WO2020211853A1 - 新型egfr三突变抑制剂及其应用 - Google Patents

新型egfr三突变抑制剂及其应用 Download PDF

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WO2020211853A1
WO2020211853A1 PCT/CN2020/085438 CN2020085438W WO2020211853A1 WO 2020211853 A1 WO2020211853 A1 WO 2020211853A1 CN 2020085438 W CN2020085438 W CN 2020085438W WO 2020211853 A1 WO2020211853 A1 WO 2020211853A1
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substituted
cancer
unsubstituted
compound
halogen
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PCT/CN2020/085438
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French (fr)
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李洪林
谢华
赵振江
丁健
李圣青
陈卓
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华东理工大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • 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
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems

Definitions

  • the present invention relates to the field of medicinal chemistry; specifically, the present invention relates to novel indole and tri-substituted imidazole compounds as EGFR triple mutation inhibitors and their application as EGFR inhibitors in the preparation of drugs for tumor-related diseases.
  • Epidermal growth factor receptor is a type of transmembrane glycoprotein that exists on the cell membrane of human tissues with a molecular weight of 170-kDa. It is compatible with ErbB-2 (HER2/Neu) and ErbB-3 (HER3). ), ErbB-4 (HER4) belong to the ErbB receptor tyrosine kinase (receptor tyrosine kinase, RTK) family. These receptors are composed of extracellular ligand binding domains, which are then connected to intracellular tyrosine kinases through transmembrane regions. The tyrosine kinase domain in ErbB receptors is highly conserved.
  • EGFR and ErbB-4 have a complete extracellular ligand binding domain and an activatable kinase domain.
  • the extracellular domain of ErbB-2 may not have ligand binding capacity.
  • ErbB-3 can bind to ATP, it is inactive.
  • Tyrosine kinase receptor its homodimer is not active.
  • the signal transmission mode of EGFR is mainly to bind specific ligands, cause dimerization, activate its intracellular tyrosine kinase activity, and autophosphorylate some tyrosine residues at the end, thereby activating downstream signal pathways.
  • tumor molecular targeted drugs for EGFR are mainly divided into two categories according to their properties: one is monoclonal antibodies that directly act on the extracellular receptor region; the other is that interferes with the intracellular EGFR tyrosine kinase activity Small molecule inhibitors.
  • Monoclonal antibody drugs interact with the extra-membrane ligand binding domain of EGFR, so that endogenous ligands such as EGF cannot bind to EGFR, thereby preventing the signal from entering cells; small molecule drugs interact with the intracellular tyrosine kinase catalytic region Bind, inhibit its catalytic activity, thereby blocking cell proliferation signals.
  • EGFR mutations are mainly concentrated on exons 18-21, which are responsible for encoding the EGFR tyrosine kinase domain.
  • the deletion of exon 19 accounts for 44% of EGFR tyrosine kinase sensitive mutations.
  • the point mutation in exon 21-L858R mutation accounted for 41% of EGFR tyrosine kinase sensitive mutations.
  • Residue 719 was mutated from glycine to serine. Alanine or cysteine accounted for 10% of the total mutations, while insertion or replication mutations in exon 20 accounted for the remaining 5%.
  • the deletion of exon 19 and the L858R point mutation are the most common sensitive mutations.
  • the first is the first generation of EGFR small molecule inhibitors, such as Gefitinib and Erlotinib. These inhibitors are mainly aimed at sensitive mutations, but with the discovery of T790M resistance mutations, patients gradually develop resistance. Therefore, the second and third generations of EGFR inhibitors were derived. This type of inhibitors mainly improve the inhibitory activity by covalently binding the Michael receptor on the molecule to the cysteine 797 residue of the protein.
  • the purpose of the present invention is to provide a novel EGFR inhibitory compound which can inhibit the triple mutation of EGFR19del/T790M/C797S.
  • Another object of the present invention is to provide a pharmaceutical composition containing the above-mentioned compound.
  • Another object of the present invention is to provide the use of the above compound in the preparation of drugs for treating EGFR-related diseases or inhibiting EGFR.
  • the present invention provides a compound represented by the general formula I or a stereoisomer or optical isomer or a pharmaceutically acceptable salt thereof:
  • W is CH or N
  • X is CH or N
  • Y is CH or N or C halogen
  • Z is CH or N
  • R 1 is any integer from 1 to 5, optionally located at 1', 2', 5', 6'or 7', and is independently selected from: H, halogen, C1-C6 substituted or unsubstituted alkane base,
  • n is an integer from 0 to 4.
  • P is 1-2 C1-C3 alkyl groups or not present
  • Q is -OH, -SH, -NH 2 , -NHCH 3 , -COOH, -CONH 2 , -NHCONH 2 , -NHCONHNH 2 , -SO 3 H, -SO 2 NH 2 ;
  • R 4 When, R 4 can have one or more Qs at each position;
  • X is O, S, NH
  • R 2 is selected from: hydrogen, C1-C5 substituted or unsubstituted alkylcarboxamido, C2-C5 substituted or unsubstituted alkenylcarboxamido;
  • R 3 is selected from: hydrogen, halogen, NR 7 R 8 , substituted N-C1-C3 alkylpiperazinyl;
  • R 7 and R 8 are independently selected from: H, C1-C6 substituted or unsubstituted alkyl, -(CH 2 ) n NR 9 R 10 , where n is an integer of 0-3;
  • R 9 and R 10 are independently selected from: H, C1-C3 substituted or unsubstituted alkyl.
  • W is CH
  • X is CH or N
  • Y is CH or N
  • R 1 is 1 or 2, optionally located at the 1'or 5'position, and is independently selected from: H, C1-C6 substituted or unsubstituted alkylhydroxyl or polyhydroxyl, halogen;
  • R 2 is selected from: hydrogen, C1-C3 substituted or unsubstituted alkylcarboxamido, C2-C3 substituted or unsubstituted alkenylcarboxamido;
  • R 3 is selected from: halogen, NR 7 R 8 ;
  • R 7 and R 8 are independently selected from: H, C1-C3 substituted or unsubstituted alkyl, -(CH 2 ) n NR 9 R 10 , where n is 1 or 2;
  • R 9 and R 10 are independently selected from: H, C1-C3 substituted or unsubstituted alkyl.
  • the present invention provides the following compounds or their stereoisomers or optical isomers or their pharmaceutically acceptable salts:
  • the present invention provides a compound represented by formula II or its stereoisomer or optical isomer or its pharmaceutically acceptable salt:
  • R 1 is selected from:
  • n is an integer from 0 to 4.
  • P is 1-2 C1-C3 alkyl groups or not present
  • Q is -OH, -SH, -NH 2 , -NHCH 3 , -COOH, -CONH 2 , -NHCONH 2 , -NHCONHNH 2 , -SO 3 H, -SO 2 NH 2 ;
  • R 4 When, R 4 can have one or more Qs at each position;
  • X is O, S, NH
  • R 4 is selected from:
  • the number of A is any integer from 0 to 4, and is independently selected from: halogen, substituted or unsubstituted C1-C3 alkoxy;
  • V, S and T are each an integer from 0 to 4, and are independently selected from halogen, substituted or unsubstituted C1-C3 alkoxy;
  • R 5 is selected from: H, substituted or unsubstituted
  • R 6 is selected from: H, substituted or unsubstituted C1-C3 alkyl.
  • R 4 is selected from:
  • the number of A is any integer from 0 to 4, and is independently selected from: halogen, substituted or unsubstituted C1-C3 alkoxy;
  • V is any integer from 0 to 4, and is independently selected from halogen, substituted or unsubstituted C1-C3 alkoxy;
  • R 5 is selected from: substituted or unsubstituted
  • R 6 is selected from: H.
  • the present invention provides a compound selected from the following or a stereoisomer or optical isomer or a pharmaceutically acceptable salt thereof:
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the compound described in the first or second aspect or its stereoisomer or optical isomer or a pharmaceutically acceptable salt thereof, And optionally a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition is a dosage form suitable for oral administration, including but not limited to tablets, solutions, suspensions, capsules, granules, and powders.
  • the present invention provides the compound described in the first or second aspect or its stereoisomer or optical isomer or its pharmaceutically acceptable salt, or the pharmaceutical composition described in the third aspect is prepared Use in the treatment or prevention of EGFR-mediated diseases or drugs that inhibit EGFR.
  • the EGFR-mediated disease is cancer.
  • the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, breast cancer, prostate cancer, glioma, ovarian cancer, head and neck cancer Squamous cell carcinoma, cervical cancer, esophageal cancer, liver cancer, kidney cancer, pancreatic cancer, colon cancer, skin cancer, leukemia, lymphoma, gastric cancer, multiple bone marrow cancer and solid tumors.
  • the present invention provides a method for treating or preventing EGFR-mediated diseases, comprising administering the compound described in the first or second aspect or the pharmaceutical composition described in the third aspect to a subject in need thereof.
  • the EGFR-mediated disease is cancer; preferably, the cancer is selected from the following group: non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, breast cancer, prostate cancer , Glioma, ovarian cancer, head and neck squamous cell carcinoma, cervical cancer, esophageal cancer, liver cancer, kidney cancer, pancreatic cancer, colon cancer, skin cancer, leukemia, lymphoma, gastric cancer, multiple bone marrow cancer and solid tumors .
  • the cancer is selected from the following group: non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, breast cancer, prostate cancer , Glioma, ovarian cancer, head and neck squamous cell carcinoma, cervical cancer, esophageal cancer, liver cancer, kidney cancer, pancreatic cancer, colon cancer, skin cancer, leukemia, lymphoma, gastric cancer, multiple bone marrow
  • Figure 1 shows the cell activity evaluation of compound 4, in which the inhibitory activity of the compound on A431 (A), H1975 (B) and BaF3/EGFR19D/T790M/C797S (C) was tested by CCK-8 assay.
  • alkyl refers to a saturated branched or straight chain alkyl with a carbon chain length of 1-10 carbon atoms, and preferred alkyl groups include 2-8, 1-6, 1-4, An alkyl group with 3-8 and 1-3 carbon atoms. Examples of alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, heptyl and the like.
  • the alkyl group may be substituted by one or more substituents, for example by halogen or haloalkyl.
  • the alkyl group may be an alkyl group substituted with 1-4 fluorine atoms, or the alkyl group may be an alkyl group substituted with a fluoroalkyl group.
  • alkoxy refers to an oxy group substituted by an alkyl group.
  • Preferred alkoxy groups are alkoxy groups having 1 to 6 carbon atoms in length, more preferably alkoxy groups having 1 to 4 carbon atoms in length. Examples of alkoxy groups include, but are not limited to: methoxy, ethoxy, propoxy and the like.
  • halogen refers to fluorine, chlorine, bromine and iodine.
  • “amido” refers to a group with the structural formula "-R'-NH-C(O)-R", wherein R'can be selected from hydrogen or alkyl, and R can be selected from alkyl and alkenyl , alkynyl, NR c R d is substituted alkyl, NR c R d is substituted alkenyl and NR c R d group substituted alkynyl, halogen substituted alkyl, alkenyl substituted with cyano group, Among them, R c and R d can be selected from alkyl and alkenyl.
