WO2022002018A1 - 一种用于抑制krasg12c突变蛋白的化合物及其制备方法和用途 - Google Patents

一种用于抑制krasg12c突变蛋白的化合物及其制备方法和用途 Download PDF

Info

Publication number
WO2022002018A1
WO2022002018A1 PCT/CN2021/102995 CN2021102995W WO2022002018A1 WO 2022002018 A1 WO2022002018 A1 WO 2022002018A1 CN 2021102995 W CN2021102995 W CN 2021102995W WO 2022002018 A1 WO2022002018 A1 WO 2022002018A1
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
pharmaceutically acceptable
compound
formula
compound represented
Prior art date
Application number
PCT/CN2021/102995
Other languages
English (en)
French (fr)
Inventor
余尚海
李本
Original Assignee
苏州闻天医药科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 苏州闻天医药科技有限公司 filed Critical 苏州闻天医药科技有限公司
Publication of WO2022002018A1 publication Critical patent/WO2022002018A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

  • the present invention relates to the field of drug synthesis, in particular, to a compound that can be used to inhibit KRASG12C mutant protein and a preparation method and use thereof.
  • RAS mutations are frequently observed in malignancies and support various hallmarks of cancer, including genomic instability, cell proliferation, inhibition of apoptosis, metabolic reprogramming, changes in the microenvironment, evasion of immune responses, and promotion of metastasis . Consistent with its pervasive effects on cancer cell function, regression of oncogenic KRAS in many established tumor models results in tumor regression. Therefore, RAS is a potentially very effective target for cancer therapy. There appear to be multiple functional classes of RAS mutations, and strategies may need to be developed for each functional class.
  • RAS family members oncogenic mutations were most frequently found in KRAS (85%), whereas NRAS (12%) and HRAS (3%) were less common. Most RAS family mutations occur at amino acid residues 12, 13, and 61, which mostly interact directly with GTP in the three-dimensional conformation. For example, mutation of glycine at amino acid residue 12 to any other amino acid other than proline creates a steric block that prevents GAP protein from entering KRAS, thereby inhibiting GTP hydrolysis and leading to a significant increase in the highly active GTP-bound form of KRAS.
  • KRAS G12C is the most common RAS mutation in non-small cell lung cancer and is the leading cause of cancer death in the United States, for which there is still no direct and effective clinical drug. But in recent years, there has been significant progress in this field.
  • WO2015054572 discloses the compound of the following general formula and the compound A as a comparison in the present invention, but no example discloses the same or similar compound with the present invention.
  • WO2020081282 discloses the compound of the following general formula and the compound B used as a comparison in the present invention, and there is also no example that discloses the same compound as the present invention.
  • the present invention provides a class of substituted piperazine compounds. Compared with Compound A disclosed in WO2015054572 and Compound B disclosed in WO2020081282, the compound has unexpectedly greatly improved biological activity and improved metabolic stability. Good pharmacokinetic properties, with better drug properties. At the same time, when the F substituent of R2 in the above general formula is replaced with an amino group, it also unexpectedly maintains a good biological activity.
  • the present invention adopts the following technical solutions:
  • the present invention provides a compound represented by the following formula (I) and an isomer thereof or a pharmaceutically acceptable salt thereof,
  • R 1 is selected from -H, -CH 3 , -CH 2 CN;
  • R 2 is selected from -H, -CH 3 ;
  • R 3 is selected from -H, -CH 3 ;
  • R 4 is selected from -F, -NH 2 , -NHCH 3 ;
  • R 1 is -CH 2 CN; R 2 is -H; R 3 is -H; R 4 is selected from -F, - NH 2.
  • R 1 is -H;
  • R 2 is -CH 3 ;
  • R 3 is -H;
  • R 4 is selected from -F, -NH 2 .
  • R 1 is -H; R 2 is -CH 3 ; R 3 is -CH 3 ; R 4 is selected from -F, - NH 2.
  • R 1 is -CH 3 ;
  • R 2 is -H;
  • R 3 is -H;
  • R 4 is selected from -F, -NH 2 .
  • R 4 is -F.
  • the compound represented by formula (I) and its isomers or pharmaceutically acceptable salts thereof are selected from the following compounds:
  • the present invention provides a compound represented by formula (I) and a pharmaceutically acceptable salt thereof and an isomer thereof or a pharmaceutically acceptable salt thereof in the manufacture of a KRASG12C mutant protein-related cancer therapy Use in medicine.
  • the KRASG12C mutant protein-related cancer disease is selected from the group consisting of: lung cancer, colorectal cancer, pancreatic cancer, pancreatic cancer, liver cancer, gastric cancer, esophageal cancer, bile duct cancer, breast cancer, ovarian cancer, cervical cancer , melanoma, glioma, lymphoma, leukemia.
  • the present invention provides a pharmaceutical composition for treating KRASG12C mutant protein-related cancer, the pharmaceutical composition comprising a therapeutically effective amount of the compound represented by formula (I) according to the present invention and Its pharmaceutically acceptable salts and its isomers or its pharmaceutically acceptable salts are used as active ingredients, and pharmaceutically acceptable auxiliary materials.
  • the KRASG12C mutant protein-related cancer disease is selected from the group consisting of: lung cancer, colorectal cancer, pancreatic cancer, pancreatic cancer, liver cancer, gastric cancer, esophageal cancer, bile duct cancer, breast cancer, ovarian cancer, Cervical cancer, melanoma, glioma, lymphoma, leukemia.
  • the present invention provides a method of treatment of KRASG12C mutant protein-related cancer, the method of treatment comprising administering to a subject an effective amount of the compound according to the present invention or comprising the compound and the same Pharmaceutical compositions with a pharmaceutically acceptable salt as the active ingredient.
  • the cancer disease is selected from the group consisting of: lung cancer, colorectal cancer, pancreatic cancer, pancreatic cancer, liver cancer, gastric cancer, esophageal cancer, bile duct cancer, breast cancer cancer, ovarian cancer, cervical cancer, melanoma, glioma, lymphoma, leukemia.
  • salt refers to a compound containing a cation and an anion, which can be produced by protonation of proton-accepting sites and/or deprotonation of proton-accepting sites.
  • protonation of proton-accepting sites results in the formation of cationic species, the charge of which is balanced by the presence of physiological anions, while deprotonation of proton-available sites results in the formation of anionic species, whose charges are balanced by the presence of physiological cations And balance.
  • pharmaceutically acceptable salt means that the salt is pharmaceutically acceptable.
  • 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, etc.; or formed 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, ethyl acetate Sulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-p-toluenesulfonic acid, camphoric acid
  • C1 -M alkyl refers to an alkyl group containing 1-M carbon atoms, eg, wherein M is an integer having the following values: 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.
  • C1-6 alkyl refers to an alkyl group containing 1-6 carbon atoms.
  • 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.
  • aryl refers to an aromatic system, which may be a single ring or multiple aromatic rings that are originally 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 group which may be optionally substituted, such a group may be substituted with 1-4 substituents selected from the group consisting of an aryl or heterocyclic group: halo, -CN, -OH, -NO 2, amino, alkyl, cycloalkyl group, alkenyl, alkynyl, alkoxy, aryloxy, substituted alkoxy, alkylcarbonyl, alkylcarboxy, alkylamino, or arylthio.
  • substituted means that the referenced group may be replaced by one or more additional groups, individually and independently selected from the group consisting of, alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic , hydroxyl, alkoxy, alkylthio, arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone, arylsulfone, cyano, halogen, carbonyl, thiocarbonyl, nitro, haloalkane radicals, fluoroalkyl and amino groups, including mono- and di-substituted amino groups and their protected derivatives.
  • the compound represented by formula (I) or a pharmaceutically acceptable salt thereof provided by the present invention can be in various forms, such as tablet, capsule, powder, syrup, solution, suspension Liquids and aerosols, etc., and may be present in a suitable solid or liquid carrier or diluent and in a suitable sterile device for injection or instillation.
  • the unit dose of the formulation contains 0.05-200 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof, preferably, the unit dose of the formulation contains 0.1 mg-100 mg of the compound of formula (I).
  • the compounds and pharmaceutical compositions represented by the general formula (I) of the present invention can be clinically used for mammals, including humans and animals, and can be administered through oral, nasal, skin, lung, or gastrointestinal tract or the like. Most preferred is oral administration.
  • the most preferred daily dose is 0.01-200 mg/kg body weight, taken in one time, or 0.01-100 mg/kg body weight in divided doses. Regardless of the method of administration, the optimal dose for an individual will depend on the specific treatment. It is common to start with a small dose and gradually increase the dose until the most suitable dose is found.
  • the term "effective amount" may refer to an effective amount in a dose and for a period of time required to achieve the desired effect. This effective amount may vary depending on factors such as the type of disease or condition of the disease at the time of treatment, the architecture of the particular target organ being administered, the size of the individual patient, or the severity of the disease or symptom.
  • One of ordinary skill in the art can empirically determine the effective amount of a particular compound without undue experimentation.
  • a typical formulation is prepared by mixing the compound represented by the general formula (I) of the present invention and a carrier, diluent or excipient.
  • Suitable carriers, diluents or excipients are well known to those skilled in the art and include, for example, carbohydrates, waxes, water-soluble and/or swellable polymers, hydrophilic or hydrophobic substances, gelatin, oils, solvents , water and other substances.
  • solvents are generally selected on the basis of solvents considered by those skilled in the art to be safe and effective for administration to mammals.
