL/min; mobile phase A:0.05% TFA+H) ₂ O, mobile phase B: CH (CH) ₃ CN) to give compound 4 and compound 5, which belong to one of the single configuration compound (shorter retention time) and the single configuration compound (longer retention time), respectively.

Single configuration compounds (shorter retention time):

MS m/z(ESI):582.9[M+1] ⁺

1mg; HPLC retention time 8.826 mins.

Single configuration compounds (longer retention time):

MS m/z(ESI):582.9[M+1] ⁺

2mg; HPLC retention time 9.239 mins.

Biological evaluation

Test example 1, determination of the ability of the inventive Compounds 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: using reaction buffer (20mM HEPES,150mM NaCl,1mM MgCl) ₂ 1mM DTT) was prepared with recombinant human KRAS G12C protein (aa 1-169) at a concentration of 4. Mu.M for use. Test compounds were dissolved in DMSO to prepare 10mM stock solution, which was then 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: 3. Mu.M) was added to the wells, followed by mixing by adding 23.5. Mu.L of the reaction buffer, followed by adding 25. Mu.L of 4. Mu.M recombinant human KRAS G12C protein, incubating for 5 minutes at room temperature, terminating the reaction by adding 5. Mu.L of acetic acid, and transferring the sample to a sample bottle. The ratio of covalent binding of the test compound to KRAS G12C protein was measured using an Agilent 1290/6530 instrument, and the sample was purified by liquid chromatography column (XBridge Protein BEH C,

3.5 μm,2.1 mm. Times.50 mm), mobile phase A was 0.1% aqueous formic acid and mobile phase B was acetonitrile during the detection procedure, mobile phase elution procedure was: 0 to 0.5 minutes, maintaining the mobile phase A:95%,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 were analyzed using MassHunter Workstation Software Bioconfirm Version B.08.00 software to obtain the covalent Binding Rate (Binding Rate) of the test compound at a concentration of 3. Mu.M under incubation for 5min with KRAS G12C protein, and the results are shown in Table 1 below.

TABLE 1 Table of the covalent binding Activity of the compounds of the invention on KRAS G12C proteins

Conclusion that the compound of the invention has better covalent binding rate with KRAS G12C protein under the condition of 3 mu M and 5 min.

Test example 2 inhibition of NCI-H358 cell proliferation assay by Compounds of the 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 Shanghai institute of life sciences, china 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

Luminescent Cell Viability Assay kit (Promega, cat# G7573).

The experimental method is operated according to the steps of the instruction book of the kit, and is briefly described as follows: test compounds were first prepared as 10mM stock solutions in DMSO, then diluted with medium, and formulatedThe final concentration of the compound was in the range of 1000nM to 0.015nM for the test sample. Cells in the logarithmic growth phase were seeded at a density of 800 cells per well in 96-well cell culture plates at 37℃with 5% CO ₂ The culture was continued overnight in the incubator, followed by the addition of the test compound and continued for 120 hours. After the incubation was completed, a volume of 50. Mu.L of CellTiter-Glo assay solution was added to each well, and after shaking for 5 minutes, the mixture was allowed to stand for 10 minutes, followed by reading the Luminescence values of each well of the sample on a microplate reader using the Luminescence mode. The percent inhibition of compounds at each concentration point was calculated by comparison with the values of the control group (0.3% dmso), followed by nonlinear regression analysis of the compound concentration log-inhibition in GraphPad Prism 5 software to obtain IC compounds that inhibited cell proliferation ₅₀ Values, results are given in the table below.

TABLE 2 inhibition of proliferation of NCI-H358 cells by the compounds of the present invention

Conclusion the compounds of the present invention have a better proliferation inhibition effect on NCI-H358 (human non-small cell lung cancer) cells.

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

The following methods were used to determine the p-ERK1/2 inhibitory activity of the compounds of the invention on NCI-H358 cells. The method uses an Advanced phospho-ERK1/2 (Thr 202/tyr 204) kit (cat No. 64 AERPEH) from Cisbio, and the detailed experimental procedure is referred to the kit instructions. NCI-H358 cells (containing KRAS G12C mutation) were purchased from the China academy of sciences of Shanghai life sciences cell resource center.