  • substituted or unsubstituted or “optionally substituted” means that the modified substituents can be optionally selected from the following by 1-5 (for example, 1, 2, 3, 4, or 5) Substituent substitution: halogen, C 1-4 aldehyde, C 1-6 linear or branched alkyl, cyano, nitro, amino, hydroxy, hydroxymethyl, halogen substituted alkyl (such as trifluoromethyl Group), halogen-substituted alkoxy (e.g. trifluoromethoxy), carboxy, C 1-4 alkoxy, ethoxyformyl, N(CH 3 ) and C 1-4 acyl.
  • 1-5 for example, 1, 2, 3, 4, or 5
  • Substituent substitution halogen, C 1-4 aldehyde, C 1-6 linear or branched alkyl, cyano, nitro, amino, hydroxy, hydroxymethyl, halogen substituted alkyl (such as trifluoromethyl Group), halogen-substit
  • substituted refers to the replacement of one or more hydrogen atoms on a specific group by a specific substituent.
  • the specific substituents may be the substituents correspondingly described in the foregoing, or may be specific substituents appearing in each embodiment. Therefore, in the present invention, the substituents in the general formula may each independently be the corresponding group in the specific compound in the embodiment; that is, the present invention includes the combination of the substituents in the above general formula, and also includes the substituents in the general formula. The combinations of some substituents and other specific substituents appearing in the examples are shown.
  • the present invention provides a series of EGFR inhibitory compounds with a completely new structure, which can significantly inhibit the EGFR19del/T790M/C797S triple mutation.
  • the present invention provides a compound represented by general formula I or II or its stereoisomer or optical isomer or its pharmaceutically acceptable salt:
  • the compound of the present invention can be reacted with an inorganic acid or an organic acid to form a conventional pharmaceutically acceptable salt.
  • the inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, aminosulfonic acid, phosphoric acid, etc.
  • the organic acids include citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, Methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, catabolic acid , Hydroxymaleic acid,
  • the structural formula described in the present invention is intended to include all isomeric forms (such as enantiomers, diastereomers and geometric isomers (or conformational isomers): for example, containing asymmetric centers The R, S configuration of the double bond (Z), (E) isomers, etc. Therefore, a single stereochemical isomer of the compound of the present invention or its enantiomers, diastereomers or geometric Mixtures of isomers (or conformational isomers) are all within the scope of the present invention.
  • composition of the present invention and method of administration
  • the compounds of the present invention can be used to prepare and treat EGFR (especially EGFR 19del/T790M/C797S triple mutation) mediated diseases, such as cancer, including but not limited to: non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous Cancer, breast cancer, prostate cancer, glioma, ovarian cancer, head and neck squamous cell carcinoma, cervical cancer, esophageal cancer, liver cancer, kidney cancer, pancreatic cancer, colon cancer, skin cancer, leukemia, lymphoma, stomach cancer, Multiple bone marrow cancers and solid tumors.
  • cancer including but not limited to: non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous Cancer, breast cancer, prostate cancer, glioma, ovarian cancer, head and neck squamous cell carcinoma, cervical cancer, esophageal cancer, liver cancer, kidney cancer, pancreatic cancer, colon cancer, skin
  • the invention also provides a pharmaceutical composition comprising the compound of the invention, the pharmaceutical composition optionally comprising a pharmaceutically acceptable excipient.
  • the pharmaceutical composition of the present invention contains the compound of the present invention or a pharmaceutically acceptable salt thereof in a safe and effective amount and a pharmaceutically acceptable excipient or carrier.
  • safe and effective amount refers to: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects.
  • “Pharmaceutically acceptable excipients or carriers” refer to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use, and must have sufficient purity and sufficiently low toxicity. "Compatibility” here means that the components in the composition can be blended with the compound of the present invention and between them without significantly reducing the efficacy of the compound.
  • pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, and solid lubricants (such as stearic acid).
  • Magnesium stearate calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers Wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • the method of administration of the compound or pharmaceutical composition of the present invention is not particularly limited.
  • Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration .
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or mixed with the following ingredients: (a) fillers or compatibilizers, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectant, For example, glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) Absorption accelerators, such as quaternary amine compounds; (g) wetting agents, such as cetyl alcohol and gly
  • Solid dosage forms such as tablets, sugar pills, capsules, pills and granules can be prepared with coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifying agents, and the active compound or the release of the compound in such a composition may be released in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microcapsules with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • the liquid dosage form may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-Butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.
  • composition may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • suspending agents for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • composition for parenteral injection may contain physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • the dosage form of the compound of the present invention for topical administration includes ointment, powder, patch, spray and inhalant.
  • the active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required if necessary.
  • the compound of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds.
  • a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage when administered is a pharmaceutically effective dosage.
  • the compounds and pharmaceutical compositions of the present invention can be administered via oral, nasal, skin, lung, or gastrointestinal tract. The most preferred is oral administration, one-time administration or divided administration. Regardless of the method of administration, the individual's optimal dose should be determined based on the specific treatment. Usually start with a small dose and gradually increase the dose until the most suitable dose is found. Of course, the specific dosage should also consider factors such as the route of administration, the patient's health status, etc., which are within the skill range of a skilled physician.
  • the compound of the present invention is an EGFR inhibitory compound with a novel structure
  • the compound of the present invention can significantly inhibit the EGFR 19del/T790M/C797S three mutations, thereby being able to be developed as a new generation of EGFR inhibitors and lay a new material foundation for the development of a new generation of EGFR inhibitors.
  • the preparation method of the compound of the present invention can be a conventional method in this field, or the synthetic route of the present invention can be adopted.
  • the reagents and conditions of each step are as follows:
  • Dissolve 2b (2.59g, 14.7mmol) in 50mL of 1,2-dichloroethane, ice-bath to 0°C, add anhydrous aluminum trichloride (2.81g, 21.0mmol), increase to room temperature for 10min and stir for 15min.
  • Compound 5 is obtained by referring to the synthetic route of compound 1. Yellow solid, yield 21%.
  • reaction was quenched with saturated ammonium chloride solution, the solvent was spin-dried, water and acetic acid Extract with ethyl ester, dry with anhydrous sodium sulfate, spin-dry the filtrate to obtain the intermediate.
  • Compound 8 is obtained by referring to the synthetic route of compound 1. Yellow-brown solid, yield 23%.
  • the reagents and conditions of each step are as follows:
  • o-phthalaldehyde (1.00g, 7.5mmol) into a 250mL single-neck flask, dissolve it with 35mL acetonitrile, add D,L-phenylglycine (1.13g, 7.5mmol) and 2mL acetic acid, replace with nitrogen 3 times, reflux for 3h, TLC monitoring, the reaction is over, suction filtration, the filter cake is rinsed and vacuum dried to obtain 1.3 g of 2w black solid, with a yield of 65%.
  • Tris(dibenzylideneacetone)dipalladium (12.00mg, 0.013mmol), pinacol diborate (1.00g, 3.9mmol), 2-dicyclohexylphosphorus-2,4,6-triisopropyl Biphenyl (13.00mg, 0.026mmol) and potassium acetate (385.00mg, 3.9mmol) were put into a 50mL three-necked flask, replaced with nitrogen three times, weighed 2x (267.00mg, 1.3mmol) dissolved in 8mL anhydrous 1,4-diox The six rings were added to a three-necked flask and reacted at 110°C for 16 hours. TLC monitored.
  • Potassium carbonate (24.00mg, 0.2mmol) and [1,1'-bis(diphenylphosphine)ferrocene] dichloropalladium dichloromethane complex (6.00mg, 0.007mmol) were sequentially filled into 25mL three mouths In the bottle, replace with nitrogen three times, add 2v, 2y solution dissolved in 4mL 1,4-dioxane and 1mL water, react at 90°C for 14h, monitor by TLC, cool to room temperature after the reaction, and extract with ethyl acetate.
  • Tyrosine kinase EGFR(WT)
  • Enzyme-Linked Immunosorbent Assay was used to detect the kinase phosphorylation substrate ability, and calculate the inhibitory effect of the compound on the kinase activity.
  • the kinase uses EGFR L858R/T790M/C797S (purchased from BPS Bioscience).
  • Enzyme reaction substrate Poly(Glu, Tyr) 4:1 is diluted with potassium ion-free PBS to 2.5 ⁇ g/well, and reacted at 37°C for 12-16h to coat the ELISA plate for use.
  • ATP final concentration 5 ⁇ M
  • reaction buffer 50mM HEPES pH 7.4, 20mM MgCl 2 , 0.1mM MnCl 2 , 0.2mM Na 3 VO 4 , 1mM DTT
  • the plate was washed three times with T-PBS, and 100 ⁇ L of antibody PY99 (T-PBS containing 5 mg/mL BSA, diluted 1:500) was added and reacted in a shaker at 37°C for 0.5 h. After washing the plate with T-PBS, 100 ⁇ L of horseradish peroxidase-labeled goat anti-mouse IgG (T-PBS containing 5 mg/mL BSA, diluted 1:2000) was added, and the plate was shaken at 37°C for 0.5 h.
  • the enzyme activity test results are as follows:
  • a Kinase activity test was performed by using an ELISA-based EGFR-TK test. The data are the average of at least two independent determinations and are expressed as the mean ⁇ SD (standard deviation). b two mutants (EGFR L858R/T790M ). c three mutants (EGFR L858R/T790M/C797S ).
  • a Kinase activity test was performed by using an ELISA-based EGFR-TK test.
  • the data are the average of at least two independent determinations and are expressed as mean ⁇ SD.
  • Tyrosine kinase EGFR T790M/19DEL/C797S
  • Test method The original mouse B cell line BaF3 cell line and EGFR 19del/T790M/C797S- BaF3 were used in the experiment. At the beginning of the experiment, cells in the logarithmic growth phase were selected. Three groups of cells expressing 19del+T790M+C797S mutation were set up in 96-well plates. The number of cells in each group was 5000. The maximum concentration of the drug was 10 ⁇ mol/L, and the ratio was 1:2. Decrease, after 72 hours, add 10ul of CCK8 to each well, use a thermo microplate reader to measure cell viability at 450nm, and finally get the IC 50 value after fitting the curve.

