CN113880827A

CN113880827A - Compound for inhibiting KRASG12C mutant protein and preparation method and application thereof

Info

Publication number: CN113880827A
Application number: CN202010636659.1A
Authority: CN
Inventors: 余尚海; 李本
Original assignee: Suzhou Wentian Pharmaceutical Technology Co ltd
Current assignee: Suzhou Wentian Pharmaceutical Technology Co ltd
Priority date: 2020-07-03
Filing date: 2020-07-03
Publication date: 2022-01-04
Also published as: WO2022002018A1

Abstract

The invention discloses a compound represented by the following formula (I) and an isomer or pharmaceutically acceptable salt thereof. The compounds may be used to inhibit the KRASG12C mutein and to treat related cancers.

Description

Compound for inhibiting KRASG12C mutant protein and preparation method and application thereof

Technical Field

The invention relates to the field of drug synthesis, in particular to a compound for inhibiting KRASG12C mutant protein, and a preparation method and application thereof.

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. 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. For example, mutation of glycine at amino acid residue 12 to any other amino acid other than proline creates a steric block that prevents the GAP protein from entering KRAS, thereby inhibiting GTP hydrolysis and resulting in a significant increase in KRAS in the highly active GTP-bound form.

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 cysteine at G12C in a small pocket near the KRAS effector domain. Small molecules that bind to this pocket can 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.

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.

WO2015054572 discloses compounds of the following general formula and compound a as a comparison in the present invention, but does not disclose any examples of compounds identical or similar to the present invention.

WO2020081282 discloses compounds of the general formula and compound B as a comparison in the present invention, as well as none of the examples disclose the same compounds as in the present invention.

Compared with the compound A disclosed by WO2015054572 and the compound B disclosed by WO2020081282, the substituted piperazine compound unexpectedly and greatly improves the biological activity, improves the metabolic stability, shows good pharmacokinetic properties and has better druggability. Meanwhile, when the F substituent of R2 on the ring is replaced by amino in the above formula, the good biological activity is also unexpectedly maintained.

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 compound represented by the following formula (I) and isomers thereof or pharmaceutically acceptable salts thereof,

wherein the content of the first and second substances,

R₁is selected from-H, -CH₃、-CH₂CN；

R₂Is selected from-H, -CH₃；

R₃Is selected from-H, -CH₃；

R₄Selected from-F, -NH₂、-NHCH₃；

When R is₄When is-F, said R₁、R₂、R₃Cannot be simultaneously-H.

According to another aspect of the present invention, preferably, in the structure represented by formula (I), R₁is-CH₂CN；R₂is-H; r₃is-H; r₄Selected from-F, -NH₂。

According to another aspect of the present invention, preferably, in the structure represented by formula (I), R₁is-H; r₂is-CH₃；R₃is-H; r₄Selected from-F, -NH₂。

According to another aspect of the present invention, preferably, in the structure represented by formula (I), R₁is-H; r₂is-CH₃；R₃is-CH₃；R₄Selected from-F, -NH₂。

According to another aspect of the present invention, preferably, in the structure represented by formula (I), R₁is-CH₃；R₂is-H; r₃is-H; r₄Selected from-F, -NH₂。

Even more preferably, R₄is-F.

Still more preferably, the compound represented by formula (I) and isomers thereof or pharmaceutically acceptable salts thereof are selected from the following compounds:

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, the present invention provides a pharmaceutical composition for treating KRASG12C mutein related cancers, 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 excipients.

Preferably, according to the pharmaceutical composition, 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.

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.

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 (solvates) or crystal forms (polymorphs) as defined herein of the same acid addition salt.

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-butylOr 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 NMR 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, room temperature and is 20 ℃ to 30 ℃.

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: NCS, N-chlorosuccinimide; PdCl₂(dtbpf), dichloro [1,1' -bis (oto-tert-butylphosphino) ferrocene palladium (II); boc, tert-butoxycarbonyl; THF, tetrahydrofuran; EA, ethyl acetate; DCM, dichloromethane; DIPEA, diisopropylethylamine; DMF, N-dimethylamide; TFA, trifluoroacetic acid; h, hours; TEA, triethylamine.

Example 1: preparation of Compound 1

Compound 1 was prepared according to the following route using starting material 1-1.

Step 1: synthesis of intermediate 1-2

The starting materials 1-1(4.0g, 0.017mol), NCS (2.5g, 0.018mol) were dissolved in DMF (40ml) and reacted overnight at 70 ℃. After the reaction, the reaction mixture was poured into 100mL of ice-water and filtered with suction to obtain a solid (4.6 g).

