CN114409653A - Bridged ring pyrimidine-fused ring compound and application thereof - Google Patents

Abstract

The invention provides a bridged pyrimido cyclic compound and application thereof, wherein the bridged pyrimido cyclic compound is a compound represented by the following formula (I) and an isomer thereof or a pharmaceutically acceptable salt thereof. The compound can be applied to a therapeutic drug aiming at KRASG12C mutant protein related cancer, wherein KRASG12C mutant protein related cancer diseases are selected from: lung cancer, colorectal cancer, pancreatic cancer, liver cancer, stomach cancer, esophageal cancer, bile duct cancer, breast cancer, ovarian cancer, cervical cancer, melanoma, brain glioma, lymphoma, and leukemia.

Description

Bridged ring pyrimidine-fused ring compound and application thereof

Technical Field

The invention relates to the technical field of drug synthesis, in particular to a bridged pyrimido-cyclic compound and application thereof in preparing a drug for treating KRASG12C mutant protein-related cancers.

Background

RAS mutations are often observed in malignancies and support a variety of markers of cancer, including genomic instability, cell proliferation, inhibition of apoptosis, reprogramming of metabolism, alteration of the microenvironment, escape of immune response and promotion of metastasis. Consistent with its general effect on cancer cell function, regression of oncogenic KRAS in many established tumor models results in tumor regression, and therefore RAS is a potentially very effective cancer therapeutic target. RAS mutations appear to have multiple functional classes, and strategies may need to be developed for each functional class.

Among RAS family members, oncogenic mutations are most common in KRAS (85%), whereas NRAS (12%) and HRAS (3%) are less common. Most RAS family mutations occur at amino acid residues 12, 13 and 61, which are mostly direct interactions with GTP in three-dimensional conformations, e.g., mutation of glycine at amino acid residue 12 to any other amino acid except proline creates a steric block that prevents GAP protein from entering KRAS, thereby inhibiting GTP hydrolysis and resulting in a significant increase in KRAS in highly active GTP-bound forms. Variant KRAS accounted for 30% of lung cancer. 97% of KRAS variations occur in exons 2 and 3, including amino acids G12 (39%), G13 and Q61. KRAS G12C is the most common RAS mutation in non-small cell lung cancer, the leading cause of cancer death in the united states, and there is still no direct and effective drug clinically. But in recent years there has been significant progress in this area.

In 2013, Shokat et al reported breakthrough results in Nature, who screened active small molecules designed to bind irreversibly to mutant cysteines at G12C to a small pocket near the KRAS effector region, and small molecules bound to this pocket could inhibit KRAS activity by locking the protein in a GDP-bound inactive state. In 2016, Wellspring corporation reported on cancer discovery targeting KRAS G12C small molecule inhibitor ARS-853. Although the activity is still in micromolar, the proof of concept of the cell and animal experiments is completed. In 2018, the company discloses a new generation of targeting KRAS G12C small molecule inhibitor ARS-1620 on cell. U.S. Pat. No. US2018334454 discloses a targeting KRAS G12C small molecule inhibitor AMG510, the structural formula of which is shown below. At the american clinical oncology society of America (ASCO) annual meeting held 6 months 2019, Amgen and Mirati, respectively, reported that their small molecule inhibitors targeting KRAS G12C have begun a phase of human clinical trials.

Inhibition of KRAS muteins inhibits the growth of cancer cells, and thus, treatment of cancers such as lung cancer, pancreatic cancer, and colorectal cancer would seem to be a promising therapeutic approach. Thus, there is a potentially great unmet medical need in this field.

Disclosure of Invention

In order to solve the technical problems, the invention adopts the following technical scheme:

according to one aspect of the present invention, there is provided a bridged pyrimido ring compound represented by the following formula (I) and isomers thereof or pharmaceutically acceptable salts thereof,

wherein,

R_1ais hydrogen;

R_1bselected from hydrogen, fluorine, -CH₃、-CH₂F、-CHF₂、-CF₃、-CH₂Cl、-CH₂OH、-CH₂OCH₃、-CH₂N(CH₃)₂、-CH₂N(CH₂CH₃)₂、

R₂Selected from hydrogen, halogen or-OC_1-3An alkyl group;

R_3a、R_3b、R_3cand R_3dEach independently selected from hydrogen or C_1-3An alkyl group; said C is_1-3The alkyl group may be substituted by 1 or more R₆Substitution; the R is₆Is cyano or halogen;

R₄is selected from C_1-3An alkyl group; said C is_1-3The alkyl group may be substituted by 1-N (C)_1-2Alkyl radical)₂Substitution;

R₅selected from phenyl or naphthyl; the phenyl or naphthyl group may be substituted by 1 or more R₇Substitution; the R is₇Selected from halogen, hydroxy, -CF₃、-OCF₃、-NH₂、C_1-6Alkyl or C_1-6An alkoxy group;

l is selected from-O-.

According to another aspect of the present invention, preferably, in the structure represented by formula (I), R is_1aIs hydrogen; r_1bIs hydrogen; r₂Selected from hydrogen or fluorine; r_3aIs selected from-CH₂CN；R_3b、R_3cAnd R_3dAre all selected from hydrogen; r₄Is composed of

R₅Is phenyl or naphthyl; the phenyl or naphthyl group may be substituted by 1 or more R₇Substitution; the R is₇Selected from fluorine, chlorine, bromine, -CF₃、-NH₂Methyl, ethyl, n-propyl, isopropyl or methoxy.