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Abstract

通式I或II的EGFR抑制性化合物，其能够抑制EGFR 19del/T790M/C797S，L858R/T790M/C797S三突变，可用于EGFR介导的相关疾病的治疗用途。

Description

新型EGFR三突变抑制剂及其应用 技术领域
本发明涉及药物化学领域；具体地说，本发明涉及新型吲哚类和三取代咪唑类化合物作为EGFR三突变抑制剂及其作为EGFR抑制剂在制备肿瘤相关疾病的药物中的应用。
背景技术
表皮生长因子受体(epidermal growth factor receptor，EGFR)是一类存在于人组织细胞膜上的跨膜糖蛋白，分子量为170-kDa，其与ErbB-2(HER2/Neu)、ErbB-3(HER3)、ErbB-4(HER4)同属ErbB受体酪氨酸激酶(receptor tyrosine kinase，RTK)家族。这些受体是由细胞外配体结合结构域组成，然后通过跨膜区与细胞内酪氨酸激酶连接。ErbB受体内的酪氨酸激酶结构域是高度保守的，相反，胞外结构域有明显的差异，这也致使受体在结合配体时显现出不同的特异性。EGFR与ErbB-4带有完整的胞外配体结合域和可激活的激酶结构域，ErbB-2的胞外域则可能不具备配体结合力，ErbB-3虽然能与ATP结合，但无活性酪氨酸激酶受体，它的同源二聚体不具备活性。EGFR的信号传递方式主要是结合特异的配体，引起二聚化，激活其胞内酪氨酸激酶活性，使末端的一些酪氨酸残基自磷酸化，从而激活下游信号通路。
研究发现，表皮生长因子受体在细胞的增殖、代谢等方面扮演着重要的角色。由于它在多种癌细胞中存在过表达现象，因而，以EGFR为药物靶点极具研究意义。目前针对EGFR的肿瘤分子靶向药物，按其性质主要分为两大类：一类是直接作用于细胞外受体区域的单克隆抗体；另一类是干扰细胞内EGFR酪氨酸激酶活性的小分子抑制剂。单克隆抗体药物通过与EGFR的膜外配体结合域作用，使EGF等内源性配体无法与EGFR结合，从而阻止信号传入细胞；小分子药物则通过与胞内酪氨酸激酶催化区结合，抑制其催化活性，从而阻断细胞增殖信号。
EGFR的突变主要集中在18-21号外显子上，这几个外显子负责编码EGFR酪氨酸激酶域。其中19号外显子缺失占据了EGFR酪氨酸激酶敏感性突变的44％。在21号外显子上的点突变-L858R突变，占据了EGFR酪氨酸激酶敏感性突变的41％。719号残基从甘氨酸突变到丝氨酸，丙氨酸或者半胱氨酸占据了总突变的10％，而20号外显子的插入或复制突变占据了剩余的5％。其中19号外显子缺失和L858R点突变是最普遍的敏感性突变。这些突变会增强EGFR激酶的活性，从而提高下游信号通路。另外报道发现，由EGFR酪氨酸激酶抑制剂治疗产生的耐药性中，50％的病人发现了20号外显子上的T790M点突变。这种突变被认为是在治疗期间产生的，因为在未治疗病人中并未检测到这种突变。针对这些不同的突变衍生出了一系列的小分子抑制剂。
首先是第一代EGFR小分子抑制剂，例如Gefitinib、Erlotinib。这些抑制剂主要针对的是敏感性突变，但随着T790M抗性突变的发现，患者逐渐产生耐药性。因此衍生出了第二代及第三代EGFR抑制剂，该类抑制剂主要通过分子上的迈克尔受体与蛋白的半胱氨酸797残基发生共价结合来提高抑制活性。
虽然利用第三代EGFR抑制剂治疗携带有T790M突变的非小细胞肺癌病人有希望，但耐药性也逐渐产生。经过研究发现其耐药性的发生主要是由于Cys797残基发生突变形成Ser797残基，破坏了小分子与激酶间的结合力，导致第三代抑制剂基本失去作用。
因此，本领域急需开发新一代的抑制剂来克服EGFR的L858R/T790M/C797S以及EGFR19del/T790M/C797S三突变。
发明内容
本发明的目的在于提供一种新型的EGFR抑制性化合物，这种化合物能够抑制EGFR19del/T790M/C797S三突变。
本发明的另一目的是提供包含上述化合物的药物组合物。
本发明还有一目的是提供上述化合物在制备治疗EGFR相关疾病或抑制EGFR的药物中的用途。
在第一方面，本发明提供通式I所示的化合物或其立体异构体或光学异构体或其药学上可接受的盐：
Figure PCTCN2020085438-appb-000001
式中，
W是CH或N；
X是CH或N；
Y是CH或N或C卤素；
Z是CH或N；
R 1的数量为1-5中任意的整数，任选位于1’、2’、5’、6’或7’位，并且独立选自：H、卤素、C1-C6取代或未取代的烷基、
Figure PCTCN2020085438-appb-000002
其中，n为0～4的整数；
P为1-2个C1-C3烷基或不存在；
Q为-OH、-SH、-NH 2、-NHCH 3、-COOH、-CONH 2、-NHCONH 2、-NHCONHNH 2、-SO 3H、-SO 2NH 2；
其中当R 4为
Figure PCTCN2020085438-appb-000003
时，在R 4各位置上可以具有一个或多个Q；
X为O、S、NH；
R 2选自：氢、C1-C5取代或未取代的烷基甲酰胺基、C2-C5取代或未取代的烯基甲酰胺基；
R 3选自：氢、卤素、NR 7R 8、取代的N-C1-C3烷基哌嗪基；
R 7和R 8独立选自：H、C1-C6取代或未取代的烷基、-(CH 2) nNR 9R 10，其中n是0-3的整数；
R 9和R 10独立选自：H、C1-C3取代或未取代的烷基。
在具体的实施方式中，
W是CH；
X是CH或N；
Y是CH或N；
Z是CH；
R 1的数量为1或2，任选位于1’或5’位，并且独立选自：H、C1-C6取代或未取代的烷基羟基或多羟基、卤素；
R 2选自：氢、C1-C3取代或未取代的烷基甲酰胺基、C2-C3取代或未取代的烯基甲酰胺基；
R 3选自：卤素、NR 7R 8；
R 7和R 8独立选自：H、C1-C3取代或未取代的烷基、-(CH 2) nNR 9R 10，其中n是1或2；
R 9和R 10独立选自：H、C1-C3取代或未取代的烷基。
在具体的实施方式中，本发明提供以下的化合物或其立体异构体或光学异构体或其药学上可接受的盐：
Figure PCTCN2020085438-appb-000004
在第二方面，本发明提供通式II所示的化合物或其立体异构体或光学异构体或其药学上可接受的盐：
Figure PCTCN2020085438-appb-000005
式中，
R 1选自：
Figure PCTCN2020085438-appb-000006
其中，n为0～4的整数；
P为1-2个C1-C3烷基或不存在；
Q为-OH、-SH、-NH 2、-NHCH 3,、-COOH、-CONH 2、-NHCONH 2、-NHCONHNH 2、-SO 3H、-SO 2NH 2；
其中当R 4为
Figure PCTCN2020085438-appb-000007
时，在R 4各位置上可以具有一个或多个Q；
X为O、S、NH；
R 4选自：
Figure PCTCN2020085438-appb-000008
A的数量为0-4的任一整数，并且独立选自：卤素、取代或未取代的C1-C3烷氧基；
V、S、T的数量分别为0-4的任一整数，并且独立选自卤素、取代或未取代的C1-C3烷氧基；
R 5选自：H、取代或未取代的
Figure PCTCN2020085438-appb-000009
R 6选自：H、取代或未取代的C1-C3烷基。
在具体的实施方式中，
R 4选自：
Figure PCTCN2020085438-appb-000010
A的数量为0-4的任一整数，并且独立选自：卤素、取代或未取代的C1-C3烷氧基；
V的数量为0-4的任一整数，并且独立选自卤素、取代或未取代的C1-C3烷氧基；
R 5选自：取代或未取代的
Figure PCTCN2020085438-appb-000011
R 6选自：H。
在具体的实施方式中，本发明提供选自以下的化合物或其立体异构体或光学异构体或其药学上可接受的盐：
Figure PCTCN2020085438-appb-000012
在第三方面，本发明提供一种药物组合物，所述药物组合物含有第一或第二方面所述的化合物或其立体异构体或光学异构体或其药学上可接受的盐，以及任选的药学上可接受的载体或赋形剂。
在优选的实施方式中，所述药物组合物是适于口服的剂型，包括但不限于片剂、溶液剂、混悬液、胶囊剂、颗粒剂、粉剂。
在第四方面，本发明提供第一或第二方面所述的化合物或其立体异构体或光学异构体或其药学上可接受的盐，或第三方面所述的药物组合物在制备治疗或预防EGFR介导的疾病或抑制EGFR的药物中的用途。
在具体的实施方式中，所述EGFR介导的疾病为癌症。
在具体的实施方式中，所述癌症选自下组：非小细胞肺癌、小细胞肺癌、肺腺癌、肺鳞癌、乳腺癌、前列腺癌、神经胶质细胞瘤、卵巢癌、头颈部鳞癌、宫颈癌、食管癌、肝癌、肾癌、胰腺癌、结肠癌、皮肤癌、白血病、淋巴瘤、胃癌、多发性骨髓癌和实体瘤。
在第五方面，本发明提供治疗或预防EGFR介导的疾病方法，包括将第一或第二方面所述的化合物或第三方面所述的药物组合物给予有此需要的对象。
在优选的实施方式中，所述EGFR介导的疾病为癌症；优选地，所述癌症选自下组：非小细胞肺癌、小细胞肺癌、肺腺癌、肺鳞癌、乳腺癌、前列腺癌、神经胶质细胞瘤、卵巢癌、头颈部鳞癌、宫颈癌、食管癌、肝癌、肾癌、胰腺癌、结肠癌、皮肤癌、白血病、淋巴瘤、胃癌、多发性骨髓癌和实体瘤。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不 再一一累述。
附图说明
图1显示了化合物4的细胞活性评价，其中通过通过CCK-8测定检测了化合物对A431(A)、H1975(B)和BaF3/EGFR19D/T790M/C797S(C)的抑制活性。
具体实施方式
发明人经过广泛而深入的研究，出乎意料地发现了一系列结构全新的EGFR抑制性化合物，所述化合物能够显著抑制EGFR 19del/T790M/C797S三突变，从而能够开发作为新一代的EGFR抑制剂并且为开发新一代的EGFR抑制剂奠定了全新的物质基础。在此基础上完成了本发明。
术语定义
本文中涉及到的一些基团定义如下：
本文中，“烷基”指碳链长度为1-10个碳原子的饱和的支链或直链烷基，优选的烷基包括长2-8个、1-6个、1-4个、3-8个、1-3个碳原子不等的烷基。烷基的例子包括但不限于：甲基、乙基、正丙基、异丙基、正丁基、异丁基、庚基等。烷基可以被1个或多个取代基取代，例如被卤素或卤代烷基取代。例如，烷基可以是被1-4个氟原子取代的烷基，或者烷基可以是被氟代烷基取代的烷基。
本文中，“烷氧基”指被烷基取代的氧基。优选的烷氧基是长1-6个碳原子的烷氧基，更优选为长1-4个碳原子的烷氧基。烷氧基的例子包括但不限于：甲氧基、乙氧基、丙氧基等。
本文中，“卤素”指氟、氯、溴和碘。
本文中，“酰氨基”指结构式为“-R’-NH-C(O)-R”的基团，其中，R’可选自氢或烷基，R可选自烷基、链烯基、炔基、被NR cR d取代的烷基、被NR cR d取代的链烯基和NR cR d取代的炔基、被卤素取代的烷基、被氰基取代的链烯基，其中，R c和R d可选自烷基和链烯基。
本文中，“取代或未取代的”或“任选取代的”指其所修饰的取代基可任选地被1-5个(例如，1、2、3、4或5个)选自以下的取代基取代：卤素、C 1-4醛基、C 1-6直链或支链烷基、氰基、硝基、氨基、羟基、羟甲基、卤素取代的烷基(例如三氟甲基)、卤素取代的烷氧基(例如三氟甲氧基)、羧基、C 1-4烷氧基、乙氧甲酰基、N(CH 3)和C 1-4酰基。
本文所用的术语“取代”是指特定基团上的一个或多个氢原子被特定的取代基所替代。特定的取代基可以是前文中相应描述的取代基，也可以是各实施例中出现的具体取代基。因此，在本发明中，通式中的取代基可以各自独立地为实施例中具体化合物中的相应基团；即，本发明包括上述通式中各取代基的组合，也包括通式中所示部分取代基与实施例中出现的其它具体取代基的组合。
本发明的化合物
本发明提供了一系列结构全新的EGFR抑制性化合物，所述化合物能够显著抑制EGFR19del/T790M/C797S三突变。在具体的实施方式中，本发明提供通式I或II所示的化合物或 其立体异构体或光学异构体或其药学上可接受的盐：
Figure PCTCN2020085438-appb-000013
式中，各取代基如上所述定义。