MS m/z(ESI):269.9[M+1].

Step 2: synthesis of intermediates 1 to 3

Compound 1-2(4.6g, 0.017mol) was dissolved in ethanol (60ml), formamidine acetate (21.4g, 0.20mmol) was added, and the reaction was heated under reflux overnight. After completion of the reaction, the dry ethanol was concentrated, 20ml of water and 60ml of EA were added for liquid separation, and the concentrated organic phase was dried to obtain a crude solid (5.2g) which was used directly in the next step.

MS m/z(ESI):276.9[M+1].

And step 3: synthesis of intermediates 1 to 4

Compound 1-3(5.2g, 0.018mol) was added to SOCl₂To (30mL) was added dropwise 6 drops of DMF, and the mixture was refluxed overnight. After completion of the reaction, the dry SOCl was concentrated₂Then, saturated sodium bicarbonate and EA were added to separate the solution, and the organic phase was concentrated by drying and purified by column chromatography to obtain a yellow solid (3.0g).

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

Compound 1-4(296mg, 1.0mmol) and compound 1-5(400mg, 2.0mmol) were dissolved in dioxane (6ml), DIPEA (387mg, 3.0mmol) was added, and the mixture was heated to 50 ℃ to react. After the reaction is finished, H is added₂O (5ml) and EA (15ml), extracting to obtain an organic phase, drying and concentrating the organic phase, and purifying by column chromatography to obtain a solid (300 mg).

MS m/z(ESI):459.0[M+1].

And 5: synthesis of intermediates 1 to 8

Compounds 1-6(200mg, 0.43mmol) were dissolved in dioxane/H₂O (6ml/2ml), boric acid 1-7(163mg, 0.52mmol, see WO2020081282 synthesis) and PdCl₂(dtbpf)(28.4mg，0.04mmol,0.1eq)，K₃PO₄(139mg，0.65mmol)，N₂Heating to 90 ℃ under protection and reacting for 2 h. After the reaction was completed, 5ml of water and 15ml of EA were added, and the organic phase was extracted, dried, concentrated, and purified to obtain a solid (78 mg).

MS m/z(ESI):647.2[M+1].

Step 6: synthesis of intermediates 1 to 9

Compound 1-8(78mg) was dissolved in DCM (6ml), and 1ml TFA was added to the mixture, and the mixture was reacted at room temperature overnight, after completion of the reaction, the reaction mixture was concentrated to dryness, EA was added, the pH was adjusted to about 8 with saturated sodium bicarbonate, and the organic phase was dried and concentrated to obtain a crude product (70.0 mg).

And 7: synthesis of Compound 1

Compounds 1-9(70.0mg, 0.156mmol) were dissolved in THF/H₂To O (2ml/2ml), K was added₂CO₃(95.3mg，0.69mmol)，N₂Cooled to 0 ℃ under protection, acryloyl chloride (14.2mg, 0.156mmol) was slowly added dropwise, and the reaction was continued for 10min at 0 ℃ after the addition. After the reaction is finished, addingAdding water, extracting with EA, drying the organic phase, concentrating, and purifying by column chromatography to obtain white solid (30.0 mg).

¹H NMR(400MHz,CDCl₃):8.80(s,1H),7.77(s,1H),7.24-7.22(m,1H),7.02-6.97(m,1H),6.65-6.56(m,1H),6.40-6.37(m,1H),5.80-5.76(m,3H),4.76-4.65(m,1H),4.20-4.04(m,2H),3.73-3.46(m,4H),1.42-1.40(m,3H).

MS m/z(ESI):501.1[M+1].

The following compounds of examples 2-4 were prepared using a synthetic route analogous to that described in example 1, substituting only the starting piperazine 1-5 involved. Example numbers, compound designations and structural characterizations are listed in the following table:

example 5: preparation of Compound 5

Compound 1 was prepared according to the following route using starting materials 5-6.

Step 1: synthesis of intermediates 5 to 7

Compound 5-6(500mg, 1.12mmol) was dissolved in DMSO (10ml), and added to 25% aqueous ammonia (160mg) in a sealed tube and heated to 90 ℃ for reaction overnight. After the reaction was completed, 5ml of water and 15ml of EA were added, and the organic phase was extracted, dried, concentrated, and purified to obtain intermediate 5-7(105 mg).

¹H NMR(400MHz,CDCl₃):8.62(s,1H),7.26(s,1H),5.57(brs,2H),3.70-3.64(m,8H),1.50(s,3H)；MS m/z(ESI):442.1[M+1].