Even more preferably, R_1aIs hydrogen; r_1bIs hydrogen.

According to another aspect of the present invention, preferably, in the structure represented by formula (I), R₂Selected from hydrogen, halogen or-OC_1-3An alkyl group.

Further preferably, R₂Selected from hydrogen or fluorine.

Further preferably, R_3a、R_3b、R_3cAnd R_3dEach independently selected from hydrogen or C_1-3An alkyl group; said C is_1-3The alkyl group may be substituted by 1 or more R₉Substitution; the R is₉Is cyano or halogen;

further preferably, R_3aSelected from hydrogen or C_1-3An alkyl group; said C is_1-3The alkyl group may be substituted by 1 or more R₉Substitution; the R is₉Is cyano or halogen; r_3b、R_3cAnd R_3dAre all selected from hydrogen;

even more preferably, R_3aIs selected from-CH₂CN；R_3b、R_3cAnd R_3dAre all selected from hydrogen.

Preferably, in the structure of formula (I), R_3a、R_3b、R_3cAnd R_3dEither or both of which are-CH₃And the remainder are hydrogen.

According to another aspect of the present invention, preferably, in the structure represented by formula (I), R₄Is selected from C_1-3An alkyl group; said C is_1-3The alkyl group may be substituted by 1-N (C)_1-2Alkyl radical)₂Substitution;

according to another aspect of the present invention, preferably, in the structure represented by formula (I), R₄Is selected from C_1-3An alkyl group; said C is_1-3Alkyl may be substituted with 1 four to eight membered saturated or unsaturated heterocyclic ring containing 1 to 3 heteroatoms selected from N, O and S; said four to eight membered saturated or unsaturated heterocyclic ring containing 1 to 3 heteroatoms selected from N, O and S may be substituted with 1 or more fluoro, chloro, methyl, methoxy or benzyl;

further preferably, R₄Is selected from

According to another aspect of the present invention, preferably, in the structure represented by formula (I), L is-O-.

According to another aspect of the present invention, preferably, the compound represented by formula (I) and isomers thereof or pharmaceutically acceptable salts thereof is composed of a compound selected from the group consisting of:

according to another aspect of the invention, the invention provides a compound represented by formula (I) and pharmaceutically acceptable salts thereof and isomers thereof or pharmaceutically acceptable salts thereof for use in the preparation of a medicament for treating a KRASG12C mutein-related cancer.

Preferably, according to said use, said KRASG12C mutein-related cancer disease is selected from the group consisting of: lung cancer, colorectal cancer, pancreatic cancer, liver cancer, stomach cancer, esophageal cancer, bile duct cancer, breast cancer, ovarian cancer, cervical cancer, melanoma, brain glioma, lymphoma, and leukemia.

According to another aspect of the present invention, there is provided a medicament for treating KRASG12C mutein-related cancer, comprising a therapeutically effective amount of a compound represented by formula (I) and pharmaceutically acceptable salts thereof and isomers thereof or pharmaceutically acceptable salts thereof according to the present invention as an active ingredient, and pharmaceutically acceptable adjuvants.

Preferably, according to the medicament, the KRASG12C mutein-related cancer diseases are selected from the group consisting of: lung cancer, colorectal cancer, pancreatic cancer, liver cancer, stomach cancer, esophageal cancer, bile duct cancer, breast cancer, ovarian cancer, cervical cancer, melanoma, brain glioma, lymphoma, and leukemia.

According to another aspect of the present invention, the present invention provides a method for treating KRASG12C mutein-related cancer, comprising administering to a subject an effective amount of the compound according to the present invention or a pharmaceutical composition comprising the compound and pharmaceutically acceptable salts thereof as an active ingredient.

Preferably, according to said method of treatment, said KRASG12C mutein related cancer is a treatment of a cancer disease selected from the group consisting of: lung cancer, colorectal cancer, pancreatic cancer, liver cancer, stomach cancer, esophageal cancer, bile duct cancer, breast cancer, ovarian cancer, cervical cancer, melanoma, brain glioma, lymphoma, and leukemia.

According to another aspect of the present invention, there is provided a method for preparing the compound represented by formula ((I): wherein X ═ CH₂CH₂The structural compound of (a) to (b) is exemplified, and the preparation method is as follows:

1) commercial or homemade starting material I-1 is dissolved in MeOH, N₂Adding NaOMe and S-methylisothiourea sulfate in the atmosphere, stirring at room temperature overnight, adding water, filtering, and drying to obtain fused ring I-2;

2) dissolving the compound I-2 in DCM (10mL), cooling to 0 ℃, adding DIEA and Tf2O, heating to room temperature, and stirring for 2h to obtain an activated ester I-3;

3) dissolving the compound I-3, cyclic ammonia and DIPEA in DMF, heating to 100 ℃, stirring for reaction for 2h, and generating an intermediate I-4 through substitution reaction;

4) dissolving the intermediate I-4 in EA and water, adding sodium bicarbonate, dropwise adding a protecting group reagent such as Cbz-Cl and the like, reacting completely, and performing post-treatment to obtain an amino-protected intermediate I-5;

5) oxidizing the methyl sulfide I-5 by using m-CPBA to obtain a sulfoxide analogue I-6;

6) dissolving sulfoxide analogues I-6 and R4-LH in toluene, adding t-BuONa, cooling to 0 ℃, stirring for reaction for 2 hours, and carrying out substitution reaction on R4-LH and an intermediate I-9 to obtain an intermediate I-7;