在本发明的化合物的基础上，本领域技术人员可以其制成药学上可接受的盐。例如，可以将本发明的化合物与无机酸或有机酸反应形成常规的可药用盐。所述无机酸包括盐酸、氢溴酸、硫酸、硝酸、胺基磺酸和磷酸等，以及所述有机酸包括柠檬酸、酒石酸、乳酸、丙酮酸、乙酸、苯磺酸、对甲苯磺酸、甲磺酸、萘磺酸、乙磺酸、萘二磺酸、马来酸、苹果酸、丙二酸、富马酸、琥珀酸、丙酸、草酸、三氟乙酸、硬酯酸、扑酸、羟基马来酸、苯乙酸、苯甲酸、水杨酸、谷氨酸、抗坏血酸、对胺基苯磺酸、2-乙酰氧基苯甲酸和羟乙磺酸等；或者将本发明的化合物与无机碱形成钠盐、钾盐、钙盐、铝盐或铵盐；或者与有机碱形成甲胺盐、乙胺盐或乙醇胺盐。
除非特别说明，本发明所描述的结构式意在包括所有的同分异构形式(如对映异构，非对映异构和几何异构体(或构象异构体)：例如含有不对称中心的R、S构型，双键的(Z)、(E)异构体等。因此，本发明化合物的单个立体化学异构体或其对映异构体、非对映异构体或几何异构体(或构象异构体)的混合物都属于本发明的范围。
本发明的药物组合物以及施用方法
本发明的化合物能够用于制备治疗EGFR(尤其是EGFR 19del/T790M/C797S三突变)介导的疾病，例如癌症，包括但不限于：非小细胞肺癌、小细胞肺癌、肺腺癌、肺鳞癌、乳腺癌、前列腺癌、神经胶质细胞瘤、卵巢癌、头颈部鳞癌、宫颈癌、食管癌、肝癌、肾癌、胰腺癌、结肠癌、皮肤癌、白血病、淋巴瘤、胃癌、多发性骨髓癌和实体瘤。
有鉴于此，在本发明的化合物及其药学上可接受的盐的基础上。本发明还提供了包含本发明化合物的药物组合物，所述药物组合物任选包含药学上可接受的赋形剂。
在具体的实施方式中，本发明的药物组合物包含安全有效量范围内的本发明化合物或其药学上可接受的盐以及药学上可以接受的赋形剂或载体。其中“安全有效量”指的是：化合物的量足以明显改善病情，而不至于产生严重的副作用。
“药学上可以接受的赋形剂或载体”是指：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的化合物以及它们之间相互掺和，而不明显降低化合物的药效。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂
Figure PCTCN2020085438-appb-000014
润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
本发明化合物或药物组合物的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、瘤内、直肠、肠胃外(静脉内、肌肉内或皮下)、和局部给药。
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，活性化合物与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性化合物或化合物的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，活性化合物也可与上述赋形剂中的一种或多种形成微胶囊形式。
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性化合物外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。
除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、娇味剂和香料。
除了活性化合物外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
用于局部给药的本发明化合物的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
本发明化合物可以单独给药，或者与其他药学上可接受的化合物联合给药。使用药物组合物时，是将安全有效量的本发明化合物适用于需要治疗的哺乳动物(如人)，其中施用时剂量为药学上认为的有效给药剂量。本发明的化合物和药物组合物可通过口、鼻、皮肤、肺或者胃肠道等的给药途径。最优选为口服，一次性服用或分次服用。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。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明的优点：
1.本发明的化合物是结构全新的EGFR抑制性化合物；和
2.本发明的化合物能够显著抑制EGFR 19del/T790M/C797S三突变，从而能够开发作为新一代的EGFR抑制剂并且为开发新一代的EGFR抑制剂奠定了全新的物质基础。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例
材料
以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
制备方法
本发明的化合物的制备方法可以是本领域的常规方法，也可以采用本发明的合成路线。
吲哚类化合物合成方案
Figure PCTCN2020085438-appb-000015
其中，各个步骤的反应试剂和条件如下所示：
(a)DMSO,KOH,25℃,1h,3-chloropropan-1-ol,8h,36％；
(b)DCE,AlCl3,0℃,2,4-dichloropyrimidine,55℃,1.5h,31％；
(c)TsOH,2-pentanol,4-fluoro-2-methoxy-5-nitroaniline,105℃,2.5h,76％；
(d)Fe,MeOH/DCM,AcOH,55℃,4h；
(e)acryloyl chloride,DCM,DIPEA,0℃,1.5h,35％；
(f)N,N,N-trimethylethane-1,2-diamine,DMF,K2CO3,110℃,3h,60％；
(g)Pd/C,H2,MeOH,25℃,5h.
实施例1：化合物1的合成
3-(1H-吲哚-1-基)丙-1-醇(2b)的合成(步骤a)
Figure PCTCN2020085438-appb-000016
称取氢氧化钾(4.48g，80.0mmol)于250mL单口瓶，加入二甲基亚砜100mL，室温搅拌15min，加入2a(4.46g，40.0mmol)，室温反应1h，向溶液中逐滴加入3-氯-1-丙醇(5.67g，60.0mmol)，室温反应，TLC监测，反应8h，反应结束，加入大量水淬灭，用乙酸乙酯萃取，收集有机相，无水硫酸钠干燥，旋干溶剂，粗品经硅胶柱层析，以石油醚:乙酸乙酯＝20:1分离，得到2c黄色液体2.59g，收率36％。
1H NMR(400MHz,Chloroform-d):δ7.55(d,J＝7.9Hz,1H),7.29(d,J＝8.2Hz,1H),7.16–7.09(m,1H),7.06–6.97(m,2H),6.41(d,J＝3.1Hz,1H),4.17(t,J＝6.7Hz,2H),3.47(t,J＝6.0Hz,2H),1.94(p,J＝6.3Hz,2H),1.59(s,1H).LC-MS:m/z:176.2(M+H) +.
3-(3-(2-氯嘧啶-4-基)-1H-吲哚-1-基)丙-1-醇(2c)的合成(步骤b)
Figure PCTCN2020085438-appb-000017
将2b(2.59g，14.7mmol)溶于50mL 1，2-二氯乙烷，冰浴至0℃，加入无水三氯化铝(2.81g，21.0mmol)，10min后升至室温搅拌15min，加入2，4-二氯嘧啶(1.73g，11.0mmol)，升温至55℃并反应1.5h，TLC监测，反应结束，静置冷却至室温，随后冰浴下加入甲醇/水(20mL/10mL)，再恢复至室温搅拌30min后，抽滤并收集滤液，并用二氯甲烷萃取，无水硫酸钠干燥，旋干溶剂经硅胶柱层析，以石油醚:乙酸乙酯＝20:1分离，得到2c淡黄色固体1.05g，收率31％。
1H NMR(400MHz,Chloroform-d):δ8.30(d,J＝5.4Hz,1H),8.26–8.19(m,1H),7.92(s,1H),7.45–7.31(m,2H),7.27–7.19(m,2H),4.27(t,J＝6.7Hz,2H),3.54(t,J＝5.7Hz,2H),2.02(p,J＝6.3Hz,2H).LC-MS:m/z:288.1(M+H) +.
3-(3-(2-((4-氟-2-甲氧基-5-硝基苯基)氨基)嘧啶-4-基)-1H-吲哚-1-基)丙-1-醇(2e)的合成 (步骤c)
Figure PCTCN2020085438-appb-000018
将4-甲基苯磺酸水合物(0.75g，4.3mmol)一次性加入到含有2c(1.05g，3.6mmol)和4-氟-2-甲氧基-5-硝基苯胺(0.75g，4.0mmol)的25mL 2-戊醇溶液中。所得混合物在105℃下搅拌2.5h。TLC监测，结束反应，将该混合物冷却至室温。过滤，滤饼用冰的2-戊醇洗涤，收集滤饼并真空干燥，得到2d黄色固体1.225g，收率76％。
1H NMR(400MHz,DMSO-d 6):δ10.21(s,1H),8.83(s,1H),8.73(d,J＝8.2Hz,1H),8.37(d,J＝6.6Hz,1H),8.16(s,1H),7.65(d,J＝8.3Hz,1H),7.58–7.48(m,2H),7.30(t,J＝7.4Hz,1H),7.11(q,J＝7.5,6.8Hz,1H),4.37(t,J＝7.0Hz,2H),4.00(s,3H),3.43(t,J＝6.0Hz,2H),2.52(p,J＝1.9Hz,1H),2.00(p,J＝6.5Hz,2H).LC-MS:m/z:438.2(M+H) +.
3-(3-(2-((5-氨基-4-氟-2-甲氧基苯基)氨基)嘧啶-4-基)-1H-吲哚-1-基)丙-1-醇(2f)的合成(步骤d)
Figure PCTCN2020085438-appb-000019
将2d(600.00mg，1.4mmol)溶于混合溶剂(二氯甲烷∶甲醇＝3:1)40mL中，加入铁粉(2.92g，52.1mmol)和冰醋酸12mL，搅拌，于55℃反应4h。TLC监测，反应结束，过滤，将滤液浓缩至干，向残余物加入饱和碳酸氢钠溶液调节pH至碱性。用乙酸乙酯萃取，收集有机相并用无水硫酸钠干燥，旋干溶剂，直接用于下一步反应。
N-(2-氟-5-((4-(1-(3-羟丙基)-1H-吲哚-3基)嘧啶-2-基)氨基)-4-甲氧基苯基)丙烯酰胺(1)的合成(步骤e)
Figure PCTCN2020085438-appb-000020
将溶于3.5mL二氯甲烷中的丙烯酰氯(128.00mg，1.4mmol)滴加到溶于16mL二氯甲烷的2e(500.00mg，1.2mmol)和N，N-二异丙基乙胺(0.24mL，1.4mmol)的溶液中，在冰水 浴中冷却。将混合物搅拌1.5h，TLC监测，反应结束，使用少量二氯甲烷稀释并用饱和碳酸氢钠水溶液洗涤。收集有机相，同时将含水层用二氯甲烷再次萃取，合并有机相并用无水硫酸钠干燥，旋干溶剂经硅胶柱层析，以二氯甲烷:甲醇＝200:1分离，得到化合物1黄色固体200mg，收率35％。
1H NMR(400MHz,DMSO-d 6):δ9.88(s,1H),8.61(d,J＝8.5Hz,1H),8.47(s,1H),8.32(dd,J＝8.7,6.5Hz,2H),8.09(s,1H),7.56(d,J＝8.2Hz,1H),7.31–7.18(m,2H),7.18–7.07(m,2H),6.59(dd,J＝17.0,10.2Hz,1H),6.26(dd,J＝17.1,2.0Hz,1H),5.76(dd,J＝10.2,2.0Hz,1H),4.69(s,1H),4.33(t,J＝7.0Hz,2H),3.87(s,3H),3.40(t,J＝6.2Hz,2H),1.96(p,J＝6.6Hz,2H). 13C NMR(151MHz,DMSO):δ163.79,162.58,160.30,151.53,149.93,148.22,137.47,133.06,132.01,127.26,125.97,125.04,122.59,122.39,121.39,118.59,117.76,113.01,110.99,107.87,100.23,58.19,56.85,43.56,33.24.HRMS(EI)(m/z):calcd for C 25H 24FN 5O 3[M] +461.1863,found 461.1862.