Step 2: synthesis of intermediates 5 to 8

Dissolve compound 5-7(55mg, 0.12mmol) in dioxane/H₂O(3ml/1ml), boric acid 1-7(60mg, 0.19mmol) and PdCl are added₂(dtbpf)(8.2mg，0.012mmol)，K₃PO₄(40mg，0.18mmol)，N₂Heating to 90 ℃ under protection and reacting for 2 h. After the reaction was completed, 5ml of water and 15ml of EA were added, and the organic phase was extracted, dried, concentrated, and purified to obtain intermediate 5-8(17 mg).

MS m/z(ESI):630.3[M+1].

And step 3: synthesis of intermediates 5 to 9

Compound 5-8(30mg) was dissolved in DCM (2ml), 0.5ml TFA was added, the reaction was allowed to proceed overnight at room temperature, after completion of the reaction, the reaction mixture was concentrated to dryness, EA was added, the pH was adjusted to about 8 with saturated sodium bicarbonate, and the organic phase was dried and concentrated to obtain a crude product (28 mg).

And 4, step 4: synthesis of Compound 5

Compound 5-9(28mg, 0.069mmol) was dissolved in THF/H₂To O (2ml/2ml), K was added₂CO₃(42.5mg，3.08mmol)，N₂Cooled to 0 ℃ under protection, acryloyl chloride (6.9mg, 0.083mmol) is slowly added dropwise, and the reaction is continued for 10min at 0 ℃ after the addition. After the reaction, water was added, and EA was used for extraction, and the organic phase was dried, concentrated, and purified by column chromatography to obtain a white solid (12.5 mg).

MS m/z(ESI):484.1[M+1].

The following compounds of examples 6-8 were prepared using a synthetic route analogous to that described in example 2, substituting only a portion of the starting materials involved. Example numbers, compound designations and structural characterizations are listed in the following table:

test example 1: evaluation of Compounds for modification of KRAS-G12C

Purpose of the experiment:

this example illustrates covalent binding of an exemplary compound of the invention to KRAS G12C and determination of the covalent adduct of a compound of the invention with KRAS G12C using LCMS

The experimental steps are as follows:

1. loading GDP onto KRAS-4B-G12C protein

1) KRAS-4B-G12C protein was diluted one-fold with low magnesium buffer to a concentration of 103 uM.

2) To 1mL of 103uM KRAS-4B-G12C protein was added 1mL of 2X GDP loading buffer, and gently and slowly mixed.

3) 2mL of the mixture was reacted at room temperature for 1.5 h.

4) Subpackaging into 100 uL/tube, quickly freezing with liquid nitrogen, and storing at-80 deg.C.

2. Covalent modification analysis of KRAS-4B-G12C protein by small molecule compound

1) KRAS-4B-G12C protein was diluted with 10 Xreaction buffer to prepare a reaction system as shown in the following table:

2) the reaction was carried out at room temperature for 3min and 30min, respectively.

3) The reaction was stopped by adding 5uL of 5% formic acid.

3. Mass spectrometric detection

A total of 55uL of the reaction was centrifuged at 15000rpm for 10min before mass spectrometric detection.

4. The coupling ratio to Kras-G12C was calculated

Coupling ratio (%). protein-compound complex peak area/(complex peak area + monomer protein peak area). 100.

The mass of each protein was calculated as a percentage of the total signal of that protein, which was then normalized to the signal of the protein without the addition of reactive compound. These normalized signals are reported as normalized control Percentage (POC). An increase in POC values indicates that the compound shows a higher degree of modification of KRASG 12C. The results of testing the exemplary compounds of formula (I) at a concentration of 3. mu.M for 30 minutes are shown in Table 2.

Specific test data are shown in table 1 below.

Compound (I)

POC

Compound (I)

POC

Compound (I)

POC

Compound (I)

POC

Compound (I)

POC

1

89.8％

2

75.5％

3

71.5％

4

60.5％

B

55.2％

And (4) conclusion: the compounds of the invention all showed better KRASG12C covalent binding capacity than control compound B.