7) intermediate I-7 was dissolved in DCM, N₂Protecting, cooling to 0 deg.C, removing Pg1 protecting group (such as Boc protecting group) with deprotection reagent such as trifluoroacetic acid, and post-treating to obtain free amine intermediate I-8;

8) reacting the amine intermediate I-8, aryl halide, CsCO₃、Pd₂(dba)₃Xantphos in a solvent, N₂Raising the temperature to 90 ℃ in the atmosphere, after the reaction is completed, filtering and removing Pd₂(dba)₃Spin-drying and purifying the filtrate to obtain a coupling intermediate I-9;

9) dissolve Compound I-9 in MeOH and add NH₃·MeOH，10％Pd/C，H₂Replacement 3 times, H₂Stirring and reacting for 1h in the atmosphere, and obtaining an intermediate I-10 after removing protecting groups such as Cbz and the like by hydrogenation;

10) dissolving the compound I-10 in DCM, adding TEA, cooling to below 0 ℃, adding allyl chloride for reaction for 2 hours, and condensing with acyl chloride to obtain the final product I.

Detailed Description

Hereinafter, the present invention will be described in detail. Before the description is made, it should be understood that the terms used in the present specification and the appended claims should not be construed as limited to general and dictionary meanings, but interpreted based on the meanings and concepts corresponding to technical aspects of the present invention on the basis of the principle that the inventor is allowed to define terms appropriately for the best explanation. Accordingly, the description proposed herein is just a preferable example for the purpose of illustrations only, not intended to limit the scope of the invention, so it should be understood that other equivalents and modifications could be made thereto without departing from the spirit and scope of the invention.

In accordance with the present invention, all terms referred to herein have the same meaning as those skilled in the art to understand the present invention, unless otherwise specified.

The term "salt" as used herein refers to a cation and anion containing compound that can be produced by protonation of an acceptable proton site and/or deprotonation of an available proton site. Notably, protonation of the acceptable proton sites results in the formation of cationic species whose charge is balanced by the presence of physiological anions, while deprotonation of the available proton sites results in the formation of anionic species whose charge is balanced by the presence of physiological cations.

The term "pharmaceutically acceptable salt" means that the salt is pharmaceutically acceptable. Examples of pharmaceutically acceptable salts include, but are not limited to: (1) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as glycolic acid, pyruvic acid, lactic acid, malonic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1, 2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-p-toluenesulfonic acid, camphoric acid, dodecylsulfuric acid, gluconic acid, glutamic acid, salicylic acid, cis-hexadiene diacid, and the like; or (2) a base addition salt, and a conjugate base of any of the above inorganic acids, wherein the conjugate base comprises a compound selected from Na⁺、K⁺、Mg²⁺、Ca²⁺、NH_xR_4-x ⁺The cationic component of (1), wherein NH_xR_4-x ⁺(R is C_1-4Alkyl, subscript x is an integer selected from 0, 1,2, 3, or 4) represents a cation in the quaternary ammonium salt. It is to be understood that all references to pharmaceutically acceptable salts include the solvent addition forms (solvents) as defined herein of the same acid addition saltCompound) or crystalline form (polymorph).

The term "C_1-MAlkyl "refers to an alkyl group containing from 1 to M carbon atoms, for example where M is an integer having the following value: 2. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. For example, the term "C_1-6Alkyl "refers to an alkyl group containing 1 to 6 carbon atoms. Examples of alkyl groups include, but are not limited to, lower alkyl groups including methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl or pentyl, isopentyl, neopentyl, hexyl, heptyl, and octyl.

The term "aryl" refers to an aromatic system that may be a single ring or multiple aromatic rings that are otherwise fused or linked together such that at least a portion of the fused or linked rings form a conjugated aromatic system. Aryl groups include, but are not limited to: phenyl, naphthyl, tetrahydronaphthyl. Aryl groups may be optionally substituted, such as aryl or heterocyclic which may be substituted with 1 to 4 groups selected from: halogen, -CN, -OH, -NO₂Amino, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, aryloxy, substituted alkoxy, alkylcarbonyl, alkylcarboxy, alkylamino or arylthio.

The term "substituted" means that the reference group may be substituted with one or more additional groups individually and independently selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic hydrocarbon, hydroxy, alkoxy, alkylthio, arylthio, alkylsulfinyl, arylsulfonyl, alkylsulfonyl, arylsulfonyl, cyano, halo, carbonyl, thiocarbonyl, nitro, haloalkyl, fluoroalkyl and amino, including mono-and di-substituted amino groups and protected derivatives thereof.

The compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition containing the compound provided by the present invention may be in various forms such as tablets, capsules, powders, syrups, solutions, suspensions, aerosols, and the like, and may be present in a suitable solid or liquid carrier or diluent, as well as in a suitable sterile device for injection or instillation.

Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional preparation methods in the pharmaceutical field. For example, a unit dose of the formulation thereof may contain from 0.05 to 200mg of a compound of formula (I) or a pharmaceutically acceptable salt thereof, preferably a unit dose of the formulation may contain from 0.1mg to 100mg of a compound of formula (I).

The compounds and pharmaceutical compositions of the present invention represented by general formula (I) can be administered to mammals clinically, including humans and animals, via oral, nasal, dermal, pulmonary, or gastrointestinal routes of administration. Most preferably oral. The optimal daily dosage is 0.01-200mg/kg body weight, and can be administered in one time or 0.01-100mg/kg body weight in several times. Regardless of the method of administration, the optimal dosage for an individual will depend on the particular treatment. Usually starting with a small dose and gradually increasing the dose until the most suitable dose is found.