实施例2：N-(2-氟-5-((4-(1-(3-羟丙基)-1H-吲哚-3-基)嘧啶-2-基)氨基)-4-甲氧基苯基)丙酰胺(2)的合成(步骤e)
Figure PCTCN2020085438-appb-000021
化合物2是参照化合物1的合成路线得到。淡黄色固体，收率为23％。
1H NMR(400MHz,DMSO-d 6):δ9.54(s,1H),8.49(d,J＝8.6Hz,1H),8.46(s,1H),8.35(d,J＝8.0Hz,1H),8.31(d,J＝5.4Hz,1H),8.00(s,1H),7.57(d,J＝8.2Hz,1H),7.27–7.21(m,2H),7.19–7.13(m,1H),7.08(d,J＝12.2Hz,1H),4.70(t,J＝4.9Hz,1H),4.34(t,J＝7.0Hz,2H),3.86(s,3H),3.42(q,J＝5.8Hz,2H),2.37(q,J＝7.5Hz,2H),1.97(p,J＝6.5Hz,2H),1.08(t,J＝7.6Hz,3H). 13C NMR(151MHz,DMSO-d 6):δ172.49,162.45,160.50,157.76,151.61,150.02,147.90,137.45,132.90,126.00,125.07,122.56,121.31,118.90,118.12,113.02,110.97,107.84,100.13,58.20,56.81,43.53,33.25,29.28,10.19.HRMS(EI)(m/z):calcd forC 25H 24FN 5O 3[M] +463.2020,found 463.2021.
3-(3-(2-((4-((2-(二甲基氨基)乙基)(甲基)氨基)-2-甲氧基-5-硝基苯基)氨基)嘧啶-4-基)-1H-吲哚-1-基)丙-1-醇(2i)的合成
Figure PCTCN2020085438-appb-000022
将N，N，N'-三甲基乙二胺(35.00mg，0.3mmol)、N,N-二异丙基乙胺(46.00mg，0.3mmol)和2.5mL N，N-二甲基乙酰胺依次装入10mL单口瓶，25℃反应0.5h，之后加入2d(108.00mg， 0.6mmol)，升温至85℃反应3h，TLC监测，停止反应，静置至室温，加入饱和食盐水和乙酸乙酯萃取，收集有机相并用无水硫酸钠干燥，抽滤，滤液旋干经硅胶柱层析，以二氯甲烷:甲醇＝20:1分离，得到2i黄红色固体84mg，收率65％。
1H NMR(400MHz,Chloroform-d):9.50(s,1H),9.00(s,1H),8.30(d,J＝5.2Hz,1H),8.03–7.94(m,1H),7.67(s,1H),7.41–7.33(m,1H),7.19(dq,J＝8.0,5.3Hz,3H),6.71(s,1H),4.54(t,J＝5.8Hz,2H),3.73(s,3H),3.51(t,J＝5.2Hz,2H),2.95–2.88(m,2H),2.62(s,3H),2.43(q,J＝7.7Hz,2H),2.32(s,6H),2.00(p,J＝5.2Hz,2H).
实施例3：N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-5-((4-(1-(3羟丙基)-1H-吲哚-3-基)嘧啶-2-基)氨基)-4-甲氧基苯基)丙酰胺(3)的合成
Figure PCTCN2020085438-appb-000023
淡橙色固体，收率26％。
1H NMR(400MHz,Chloroform-d):δ9.84(s,1H),9.50(s,1H),9.00(s,1H),8.30(d,J＝5.2Hz,1H),8.03–7.94(m,1H),7.67(s,1H),7.41–7.33(m,1H),7.19(dq,J＝8.0,5.3Hz,3H),6.67(s,1H),4.44(t,J＝5.8Hz,2H),3.79(s,3H),3.48(t,J＝5.2Hz,2H),2.95–2.88(m,2H),2.59(s,3H),2.49(q,J＝7.7Hz,2H),2.38(s,2H),2.29(s,6H),2.00(p,J＝5.2Hz,2H),1.28(t,J＝7.6Hz,3H). 13C NMR(151MHz,CDCl 3-d 6)δ173.20,161.94,159.52,158.07,144.35,137.12,134.86,134.36,129.30,127.77,126.26,121.84,121.03,120.37,113.58,110.55,109.76,107.95,104.66,57.49,57.23,56.11,55.79,45.29,44.32,42.54,32.54,31.26,10.90.HRMS(EI)(m/z):calcd for C 30H 39N 7O 3[M] +545.3114,found 545.3111.
N1-(3-甲氧基-4-硝基苯基)-N1，N2，N2-三甲基乙烷-1,2-二胺(2g)的合成(步骤f)
Figure PCTCN2020085438-appb-000024
将2f(425.00mg，2.5mmol)溶于5mL N，N-二甲基甲酰胺，依次向其中加入N，N，N'-三甲基乙二胺(380.00mg，3.72mmol)和碳酸钾(750.00mg，4.9mmol)，于110℃下反应3h，TLC监测，反应结束冷却至室温，加入水，并用乙酸乙酯萃取，收集有机相，无水硫酸钠干燥，抽滤，旋干滤液经硅胶柱层析，以二氯甲烷:甲醇＝100:1分离，得到2g黄绿色液体370mg，收率60％。
1H NMR(400MHz,Chloroform-d):δ8.00–7.86(m,1H),6.17(ddd,J＝9.5,6.8,2.4Hz,1H),6.11–6.00(m,1H),3.89(dd,J＝5.8,1.8Hz,3H),3.48(q,J＝6.8Hz,2H),3.09–2.98(m, 3H),2.45(dt,J＝10.3,6.4Hz,2H),2.24(dd,J＝5.6,1.8Hz,6H)。LC-MS:m/z:254.3(M+H) +.
N1-(3-甲氧基-4-胺基苯基)-N1，N2，N2-三甲基乙烷-1,2-二胺(2h)的合成(步骤g)
Figure PCTCN2020085438-appb-000025
将2g(250.00mg，1.0mmol)装入50mL三口瓶，加入10mL甲醇，溶解后加入钯碳(50.00mg，20％)，氢气置换三次，于25℃反应5h，硅藻土助滤，收集滤液，旋干，直接用于下一步反应。
实施例4：3-(3-(2-((4-((2-(二甲基氨基)乙基)(甲基)氨基)-2-甲氧基苯基)氨基)嘧啶-4-基)-1H-吲哚-1-基)丙-1-醇(4)的合成
Figure PCTCN2020085438-appb-000026
绿色固体，收率14％。
1H NMR(400MHz,Chloroform-d):δ8.30–8.25(m,1H),8.23–8.17(m,2H),7.84(s,1H),7.38–7.31(m,1H),7.25(s,1H),7.22–7.13(m,3H),6.91(d,J＝5.3Hz,1H),6.38(dd,J＝8.8,2.7Hz,1H),6.32(d,J＝2.7Hz,1H),4.27(t,J＝6.7Hz,2H),3.80(s,3H),3.54(t,J＝5.8Hz,2H),3.47(t,J＝7.4Hz,2H),2.87(s,3H),2.63(t,J＝7.3Hz,2H),2.39(s,6H),2.01(p,J＝6.4Hz,2H). 13C NMR(151MHz,DMSO-d 6):δ162.59,161.61,157.33,152.88,146.91,146.10,138.16,132.17,126.23,125.98,123.18,122.52,121.02,119.12,113.22,110.77,106.71,104.46,97.44,58.15,55.92,54.72,49.35,44.68,43.46,39.07,33.18,21.26.HRMS(EI)(m/z):calcd for C 27H 34N 6O 2[M] +474.2743,found 474.2745.
7-乙烯基-1H-吲哚(2k)的合成
Figure PCTCN2020085438-appb-000027
将甲基三苯基碘化(1.30g，3.2mmol)装入100mL三口瓶，氮气置换三次，加入10mL无水四氢呋喃，通过恒压滴液漏斗向三口瓶中逐滴加入双(三甲基硅烷基)氨基钾(3.22mL，3.2mmol)和10mL无水四氢呋喃混合液，25℃反应1h，加入2j(188.00mg，1.3mmol)并于 25℃反应2h，TLC监测，停止反应，饱和氯化铵溶液淬灭，加入水与乙酸乙酯萃取，无水硫酸钠干燥，抽滤，旋干滤液经硅胶柱层析，以石油醚:乙酸乙酯＝15:1分离，得到中间体淡绿色液体171mg，收率90％。冰浴下，将中间体装入100mL三口瓶，加入10mL无水四氢呋喃混合，氮气置换三次，逐滴加入硼烷四氢呋喃(5mL，5.0mmol)，滴加完毕，恢复至室温，反应1h，之后再逐滴缓慢加入氢氧化钠溶液(10mL，1mol/L)以及双氧水(10mL，30％)，回流反应1h，TLC监测，反应结束，冷却至室温并加入15mL饱和亚硫酸钠溶液，乙酸乙酯萃取，收集有机相，无水硫酸钠干燥，抽滤，滤液经硅胶柱层析，以石油醚；乙酸乙酯＝20:1分离，得到2k淡绿色液体38mg，收率20％。
1H NMR(400MHz,Chloroform-d):δ8.91(s,1H),7.46(d,J＝7.8Hz,1H),7.09(t,J＝2.8Hz,1H),6.98(t,J＝7.5Hz,1H),6.89(d,J＝7.0Hz,1H),6.47(dd,J＝3.2,2.0Hz,1H),3.87(t,J＝5.8Hz,2H),2.98(t,J＝5.7Hz,2H).LC-MS:m/z:162.2(M+H) +.
实施例5：2-(3-(2-((4-((2-(二甲基氨基)乙基)(甲基)氨基)-2-甲氧基苯基)氨基)嘧啶-4-基)-1H-吲哚-7-基)乙醇(10)的合成
Figure PCTCN2020085438-appb-000028
化合物10是参照化合物4的合成路线得到。淡黄色固体，收率23％。
1H NMR(400MHz,DMSO-d 6):δ11.69(d,J＝3.0Hz,1H),8.24(d,J＝6.7Hz,1H),8.21(d,J＝3.0Hz,1H),8.18(d,J＝5.3Hz,1H),7.78(s,1H),7.63(d,J＝8.7Hz,1H),7.15(d,J＝5.3Hz,1H),6.99(q,J＝4.2Hz,2H),6.42(d,J＝2.6Hz,1H),6.32(dd,J＝8.8,2.6Hz,1H),4.71(t,J＝5.1Hz,1H),3.80(s,3H),3.71(td,J＝7.0,4.8Hz,2H),3.49(t,J＝7.2Hz,2H),3.03(t,J＝7.0Hz,2H),2.94(s,3H),2.57(s,2H),2.32(s,6H). 13C NMR(151MHz,DMSO-d 6):δ167.54,164.71,156.53,153.88,147.54,144.32,139.21,134.32,127.63,126.32,122.78,122.62,120.32,119.54,113.73,110.81,106.82,103.86,97.74,60.25,55.52,54.42,49.43,44.78,43.46,33.32.HRMS(EI)(m/z):calcd for C 26H 32N 6O 2[M] +460.2587,found 460.2581.