  • safe solvents are non-toxic aqueous solvents such as water, and 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 (eg, 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, Coloring agents, sweetening agents, flavoring agents, flavoring agents, or other known additives to bring the drug into an acceptable form for manufacture or use.
  • the two or more drugs can be used separately or in combination, preferably in the form of a pharmaceutical composition.
  • the compounds or pharmaceutical compositions of formula (I) of the present invention can be administered separately or together in any of the known forms of oral, intravenous, rectal, vaginal, transdermal, other topical or systemic administration drug to subjects.
  • compositions may also contain one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifying agents, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids agents, coloring agents, sweetening agents, flavoring agents, flavoring agents, or other known additives to bring the pharmaceutical compositions into an acceptable form for manufacture or use.
  • Solid dosage forms for oral administration may include capsules, tablets, powders or granules.
  • the compounds or pharmaceutical compositions of the present invention are admixed with at least one inert excipient, diluent, or carrier.
  • Suitable excipients, diluents or carriers include substances such as sodium citrate or dicalcium phosphate, or starch, lactose, sucrose, mannitol, silicic acid, etc.; binders such as carboxymethyl cellulose, alginic acid Salt, gelatin, polyvinylpyrrolidone, sucrose, gum arabic, etc.; wetting agents such as glycerol, etc.; disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, specific complex silicates, sodium carbonate, etc.; Solution blockers such as paraffin, etc.; absorption enhancers such as quaternary ammonium compounds, etc.; adsorbents such as kaolin, bentonite, etc.; lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate Wait.
  • the dosage form may also include substances such as sodium cit
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs.
  • 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-butanediol, dimethylformamide; oils (such as cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, sesame oil etc.); glycerin; tetrahydrofurfuryl alcohol; fatty acid esters of polyethylene glycol and sorbitan; or mixtures of several of these substances, etc.
  • inert diluents commonly used in the art, such as water or other solvents
  • solubilizers and emulsifiers
  • compositions may also include excipients such as one or more of wetting agents, emulsifying agents, suspending agents, sweetening, flavoring, and perfuming agents.
  • excipients such as one or more of wetting agents, emulsifying agents, suspending agents, sweetening, flavoring, and perfuming agents.
  • a carrier such as a suspending agent such as ethoxyl may be further contained Polyisostearyl alcohol, polyoxyethylene sorbitol, sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, or a mixture of several of these substances, etc.
  • the compound represented by general formula (I) or a pharmaceutically acceptable salt thereof of the present invention or a pharmaceutical composition comprising the same can be administered in other topical dosage forms, including creams, powders, sprays and inhalants.
  • the drug may be mixed under sterile conditions with a pharmaceutically acceptable excipient, diluent or carrier and any required preservatives, buffers or propellants.
  • Ophthalmic formulations, ophthalmic ointments, powders and solutions are also intended to be encompassed within the scope of the present invention.
  • kits eg., pharmaceutical packaging are also encompassed by the present disclosure.
  • Kits are provided that can contain a pharmaceutical composition or compound described herein and a container (e.g., a vial, ampule, bottle, syringe, and/or subpack or other suitable container).
  • a container e.g., a vial, ampule, bottle, syringe, and/or subpack or other suitable container.
  • kits can optionally further comprise a second container comprising a pharmaceutically acceptable excipient for diluting or suspending a pharmaceutical composition or compound described herein.
  • the pharmaceutical compositions or compounds described herein disposed in the first container and the second container are combined to form one unit dosage form.
  • kits described herein further comprise instructions for using the compounds or pharmaceutical compositions contained in the kits.
  • the kits described herein may also include information required by regulatory agencies such as the US Food and Drug Administration (FDA).
  • FDA US Food and Drug Administration
  • the information included in the kit is prescribing information.
  • the kits and instructions provide for treating and/or preventing a proliferative disease in a subject in need thereof.
  • the kits described herein may contain one or more additional pharmaceutical formulations as separate compositions.
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • MS was measured with a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).
  • the thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate, the size of the silica gel plate used for thin layer chromatography (TLC) is 0.15mm-0.2mm, and the size of the TLC separation and purification products is 0.4mm -0.5mm.
  • the temperature of the reaction is room temperature, which is 20 degrees Celsius to 30 degrees Celsius.
  • the detection of the reaction progress in the embodiment adopts thin layer chromatography (TLC), the used developing solvent system, and the eluent system of the column chromatography used for purifying the compound include: 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.
  • TLC thin layer chromatography
  • NCS N-chlorosuccinimide
  • PdCl 2 dtbpf
  • Boc tert-Butoxycarbonyl
  • THF tetrahydrofuran
  • EA ethyl acetate
  • DCM dichloromethane
  • DIPEA diisopropylethylamine
  • DMF N,N-dimethylamide
  • TFA trifluoroacetic acid
  • h hour
  • TEA triethylamine.
  • Compound 1 was prepared using starting material 1-1 according to the following route.
  • Example numbers, compound designations and structural characterizations are listed in the following table:
  • Compound 1 was prepared using starting materials 5-6 according to the following routes.
  • Example numbers, compound designations and structural characterizations are listed in the following table:
  • the KRAS-4B-G12C protein was diluted one-fold with low magnesium buffer to a concentration of 103uM.
  • a total of 55 uL of the reaction was centrifuged at 15000 rpm for 10 min before mass spectrometry detection.
  • Coupling ratio (%) protein-compound complex peak area/(complex peak area+monomeric protein peak area)*100.
  • Test Example 2 Compound activity assay on KRAS G12C mutant cells
  • the purpose of this experiment is to verify the inhibition of ERK phosphorylation in KRAS G12C-mutated NCI-H358 human non-small cell lung cancer cells by the compounds of the present invention.
  • NCI-H358 cells purchased from ATCC
  • RPMI 1640 both purchased from Gibco
  • FBS penicillin-streptomycin
  • 37 degrees, 5% CO 2 the medium was discarded and washed once with DPBS.
  • 2 ml of TrypLE TM Express Enzyme purchased from Gibco was added to digest at room temperature until the cells became round. Add 5 ml of fresh medium, pipet and collect cells. Centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, resuspend the cells in fresh medium and count.
  • 200nl of each well of serially diluted compounds (0.5% DMSO, initial concentration of 1000nm, 3-fold dilution, 10 concentration points in total) were added with Echo 550, and the incubation was continued for 3 hours.
  • 40 ⁇ l of 8% fixative solution purchased from Solarbio
  • 40 ⁇ l of 100% cold methanol was added and incubated for 10 minutes at room temperature. Wash once with 40 ⁇ l PBS.
  • Test Example 3 Evaluation of the activity of compounds on cells
  • the purpose of this experiment is to verify that the compounds of the present invention inhibit the proliferation of KRAS G12C-mutated NCI-H358 human non-small cell lung cancer cells.
  • Test Example 4 Stability test of compound in mouse whole blood
  • Test Example 5 Stability test of compound in human whole blood
  • mice Using mice as the test animals, the drug concentrations in plasma at different times after the mice were given compound 1, compound 3 and compound B by gavage were tested.
  • Each group of examples selected 9 mice with similar body weight, and the oral administration dose was 10 mg/kg, single administration. Blood was collected at 15min, 30min, 1h, 2h, 4h, 6h, 8h, 12h, and 24h after the animals were administered (each mouse was sampled at 2 time points, a total of 3 time points).
  • the LC-MS/MS analysis method was used to detect the content of the compounds in plasma, and the lower limit of quantification of the method was 20ng/mL.
  • Metabolic kinetic data analysis software WinNonlin 7.0 was used to perform statistics on plasma concentration data, and non-compartmental model (NCA) was used to calculate pharmacokinetic parameters, as shown in Table 2 below.
  • Drug preparation Take a certain amount of drug, add 2% Klucel LF + 0.1% Tween 80 aqueous solution, and prepare a clear solution or uniform suspension.
  • mice were fasted overnight and then administered by gavage at a dose of 10 mg/kg.
  • mice were given intragastric administration, and blood was collected from the tail vein before administration and 15min, 30min, 1h, 2h, 4h, 6h, 8h, 12h, and 24h after administration, and placed in a heparinized sample tube, 4 degrees Celsius, 3500 Plasma was separated by centrifugation at rpm for 10 minutes, stored at -20°C, and fed 2 hours after administration.
  • Determination of the content of the test compound in mouse plasma after intragastric administration of different concentrations of drugs 50 ⁇ L of plasma samples were thawed at room temperature, and 130 ⁇ L of internal standard working solution (1000ng/mL, acetonitrile, tolbutamide) was added, and vortexed. After about 1 min, centrifuge for 10 min at 4°C and 13000 rpm. 50 ⁇ L of supernatant was mixed with 100 ⁇ L of 50% acetonitrile water and injected into LC/MS/MS for analysis.
  • internal standard working solution 1000ng/mL, acetonitrile, tolbutamide
  • the compounds of the present invention have good pharmacokinetic absorption, especially compound 3, compared with the control compound B, not only the half-life is greatly prolonged, but also the drug exposure in blood is greatly improved.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oncology (AREA)
  • Hematology (AREA)
  • Epidemiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