In the present invention, the term "effective amount" may refer to an effective amount in a dosage and for a period of time required to achieve a desired effect. This effective amount may vary depending on factors such as the type of disease or the condition of the disease being treated, the particular target organ being administered, the size of the individual patient, or the severity of the disease or symptoms. One of ordinary skill in the art can empirically determine the effective amount of a particular compound without undue experimentation.

Typical formulations are prepared by mixing a compound of the invention of formula (I) with a carrier, diluent or excipient. Suitable carriers, diluents or excipients are well known to those skilled in the art and include materials such as carbohydrates, waxes, water soluble and/or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like.

The particular carrier, diluent or excipient employed will depend upon the mode of use and the purpose of the compound of the invention. The solvent is generally selected based on the solvent that one of skill in the art would consider safe and effective for administration to mammals. Generally, safe solvents are non-toxic aqueous solvents such as water, as well as other non-toxic solvents that are soluble or miscible with water. Suitable aqueous solvents include one or more of water, ethanol, propylene glycol, polyethylene glycol (e.g., PEG400, PEG300), and the like. The formulation may also include one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, flavoring agents or other known additives to make or use the drug in an acceptable form.

When the compound of formula (I) according to the present invention is used in combination with at least one other drug, the two drugs or more may be used separately or in combination, preferably in the form of a pharmaceutical composition. The compounds or pharmaceutical compositions of the invention according to formula (I) can be administered to a subject separately or together in any known oral, intravenous, rectal, vaginal, transdermal, other topical or systemic administration form.

These pharmaceutical compositions may also contain one or more buffering agents, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifying agents, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, flavoring agents or other known additives to allow the pharmaceutical composition to be manufactured or used in an acceptable form.

The route of oral administration is preferred for the medicaments of the invention. Solid dosage forms for oral administration may include capsules, tablets, powders or granules. In solid dosage forms, the compounds or pharmaceutical compositions of the present invention are mixed with at least one inert excipient, diluent or carrier. Suitable excipients, diluents or carriers include substances such as sodium citrate or dicalcium phosphate, or starches, lactose, sucrose, mannitol, silicic acid and the like; binders such as carboxymethyl cellulose, alginate, gelatin, polyvinyl pyrrolidone, sucrose, gum arabic, etc.; humectants such as glycerin, etc.; disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, specific complex silicate, sodium carbonate, etc.; solution retarding agents such as paraffin, etc.; absorption accelerators such as quaternary ammonium compounds and the like; adsorbents such as kaolin, bentonite, etc.; lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and the like. In the case of capsules and tablets, the dosage form may also include buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using lactose and high molecular weight polyethylene glycols and the like as excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the compounds of the present invention or pharmaceutical compositions thereof, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents; solubilizers and emulsifiers such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide; oils (e.g., cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, sesame oil, etc.); glycerol; tetrahydrofurfuryl alcohol; fatty acid esters of polyethylene glycol and sorbitan; or mixtures of several of these substances, and the like.

In addition to these inert diluents, the compositions can also include excipients such as one or more of wetting agents, emulsifying agents, suspending agents, sweetening, flavoring, and perfuming agents.

The suspension may further contain a carrier such as a suspending agent, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol, sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, or a mixture of several of these substances, in addition to the compound represented by the general formula (I) of the present invention or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same.

The compound represented by the general formula (I) or the pharmaceutically acceptable salt thereof or the pharmaceutical composition containing the same can be administered by other topical administration dosage forms, including ointments, powders, sprays and inhalants. The medicament may be mixed under sterile conditions with a pharmaceutically acceptable excipient, diluent or carrier, and any preservatives, buffers or propellants which may be required. Ophthalmic formulations, ophthalmic ointments, powders and solutions are also intended to be within the scope of the present invention.

In addition, kits (e.g., pharmaceutical packages) are also contemplated by the present disclosure. The provided kits can comprise a pharmaceutical composition or compound described herein and a container (e.g., a vial, ampoule, bottle, syringe, and/or split-pack or other suitable container). In some embodiments, the provided kits may optionally further comprise a second container comprising a pharmaceutically acceptable excipient for diluting or suspending a pharmaceutical composition or compound described herein. In some embodiments, the pharmaceutical compositions or compounds described herein disposed in the first and second containers are combined to form one unit dosage form.

In certain embodiments, the kits described herein further comprise instructions for using the compounds or pharmaceutical compositions contained in the kit. The kits described herein may also include information required by regulatory agencies such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kit is prescription information. In certain embodiments, the kits and instructions provide for the treatment and/or prevention of a proliferative disease in a subject in need thereof. The kits described herein may comprise one or more additional pharmaceutical agents as separate compositions.

The present invention is described in further detail below with reference to specific examples, but the present invention is not limited to the following examples, which are intended to better illustrate certain embodiments of the present invention and should not be construed as limiting the scope of the present invention in any way. Conditions not noted in the examples are conventional conditions. Unless otherwise specified, reagents and equipment used in the following examples are commercially available products.

The structures of the compounds in the following examples were determined by Nuclear Magnetic Resonance (NMR) or/and Mass Spectrometry (MS). NMR shifts (. delta.) are given in units of 10-6(ppm), NMR was measured using a Bruker AVANCE-400 nuclear magnetic spectrometer using deuterated dimethylsulfoxide (DMSO-d6), deuterated chloroform (CDCl3), deuterated methanol (CD3OD), and Tetramethylsilane (TMS) as an internal standard.