实施例6：N-(2-氟-5-((4-(6-氟-1-(3-羟基丙基)-1H-吲哚-3-基)嘧啶-2-基)氨基)-4-甲氧基苯基)丙酰胺(5)的合成
Figure PCTCN2020085438-appb-000029
化合物5是参照化合物1的合成路线得到。黄色固体，收率21％。
1H NMR(400MHz,DMSO-d 6):δ9.54(s,1H),8.45(s,1H),8.33(dd,J＝21.5,6.9Hz,3H),8.17(s,1H),7.46(dd,J＝10.2,2.4Hz,1H),7.23(d,J＝5.5Hz,1H),7.09(d,J＝12.1Hz,1H),6.99(td,J＝9.2,2.3Hz,1H),4.30(t,J＝6.9Hz,2H),3.85(s,3H),3.41(t,J＝6.1Hz,2H),2.35(q,J＝7.5Hz,2H),1.96(p,J＝6.6Hz,2H),1.07(t,J＝7.6Hz,3H). 13C NMR(151MHz,DMSO):δ172.50,162.42,160.42,158.85,152.08,150.45,148.58,137.74,133.58,124.70,123.93,122.68,119.81,118.12,113.25,109.64,107.61,100.35,97.58,58.09,56.79,43.67,33.09,29.24,10.13.HRMS(EI)(m/z):calcd for C 25H 25F 2N 5O 3[M] +481.1925,found 481.1927.
2-(1H-吲哚-1-基)乙醇(2l)的合成
Figure PCTCN2020085438-appb-000030
将2-溴乙醇(230.00mg，1.8mmol)装入25mL单口瓶，加入4mL二氯甲烷溶解，随后依次加入叔丁基二甲基氯硅烷(300.00mg，2.0mmol)和咪唑(140.00mg，2.0mmol)，于25℃反应3h，抽滤，二氯甲烷洗涤，收集滤液，旋干得到中间体，称取2a(108.00mg，0.9mmol)和氢化钠(72.00mg，1.8mmol)置于50mL两口瓶中，氮气置换3次，冰浴下，缓慢加入无水7mL N，N-二甲基甲酰胺溶液，搅拌15分钟后，加入中间体，升温至100℃反应4h，TLC监测，反应结束冷却至室温，加水淬灭并用二氯甲烷萃取，收集有机相，无水硫酸钠干燥，旋干得到2l黄色液体230mg，收率80％。
1H NMR(400MHz,Chloroform-d):δ7.55(d,J＝7.9Hz,1H),7.29(d,J＝8.2Hz,1H),7.16–7.09(m,1H),7.06–6.97(m,2H),6.41(d,J＝3.1Hz,1H),4.09(t,J＝6.7Hz,2H),3.50(t,J＝6.0Hz,2H),.LC-MS:m/z:162.2(M+H) +.
实施例7：N-(2-氟-5-((4-(1-(2-羟乙基)-1H-吲哚-3-基)嘧啶-2-基)氨基)-4-甲氧基苯基)丙酰胺(6)的合成
Figure PCTCN2020085438-appb-000031
化合物6是参照化合物1的合成路线得到。白色固体，收率23％。
1H NMR(400MHz,DMSO-d 6):δ9.51(s,1H),8.48(d,J＝8.4Hz,1H),8.42(s,1H),8.32(dd,J＝13.6,6.7Hz,2H),7.97(s,1H),7.57(d,J＝8.1Hz,1H),7.22(t,J＝6.2Hz,2H),7.15(t,J＝7.5Hz,1H),7.07(d,J＝12.0Hz,1H),4.96(t,J＝5.1Hz,1H),4.32(t,J＝5.5Hz,2H),3.86(s,3H),3.79(q,J＝5.4Hz,2H),2.36(q,J＝7.5Hz,2H),1.07(t,J＝7.5Hz,3H). 13C NMR(151MHz,DMSO):δ172.48,172.45,162.52,160.49,157.72,151.66,150.06,147.97,147.90,137.73,133.47,126.04,125.08,122.40,121.25,118.94,118.09,118.00,112.86,111.19,107.80,100.21,100.05,60.44,56.81,49.25,29.25,10.16.HRMS(EI)(m/z):calcd for C 24H 24FN 5O 3[M] +449.1863,found 449.1862.
实施例8：N-(2-氟-5-((4-(1-(4-羟基丁基)-1H-吲哚-3-基)嘧啶-2-基)氨基)-4-甲氧基苯基)丙酰胺(7)的合成
Figure PCTCN2020085438-appb-000032
化合物7是参照化合物1的合成路线得到。土黄色固体，收率10％。
1H NMR(400MHz,DMSO-d 6):δ9.54(s,1H),8.52(s,1H),8.48(d,J＝8.6Hz,1H),8.31(t,J＝7.3Hz,2H),8.13(s,1H),7.58(d,J＝8.2Hz,1H),7.28–7.21(m,2H),7.16(t,J＝7.5Hz,1H),7.08(d,J＝12.2Hz,1H),4.30(t,J＝7.0Hz,2H),3.86(s,3H),3.41(t,J＝6.4Hz,2H),2.37(q,J＝7.6Hz,2H),1.92–1.79(m,2H),1.51–1.35(m,2H),1.08(t,J＝7.6Hz,3H). 13C NMR(151MHz,DMSO):δ172.52,137.54,126.01,122.79,122.52,121.61,112.83,111.21,107.61,100.44,100.27,60.71,56.84,46.54,30.14,29.29,26.88,10.18.HRMS(EI)(m/z):calcd for C 26H 28FN 5O 3[M] +477.2176,found 477.2179.
1H-吲哚-2-甲醛(2n)的合成
Figure PCTCN2020085438-appb-000033
冰浴下，将2m(806.00mg，5.0mmol)装入100mL三口瓶，加入30mL无水四氢呋喃溶解，边搅拌边分批加入四氢铝锂(380.00mg，10.0mmol)，加入完毕恢复至室温，反应14h，反应结束，饱和氯化铵溶液淬灭，抽滤，收集滤液并用乙酸乙酯萃取，无水硫酸钠干燥，旋干溶剂得到中间体。将中间体溶解于10mL二甲基亚砜，加入2-碘酰基苯甲酸(1.70g，6.0mmol)，于25℃反应10h，TLC监测，结束反应加入大量水并乙酸乙酯萃取，无水硫酸钠干燥，旋干滤液经硅胶柱层析，以石油醚:乙酸乙酯＝20:1分离，得到2n白色固体650mg，收率89％。
1H NMR(400MHz,Chloroform-d):δ9.78(s,1H),9.30(s,1H),7.68(d,J＝8.0Hz,1H),7.43–7.37(m,1H),7.32(ddd,J＝8.2,6.8,1.1Hz,1H),7.23–7.16(m,1H),7.11(ddd,J＝8.0,6.9,1.0Hz,1H).LC-MS:m/z:146.1(M+H) +.
3-(1H-吲哚-2-基)丙-1-醇(2o)的合成
Figure PCTCN2020085438-appb-000034
冰浴下，将磷酰基乙酸三乙酯(6.67g，29.7mmol)和氢化钠(1.5g，37.5mmol)装入100mL三口瓶，缓慢加入20mL无水四氢呋喃溶解，搅拌1h后，通过恒压滴液漏斗逐滴加入溶于20mL无水四氢呋喃的2n(3.60g，24.8mmol)溶液，于25℃反应10h，TLC监测，结束反 应用饱和氯化铵溶液淬灭反应，旋干溶剂，用水与乙酸乙酯萃取，无水硫酸钠干燥，旋干滤液得到中间体。冰浴下，将四氢铝锂(2.00g，52.6mmol)缓慢加入到30mL无水四氢呋喃溶液中，接着向其中缓慢加入溶于30mL无水四氢呋喃的中间体溶液，于20℃反应8h，TLC监测，结束反应，加水淬灭并用乙酸乙酯萃取，无水硫酸钠干燥，旋干滤液经硅胶柱层析，以石油醚:乙酸乙酯＝8:1分离得到2o白色固体2.58g，收率60％。
1H NMR(400MHz,Chloroform-d):δ8.15(s,1H),7.44(dd,J＝7.2,1.6Hz,1H),7.20–7.13(m,1H),7.00(dtd,J＝16.2,7.2,1.3Hz,2H),6.16–6.09(m,1H),3.57(t,J＝6.1Hz,2H),2.70(t,J＝7.3Hz,2H),1.81(tt,J＝7.3,6.1Hz,2H).LC-MS:m/z:176.2(M+H) +.
实施例9：N-(2-氟-5-((4-(2-(3-羟基丙基)-1H-吲哚-3-基)嘧啶-2-基)氨基)-4-甲氧基苯基)丙酰胺(8)的合成
Figure PCTCN2020085438-appb-000035
化合物8是参照化合物1的合成路线得到。黄褐色固体，收率23％。
1H NMR(400MHz,DMSO-d 6):δ11.56(s,1H),9.45(s,1H),8.36(d,J＝5.3Hz,1H),8.29(d,J＝8.5Hz,1H),8.07–7.94(m,2H),7.36(d,J＝7.9Hz,1H),7.14–7.01(m,4H),4.60(d,J＝5.2Hz,1H),3.85(s,3H),3.44(q,J＝5.6Hz,2H),3.11(t,J＝7.8Hz,2H),2.31(q,J＝7.5Hz,2H),1.85(p,J＝6.7Hz,2H),1.05(t,J＝7.5Hz,3H). 13C NMR(151MHz,DMSO):δ172.41,163.18,160.64,157.83,143.57,135.84,126.99,125.08,121.73,120.60,119.76,111.56,110.31,109.85,100.17,60.76,56.82,32.64,29.12,24.61,10.17.HRMS(EI)(m/z):calcd forC 25H 26FN 5O 3[M] +463.2020,found 463.2023.
实施例10：N-(2-氟-5-((4-(7-(2-羟基乙基)-1H-吲哚-3-基)嘧啶-2-基)氨基)-4-甲氧基苯基)丙酰胺(9)的合成
Figure PCTCN2020085438-appb-000036
化合物9是参照化合物1的合成路线得到。浅黄色固体，收率22％。
1H NMR(400MHz,DMSO-d 6):δ11.73(d,J＝3.0Hz,1H),9.50(s,1H),8.36(d,J＝8.6Hz,1H),8.30(d,J＝3.0Hz,1H),8.27(d,J＝5.4Hz,1H),8.25–8.19(m,1H),7.99(s,1H),7.29(d,J＝5.4Hz,1H),7.10–6.97(m,3H),4.71(t,J＝5.0Hz,1H),3.85(s,3H),3.72(td,J＝7.0,4.7Hz,2H),3.04(t,J＝7.0Hz,2H),2.33(q,J＝7.6Hz,2H),1.06(t,J＝7.5Hz,3H). 13C NMR(151MHz,DMSO):δ172.48,172.45,162.52,160.49,157.72,151.66,150.06,147.97,148.91,137.73,133.47,126.04,125.08,122.40,121.25,118.95,118.06,118.00,112.86,112.19,107.80,100.21,99.05,60.44,56.81,49.25,29.25,10.16.HRMS(EI)(m/z):calcd for C 24H 24FN 5O 3[M] +449.1863,found 449.1862.