本发明公开了一种式(I)表示的化合物及其异构体或药学上可接受的盐。所述化合物可以用于抑制KRASG12C突变蛋白和治疗相关的癌症。

Description

一种用于抑制KRASG12C突变蛋白的化合物及其制备方法和用途 技术领域
本发明涉及药物合成领域，具体而言，涉及一种可以用于抑制KRASG12C突变蛋白的化合物及其制备方法和用途。
背景技术
RAS突变经常在恶性肿瘤中观察到并且支持癌症的各种标志，包括基因组不稳定性，细胞增殖，细胞凋亡的抑制，代谢的重编程，微环境的改变，免疫应答的逃避和转移的促进。与其对癌细胞功能的普遍影响一致，许多已建立的肿瘤模型中致癌KRAS的消退导致肿瘤消退。因此，RAS是一个潜在的非常有效的癌症治疗靶点。RAS突变看起来有多种功能类别，可能需要针对每个功能类别制定策略。
在RAS家族成员中，致癌突变最常见于KRAS(85％)，而NRAS(12％)和HRAS(3％)则较为少见。大多数RAS家族突变发生在氨基酸残基12、13和61处，在三维空间构象中，这些氨基酸残基多与GTP有直接相互作用。比如，氨基酸残基12处的甘氨酸突变为除脯氨酸以外的任何其它氨基酸均会产生空间阻断，阻止GAP蛋白进入KRAS，从而抑制GTP水解并导致高活性的GTP结合形式的KRAS显著增加。
变异的KRAS占肺癌的30％。97％的KRAS变异发生在外显子2和3，包括氨基酸G12(39％),G13和Q61。KRAS G12C是非小细胞肺癌中最常见的RAS突变，是美国癌症死亡的主要原因，临床上仍然没有直接和有效的药物。但近年该领域开始有了显著的进展。
2013年，Shokat等在《Nature》报道了突破性的结果，他们筛选设计的活性小分子能够在G12C处不可逆地与突变体半胱氨酸结合到KRAS效应区附近的一个小口袋。结合到这个口袋的小分子可以通过将蛋白质锁定在GDP结合的非活性状态来抑制KRAS活性。
2016年，Wellspring公司在《癌症发现》上报道了靶向KRAS G12C小分子抑制剂ARS-853。虽然活性仍在微摩尔级，但完成了细胞及动物试验的概念验证。2018年，该公司又在《细胞》上公开了新一代靶向KRAS G12C小分子抑制剂ARS-1620。
通过抑制KRAS突变蛋白，从而抑制癌症细胞的生长，进而治疗肺癌、胰腺癌、结直肠癌等癌症看起来是一个很有希望的治疗手段。因此，该领域存在潜在的远未被满足的巨大药物需求。
WO2015054572公开了如下通式化合物及在本发明中作为对比的化合物A，但没有任何实施例公开与本发明相同或相近的化合物。
Figure PCTCN2021102995-appb-000001
WO2020081282公开了如下通式化合物及在本发明中作为对比的化合物B，同样也没有任何实施例公开与本发明相同的化合物。
Figure PCTCN2021102995-appb-000002
本发明提供了一类取代的哌嗪化合物，该类化合物与WO2015054572公开的化合物A及WO2020081282公开的化合物B相比，出乎意料的大大提高了生物活性，同时提高了代谢稳定性，也显示了良好的药代性质，具有更好的成药性质。同时，当以上通式中并环上R2的F取代基用氨基替换后，也不可预料地保持了很好的生物活性。
发明内容
为解决以上技术问题，本发明采取如下技术方案：
根据本发明的一个方面，本发明提供了一种以下式(I)表示的化合物及其异构体或其药学上可接受的盐，
Figure PCTCN2021102995-appb-000003
其中，
R 1选自-H、-CH 3、-CH 2CN；
R 2选自-H、-CH 3；
R 3选自-H、-CH 3；
R 4选自-F、-NH 2、-NHCH 3；
当R 4为-F时，所述R 1、R 2、R 3不能同时为-H。
根据本发明的另一个方面，优选地，在式(I)所示的结构中，R 1为-CH 2CN；R 2为-H；R 3为-H；R 4选自-F、-NH 2。
根据本发明的另一个方面，优选地，在式(I)所示的结构中，R 1为-H；R 2为-CH 3；R 3为-H；R 4选自-F、-NH 2。
根据本发明的另一个方面，优选地，在式(I)所示的结构中，R 1为-H；R 2为-CH 3；R 3为-CH 3；R 4选自-F、-NH 2。
根据本发明的另一个方面，优选地，在式(I)所示的结构中，R 1为-CH 3；R 2为-H；R 3为-H；R 4选自-F、-NH 2。
更进一步优选地，R 4为-F。
更进一步优选地，式(I)表示的化合物及其异构体或其药学上可接受的盐选自以下化合物：
Figure PCTCN2021102995-appb-000004
根据本发明的另一个方面，本发明提供了由式(I)表示的化合物及其药学上可接受的盐及其异构体或其药学上可接受的盐在制备针对KRASG12C突变蛋白相关癌症治疗药物中的用途。
优选地，根据所述用途，所述KRASG12C突变蛋白相关癌症疾病选自：肺癌、结直肠癌、胰腺癌、胰腺癌、肝癌、胃癌、食道癌、胆管癌、乳腺癌、卵巢癌、子宫颈癌、黑色素瘤、脑胶质瘤、淋巴癌、白血病。
根据本发明的另一个方面，本发明提供了一种用于治疗KRASG12C突变蛋白相关癌症的药物组合物，所述药物组合物包括治疗有效量的根据本发明的由式(I)表示的化合物及其药学上可接受的盐及其异构体或其药学上可接受的盐作为活性成分，以及药学上可接受的辅料。
优选地，根据所述药物组合物，所述KRASG12C突变蛋白相关癌症疾病选自：肺癌、结直肠癌、胰腺癌、胰腺癌、肝癌、胃癌、食道癌、胆管癌、乳腺癌、卵巢癌、子宫颈癌、黑色素瘤、脑胶质瘤、淋巴癌、白血病。
根据本发明的另一个方面，本发明提供了一种KRASG12C突变蛋白相关癌症的治疗方法，所述治疗方法包括向受试者施用有效量的根据本发明的所述化合物或包含所述化合物及其药学上可接受的盐作为活性成分的药物组合物。
优选地，根据所述治疗方法，所述KRASG12C突变蛋白相关癌症的治疗方法，所述癌症疾病选自：肺癌、结直肠癌、胰腺癌、胰腺癌、肝癌、胃癌、食道癌、胆管癌、乳腺癌、卵巢癌、子宫颈癌、黑色素瘤、脑胶质瘤、淋巴癌、白血病。
具体实施方式
以下，将详细地描述本发明。在进行描述之前，应当理解的是，在本说明书和所附的权利要求书中使用的术语不应解释为限制于一般含义和字典含义，而应当在允许发明人适当定义术语以进行最佳解释的原则的基础上，根据与本发明的技术方面相应的含义和概念进行解释。因此，这里提出的描述仅仅是出于举例说明目的的优选实例，并非意图限制本发明的范围，从而应当理解的是，在不偏离本发明的精神和范围的情况下，可以由其获得其他等价方式或改进方式。
根据本发明，如果无另外说明，这里引用的所有术语具有与那些本领域的熟练人员理解本发明相同的含义。
如本文所用的术语“盐”是指含阳离子和阴离子的化合物，其可通过可接受质 子部位的质子化和/或可供质子部位的去质子化来产生。值得注意的是，可接受质子部位的质子化导致形成阳离子类物质，其电荷通过生理阴离子的存在而平衡，而可供质子部位的去质子化导致形成阴离子类物质，其电荷通过生理阳离子的存在而平衡。
术语“药学上可接受的盐”指所述盐是药学上可接受的。药学上可接受的盐的例子包括但不限于：(1)酸加成盐，与无机酸形成，如盐酸、氢溴酸、硫酸、硝酸、磷酸等等；或与有机酸形成，如羟基乙酸、丙酮酸、乳酸、丙二酸、苹果酸、马来酸、富马酸、酒石酸、柠檬酸、3-(4-羟基苯甲酰基)苯甲酸、肉桂酸、扁桃酸、甲磺酸、乙磺酸、1,2-乙烷-二磺酸、2-羟基乙烷磺酸、苯磺酸、4-氯苯磺酸、2-萘磺酸、4对甲苯磺酸、樟脑酸、十二烷基硫酸、葡萄糖酸、谷氨酸、水杨酸、顺式-已二烯二酸等等；或(2)碱加成盐，和上述无机酸的任一种的共轭碱形成，其中共轭碱包含选自Na +、K +、Mg 2+、Ca 2+、NH xR 4-x +中的阳离子组分，其中NH xR 4-x +(R是C 1-4烷基，下标x是选自0、1、2、3或4的整数)表示季铵盐中的阳离子。应该理解，所有涉及药学上可接受的盐都包括相同酸加成盐的本文中所定义的溶剂加成形式(溶剂化物)或晶体形式(多晶型物)。
术语“C 1-M烷基”是指包含1-M个碳原子的烷基，例如其中M是具有下列数值的整数：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或30。例如术语“C 1-6烷基”是指含有1-6个碳原子的烷基。烷基的例子包括但不限于低级烷基，包括甲基、乙基、丙基、异丙基、正丁基、异丁基、叔丁基或戊基、异戊基、新戊基、己基、庚基和辛基。