MS was determined using a FINNIGAN LCQAD (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).

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

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

In the examples, the reaction temperature is, unless otherwise specified, from 20 ℃ to 30 ℃ at room temperature.

The reaction progress in the examples was checked by Thin Layer Chromatography (TLC), the developing solvent system used, and the elution system for column chromatography used for purifying the compounds included: a: dichloromethane and methanol system, B: n-hexane and ethyl acetate system, C: petroleum ether and ethyl acetate system, D: acetone and petroleum ether system, the volume ratio of the solvent is adjusted according to the polarity of the compound.

Abbreviations used in the experiments: NaOMe, sodium methoxide; EA, ethyl acetate; DCM, dichloromethane; h, h; DMF, N-dimethylamide; THF, tetrahydrofuran; TEA, triethylamine; DIPEA, diisopropylethylamine; tf₂O, trifluoromethanesulfonic anhydride; boc, tert-butoxycarbonyl; cbz, benzyloxycarbonyl; mCPBA, m-chloroperoxybenzoic acid.

Example 1: preparation of Compound 1

Compound 1 was prepared according to the following route.

Step 1: synthesis of intermediates 1 to 3

NaH (3.2g, 0.0793mol) was added to THF (80mL), cooled to 0 deg.C, 1-2(18.97g, 0.0846mol) was slowly added dropwise, the temperature was maintained at < 5 deg.C, the 0 deg.C addition was completed and stirred for 3.0h, and then a solution of aldehyde 1-1(8.1g, 0.0529mol) in THF (25mL) was slowly added dropwise into the system, the temperature was maintained at < 5 deg.C. After the addition, the reaction solution was allowed to naturally warm to room temperature overnight. After the reaction was completed, 20mL of saturated ammonium chloride solution was added to quench the reaction, the solution was separated, the aqueous phase was extracted 2 times with EA, the organic phases were combined, washed once with saturated sodium chloride, dried over anhydrous sodium sulfate, the solvent was spin-dried, n-hexane was added to the mixture for beating, and suction filtration was performed to obtain a pale red solid (10.0g, 84%).

¹H NMR(400MHz,CDCl₃):10.14(s,1H),7.60(d,J＝16.0Hz,1H),6.92-6.90(m,1H),6.55-6.54(m,1H),6.40(d,J＝16.0Hz,1H),4.28(q,J＝8.0Hz,2H),3.94(s,3H),1.34(t,J＝8.0Hz,3H)；

MS m/z(ESI):224.0[M+1]。

Step 2: synthesis of intermediates 1 to 4

Raney Ni (2.0g) is taken and washed with absolute ethyl alcohol for 2 times, the intermediate 1-3(10.0g) is added to react at room temperature overnight, the system is gradually dissolved and cleared, the reaction is completed, the filtration is carried out, and the filtrate is dried by spinning to obtain a solid crude product (9.6g, 96%).

MS m/z(ESI):226.1[M+1].

And step 3: synthesis of intermediates 1 to 5

Intermediate 1-4(13.0g, 0.058mol) was dissolved in acetonitrile (150mL), triethylamine (14.7g, 0.145mol), DMAP (7.1g, 0.058mol) were added, and Boc was added in portions₂O (25.2g, 0.116mol), heated to 60 ℃ and reacted for 3.0 h. After the reaction was complete, acetonitrile was spin-dried, 50ml of lcm was added, washed with dilute hydrochloric acid for 2 times, separated, dried and concentrated in the organic phase, and purified by column chromatography to obtain a solid (13.0g, 85%).

¹H NMR(400MHz,CDCl₃):6.78(d,J＝4.0Hz,1H),5.96(d,J＝4.0Hz,1H),4.15(q,J＝8.0Hz,2H),3.82(s,3H),3.06(t,J＝8.0Hz,2H),2.67(t,J＝8.0Hz,2H),1.53(s,9H),1.25(t,J＝8.0Hz,3H).

And 4, step 4: synthesis of intermediates 1 to 6

Intermediate 1-5(13g) was dissolved in methanol (200mL), and 10% Pd/C1.3 g was added to the solution to react overnight at room temperature under a hydrogen atmosphere. Filtering, concentrating methanol, and purifying by column chromatography to obtain 8.4g of reduction product.

And 5: synthesis of intermediates 1 to 7

The intermediates 1-6(7.8g, 1.0eq) and sodium ethoxide (4.84g, 3.0eq) were suspended in 80mL of toluene, and reacted at 90 ℃ for 6h under nitrogen protection. The system is cooled to room temperature, diluted HCl is added to adjust the pH value to 6, liquid is separated, the organic phase is added with DCM for extraction, the organic phases are combined, saturated saline is washed once, anhydrous sodium sulfate is dried, and column chromatography purification is carried out to obtain a pure product of 3.0 g.

¹H NMR(400MHz,CDCl₃):11.95(s,1H),4.50-4.35(m,2H),4.21(q,J＝8.0Hz,2H),2.80-2.70(m,1H),2.13-1.95(m,4H),1.64-1.60(m,1H),1.45(s,9H),1.29(t,J＝8.0Hz,3H).