Test example 2: 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:

4 x 10⁶NCI-H358 cells (purchased from ATCC) were inoculated into T75 flasks, cultured for 2 days at 37 degrees, 5% CO with RPMI1640 (both purchased from Gibco) supplemented with 10% FBS (purchased from Transgene) and 1% penicillin streptomycin₂. On day 3, the medium was decanted and washed once with DPBS. Adding 2ml TrypLE^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 minutes. 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 ℃ in 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 was continued for 3 hours. Add 40. mu.l of 8% fixative (from Solarbio) to each well and incubate for 20 min at room temperature. Add 40 u l PBS washing once. Then 40. mu.l of 100% cold methanol was added and incubated for 10 minutes at room temperature. Wash once with 40. mu.l PBS. Mu.l of blocking solution (purchased from Licor) was added to each well and incubated for 1 hour at room temperature. The blocking solution was aspirated and 20 μ l of primary antibody cocktail, rabbit anti-pERK (purchased from CST, 1:1000 dilution) and mouse anti-GAPDH (purchased from CST, 1:2000 dilution), 4 degrees overnight. 40 μ l each well of PBST containing 0.05% Tween was washed 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) were added to each well and incubated for 45 minutes at room temperature. Add 40. mu.l PBST per well and wash 3 times. The plates were centrifuged at 1000rpm for 1 minute 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 2 below.

And (4) conclusion: the compounds of the present invention all showed stronger inhibitory activity than control compound a and control compound B.

Test example 3: evaluation of activity of compounds on cells for experimental purposes:

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-Glo 3D Cell Viability Assay kit main consumables and instruments:

t75 cell culture bottle, 384 ultra-low adhesion cell culture round bottom micro-porous plate, CO₂The experimental method comprises the following steps of (1) a constant-temperature incubator, an Eppendorf centrifuge, an Echo 550 liquid workstation and an Envision multi-label analyzer:

200nl of a gradient dilution compound (starting concentration 1000nM,3 fold dilution, 10 concentration points) was added to 384 ultra low adhesion cell culture round bottom microplates and 40. mu.l of 800 freshly digested NCI-H358 cells in logarithmic growth phase, 37 ℃ 5% CO was seeded₂The 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-Glo 3D Cell Viability reagent (from Promega) was added, shaken at room temperature for 1 hour, 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 3 below.

And (4) conclusion: most of the compounds of the present invention unexpectedly show stronger cellular tumor inhibiting activity than control compound a and control compound B.

Test example 4: stability test of Compounds in Whole blood of mice

This experiment was conducted to examine the stability of the compounds of the present invention in whole blood samples from mice.

Samples (n-3) were prepared at room temperature using ICR male EDTA-K2 whole blood as a blank medium at 100 ng/mL. A stability sample is taken out at a predetermined time point (for example, immediately after preparation, 1 hour), and centrifuged (centrifugation conditions: 1500. + -.20 g, temperature: 2 to 8 ℃ C., centrifugation 10min) to obtain a plasma sample.

Adding the plasma sample into an internal standard solution, performing vortex for about 1min, 15400g and 4 ℃, centrifuging for 10min, taking supernate, performing sample injection analysis, calculating a result according to the peak area ratio of an 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 1h stability results are shown in table 4 below.

Compound (I)

Deviation of

Compound (I)

Deviation of

Compound (I)

Deviation of

Compound (I)

Deviation of

Compound (I)

Deviation of

1

-1.33％

2

-12.8％

3

+14.0％

4

+9.4％

B

-27.1％

And (4) conclusion: the compounds of the invention unexpectedly show better stability in whole mouse blood compared to the disclosed comparative compound B.

Test example 5: stability test of Compounds in human Whole blood

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

Samples (n-3) were prepared at room temperature using human whole blood as a blank medium at a concentration of 100 ng/mL. A stability sample is taken out at a predetermined time point (for example, immediately after preparation, 1 hour), and centrifuged (centrifugation conditions: 1500. + -.20 g, temperature: 2 to 8 ℃ C., centrifugation 10min) to obtain a plasma sample.

Adding the plasma sample into an internal standard solution, performing vortex for about 1min, 15400g and 4 ℃, centrifuging for 10min, taking supernate, performing sample injection analysis, calculating a result according to the peak area ratio of an 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 1h stability results are shown in table 5 below.

Compound (I)	Deviation of	Compound (I)	Deviation of	Compound (I)	Deviation of
						3	+3.2％	4	+5.8％	B	-35.4％

And (4) conclusion: the compounds of the present invention unexpectedly show better stability in human whole blood compared to the disclosed comparative compound B.