The experimental procedure is briefly described as follows: NCI-H358 cells were cultured on a medium containing 10% fetal bovine serum and 100U penicillin100. Mu.g/mL streptomycin and 1mM Sodium Pyruvate RPMI 1640 complete medium. NCI-H358 cells were plated in 96-well plates 30000 per well, with medium being complete medium, at 37℃with 5% CO ₂ The cells were incubated overnight in an incubator. Test compounds were dissolved in DMSO to prepare a 10mM stock solution, then diluted with RPMI 1640 basal medium, and 90. Mu.L of RPMI 1640 basal medium containing the test compound at the corresponding concentration was added to each well, and the test compound was placed in a cell culture incubator for 3 hours and 40 minutes at a final concentration in the reaction system in the range of 1000nM to 0.015 nM. Subsequently, 10. Mu.L of hEGF (available from Roche under accession number 11376454001) in RPMI 1640 basal medium was added to a final concentration of 5nM and incubated in an incubator for 20 minutes. Cell supernatants were discarded, cells were washed with ice-bath PBS, after which 45. Mu.L of 1 Xcelphos/total protein lysis buffer (Advanced phospho-ERK1/2 kit component) was added to each well for lysis, and 96-well plates were placed on ice for half an hour, followed by detection of lysates with reference to Advanced phospho-ERK1/2 (Thr 202/tyr 204) kit instructions. Finally, the fluorescence intensities of the wells at excitation wavelengths of 304nM, at which the emission wavelengths of 620nM and 665nM are measured on an microplate reader in TF-FRET mode, and the fluorescence intensity ratio of the wells 665/620 is calculated. The percent inhibition of the test compounds at each concentration was calculated by comparison with the fluorescence intensity ratio of the control group (0.1% dmso) and nonlinear regression analysis was performed by GraphPad Prism5 software with the test compound concentration log-inhibition to obtain compound IC ₅₀ Values, results are shown in Table 3 below.

TABLE 3 inhibition of p-ERK1/2 Activity of the compounds of the invention on NCI-H358 cells

Conclusion that the compounds of the present invention have better proliferation inhibition effect on p-ERK1/2 in NCI-H358 cells.

Test example 4 hERG Potassium channel inhibitory Activity of Compounds of the invention

1. Cell culture

1.1 cells used in this assay were CHO cell lines transfected with hERG cDNA and stably expressing hERG channels (supplied by Denmark Sophion Bioscience company) with a cell number of P17. Cells were cultured in medium containing the following ingredients (all from Invitrogen): ham's F medium, 10% (v/v) inactivated fetal bovine serum, 100 μg/mL hygromycin B,100 μg/mL Geneticin.

1.2 CHO hERG cells were grown in dishes containing the above medium and at 37 ℃ with 5% co ₂ Is cultured in an incubator of (a). 24 to 48 hours prior to electrophysiological experiments, CHO hERG cells were transferred to round glass plates placed in petri dishes and grown in the same culture broth and culture conditions as above. The density of CHO hERG cells on each circular slide needs to be such that the vast majority of cells are independent, single.

2. Experimental solution

The following solutions (recommended by Sophion) were used for electrophysiological recording.

Composition of intracellular and extracellular fluids

3. Electrophysiological recording procedure

3.1 electrophysiological recording System

A fully automated QPatch system (Sophion, denmark) was used to record whole cell currents. The cells were clamped at a voltage of-80 mV. The cell clamp voltage depolarized to +20mV to activate the hERG potassium channel, and after 2.5 seconds was clamped to-50 mV to eliminate inactivation and generate an outward tail current. The tail current peak is used as a value for hERG current magnitude.

3.2 QPatch Experimental procedure

After the initial stage of achieving the whole cell arrangement state of rupture of membranes, the cells were recorded for at least 120 seconds to reach stability. The voltage pattern described above was then applied to the cells every 15 seconds throughout the process. Only stable cells are allowed into the course of drug treatment in the above parameter threshold record. An external solution containing 0.1% dimethyl sulfoxide (solvent) was applied to the cells, a baseline was established, and the current was allowed to stabilize for 3 minutes. After the addition of the compound solution, the cells remain in the test environment until the effect of the compound reaches a steady state or is limited to 4 minutes. In test experiments with different gradients of compound concentration, the compound was added to the clamped cells from low to high concentration. After completion of the compound test, the cells were washed with an external solution until the current was restored to a stable state.

4. Preparation of Compounds

4.1 10mM stock solution of compound was diluted in a gradient dilution with extracellular fluid to a final mu M concentration.