术语“芳香基”指芳族体系，可以是单环或原本稠合的或连接在一起的多芳环，从而使至少一部分稠合或连接的环形成共轭的芳系。芳基基团包括但不限制于：苯基、萘基、四氢萘基。芳基可被任选取代，如可被1-4个选自下组的基团所取代的芳基或杂环：卤素、-CN、-OH、-NO 2、氨基、烷基、环烷基、链烯基、炔基、烷氧基、芳氧基、取代的烷氧基、烷基羰基、烷基羧基、烷基氨基或芳硫基。
术语“取代”指参考基团可以被一个或多个额外基团所取代，额外基团单独地且独立的选自于，烷基，环烷基，芳基，杂芳基，杂脂环烃，羟基，烷氧基，烷硫基，芳硫基，烷亚砜基，芳亚砜基，烷砜基，芳砜基，氰基，卤基，羰基，硫代羰基，硝基，卤烷基，氟烷基和氨基，包括单取代和双取代的氨基基团及其被保护的衍生物。
本发明所提供的由式(I)表示的化合物或其药学上可接受的盐，和包含该化合物的药物组合物可以是多种形式，如片剂、胶囊、粉剂、糖浆、溶液状、悬浮液和气雾剂等，并可以存在于适宜的固体或液体的载体或稀释液中以及适宜的用于注射或滴注的消毒器具中。
本发明的药物组合物的各种剂型可按照药学领域的常规制备方法制备。例如其制剂配方的单位剂量中包含0.05-200mg式(I)的化合物或其药学上可接受的盐，优选地，制剂配方的单位剂量中包含0.1mg-100mg式(I)的化合物。
本发明的由通式(I)表示的化合物和药物组合物可对哺乳动物临床使用，包括人和动物，可以通过口、鼻、皮肤、肺、或者胃肠道等的给药途径。最优选为口服。最佳优选日剂量为0.01-200mg/kg体重，一次性服用，或0.01-100mg/kg体重分次服用。不管用何种服用方法，个人的最佳剂量应依据具体的治疗而定。通常情况下是从小剂量开始，逐渐增加剂量一直到找到最适合的剂量。
本发明中，术语“有效量”可指为实现预期的效果所需的剂量和时段的有效的量。此有效量可能因某些因子而产生不同的变化，如疾病的种类或治疗时疾病的病症、被施用的特定标的器官的构造、病人个体大小、或疾病或症状的严重性。本领域具有通常知识者不需要过度实验即可凭经验决定特定化合物的有效量。
典型的配方是通过混合本发明的通式(I)表示的化合物及载体、稀释剂或赋形剂制备而成。适宜的载体、稀释剂或赋形剂是本领域技术人员所熟知的，包括诸如碳水化合物、蜡、水溶性及/或可膨胀性聚合物、亲水性或疏水性物质、明胶、油、溶剂、水等物质。
所用的特定载体、稀释剂或赋形剂，将根据本发明的化合物的使用方式和目的而定。一般以本领域技术人员认为可安全有效地给药至哺乳类动物的溶剂为基础而选择溶剂。一般而言，安全的溶剂是无毒性含水溶剂诸如水，以及其他可溶于水或与水混溶的无毒性溶剂。适宜的含水溶剂包括水、乙醇、丙二醇、聚乙二醇(如PEG400、PEG300)等中的一种或多种。该配方也可包括一种或多种缓冲剂、安定剂、表面活性剂、润湿剂、润滑剂、乳化剂、悬浮剂、防腐剂、抗氧化剂、遮光剂、助流剂、加工助剂、着色剂、增甜剂、香料剂、调味剂或其它已知的添加剂，使该药物以可被接受的形式制造或使用。
本发明所述的如式(I)的化合物与至少一种其它药物的组合使用时，两种药物或多种药物可以分开使用也可以组合使用，优选以药学组合物的形式给药。本发明的如式(I)的化合物或药物组合物能以任一已知的口服、静脉注射、直肠给药、阴道给药、透皮吸收、其它局部或全身给药形式，分开或一起给药至受试者。
这些药物组合物亦可含有一种或多种缓冲剂、安定剂、表面活性剂、润湿剂、润滑剂、乳化剂、悬浮剂、防腐剂、抗氧化剂、遮光剂、助流剂、加工助剂、着色剂、增甜剂、香料剂、调味剂或其它已知的添加剂，使该药物组合物以可被接受的形式制造或使用。
本发明药物优选口服给药途径。用于口服给药的固态剂型可包括胶囊、片剂、粉末或颗粒制剂。在固态剂型中，本发明的化合物或药物组合物与至少一种惰性赋形剂、稀释剂或载剂混合。适宜的赋形剂、稀释剂或载剂包括诸如柠檬酸钠或磷酸二钙的物质，或淀粉、乳糖、蔗糖、甘露糖醇、硅酸等；粘合剂如羧甲基纤维素、褐藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖、阿拉伯胶等；湿润剂如甘油等；崩解剂如琼脂、碳酸钙、马铃薯或木薯淀粉、褐藻酸、特定的络合硅酸盐、碳酸钠等；溶液阻滞剂如石蜡等；吸收促进剂如季铵化合物等；吸附剂如高岭土、膨润土等；润滑剂如滑石、硬脂酸钙、硬脂酸镁、固态聚乙二醇、月桂基硫酸钠等。在胶囊与片剂的情况下，该剂型亦可包括缓冲剂。类似类型的固态组合物亦可作为软式与硬式填充明胶胶囊中的填料，其使用乳糖以及高分子量聚乙二醇等作为赋形剂。
用于口服给药的液态剂型包括药学上可接受的乳化液、溶液、悬浮液、糖浆液与酏剂。除了本发明的化合物或其药物组合物之外，该液态剂型可含有本领域中常用的惰性稀释剂，诸如水或其他溶剂；增溶剂及乳化剂诸如乙醇、异丙基醇、碳酸乙酯、乙酸乙酯、苄醇、苯甲酸苄基酯、丙二醇、1,3-丁二醇、二甲基甲酰胺；油类(如棉籽油、落花生油、玉米胚芽油、橄榄油、蓖麻油、芝麻油等)；甘油；四氢糠基醇；聚乙二醇与脱水山梨糖醇的脂肪酸酯；或这些物质中的几种的混合物等。
除了这些惰性稀释剂之外，该组合物也可包括赋形剂，诸如润湿剂、乳化剂、悬浮剂、增甜剂、调味剂与香料剂中的一种或多种。
就悬浮液而言，除了本发明的由通式(I)表示的化合物或其药学上可接受的盐或者包含其的药物组合物之外，可进一步含有载剂诸如悬浮剂，如乙氧基化异硬脂醇、聚氧乙烯山梨醣醇、脱水山梨醣醇酯、微晶纤维素、偏氢氧化铝、膨润土、琼脂及黄耆胶，或这些物质中几种的混合物等。
本发明的由通式(I)表示的化合物或其药学上可接受的盐或者包含其的药物组合物可采用其它局部给药剂型给药，包括膏、粉末、喷剂及吸入剂。该药物可在无菌条件下与药学上可接受的赋形剂、稀释剂或载剂以及所需要的任一防腐剂、缓冲剂或推进剂混合。眼用配方、眼用油膏、粉末与溶液，亦意欲涵盖于本发明的范围内。
此外，本公开还涵盖了试剂盒(例如制药包装)。提供的试剂盒可以包含本文所述的药物组合物或化合物和容器(例如，药瓶、安瓿、瓶子、注射器和/或分装包装或其它合适 的容器)。在一些实施方式中，提供的试剂盒可以任选地进一步包括第二容器，其包含用于稀释或悬浮本文所述的药物组合物或化合物的药用赋形剂。在一些实施方式中，设置在第一容器和第二容器中的本文所述的药物组合物或化合物组合形成一个单元剂量形式。
在某些实施方式中，本文所述试剂盒进一步包括包含于试剂盒中的用于使用所述化合物或药物组合物的用法说明。本文所述的试剂盒还可以包括管理机构(如美国食品药品监督管理局(FDA))所要求的信息。在某些实施方式中，在试剂盒中包括的信息为处方信息。在某些实施方式中，试剂盒和用法说明提供用于治疗需要其的受试者的增殖性疾病和/或预防需要其的受试者的增殖性疾病。本文所述的试剂盒可以包含一种或多种额外的药物制剂作为单独的组合物。
以下结合具体的实施例对本发明做进一步详细的说明，但本发明不限于以下实施例，实施例是为了更好的阐释本发明的某些具体体现而不能被解释为以任何方式限定本发明的范围。实施例中未注明的条件为常规条件。除非特别说明，以下实施例中使用的试剂和仪器均为市售可得产品。
以下实施例中化合物的结构是通过核磁共振(NMR)或/和质谱(MS)来确定的。NMR位移(δ)以10-6(ppm)的单位给出。NMR的测定使用Bruker AVANCE-400核磁仪，测定溶剂为氘代二甲亚砜(DMSO-d6)、氘代氯仿(CDCl3)、氘代甲醇(CD3OD)，内标为四甲基硅烷(TMS)。
MS的测定用FINNIGAN LCQAd(ESI)质谱仪(生产商：Thermo，型号：Finnigan LCQ advantage MAX)。
薄层层析硅胶板使用烟台黄海HSGF254或青岛GF254硅胶板，薄层色谱法(TLC)使用的硅胶板采用的规格是0.15mm-0.2mm，薄层层析分离纯化产品采用的规格是0.4mm-0.5mm。
柱层析一般使用烟台黄海硅胶200-300目硅胶为载体。
实施例中无特殊说明，反应的温度为室温，为20摄氏度-30摄氏度。