Test example 6: evaluation of pharmacokinetics:

the drug concentrations in plasma of mice at different times after intragastric administration of compound 1, compound 3 and compound B were tested using the mice as test animals. The pharmacokinetic behavior of the compound of the invention in mice is studied, and the drug metabolism characteristics are evaluated. In each group of examples, 9 mice of similar body weight were used, and the oral administration dose was 10mg/kg, and the administration was a single dose. Blood was collected at 15min, 30min, 1h, 2h, 4h, 6h, 8h, 12h, 24h time points after administration to the animals (2 time point samples per mouse for 3 time points). The content of the compound in the plasma is detected by adopting an LC-MS/MS analysis method, and the lower limit of the quantification of the method is 20 ng/mL. The plasma concentration data were statistically analyzed using the pharmacokinetic data analysis software WinNonlin 7.0, and the pharmacokinetic parameters were calculated using the non-compartmental model (NCA), as shown in table 2 below.

The experimental scheme is as follows:

experimental drugs: compound 1, compound 3, compound B.

The preparation of the medicine comprises the following steps: a certain amount of the medicine is taken, and 2% Klucel LF + 0.1% Tween 80 aqueous solution is added to prepare a clear solution or a uniform suspension.

Administration: the mice were fasted overnight and then gavaged at a dose of 10 mg/kg.

The operation is as follows: the mouse is administrated by gastric lavage, tail vein blood is collected before administration and 15min, 30min, 1h, 2h, 4h, 6h, 8h, 12h and 24h after administration, the blood plasma is separated by centrifugation for 10 minutes at 3500 rpm at 4 ℃ in a heparinized sample tube, the blood plasma is stored at-20 ℃, and the mouse is taken 2 hours after administration.

Determining the content of the compound to be tested in the plasma of the mouse after the drug with different concentrations is administered by gavage: after thawing the plasma samples at room temperature, respectively taking 50 mu L, adding 130 mu L of internal standard working solution (1000ng/mL, acetonitrile, tolbutamide), vortexing for about 1min, and centrifuging for 10min at 4 ℃ and 13000 rpm. mu.L of the supernatant was mixed with 100. mu.L of 50% acetonitrile water and analyzed by LC/MS/MS.

The pharmacokinetic parameter results are shown in table 6.

Table 4: drug metabolism data in mice

And (4) conclusion: the compound of the invention has good drug absorption, especially the compound 3, compared with the control compound B, not only greatly prolongs the half-life period, but also greatly improves the drug exposure in blood.

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

1. A compound represented by the following formula (I) and isomers thereof or pharmaceutically acceptable salts thereof,

wherein the content of the first and second substances,

R₁is selected from-H, -CH₃、-CH₂CN；

R₂Is selected from-H, -CH₃；

R₃Is selected from-H, -CH₃；

R₄Selected from-F, -NH₂、-NHCH₃；

When R is₄When is-F, said R₁、R₂、R₃Cannot be simultaneously-H.

2. The compound of formula (I) and isomers or pharmaceutically acceptable salts thereof according to claim 1, wherein R is₁is-CH₂CN；R₂is-H; r₃is-H; r₄Selected from-F, -NH₂。

3. The compound of formula (I) and isomers or pharmaceutically acceptable salts thereof according to claim 1, wherein R is₁is-H; r₂is-CH₃；R₃is-H; r₄Selected from-F, -NH₂。

4. The compound of formula (I) and isomers or pharmaceutically acceptable salts thereof according to claim 1, wherein R is₁is-H; r₂is-CH₃；R₃is-CH₃；R₄Selected from-F, -NH₂。

5. The compound of formula (I) and isomers or pharmaceutically acceptable salts thereof according to claim 1, wherein R is₁is-CH₃；R₂is-H; r₃is-H; r₄Selected from-F, -NH₂。

6. The compound represented by the formula (I) and isomers thereof or pharmaceutically acceptable salts thereof according to any one of claims 1 to 5, wherein R is₄is-F.

7. The compound represented by the formula (I) and isomers thereof or pharmaceutically acceptable salts thereof according to claim 1, wherein the compound represented by the formula (I) and isomers thereof or pharmaceutically acceptable salts thereof is selected from the following compounds:

8. use of a compound represented by formula (I) according to any one of claims 1 to 7, and isomers thereof or pharmaceutically acceptable salts thereof, for the manufacture of a medicament for the treatment of KRASG12C mutein-related cancer 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.

9. A pharmaceutical composition for treating KRASG12C mutein-related cancer comprising a therapeutically effective amount of a compound represented by formula (I) according to any one of claims 1 to 7 and pharmaceutically acceptable salts and isomers or pharmaceutically acceptable salts thereof as an active ingredient, together with pharmaceutically acceptable excipients, said KRASG12C mutein-related cancer disease being 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.

10. 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 according to any one of claims 1 to 7 as an active ingredient in a pharmaceutical composition, 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.