The highest test concentration of 4.2 was 30. Mu.M, followed by a total of 6 concentrations of 30,10,3,1,0.3 and 0.1. Mu.M.

4.3 the final concentration of DMSO in the compound solutions of other concentrations was 0.1% except for the 30. Mu.M compound DMSO of 0.3%. All compound solutions were subjected to conventional 5 to 10 minute sonication and shaking to ensure complete dissolution of the compound.

5. Data analysis

Test data were analyzed by QPatch analysis software supplied by Sophion, excel, graphpad Prism, etc.

6. Experimental results

The results of inhibition of hERG current by the compounds of the present invention are shown in table 4.

TABLE 4 inhibition of hERG current by the inventive compounds

Conclusion: inhibition of the cardiac hERG potassium channel by drugs is the primary cause of QT prolongation syndrome by drugs. From the experimental results, the compounds with retention time of 9.239 minutes in the invention 4 or 5 have no obvious inhibition effect on cardiac hERG potassium ion channels, and can avoid toxic and side effects of the heart at high doses.

Test example 5 SD rat pharmacokinetic study of the Compounds of the invention

1. Purpose of experiment

The compound is taken as a tested animal by SD rat, and the compound is administrated by intravenous injection and/or intragastric administration, and the drug concentration in plasma at different times is measured by adopting an LC/MS/MS method, so that the pharmacokinetic characteristics of the compound in SD rat are studied.

2. Experimental protocol

2.1 Experimental drugs and animals

Compound of compound 4 or 5 having a retention time of 9.239 minutes

SD rats, male, 195-235g,6-8 weeks purchased from Shanghai Jieshike laboratory animal Co.

2.2 pharmaceutical formulation

Intravenous administration preparation: an appropriate amount of the test compound was weighed, and an appropriate amount of DMSO was added to 30% of solvent HS15 (solvent HS15: water=3:7): saline=5%: 5%:90%, and vortexing was performed to prepare a solution having a final preparation concentration of 0.2 mg/mL.

The stomach-filling administration preparation is prepared by: weighing a proper amount of a compound to be tested, adding a proper amount of DMSO (methyl methacrylate) and (PEG) 400=5:95 (v/v), and carrying out vortex oscillation to prepare a solution with the final preparation concentration of 0.5 mg/mL.

2.3 administration of drugs

SD rats were intravenously injected (3/group) and intragastrically (3/group) with the test compound.

Intravenous injection group: is administered by intravenous injection (administration dose 1mg/kg, administration volume 5 mL/kg) without fasting.

Gastric lavage group: after overnight fast, the medicine was administered by stomach irrigation (administration dose: 5mg/kg, administration volume: 10 mL/kg), and after 4 hours of administration, the medicine was fed.

3. Operation of

Intravenous injection group: about 150. Mu.L of blood was collected into EDTA-K2 anticoagulant tubes via the jugular vein at 0.083 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours and 24 hours post-administration.

Gastric lavage group: about 100. Mu.L of blood was collected into EDTA-K2 anticoagulant tubes via the jugular vein at 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours and 24 hours post-administration.

The blood samples were first kept in wet ice and the plasma was centrifuged within 15 minutes after sampling (centrifugation conditions: 7000rpm,4 ℃ C., 5 minutes). The collected upper plasma was stored at-70℃until analysis.

LC-MS/MS was used to determine the amount of test compound in rat plasma after intravenous and intragastric administration.

4. Pharmacokinetic parameter results

The pharmacokinetic parameter results of this test example are shown in table 5.

TABLE 5 results of pharmacokinetic parameters in rats

Test example 6 pharmacodynamics test of the inventive Compounds in the NCI-H358 cell BALB/c nude mice subcutaneous transplantation model 1. Experimental purposes

This test was used to evaluate the antitumor effect and safety of the compounds of the present invention (compounds with retention time of 9.239 minutes in 4 or 5) in a BALB/c nude mice animal model transplanted subcutaneously with NCI-H358 (human non-small cell lung cancer) cell line once daily for 11 days of oral gavage administration.

2. Test object preparation

2.1 blank dosing formulation:

an appropriate volume of a formulation containing DMA (dimethylacetamide): 30%Solutol HS 15:Saline (physiological saline) =10:10:80 (v/v/v) was prepared as a blank administration test solution.