实施例11：N-(2-氟-5-((6-(6-氟-1-(3-羟基丙基)-1H-吲哚-3-基)嘧啶-4-基)氨基)-4-甲氧基苯基)丙酰胺(16)的合成
Figure PCTCN2020085438-appb-000037
化合物16是参照化合物1的合成路线得到。白色固体，收率30％。
1H NMR(400MHz,DMSO-d 6):δ9.53(s,1H),8.72(s,1H),8.54(d,J＝1.0Hz,1H),8.31(dd,J＝8.9,5.6Hz,1H),8.26(d,J＝8.5Hz,1H),8.15(s,1H),7.47(dd,J＝10.2,2.4Hz,1H),7.29(s,1H),7.12–6.98(m,2H),4.67(t,J＝5.0Hz,1H),4.29(t,J＝6.8Hz,2H),3.87(s,3H),2.35(q,J＝7.5Hz,2H),1.92(q,J＝6.5Hz,2H),1.08(t,J＝7.6Hz,3H). 13C NMR(151MHz,DMSO):δ172.59,161.36,158.75,158.30,137.65,131.87,124.23,122.90,122.51,120.49,117.99,113.68,109.42,109.27,100.36,97.58,58.04,56.80,43.43,33.03,29.15,10.18.HRMS(EI)(m/z):calcd for C 25H 25F 2N 5O 3[M] +481.1925,found 481.1927.
三取代咪唑类化合物的合成方案
Figure PCTCN2020085438-appb-000038
其中，各个步骤的反应试剂和条件如下所示：
(h)SeO2,1,4-dioxane,H2O,100℃,overnight；
(i)HCl,H2O,25℃,5h；
(j)NH4OAc,MeOH,25℃,overnight,6％；
(k)Pd/C,H2,MeOH,25℃,6h,69％；
(l)2-(1-oxoisoindolin-2-yl)-2-phenylacetic acid,HATU,DIPEA,DMF,25℃,6h,5％；
(m)NIS,MeCN,80℃,16h,30％；
(n)1,4-dioxane,K2CO3,H2O,Pd(dppf)Cl2·CH2Cl2,N-phenyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine,90℃,12h,32％.
3-(4-(4-硝基苯基)-1H-咪唑-2-基)丙-1-醇(2t)的合成(步骤j)
Figure PCTCN2020085438-appb-000039
将二氧化硒(5.36g，48.7mmol)装入250mL三口瓶，加入60mL 1，4-二氧六环和20mL水于55℃搅拌溶解，待彻底溶解后，加入2p(2.00g，12.1mmol)，升温至100℃，反应10h，结束反应冷却至室温，抽滤，并用硅藻土助滤，收集滤液并用乙酸乙酯萃取，无水硫酸钠干燥，旋干滤液经硅胶柱层析，得到2q中间体。冰浴下，称取盐酸(14mL，2mol/L)装入50mL三口瓶中，将2r(1.40g，20.0mmol)逐滴加入，搅拌0.5h，恢复至室温继续反应5h，TLC检测，结束反应用二氯甲烷萃取，收集有机相，旋干溶剂，得到中间2s。将2s和醋酸铵(4.90g，63.6mmol)依次装入250mL单口瓶，加入30mL甲醇溶解，2p溶于60mL甲醇中，并将其逐滴滴加至单口瓶中，25℃反应过夜，TLC检测，结束反应将溶剂浓缩，使用饱和碳酸氢钠调节pH值至7，加入乙酸乙酯萃取，无水硫酸钠干燥，抽滤，滤液经硅胶柱层析，以二氯甲烷:甲醇＝150:1分离，得到2t黄色液体260mg，收率6％。
1H NMR(400MHz,DMSO-d 6):δ8.25–8.17(m,2H),8.02–7.94(m,2H),7.81(s,1H),3.48(t,J＝6.3Hz,2H),2.72(dd,J＝8.2,7.1Hz,2H),1.85(dq,J＝8.1,6.4Hz,2H)..LC-MS:m/z:248.1(M+H) +.
3-(4-(4-氨基苯基)-1H-咪唑-2-基)丙-1-醇(2u)的合成(步骤k)
Figure PCTCN2020085438-appb-000040
将2t(184.00mg，0.8mmol)、钯碳(40.00mg，20％)和12mL甲醇依次装入100ml单口瓶，氢气置换三次，25℃，反应6h，TLC监测，结束反应，硅藻土助滤，滤液经硅胶柱层 析，以二氯甲烷:甲醇＝40:1分离，得到2u橙黄色液体112mg，收率69％。
1H NMR(400MHz,DMSO-d 6):δ7.39–7.31(m,2H),7.09(s,1H),6.60–6.51(m,2H),3.47(t,J＝6.3Hz,2H),2.67(t,J＝7.6Hz,2H),1.89–1.73(m,2H).LC-MS(m/z):218.1(M+H) +.
2-(1-氧代异吲哚啉-2-基)-2-苯基乙酸(2w)的合成
Figure PCTCN2020085438-appb-000041
将邻苯二甲醛(1.00g，7.5mmol)装入250mL单口瓶，用35mL乙腈溶解，加入D,L-苯甘氨酸(1.13g，7.5mmol)和2mL醋酸，氮气置换3次，回流反应3h，TLC监测，反应结束，抽滤，淋洗滤饼并真空干燥得到2w黑色固体1.3g，收率65％。
1H NMR(400MHz,DMSO-d 6):δ13.39(s,1H),7.75(d,J＝7.5Hz,1H),7.60(td,J＝7.4,1.2Hz,1H),7.57–7.49(m,2H),7.50–7.37(m,5H),6.00(s,1H),4.64(d,J＝17.4Hz,1H),3.92(d,J＝17.4Hz,1H).LC-MS:m/z:268.1(M+H) +.
实施例12：N-(3-(2-(3-羟丙基)-1H-咪唑-4-基)苯基)-2-(1-氧代异吲哚啉-2-基)-2-苯基乙酰胺(11)的合成
Figure PCTCN2020085438-appb-000042
将2u(176.00mg，0.8mmol)、2w(108.00mg，0.4mmol)和2-(7-氧化苯并三氮唑)-N,N,N’,N’-四甲基脲六氟磷酸盐(308.00mg，0.8mmol)装入25mL单口瓶中，加入2.5mL N，N-二甲基甲酰胺溶解，搅拌下加入N，N-二异丙基乙胺(212.00mg，1.6mmol)，25℃反应6h，TLC监测，反应结束用水与乙酸乙酯萃取，收集有机相，无水硫酸钠干燥，抽滤，滤液经硅胶柱层析，以二氯甲烷:甲醇＝200:1分离，得到11白色固体20mg，收率5％。
1H NMR(400MHz,DMSO-d 6):δ10.57(s,1H),8.01(d,J＝1.8Hz,1H),7.76(d,J＝7.5Hz,1H),7.65–7.35(m,12H),7.29(t,J＝7.9Hz,1H),6.24(s,1H),4.85(d,J＝17.7Hz,1H),3.97(d,J＝17.6Hz,1H),3.45(t,J＝6.3Hz,2H),2.70(t,J＝7.6Hz,2H),1.82(p,J＝6.8Hz,2H). 13C NMR(151MHz,DMSO):δ168.63,168.18,149.08,142.88,139.32,135.93,132.16,132.00,129.49,129.08,128.93,128.43,124.12,123.41,120.28,117.77,115.72,60.62,59.02,48.90,31.58,24.94.HRMS(ESI)(m/z):calcd for C 39H 34N 6O 3[M+H] +467.2083,found 467.2084.
实施例13：N-(4-(2-(3-羟丙基)-1H-咪唑-4-基)苯基)-2-(1-氧代异吲哚啉-2-基)-2-苯基乙酰胺(12)的合成
Figure PCTCN2020085438-appb-000043
化合物12是参照化合物11的合成路线得到。土黄色固体，收率8％。
1H NMR(400MHz,DMSO-d 6):δ10.55(s,1H),7.76(d,J＝7.5Hz,1H),7.70–7.55(m,6H),7.54–7.37(m,7H),6.24(s,1H),4.85(d,J＝17.7Hz,1H),3.98(d,J＝17.7Hz,1H),3.46(t,J＝6.3Hz,2H),2.70(t,J＝7.6Hz,2H),1.83(p,J＝6.6Hz,2H). 13C NMR(151MHz,DMSO)δ168.74,168.23,150.06,148.90,147.73,142.88,138.65,135.71,133.80,132.21,131.94,129.56,129.05,128.46,127.57,126.69,125.79,124.14,123.41,120.11,114.30,112.01,60.23,59.05,48.89,30.85,23.80.HRMS(ESI)(m/z):calcd for C 39H 34N 6O 3[M+H] +467.2083,found 467.2084.
3-氯-N-苯基吡啶-2-胺(2x)的合成
Figure PCTCN2020085438-appb-000044
将醋酸钯(13.5mg，0.1mmol)和2，2'-双-(二苯膦基)-1，1'-联萘(37.5mg，0.1mmol)装入50mL单口瓶，氮气置换三次，用10mL甲苯溶解，25℃，搅拌10min，随后将溶液转移至含2，3-二氯吡啶(441.00mg，3.0mmol)、苯胺(329.00uL，3.6mmol)、碳酸钾(8.30g，60.0mmol)和17mL甲苯的溶液中，氮气置换3次，回流4h，TLC监测，反应结束冷却至室温，硅藻土助滤，滤液用二氯甲烷萃取，无水硫酸钠干燥，抽滤，收集滤液，旋干滤液经硅胶柱层析，以石油醚:乙酸乙酯＝100:1分离，得到2x淡橙色液体267mg，收率43％。
1H NMR(400MHz,Chloroform-d):δ8.03(dd,J＝4.8,1.7Hz,1H),7.57–7.51(m,2H),7.46(dd,J＝7.7,1.7Hz,1H),7.29–7.22(m,2H),7.00–6.93(m,1H),6.90(s,1H),6.60(dd,J＝7.7,4.8Hz,1H).LC-MS:m/z:205.1(M+H) +.