Step 6: synthesis of intermediates 1 to 8

The intermediates 1 to 7(3.0g, 1.0eq), sodium methoxide (2.86g, 5.0eq), methylthiourea (5.3g, 1.8eq) and 40mL methanol were added to a reaction flask, and stirred overnight at room temperature under nitrogen. Dilute HCl is added to adjust pH to 6, methanol is concentrated, EA and water are added to the remaining material and slurried, and the crude product is dried to obtain 1.4g, which is used directly in the next step.

MS m/z(ESI):324.1[M+1].

And 7: synthesis of intermediates 1 to 9

Dissolving the intermediate 1-8(0.48g, 1.48mmol) in dichloromethane (10mL), cooling to 0 deg.C, slowly adding DIPEA (0.38g, 2.9mmol) dropwise while maintaining the temperature at less than 5 deg.C, and slowly adding Tf2O (0.63g, 2.2mmol,1.5eq) dropwise while maintaining the temperature at less than 5 deg.C. After the addition, the reaction mixture is warmed to room temperature (20-25 ℃ C.). After completion of the reaction, the system was directly concentrated to dryness and purified by column chromatography (PE: EA ═ 10: 1) to obtain a solid (0.24g, 36%).

¹H NMR(400MHz,CDCl₃):4.97(s,1H),4.66-4.55(m,1H),3.20-3.13(m,1H),2.53(s,3H),2.48(d,J＝20.0Hz,1H),2.29-2.22(m,2H),2.00-1.96(m,1H),1.65-1.60(m,1H),1.43(s,9H).

And 7: synthesis of intermediates 1 to 11

Intermediates 1-9(240mg, 0.9eq), piperazine 1-10(120mg, 1.0eq), were dissolved in 4mL dioxane, followed by the addition of DIPEA (378mg), heating to 50 deg.C and stirring for 4 h. After the reaction was complete, 50mL of water was added and the reaction mixture was washed 2 times, separated, dried and concentrated to give 310mg of crude product, which was used directly in the next step.

And 8: synthesis of intermediates 1 to 12

Intermediate 1-11(1.5g, 3.70mmol) was dissolved in EA (20mL), water (10mL), sodium bicarbonate (0.93g, 11.1mmol) was added dropwise, Cbz-Cl (0.82g, 4.8mmol) was added dropwise, and the reaction was allowed to proceed at room temperature for 0.5 h. After completion of the reaction, 10mL of water was added and washed 2 times, the organic phase was separated, dried and concentrated to give a crude solid (1.70g) which was used directly in the next step.

And step 9: synthesis of intermediates 1 to 13

Intermediate 1-12(380mg, 0.67mmol) was dissolved in EA (6mL), cooled to 0 ℃ under nitrogen, 85% m-CPBA (137mg, 0.67mmol) dissolved in EA (5mL) was added dropwise over 0.5 h. After completion of the reaction, the reaction was quenched by addition of aqueous sodium thiosulfate (5mL) at 0 deg.C, separated, the organic phase dried and concentrated to give a crude yellow solid (480mg) which was used directly in the next step.

Step 10: synthesis of intermediates 1 to 15

1-14(0.10g, 1.66mmol) of alcohol,^tBuONa (0.24g, 2.49mmol) was dissolved in toluene (6mL) under nitrogen, cooled to 0 deg.C, stirred for 0.5h and intermediate 1-13(0.48g, 0.83mmol) diluted with toluene (4mL) was added dropwise. After the reaction was complete (0.5h), water (10mL) and EA (10mL) were added and the organic phase was extracted, the aqueous phase was extracted with EA (10mL), the organic phases were combined, dried, filtered, concentrated and purified to give 1-12(240mg) yellow solid.

MS m/z(ESI):632.5[M+1].

Step 11: synthesis of intermediates 1 to 16

Intermediate 1-15(0.40g, 0.6mmol) was dissolved in DCM (10mL), cooled to 0 deg.C, and 10mL of TFA (1.04g) diluted with LDCM was added dropwise slowly, rising to room temperature naturally and reacting for 5 h. After the reaction was complete, the system was concentrated to dryness and saturated NaHCO was added₃The reaction was quenched with solution, the pH was adjusted to 8, the layers were separated, the aqueous phase was extracted 3 times with EA (10mL), and the organic phases were combined and dried to give a pale yellow solid (0.35 g).

Step 12: synthesis of intermediates 1 to 18

Intermediate 1-16(210mg, 0.39mmol), methylbromonaphthalene 1-17(349mg, 1.58mmol), Cs₂CO₃(451mg，1.38mmol)、Pd₂(dba)₃(72mg, 0.08mmol) and Xantphos (92mg, 0.16mmol) were suspended in toluene (10mL), purged with nitrogen three times, and then warmed to 90 ℃ for 10 h. After the reaction is finished, Pd is removed by suction filtration₂(dba)₃And extracting the filtrate for three times by using EA (10mL), combining organic phases, drying, concentrating, and purifying by column chromatography to obtain a yellow solid 60 mg.

MS m/z(ESI):672.5[M+1].

Step 13: synthesis of intermediates 1 to 19

Compounds 1-14(60mg, 0.089mmol) were dissolved in methanol (6mL) and 7M NH₃To a MeOH solution (0.6mL) was added 10% Pd/C (30mg), and the reaction mixture was replaced with hydrogen gas 3 times and stirred at room temperature. After the reaction was completed, filtration, concentration and column chromatography were carried out to obtain intermediates 1 to 18(30mg, yellow solid).

MS m/z(ESI):538.5[M+1].