2.2 preparation of oral administration of Compound (Compound having a retention time of 9.239 minutes in 4 or 5)

Weighing a proper amount of compound in a 10mL centrifuge tube, adding a proper amount of DMA (direct memory access), carrying out vortex oscillation to completely dissolve solid substances, adding a proper amount of 30% solutol HS 15 in volume, carrying out vortex oscillation, and uniformly mixing; then, physiological saline was added so that the ratio of DMA to 30%Solutol HS 15:Saline was 10:10:80 (v/v/v), and a drug administration formulation was prepared at a concentration of 1 mg/mL.

3. Experimental animal

BALB/c nude mice, females, 6-7 weeks (week of mice at tumor cell inoculation), 12, purchased from Jiangsu Jiuyaokang Biotech Co., ltd., license number: SCXK 2019-0009.

4. Cell culture

NCI-H358 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 1% sodium pyruvate and 1% glutamine. NCI-H358 cells in exponential growth phase were collected, and cells were resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in nude mice.

5. Animal inoculation and grouping

Female BALB/c nude mice were inoculated subcutaneously on the right dorsal side with about 6.0X10 ⁶ NCI-H358 cells, until the average tumor volume reaches about 100-150mm ³ At this time, 6 groups were randomly grouped according to tumor size, and divided into 2 groups.

6. Animal dosing and observation

After tumor inoculation, a subcutaneous graft tumor model was established. Each treatment group and vehicle control group were orally administered by intragastric administration for 11 days. Animals were weighed daily and tumor volumes were measured 2 times per week.

Tumor Volume (TV), relative tumor proliferation rate (T/C), relative tumor inhibition rate (TGI) and percent tumor Inhibition (IR) were calculated as follows:

(1) TV (tumor volume) =1/2×a×b ² Wherein a and b respectively represent the length and width of the tumor;

(2) T/C (relative tumor proliferation rate,%) =t _RTV /C _RTV X 100%, where T _RTV RTV, C for treatment group _RTV RTV as control group;

(3) TGI% (tumor growth inhibition) = (1-T/C) ×100%; wherein T and C are the relative tumor volumes at a particular time point for the treatment group and the control group, respectively.

(4) IR (%) (tumor weight inhibition rate) = (1-TW _t /TW _c ) X 100%, TW therein _t TW for treating group tumor weight _c Is the tumor weight of the control group.

7. Results

Table 6 table of the pharmacodynamic analyses of groups of compounds of the invention in NCI-H358 cell subcutaneous transplantation tumor model at day 11 post-dose

Remarks: tumor volume data are expressed as "mean ± standard error";

TABLE 7 tumor weights of groups of nude mice in NCI-H358 cell subcutaneous transplantation tumor model

Remarks: tumor volume data are expressed as "mean ± standard error";

from tables 6 to 7, it is understood that the compound of the present invention (exemplified by the compound having a retention time of 9.239 minutes in 4 or 5) has a remarkable growth inhibitory effect on establishment of a mouse tumor model based on NCI-H358 cells within 11 days at a dose of 10mg/kg (po, qd), and has no remarkable change in body weight, good safety and tolerability.

Claims

A compound of formula (I) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof:

wherein:

ring a is selected from aryl, heteroaryl or fused ring;

R ¹ the same OR different are each independently selected from hydrogen atom, alkyl, halogen, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, =o, -OR ² 、-C(O)R ² 、-C(O)OR ² 、-NHC(O)R ² 、-NHC(O)OR ² 、-NR ³ R ⁴ 、-C(O)NR ³ R ⁴ 、-CH ₂ NHC(O)OR ² 、-CH ₂ NR ³ R ⁴ or-S (O) _r R ² Is substituted by a substituent of (2); wherein said alkyl, cycloalkyl, heterocyclyl, aryl OR heteroaryl is optionally further substituted with one OR more substituents selected from alkyl, halo, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, =o, -OR ² 、-C(O)R ² 、-C(O)OR ² 、-NHC(O)R ² 、-NHC(O)OR ² 、-NR ³ R ⁴ 、-C(O)NR ³ R ⁴ 、-CH ₂ NHC(O)OR ² 、-CH ₂ NR ³ R ⁴ or-S (O) _r R ² Is substituted by a substituent of (2);