实施例中的反应进程的检测采用薄层色谱法(TLC)，所使用的展开剂体系，以及纯化化合物采用的柱层析的洗脱机体系包括有：A：二氯甲烷和甲醇体系，B：正己烷和乙酸乙酯体系，C：石油醚和乙酸乙酯体系，D：丙酮和石油醚体系，溶剂的体积比例根据化合物的极性不同而进行调节。
实验中所用缩写语：NCS，N-氯代丁二酰亚胺；PdCl 2(dtbpf)，二氯[1,1'-双(耳叔丁基膦)二茂铁钯(II)；Boc，叔丁氧羰基；THF，四氢呋喃；EA，乙酸乙酯；DCM，二氯甲烷；DIPEA，二异丙基乙胺；DMF，N,N-二甲基酰胺；TFA，三氟醋酸；h，小时；TEA，三乙胺。
实施例1：化合物1的制备
Figure PCTCN2021102995-appb-000005
按照如下路线利用起始原料1-1制备化合物1。
Figure PCTCN2021102995-appb-000006
步骤1：中间体1-2的合成
将原料1-1(4.0g，0.017mol)、NCS(2.5g，0.018mol)溶于DMF(40ml)中，升温到70℃反应过夜。反应完毕直接倒入100mL冰水中，抽滤得到固体(4.6g)。
MS m/z(ESI):269.9[M+1].
步骤2：中间体1-3的合成
将化合物1-2(4.6g，0.017mol)溶于乙醇(60ml)中，加入乙酸甲脒(21.4g，0.20mmol)，加热回流反应过夜。反应完全后，浓缩干乙醇，加入20ml水和60ml EA分液，干燥浓缩有机相得粗品固体(5.2g)，直接用于下一步。
MS m/z(ESI):276.9[M+1].
步骤3：中间体1-4的合成
将化合物1-3(5.2g，0.018mol)加入SOCl 2(30mL)中，滴入6滴DMF，加热回流过夜。反应完全后，浓缩干SOCl 2，加入饱和碳酸氢钠和EA分液，有机相干燥浓缩，柱层析纯化得黄色固体(3.0g).
步骤4：中间体1-6的合成
将化合物1-4(296mg，1.0mmol)、化合物1-5(400mg，2.0mmol)溶于二氧六环(6ml)中，加入DIPEA(387mg，3.0mmol)，加热到50℃反应。反应结束后，加入H 2O(5ml)、EA(15ml),萃取得有机相，有机相干燥浓缩，柱层析纯化得固体(300mg)。
MS m/z(ESI):459.0[M+1].
步骤5：中间体1-8的合成
将化合物1-6(200mg，0.43mmol)溶于二氧六环/H 2O(6ml/2ml)中，加入硼酸1-7(163mg，0.52mmol，参照WO2020081282合成)及PdCl 2(dtbpf)(28.4mg，0.04mmol,0.1eq)，K 3PO 4(139mg，0.65mmol)，N 2保护下加热到90℃反应2h。反应结束后，加入5ml水和15ml EA，萃取得有机相，干燥浓缩，纯化得到固体(78mg)。
MS m/z(ESI):647.2[M+1].
步骤6：中间体1-9的合成
将化合物1-8(78mg)溶于DCM(6ml)中，加入1ml TFA,室温反应过夜，反应结束后，浓缩干，加入EA，用饱和碳酸氢钠调pH＝8左右，将有机相干燥浓缩得粗品(70.0mg)。
步骤7：化合物1的合成
将化合物1-9(70.0mg，0.156mmol)溶于THF/H 2O(2ml/2ml)中，加入K 2CO 3(95.3mg，0.69mmol)，N 2保护下冷至0℃，缓慢滴加丙烯酰氯(14.2mg，0.156mmol)，加完0℃继续反应10min。反应完毕加入水，用EA萃取，将有机相干燥，浓缩，柱层析 纯化得白色固体(30.0mg)。
1H NMR(400MHz,CDCl 3):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].
以下实施例2-4的化合物使用类似于实施例1描述的合成路线制备，仅对涉及到的原料哌嗪1-5进行替换。实施例编号、化合物标示和结构表征列于下表：
Figure PCTCN2021102995-appb-000007
实施例5：化合物5的制备
Figure PCTCN2021102995-appb-000008
按照如下路线利用起始原料5-6制备化合物1。
Figure PCTCN2021102995-appb-000009
步骤1：中间体5-7的合成
将化合物5-6(500mg，1.12mmol)溶于DMSO(10ml)中，加入25％氨水(160mg)封管中加热到90℃反应过夜。反应结束后，加入5ml水和15ml EA，萃取得有机相，干燥浓缩，纯化得到中间体5-7(105mg)。
1H NMR(400MHz,CDCl 3):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].
步骤2：中间体5-8的合成
将化合物5-7(55mg，0.12mmol)溶于二氧六环/H 2O(3ml/1ml)中，加入硼酸1-7(60mg，0.19mmol)及PdCl 2(dtbpf)(8.2mg，0.012mmol)，K 3PO 4(40mg，0.18mmol)，N 2保护下加热到90℃反应2h。反应结束后，加入5ml水和15ml EA，萃取得有机相，干燥浓缩，纯化得到中间体5-8(17mg)。
MS m/z(ESI):630.3[M+1].
步骤3：中间体5-9的合成
将化合物5-8(30mg)溶于DCM(2ml)中，加入0.5ml TFA,室温反应过夜，反应结束后，浓缩干，加入EA，用饱和碳酸氢钠调pH＝8左右，将有机相干燥浓缩得粗品(28mg)。
步骤4：化合物5的合成
将化合物5-9(28mg，0.069mmol)溶于THF/H 2O(2ml/2ml)中，加入K 2CO 3(42.5mg，3.08mmol)，N 2保护下冷至0℃，缓慢滴加丙烯酰氯(6.9mg，0.083mmol)，加完0℃继续反应10min。反应完毕加入水，用EA萃取，将有机相干燥，浓缩，柱层析纯化得白色固体(12.5mg)。
MS m/z(ESI):484.1[M+1].
以下实施例6-8的化合物使用类似于实施例2描述的合成路线制备，仅对涉及到的部分原料进行替换。实施例编号、化合物标示和结构表征列于下表：
Figure PCTCN2021102995-appb-000010
测试实施例1：化合物对KRAS-G12C修饰的评价
实验目的：
该实施例说明本发明的实施例化合物共价结合到KRAS G12C，使用LCMS测定本发明化合物与KRas G12C的共价加成物
实验步骤：
1、将GDP load到KRAS-4B-G12C蛋白上
1)将KRAS-4B-G12C蛋白用低镁缓冲液稀释一倍至浓度为103uM。
2)在1mL 103uM KRAS-4B-G12C蛋白中加入1mL 2*GDP loading buffer，轻柔缓慢混匀。
3)将2mL的混合物在室温下反应1.5h。
4)分装成100uL/管，用液氮速冻并存储到-80℃条件下。
2、小分子化合物对KRAS-4B-G12C蛋白的共价修饰分析
1)将KRAS-4B-G12C蛋白用10X反应缓冲液稀释，如下表所示配制反应体系：
Figure PCTCN2021102995-appb-000011
2)分别在室温下反应3min和30min。
3)加入5uL的5％甲酸终止反应。
3、质谱检测
在质谱检测前将总共55uL的反应体系15000rpm离心10min。
4、计算与Kras-G12C的耦联率
耦联率(％)＝蛋白-化合物复合物峰面积/(复合物峰面积+单体蛋白峰面积)*100。
每种蛋白质的质量计算为该蛋白质总信号的百分比，然后将该百分比与不加反应性化合物的蛋白质信号标准化。这些归一化的信号报告为归一化对照百分比(normalized percent of control，POC)。POC值增加表示化合物显示出更高的KRASG12C修饰程度。式(I)的示例化合物在3μM浓度下测试30分钟的结果如表2所示。
具体测试数据见下表1。
化合物 POC 化合物 POC 化合物 POC 化合物 POC 化合物 POC
1 89.8％ 2 75.5％ 3 71.5％ 4 60.5％ B 55.2％
结论：本发明中的化合物都显示了比对照化合物B更好的KRASG12C共价结合能力。
测试实施例2：化合物对KRAS G12C突变细胞活性试验
本实验旨在验证本发明化合物对KRAS G12C突变的NCI-H358人非小细胞肺癌细胞中ERK磷酸化的抑制。