Step 14: synthesis of Compound 1

Intermediate 1-19(30mg, 0.058mmol) was dissolved in DMF (6mL) and cooled to 0 deg.C, followed by the addition of DIPEA (36mg, 0.278mmol), 2-fluoroacrylic acid (15mg, 0.167mmol), 50% PPAA (71mg, 0.11 mmol). After the reaction (6.h) at 0 ℃ is finished, the reaction mixture is transferred to room temperature for reaction for 12h, and after the reaction is finished, 5 percent NaHCO is added₃The reaction was quenched (20mL), extracted 2 times with EA (10mL), the organic phases combined, dried, filtered, concentrated, and purified by column chromatography to give compound 1(5.0mg) as a pale yellow solid.

MS m/z(ESI):6103[M+1]。

The following compounds of examples 2-6 were prepared using a synthetic route analogous to that described in example 1, substituting only a portion of the starting materials involved. The example numbers, compound designations and MS details are listed in the following table:

test example 1: compounds for KRAS G12C mutant cell Activity assay

This experiment was conducted to verify the inhibition of ERK phosphorylation in KRAS G12C mutant NCI-H358 human non-small cell lung cancer cells by the compounds of the invention.

The main reagents are as follows:

cell line NCI-H358, RPMI1640 culture medium, FBS, TrypLE^TMExpress Enzyme, PBS, 8% fixative, blocking solution, 100% methanol, rabbit anti-pERK, mouse anti-GAPDH, IRDye 800CW goat anti-rabbit IgG, IRDye680RD goat anti-mouse IgG

Main consumables and instrument:

t75 cell culture bottle, 384 cell culture micro-plate, CO₂Constant temperature incubator, Eppendorf centrifuge, Echo 550 liquid workstation, infrared laser imaging system Odyssey CLx

The experimental method comprises the following steps:

4x 10⁶NCI-H358 cells (purchased from ATCC) were inoculated into T75 flasks, cultured for 2 days with RPMI1640 (both purchased from Gibco) supplemented with 10% FBS (purchased from Transgene) and 1% penicillin streptomycin, and incubated at 37 ℃ with 5% CO₂. On day 3, the medium was decanted and washed once with DPBS. Adding 2mltrypLE^TMExpress Enzyme (from Gibco) was digested at room temperature until the cells became round. 5mL of fresh medium was added, the cells were aspirated and collected. Centrifuge at 1000rpm for 5 min. The supernatant was discarded, and the cells were resuspended in fresh medium and counted. mu.L of 6000 cells per well in 384-well plates, 37 ℃ C, 5% CO₂The culture was carried out overnight. The next day, 200nl per well of gradient diluted compound (0.5% DMSO, starting concentration 1000nm,3 fold dilution, 10 concentration points) was added with Echo 550 and incubation continued for 3 h. Add 40. mu.L of 8% fixative (from Solarbio) to each well and incubate for 20min at room temperature. Add 40 u L PBS washing once. Then 40. mu.L of 100% cold methanol was added and incubated for 10min at room temperature. Wash once with 40. mu.L PBS. Add 20. mu.L of blocking solution (from Licor) per well and incubate for 1h at room temperature. The blocking solution was aspirated and 20. mu.L of primary antibody cocktail, rabbit anti-pERK (from CST, 1:1000 dilution) and mouse anti-GAPDH (from CST, 1:2000 dilution) were added and incubated overnight at 4 ℃.40 u L each hole containing 0.05% Tween PBST washing 3 times. mu.L of secondary antibody mixture, IRDye 800CW goat anti-rabbit IgG and IRDye680RD goat anti-mouse IgG (both purchased from Li-COR, both diluted 1: 2000) was added to each well and incubated at room temperature for 45 min. Wash 3 times with 40 μ LPBST per well. The plates were centrifuged at 1000rpm for 1min and the plates were read with an Odyssey CLx.

And (3) data analysis: IC (integrated circuit)₅₀Results were analyzed by GraphPad Prism 5.0 software.

Specific test data are shown in table 1 below.

And (4) conclusion: most of the compounds of the present invention showed good binding activity.

Test example 2: evaluation of Activity of Compounds on cells

Purpose of the experiment:

this experiment was conducted to verify the inhibition of KRAS G12C mutant NCI-H358 human non-small cell lung cancer cell proliferation by the compounds of the present invention.

The main reagents are as follows:

cell line NCI-H358, RPMI1640 culture medium, FBS, TrypLE^TMExpress Enzyme, PBS, CellTiter-Glo3D Cell ViabilityAssay kit

Main consumables and instrument:

t75 cell culture bottle, 384 ultra-low adhesion cell culture round bottom micro-porous plate, CO₂Constant temperature incubator, Eppendorf centrifuge, Echo 550 liquid workstation, Envision multi-label analyzer

The experimental method comprises the following steps:

200nl of a gradient dilution of the compound (starting concentration 1000nM,3 fold dilution, 10 concentration points) was added to 384 ultra low adhesion cell culture round bottom microplates and inoculated with 40. mu.L of 800 freshly digested NCI-H358 cells in logarithmic growth phase at 37 ℃ with 5% CO₂The culture was carried out for 3 days in RPMI1640 supplemented with 10% FBS and 1% penicillin streptomycin. On day 4, 20. mu.L of CellTiter-Glo3D Cell Viability reagent (from Promega) was added, shaken at room temperature for 1h, and then read with an Envision multi-label analyzer.

And (3) data analysis: IC50 results were analyzed by GraphPad Prism 5.0 software.

Specific test data are shown in table 2 below.

And (4) conclusion: most of the compounds of the present invention showed good cell tumor inhibitory activity.