R ² selected from a hydrogen atom, an alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group, wherein the alkyl group, the cycloalkyl group, the heterocyclic group, the aryl group or the heteroaryl group is optionally further substituted with one or more groups selected from a hydroxyl group, a halogen group, a nitro group, a cyano group, an alkyl group, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyl group, a heterocyclic group, an aryl group, a heteroaryl group, =o, -C (O) R ⁵ 、-C(O)OR ⁵ 、-OC(O)R ⁵ 、-NR ⁶ R ⁷ 、-C(O)NR ⁶ R ⁷ 、-SO ₂ NR ⁶ R ⁷ or-NR ⁶ C(O)R ⁷ Is substituted by a substituent of (2);

R ³ and R is ⁴ Each independently of the otherSelected from a hydrogen atom, a hydroxyl group, a halogen, an alkyl group, an alkoxy group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group, wherein the alkyl group, the alkoxy group, the cycloalkyl group, the heterocyclic group, the aryl group or the heteroaryl group is optionally further substituted with one or more groups selected from a hydroxyl group, a halogen, a nitro group, a cyano group, an alkyl group, an alkoxy group, a cycloalkyl group, a heterocyclic group, an aryl group, a heteroaryl group, =o, -C (O) R ⁵ 、-C(O)OR ⁵ 、-OC(O)R ⁵ 、-NR ⁶ R ⁷ 、-C(O)NR ⁶ R ⁷ 、-SO ₂ NR ⁶ R ⁷ or-NR ⁶ C(O)R ⁷ Is substituted by a substituent of (2);

alternatively, R ³ And R is ⁴ Together with the atoms to which they are attached form a 4-8 membered heterocyclic group, wherein the 4-8 membered heterocyclic group contains one or more of N, O or S (O) _r And said 4-8 membered heterocyclyl is optionally further substituted with one or more substituents selected from hydroxy, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, =o, -C (O) R ⁵ 、-C(O)OR ⁵ 、-OC(O)R ⁵ 、-NR ⁶ R ⁷ 、-C(O)NR ⁶ R ⁷ 、-SO ₂ NR ⁶ R ⁷ or-NR ⁶ C(O)R ⁷ Is substituted by a substituent of (2);

R ⁵ 、R ⁶ and R is ⁷ Each independently selected from the group consisting of hydrogen, alkyl, amino, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein said alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally further substituted with one or more substituents selected from the group consisting of hydroxy, halo, nitro, amino, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxyl, or carboxylate;

m is selected from 0, 1 or 2;

r is selected from 0, 1 or 2.
A compound according to claim 1, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein ring a is selected from phenyl, naphthyl, pyridinyl, benzothiazolyl, or benzothienyl, preferably benzothiazolyl or benzothienyl.
A compound according to claim 1, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein
Selected from:
a compound according to claim 1, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein the compound comprises:
a process for the preparation of a compound of general formula (I) according to claim 1, or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, which process comprises:

Carrying out Suzuki coupling reaction on a compound shown in a general formula (IA) and a compound shown in a general formula (IB) under the action of a palladium catalyst and an alkaline reagent, further removing a protecting group to obtain a compound shown in a general formula (IC), and reacting the compound shown in the general formula (IC) with acryloyl chloride under an alkaline condition, and optionally further removing the protecting group to obtain a compound shown in the general formula (I);

wherein:

X ¹ a leaving group, preferably bromine;

m is selected from-B (OH) ₂ 、-BF ₃ K or

PG is a protecting group, preferably t-butoxycarbonyl;

ring A, R ¹ And m is as defined in claim 1.
A compound of formula (IC) or a stereoisomer, tautomer thereof, pharmaceutically acceptable salt thereof,

wherein: ring A, R ¹ And m is as defined in claim 1.
The compound according to claim 6, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein the compound is:
a pharmaceutical composition comprising an effective amount of a compound according to any one of claims 1 to 4, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or combination thereof.
Use of a compound according to any one of claims 1 to 4, or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 8, for the preparation of a K-Ras gtpase inhibitor, wherein the K-Ras gtpase inhibitor is preferably a KRAS G12C inhibitor.
Use of a compound according to any one of claims 1 to 4, or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 8, 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 preferably selected from cancer, wherein the cancer is preferably 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 carcinoma, more preferably pancreatic cancer, colorectal cancer and lung cancer, wherein the KRAS mutation is preferably a KRAS G12C mutation.
Use of a compound according to any one of claims 1 to 4, or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 8, for the manufacture of a medicament for the treatment of cancer, wherein the cancer is preferably 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 carcinoma, more preferably pancreatic cancer, colorectal cancer and lung cancer.
The use according to claim 10 or 11, wherein the lung cancer is non-small cell lung cancer.