主要试剂：
细胞株NCI-H358、RPMI1640培养基、FBS、TrypLE TM Express Enzyme、PBS、8％固定液、阻断溶液、100％甲醇、兔抗pERK、鼠抗GAPDH、IRDye 800CW山羊抗兔IgG、IRDye 680RD山羊抗鼠IgG
主要耗材与仪器：
T75细胞培养瓶、384细胞培养微孔板、CO 2恒温培养箱、Eppendorf离心机、Echo 550液体工作站、红外激光成像系统Odyssey CLx
实验方法：
4 x 10 6NCI-H358细胞(购自ATCC)接种于T75培养瓶，用加10％FBS(购自Transgene)和1％青霉素链霉素的RPMI 1640(均购自Gibco)培养2天，37度，5％CO 2。第3天时，倒去培养基，并用DPBS洗一次。加入2ml TrypLE TM Express Enzyme(购自Gibco)在室温消化至细胞变圆。加入5ml新鲜培养基，吹打并收集细胞。1000rpm离心5分钟。弃去上清，用新鲜培养基重悬细胞并计数。在384孔板中每孔接种40μl 6000个细胞，37度，5％CO 2培养过夜。第二天，用Echo 550加入200nl每孔的梯度稀释化合物(0.5％DMSO，起始浓度1000nm，3倍稀释，共10个浓度点)，继续培养3小时。在每孔中加入40μl 8％固定液(购自Solarbio)，室温孵育20分钟。加入40μl PBS洗涤一次。然后加入40μl 100％冷甲醇室温孵育10分钟。用40μl PBS洗涤一次。每孔加入20μl阻断溶液(购自Licor)室温孵育1小时。吸去阻断溶液并加入20μl一抗混合物，兔抗pERK(购自CST，1:1000稀释)和鼠抗GAPDH(购自CST，1：2000稀释)，4度孵育过夜。40μl每孔的含0.05％吐温的PBST洗涤3次。每孔加入20μl二抗混合液，IRDye 800CW山羊抗兔IgG和IRDye680RD山羊抗鼠IgG(均购自Li-COR，均按1:2000稀释)，室温孵育45分钟。每孔加入40μl PBST洗涤3次。将孔板在1000rpm离心1分钟，并用Odyssey CLx读板。
数据分析：IC 50结果由GraphPad Prism 5.0软件进行分析。
具体测试数据见下表2。
Figure PCTCN2021102995-appb-000012
结论：本发明中的化合物都显示了比对照化合物A和对照化合物B更强的抑制活性。
测试实施例3：化合物对细胞的活性评价
实验目的：
本实验旨在验证本发明化合物对KRAS G12C突变的NCI-H358人非小细胞肺癌细胞增殖的抑制。
主要试剂：
细胞株NCI-H358、RPMI1640培养基、FBS、TrypLE TM Express Enzyme、PBS、CellTiter-Glo 3D Cell Viability Assay试剂盒
主要耗材与仪器：
T75细胞培养瓶，384超低粘附细胞培养圆底微孔板、CO 2恒温培养箱、Eppendorf离心机、Echo 550液体工作站、Envision多标记分析仪
实验方法：
在384超低粘附细胞培养圆底微孔板中加入200nl梯度稀释化合物(起始浓度1000nM,3倍稀释，10个浓度点)，并接种40μl 800个新消化的处于对数生长期的NCI-H358细胞，37度，5％CO 2培养3天，培养基为加10％FBS和1％青霉素链霉素的RPMI 1640。第4天，加入20μl CellTiter-Glo 3D Cell Viability试剂(购自Promega)，室温振荡1小时，然后用Envision多标记分析仪读数。
数据分析：IC50结果由GraphPad Prism 5.0软件进行分析。
具体测试数据见下表3。
化合物 NCI-H358 化合物 NCI-H358 化合物 NCI-H358 化合物 NCI-H358
  IC 50(uM)   IC 50(uM)   IC 50(uM)   IC 50(uM)
1 0.0019 2 0.0046 3 0.0038 4 0.023
5 0.055 A >0.500 B 0.0123    
结论：本发明中的大多数化合物出乎意料地显示了比对照化合物A和对照化合物B更强的细胞肿瘤抑制活性。
测试实施例4：化合物在小鼠全血中的稳定性试验
本实验旨在考察本发明化合物在小鼠全血样品中的稳定性。
以ICR雄性EDTA-K2全血为空白基质在室温条件下分别配制100ng/mL的样品(n＝3)。在预定考察时间点(例如配制后即刻、1小时)取出稳定性样品，进行离心处理(离心条件：1500±20g，温度：2～8℃，离心10min)，获取血浆样品。
取上述血浆样本加入内标溶液，涡旋约1min，15400g，4℃，离心10min，取上清液进样分析，以分析物与内标的峰面积比值来计算结果，将每个时间点稳定性样品的响应平均峰面积的比值与配制后即刻样品比较，稳定性样品测定值与配制后即刻样品的偏差应不超过±15.0％。1h稳定性结果见下表4。
化合物 偏差 化合物 偏差 化合物 偏差 化合物 偏差 化合物 偏差
1 -1.33％ 2 -12.8％ 3 +14.0％ 4 +9.4％ B -27.1％
结论：同公开的对比化合物B相比，本发明中的化合物在小鼠全血中出乎意料的显示了更好的稳定性。
测试实施例5：化合物在人全血中的稳定性试验
本实验旨在考察本发明化合物在人全血样品中的稳定性。
以人全血为空白基质在室温条件下分别配制100ng/mL的样品(n＝3)。在预定考察时间点(例如配制后即刻、1小时)取出稳定性样品，进行离心处理(离心条件：1500±20g，温度：2～8℃，离心10min)，获取血浆样品。
取上述血浆样本加入内标溶液，涡旋约1min，15400g，4℃，离心10min，取上清液进样分析，以分析物与内标的峰面积比值来计算结果，将每个时间点稳定性样品的响应平均峰面积的比值与配制后即刻样品比较，稳定性样品测定值与配制后即刻样品的偏差应不超过±15.0％。1h稳定性结果见下表5。
化合物 偏差 化合物 偏差 化合物 偏差
3 +3.2％ 4 +5.8％ B -35.4％
结论：同公开的对比化合物B相比，本发明中的化合物在人全血中出乎意料的显示了更好的稳定性。
测试实施例6：药代动力学评价：
以小鼠为受试动物，测试了小鼠鼠灌胃给予化合物1、化合物3、化合物B后不同时刻血浆中药物浓度。研究本发明化合物在小鼠鼠体内药物代谢动力学行为，评价其药物代谢特征。每组实施例选用9只体重相近的小鼠，口服给予剂量为10mg/kg，单次给药。动物给药后在15min、30min、1h、2h、4h、6h、8h、12h、24h时间点采集血液(每只小鼠间隔2个时间点采样，共采样3个时间点)。采用LC-MS/MS分析方法检测血浆中化合物含量，方法的定量下限均为20ng/mL。使用代谢动力学数据分析软件WinNonlin 7.0对血浆中的浓度数据进行统计，利用非房室模型法(NCA)计算药代参数，具体见下表2。
实验方案：
实验药品：化合物1、化合物3、化合物B。
药物配置：取一定量药物，加入2％Klucel LF+0.1％Tween 80水溶液，配制成呈澄清溶液或均匀悬浮液。
给药：小鼠禁食过夜后灌胃给药，给药剂量为10mg/kg。
操作：小鼠灌胃给药，于给药前及给药后15min、30min、1h、2h、4h、6h、8h、12h、24h尾静脉采血，置于肝素化样品管中，4摄氏度，3500转/分钟离心10分钟分离血浆，于-20摄氏度保存，给药后2小时进食。
测定不同浓度药物灌胃给药后小鼠血浆中待测化合物的含量：血浆样品室温解冻后分别取50μL，加入130μL的内标工作液(1000ng/mL，乙腈，甲苯磺丁脲)，涡旋约1min后，4℃，13000rpm条件下离心10min。取50μL上清液与100μL 50％乙腈水混合后进样LC/MS/MS分析。
药代动力学参数结果见表6。
表4：小鼠药物代谢数据
Figure PCTCN2021102995-appb-000013
结论：本发明的化合物药代吸收良好，尤其是化合物3，相比于对照化合物B，不但大大延长了半衰期，而且血液中药物暴露量得到了极大的提高。
上述实施例只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围之内。