Test example 3: stability test of Compounds in Whole blood of mice

This experiment was intended to investigate the stability of the compounds of the invention in whole blood samples.

Samples (n-3) were prepared at room temperature using ICR male EDTA-K2 whole blood as a blank medium at 100 ng/mL. Stability samples were taken at predetermined time points (e.g., immediately after preparation, 1 hour, 4 hours) and centrifuged (centrifugation conditions: 1500. + -.20 g, temperature: 2 to 8 ℃ C., centrifugation 10min) to obtain plasma samples.

Adding the plasma sample into an internal standard solution, carrying out vortex for 1min, 15400g and 4 ℃, centrifuging for 10min, taking supernatant, carrying out sample injection analysis, calculating a result according to the peak area ratio of the analyte to the internal standard, comparing the ratio of the response average peak area of the stability sample at each time point with the prepared immediate sample, wherein the deviation of the measured value of the stability sample and the prepared immediate sample is not more than +/-15.0%.

The WinNonlin7.0 software non-compartmental model method (NCA) performed the calculation of the T1/2 pharmacokinetic parameters. Specific test data are shown in table 3 below.

Examples	T1/2(h)	Examples	T1/2(h)	Examples	T1/2(h)
						1	>41.5	2	>41.5	3	>41.5
4	>41.5	5	>41.5	6	>41.5
						MRTX1257	6.11	MRTX849	20	-	-

And (4) conclusion: the compounds of the invention unexpectedly showed higher half-lives in whole mouse blood compared to the disclosed comparative compounds MRTX1257 and MRTX849, indicating that the compounds of the invention have better metabolic stability.

Wherein the control compounds MRTX1257(WO2017/201161) and MRTX849 have the following structures:

the above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims (7)

1. A bridged pyrimido ring compound is characterized in that the compound is represented by the following formula (I) and an isomer thereof or a pharmaceutically acceptable salt thereof,

wherein,

R_1ais hydrogen;

R_1bselected from hydrogen, fluorine, -CH₃、-CH₂F、-CHF₂、-CF₃、-CH₂Cl、-CH₂OH、-CH₂OCH₃、-CH₂N(CH₃)₂、-CH₂N(CH₂CH₃)₂、

R₂Selected from hydrogen, halogen or-OC_1-3An alkyl group;

R_3a、R_3b、R_3cand R_3dEach independently selected from hydrogen or C_1-3An alkyl group; said C is_1-3The alkyl group may be substituted by 1 or more R₆Substitution; the R is₆Is cyano or halogen;

R₄is selected from C_1-3An alkyl group; said C is_1-3The alkyl group may be substituted by 1-N (C)_1-2Alkyl radical)₂Substitution;

R₅selected from phenyl or naphthyl; the phenyl or naphthyl group may be substituted by 1 or more R₇Substitution; the R is₇Selected from halogen, hydroxy, -CF₃、-OCF₃、-NH₂、C_1-6Alkyl or C_1-6An alkoxy group;

l is selected from-O-.

2. A bridged pyrimido ring compound according to claim 1, wherein R is_1aIs hydrogen; r_1bIs hydrogen; r₂Selected from hydrogen or fluorine; r_3aIs selected from-CH₂CN；R_3b、R_3cAnd R_3dAre all selected from hydrogen;R₄is composed of

R₅Is phenyl or naphthyl; the phenyl or naphthyl group may be substituted by 1 or more R₇Substitution; the R is₇Selected from fluorine, chlorine, bromine, -CF₃、-NH₂Methyl, ethyl, n-propyl, isopropyl or methoxy.

3. A bridged pyrimido ring compound according to claim 1, selected from the group consisting of:

4. use of a bridged pyrimido ring compound according to any one of claims 1 to 3 for the preparation of a medicament for the treatment of a cancer related to the KRASG12C mutein.

5. The use of a bridged pyrimido ring compound according to claim 4 for the preparation of a medicament for the treatment of KRASG12C mutein related cancer, wherein the KRASG12C mutein related cancer disease is selected from the group consisting of: lung cancer, colorectal cancer, pancreatic cancer, liver cancer, stomach cancer, esophageal cancer, bile duct cancer, breast cancer, ovarian cancer, cervical cancer, melanoma, brain glioma, lymphoma, and leukemia.

6. A therapeutic agent against KRASG12C mutein-related cancer, comprising as an active ingredient a therapeutically effective amount of a compound of formula (I) according to any one of claims 1 to 3 and pharmaceutically acceptable salts and isomers or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable adjuvants, wherein the KRASG12C mutein-related cancer disease is selected from the group consisting of: lung cancer, colorectal cancer, pancreatic cancer, liver cancer, stomach cancer, esophageal cancer, bile duct cancer, breast cancer, ovarian cancer, cervical cancer, melanoma, brain glioma, lymphoma, and leukemia.

7. A method of treating KRASG12C mutant protein-associated cancer, the method comprising administering to a subject an effective amount of a compound represented by formula (I) and pharmaceutically acceptable salts and isomers thereof or pharmaceutically acceptable salts thereof as an active ingredient according to any one of claims 1 to 5, wherein the KRASG12C mutant protein-associated cancer disease is selected from the group consisting of: lung cancer, colorectal cancer, pancreatic cancer, liver cancer, stomach cancer, esophageal cancer, bile duct cancer, breast cancer, ovarian cancer, cervical cancer, melanoma, brain glioma, lymphoma, and leukemia.