Claims (10)

  1. 一种以下式(I)表示的化合物及其异构体或其药学上可接受的盐，
    Figure PCTCN2021102995-appb-100001
    其中，
    R 1选自-H、-CH 3、-CH 2CN；
    R 2选自-H、-CH 3；
    R 3选自-H、-CH 3；
    R 4选自-F、-NH 2、-NHCH 3；
    当R 4为-F时，所述R 1、R 2、R 3不能同时为-H。
  2. 根据权利要求1所述的由式(I)表示的化合物及其异构体或其药学上可接受的盐，其特征在于，R 1为-CH 2CN；R 2为-H；R 3为-H；R 4选自-F、-NH 2。
  3. 根据权利要求1所述的由式(I)表示的化合物及其异构体或其药学上可接受的盐，其特征在于，R 1为-H；R 2为-CH 3；R 3为-H；R 4选自-F、-NH 2。
  4. 根据权利要求1所述的由式(I)表示的化合物及其异构体或其药学上可接受的盐，其特征在于，R 1为-H；R 2为-CH 3；R 3为-CH 3；R 4选自-F、-NH 2。
  5. 根据权利要求1所述的由式(I)表示的化合物及其异构体或其药学上可接受的盐，其特征在于，R 1为-CH 3；R 2为-H；R 3为-H；R 4选自-F、-NH 2。
  6. 根据权利要求1-5任意一项所述的由式(I)表示的化合物及其异构体或其药学上可接受的盐，其特征在于，R 4为-F。
  7. 根据权利要求1所述的由式(I)表示的化合物及其异构体或其药学上可接受的盐，其特征在于，由式(I)表示的化合物及其异构体或其药学上可接受的盐选自以下化合物：
    Figure PCTCN2021102995-appb-100002
    Figure PCTCN2021102995-appb-100003
  8. 根据权利要求1至7中任意一项所述由式(I)表示的化合物及其异构体或其药学上可接受的盐在制备针对KRASG12C突变蛋白相关癌症治疗药物中的用途，所述KRASG12C突变蛋白相关癌症疾病选自：肺癌、结直肠癌、胰腺癌、胰腺癌、肝癌、胃癌、食道癌、胆管癌、乳腺癌、卵巢癌、子宫颈癌、黑色素瘤、脑胶质瘤、淋巴癌、白血病。
  9. 一种用于治疗KRASG12C突变蛋白相关癌症药物组合物，所述药物组合物包括治疗有效量的根据权利要求1至7中任意一项所述由式(I)表示的化合物及其药学上可接受的盐及其异构体或其药学上可接受的盐作为活性成分，以及药学上可接受的辅料，所述KRASG12C突变蛋白相关癌症疾病选自：肺癌、结直肠癌、胰腺癌、胰腺癌、肝癌、胃癌、食道癌、胆管癌、乳腺癌、卵巢癌、子宫颈癌、黑色素瘤、脑胶质瘤、淋巴癌、白血病。
  10. 一种KRASG12C突变蛋白相关癌症的治疗方法，所述治疗方法包括向受试者施用有效量的根据权利要求1至7中任意一项所述由式(I)表示的化合物及其药学上可接受的盐及其异构体或其药学上可接受的盐作为活性成分的药物组合物，所述KRASG12C突变蛋白相关癌症疾病选自：肺癌、结直肠癌、胰腺癌、胰腺癌、肝癌、胃癌、食道癌、胆管癌、乳腺癌、卵巢癌、子宫颈癌、黑色素瘤、脑胶质瘤、淋巴癌、白血病。
PCT/CN2021/102995 2020-07-03 2021-06-29 一种用于抑制krasg12c突变蛋白的化合物及其制备方法和用途 WO2022002018A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010636659.1 2020-07-03
CN202010636659.1A CN113880827B (zh) 2020-07-03 2020-07-03 一种用于抑制krasg12c突变蛋白的化合物及其制备方法和用途

Publications (1)

Publication Number Publication Date
WO2022002018A1 true WO2022002018A1 (zh) 2022-01-06

Family

ID=79013292

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/102995 WO2022002018A1 (zh) 2020-07-03 2021-06-29 一种用于抑制krasg12c突变蛋白的化合物及其制备方法和用途

Country Status (2)

Country Link
CN (1) CN113880827B (zh)
WO (1) WO2022002018A1 (zh)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022177917A3 (en) * 2021-02-16 2022-09-22 Theras, Inc. Compositions and methods for inhibition of ras
WO2022235870A1 (en) 2021-05-05 2022-11-10 Revolution Medicines, Inc. Ras inhibitors for the treatment of cancer
WO2022235864A1 (en) 2021-05-05 2022-11-10 Revolution Medicines, Inc. Ras inhibitors
WO2023172940A1 (en) 2022-03-08 2023-09-14 Revolution Medicines, Inc. Methods for treating immune refractory lung cancer
WO2023183585A1 (en) * 2022-03-25 2023-09-28 Eli Lilly And Company Kras inhibitors
WO2023240263A1 (en) 2022-06-10 2023-12-14 Revolution Medicines, Inc. Macrocyclic ras inhibitors
WO2024206858A1 (en) 2023-03-30 2024-10-03 Revolution Medicines, Inc. Compositions for inducing ras gtp hydrolysis and uses thereof
WO2024211663A1 (en) 2023-04-07 2024-10-10 Revolution Medicines, Inc. Condensed macrocyclic compounds as ras inhibitors
WO2024211712A1 (en) 2023-04-07 2024-10-10 Revolution Medicines, Inc. Condensed macrocyclic compounds as ras inhibitors
WO2024216048A1 (en) 2023-04-14 2024-10-17 Revolution Medicines, Inc. Crystalline forms of ras inhibitors, compositions containing the same, and methods of use thereof
WO2024216016A1 (en) 2023-04-14 2024-10-17 Revolution Medicines, Inc. Crystalline forms of a ras inhibitor

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106488910A (zh) * 2013-10-10 2017-03-08 亚瑞克西斯制药公司 Kras g12c的抑制剂
WO2019141250A1 (zh) * 2018-01-19 2019-07-25 南京明德新药研发股份有限公司 作为krasg12c突变蛋白抑制剂的吡啶酮并嘧啶类衍生物
WO2020081282A1 (en) * 2018-10-15 2020-04-23 Eli Lilly And Company Kras g12c inhibitors

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113754653A (zh) * 2020-06-05 2021-12-07 明慧医药（上海）有限公司 一种kras g12c抑制剂化合物及其用途

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106488910A (zh) * 2013-10-10 2017-03-08 亚瑞克西斯制药公司 Kras g12c的抑制剂
WO2019141250A1 (zh) * 2018-01-19 2019-07-25 南京明德新药研发股份有限公司 作为krasg12c突变蛋白抑制剂的吡啶酮并嘧啶类衍生物
WO2020081282A1 (en) * 2018-10-15 2020-04-23 Eli Lilly And Company Kras g12c inhibitors

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022177917A3 (en) * 2021-02-16 2022-09-22 Theras, Inc. Compositions and methods for inhibition of ras
WO2022235870A1 (en) 2021-05-05 2022-11-10 Revolution Medicines, Inc. Ras inhibitors for the treatment of cancer
WO2022235864A1 (en) 2021-05-05 2022-11-10 Revolution Medicines, Inc. Ras inhibitors
WO2023172940A1 (en) 2022-03-08 2023-09-14 Revolution Medicines, Inc. Methods for treating immune refractory lung cancer
WO2023183585A1 (en) * 2022-03-25 2023-09-28 Eli Lilly And Company Kras inhibitors
WO2023240263A1 (en) 2022-06-10 2023-12-14 Revolution Medicines, Inc. Macrocyclic ras inhibitors
WO2024206858A1 (en) 2023-03-30 2024-10-03 Revolution Medicines, Inc. Compositions for inducing ras gtp hydrolysis and uses thereof
WO2024211663A1 (en) 2023-04-07 2024-10-10 Revolution Medicines, Inc. Condensed macrocyclic compounds as ras inhibitors
WO2024211712A1 (en) 2023-04-07 2024-10-10 Revolution Medicines, Inc. Condensed macrocyclic compounds as ras inhibitors
WO2024216048A1 (en) 2023-04-14 2024-10-17 Revolution Medicines, Inc. Crystalline forms of ras inhibitors, compositions containing the same, and methods of use thereof
WO2024216016A1 (en) 2023-04-14 2024-10-17 Revolution Medicines, Inc. Crystalline forms of a ras inhibitor

Also Published As

Publication number Publication date
CN113880827B (zh) 2024-10-01
CN113880827A (zh) 2022-01-04

Similar Documents

Publication Publication Date Title
WO2022002018A1 (zh) 一种用于抑制krasg12c突变蛋白的化合物及其制备方法和用途
CN112390788A (zh) 一种用于抑制krasg12c突变蛋白的化合物及其制备方法和用途
CN114195788B (zh) 一类四环化合物及其用途
US10640512B2 (en) Imidazopyrazinamine phenyl derivative and use thereof
JP6407955B2 (ja) クマリン誘導体、ならびに過剰増殖疾患を治療する際の使用方法
RU2633694C2 (ru) Дейтерированный фениламинопиримидин и фармацевтическая композиция, содержащая такое соединение
US11192900B2 (en) Substituted 1,6-dihydropyridinones and 1,2-dihydroisoquinolinones as bet inhibitors
US9738613B2 (en) Substituted 1,2,3-triazoles as antitumor agents
KR20240054339A (ko) Tyk2/jak1 슈도키나아제 도메인 억제제로서의 화합물 및 합성과 사용 방법
AU2020255702B2 (en) Quinolyl-containing compound and pharmaceutical composition, and use thereof
CN107793371B (zh) 一类溴结构域识别蛋白抑制剂及其制备方法和用途
CN112243437A (zh) 含丙烯酰基的核转运调节剂及其用途
US11712434B2 (en) Compound having anti-cancer effect, and preparation method therefor and use thereof
CN114539246A (zh) 一种哌啶并嘧啶类化合物及其用途
CN114409653A (zh) 一种桥环并嘧啶并环类化合物及其用途
CN110143995A (zh) 氮杂环取代18β-甘草次酸衍生物及其制备和应用
TW202348235A (zh) 吉西他濱抗癌衍生物及抗癌醫藥用途
KR20170073699A (ko) 신규한 유형의 시티딘 유도체 이량체 및 그의 용도
US11351151B2 (en) Compound having anticancer activity and preparation method and application
RU2803116C2 (ru) Хинолинил-содержащее соединение и его фармацевтическая композиция и применение
WO2021092892A1 (zh) 喹啉或喹唑啉类化合物在制备抗肿瘤药物中的应用
CN111233837A (zh) 一种缩氨基硫脲的化合物及其制备方法和用途
CN117534658A (zh) 一种靶向降解CK1α蛋白并激活P53蛋白的化合物及其应用
TW202402272A (zh) 含有三氟甲基基團的化合物
CN113646313A (zh) 一种a2a受体拮抗剂的盐型、晶型及其制备方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21834159

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21834159

Country of ref document: EP

Kind code of ref document: A1