N-苯基-3-(4，4，5，5-四甲基-1，3，2-二氧硼杂环戊烷-2-基)吡啶-2-胺(2y)的合成
Figure PCTCN2020085438-appb-000045
将三(二亚苄基丙酮)二钯(12.00mg，0.013mmol)、联硼酸频那醇酯(1.00g，3.9mmol)、2-二环己基磷-2，4，6-三异丙基联苯(13.00mg，0.026mmol)和醋酸钾(385.00mg，3.9mmol)装入50mL三口瓶，氮气置换三次，称取2x(267.00mg，1.3mmol)溶于8mL无水1，4-二氧六环，并将其加入到三口瓶中，于110℃反应16h，TLC监测，反应结束冷却至室温，抽滤，硅藻土助滤，滤液用乙酸乙酯萃取，无水硫酸钠干燥，旋干滤液经硅胶柱层析，以石 油醚:乙酸乙酯＝20:1分离，得到2y淡橙色液体直接用于下一步的反应。
N-(3-(2-(3-羟丙基)-5-碘-1H-咪唑-4-基)苯基)-2-(1-氧代异吲哚啉-2-基)-2-苯基乙酰胺(2v)的合成(步骤m)
Figure PCTCN2020085438-appb-000046
将11(104.00mg，0.2mmol)装入25mL单口瓶中，加入6mL乙腈溶解，搅拌下分批加入N-碘代丁二酰亚胺(61.00mg，0.3mmol)，升温至80℃，反应16h，TLC监测，停止反应，冷却至室温，乙酸乙酯萃取，无水硫酸钠干燥，旋干滤液经硅胶柱层析，以二氯甲烷:甲醇＝100:1分离，得到2v淡黄色固体40mg，收率30％。
1H NMR(400MHz,DMSO-d 6):δ10.51(s,1H),8.11(d,J＝1.8Hz,1H),7.76(d,J＝7.5Hz,1H),7.65–7.35(m,11H),7.29(t,J＝7.9Hz,1H),6.25(s,1H),4.82(d,J＝17.7Hz,1H),3.82(d,J＝17.6Hz,1H),3.41(t,J＝6.3Hz,2H),2.81(t,J＝7.6Hz,2H),1.91(p,J＝6.8Hz,2H).LC-MS:m/z:593.1(M+H) +.
实施例14：N-(3-(2-(3-羟基丙基)-5-(2-(苯基氨基)吡啶-3-基)-1H-咪唑-4-基)苯基)-2-(1-氧代异吲哚啉-2-基基)-2-苯基乙酰胺(13)的合成
Figure PCTCN2020085438-appb-000047
将碳酸钾(24.00mg，0.2mmol)和[1，1′-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(6.00mg，0.007mmol)依次装入25mL三口瓶中，氮气置换三次，加入溶于4mL 1，4-二氧六环与1mL水的2v、2y溶液，于90℃反应14h，TLC监测，反应结束冷却至室温，用乙酸乙酯萃取，无水硫酸钠干燥，抽滤，收集有机相，旋干滤液经硅胶柱层析，以二氯甲烷:甲醇＝100:1分离，得到13白色固体14mg，收率32％。
1H NMR(400MHz,Chloroform-d):δ9.95(s,1H),8.98(s,1H),8.03(d,J＝4.8Hz,1H),7.69(d,J＝7.6Hz,1H),7.61–7.32(m,11H),7.24–7.10(m,3H),6.97–6.73(m,3H),6.49(s,1H),6.44(q,J＝7.1,6.5Hz,1H),4.86(d,J＝17.5Hz,1H),4.00(d,J＝17.5Hz,1H),3.56(t,J＝5.5Hz,2H),2.83(d,J＝7.0Hz,2H),1.91(s,2H). 13C NMR(151MHz,DMSO):δ168.54, 168.31,154.02,149.33,144.23,143.29,141.73,139.49,135.23,132.71,132.11,131.84,129.37,129.17,129.07,129.01,128.85,128.31,128.10,126.36,124.35,124.11,120.72,120.33,118.34,116.37,114.64,60.24,59.34,48.69,31.70,23.87.HRMS(ESI)(m/z):calcd for C 39H 34N 6O 3[M+H] +635.2771,found 635.2772.
实施例15：N-(4-(2-(3-羟基丙基)-5-(2-(苯基氨基)吡啶-3-基)-1H-咪唑-4-基)苯基)-2-(1-氧代异吲哚啉-2-基基)-2-苯基乙酰胺(14)的合成
Figure PCTCN2020085438-appb-000048
化合物14是参照化合物13的合成路线得到。白色固体，收率25％。
1H NMR(400MHz,DMSO-d 6):δ12.41(s,1H),10.72(s,1H),10.66(s,1H),8.05(dd,J＝4.8,1.9Hz,1H),7.76(d,J＝7.6Hz,1H),7.68(dd,J＝15.3,8.2Hz,3H),7.63–7.54(m,2H),7.54–7.40(m,5H),7.36(dd,J＝8.4,2.0Hz,3H),7.28(t,J＝7.7Hz,2H),6.89(t,J＝7.4Hz,1H),6.62(dd,J＝7.5,4.9Hz,1H),6.23(s,1H),4.83(d,J＝17.7Hz,1H),4.61(t,J＝5.1Hz,1H),3.98(d,J＝17.7Hz,1H),3.56(q,J＝6.0Hz,2H),2.82(t,J＝7.6Hz,2H),1.97(p,J＝6.9Hz,2H). 13C NMR(151MHz,DMSO):δ168.74,168.21,153.08,148.23,145.73,142.89,141.89,138.39,135.73,132.20,132.01,131.94,129.57,129.17,129.08,129.02,128.95,128.46,128.00,126.66,124.16,123.41,120.88,120.00,118.60,115.27,114.34,60.60,59.04,48.89,31.40,24.77.HRMS(ESI)(m/z):calcd for C 39H 34N 6O 3[M+H] +635.2771,found 635.2772.
N-苯基-3-(4，4，5，5-四甲基-1，3，2-二氧硼杂环戊烷-2-基)吡啶-2-胺(2z)的合成
Figure PCTCN2020085438-appb-000049
将[1，1′-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(31.00mg，0.04mmol)、联硼酸频那醇酯(480.00mg，2.0mmol)、4-溴-7-氮杂吲哚(184.00mg，1.0mmol)和醋酸钾(240.00mg，3.0mmol)依次装入25mL三口瓶，氮气置换三次，加入无水1，4-二氧六环，于90℃反应18h，TLC监测，停止反应，冷却至室温，抽滤，硅藻土助滤，用乙酸乙酯萃取，无水硫酸钠干燥，抽滤，滤液旋干，得到2z直接用于下一步的反应。
实施例16：N-(3-(2-(3-羟基丙基)-5-(2-(苯基氨基)吡啶-3-基)-1H-咪唑-4-基)苯基)-2-(1-氧代异吲哚啉-2-基基)-2-苯基乙酰胺(15)的合成
Figure PCTCN2020085438-appb-000050
将碳酸钾(59.00mg，0.4mmol)、[1，1′-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(14.00mg，0.017mmol)依次装入25mL三口瓶中，氮气置换三次，加入溶于5mL 1,4-二氧六环与1.25mL水的2v、2z溶液，于90℃反应12h，TLC监测，反应结束冷却至室温，用乙酸乙酯萃取，无水硫酸钠干燥，抽滤，旋干滤液经硅胶柱层析，以二氯甲烷:甲醇＝100:1分离，得到15白色固体10mg，收率10％。
1H NMR(400MHz,Chloroform-d):δ9.95(s,1H),8.98(s,1H),8.03(d,J＝4.8Hz,1H),7.69(d,J＝7.6Hz,1H),7.61–7.32(m,11H),7.24–7.10(m,3H),6.97–6.73(m,3H),6.49(s,1H),6.44(q,J＝7.1,6.5Hz,1H),4.86(d,J＝17.5Hz,1H),4.00(d,J＝17.5Hz,1H),3.56(t,J＝5.5Hz,2H),2.83(d,J＝7.0Hz,2H),1.91(s,2H). 13C NMR(151MHz,DMSO):δ168.68,168.17,149.57,142.86,139.22,136.37,135.79,132.18,131.93,130.07,129.53,128.97,128.44,126.64,124.37,124.13,123.40,118.83,117.59,115.26,100.73,60.53,58.87,48.84,29.48,22.56,14.43.HRMS(ESI)(m/z):calcd for C 39H 34N 6O 3[M+H] +635.2771,found 635.2772.
生物评价方法：
酪氨酸激酶：EGFR(WT)
EGFRT790M/L858R(LR/TM)
EGFRT790M/L858R/C797S(LR/TM/CS)
ELISA激酶活性检测
用酶联免疫吸附法(Enzyme-Linked Immunosorbent Assay,ELISA)检测激酶磷酸化底物的能力，计算化合物对激酶活性的抑制作用。激酶采用EGFR L858R/T790M/C797S(购自BPS Bioscience)。
ELISA主要步骤如下：酶反应底物Poly(Glu,Tyr)4:1用无钾离子的PBS稀释成2.5μg/孔，37℃反应12-16h包被酶标板，备用。每孔加入用反应缓冲液(50mM HEPES pH 7.4，20mM MgCl 2，0.1mM MnCl 2，0.2mM Na 3VO 4，1mM DTT)稀释的ATP(终浓度5μM)溶液，加入化合物或溶剂对照，然后加入激酶启动反应，37℃摇床反应1h。T-PBS洗板三次，加入抗体PY99(含5mg/mL BSA的T-PBS，1:500稀释)100μL于37℃摇床反应0.5h。T-PBS洗板后，加入辣根过氧化物酶标记的羊抗鼠的IgG(含5mg/mL BSA的T-PBS，1:2000稀 释)100μL，37℃摇床反应0.5h。再次洗板后，加入含0.03％H 2O 2，2mg/mL的OPD(0.1mol/L，pH 5.4柠檬酸盐缓冲液配制)显色液100μL/孔，25℃避光反应1-10min。加入50μL/孔2M H 2SO 4终止反应，用可调波长式微孔板酶标仪(SpectraMax Plus384，Molecular Devices)读数，波长为490nm。IC50值由抑制曲线得到。
酶活性测试结果如下表：
Figure PCTCN2020085438-appb-000051
Figure PCTCN2020085438-appb-000052
a激酶活性测试通过使用ELISA基EGFR-TK测试进行。数据是至少两次独立测定的平均值并以平均值±SD(标准偏差)表示。 b二突变体(EGFR L858R/T790M). c三突变体(EGFR L858R/T790M/C797S).
Figure PCTCN2020085438-appb-000053
Figure PCTCN2020085438-appb-000054
a激酶活性测试通过使用ELISA基EGFR-TK测试进行。数据是至少两次独立测定的平均值并以平均值±SD表示。
细胞增殖抑制测试
酪氨酸激酶：EGFR T790M/19DEL/C797S
测试方法：实验选用小鼠原B细胞株BaF3细胞株和EGFR 19del/T790M/C797S-BaF3。实验起始选取对数生长期的细胞，96孔板中设立3组表达19del+T790M+C797S突变的细胞，每组细胞的数量为5000，分别使药物最高浓度为10μmol/L，按1:2递减，72小时后，每孔加入CCK8 10ul，用thermo的酶标仪，在450nm测细胞活性，最后经过拟合曲线得到IC 50值。
Figure PCTCN2020085438-appb-000055
化合物4的细胞活性评价如下表和图1所示(通过CCK-8测定检测了A431(A)、H1975(B)和BaF3/EGFR19D/T790M/C797S(C))
Figure PCTCN2020085438-appb-000056
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 通式I所示的化合物或其立体异构体或光学异构体或其药学上可接受的盐：
    Figure PCTCN2020085438-appb-100001
    式中，
    W是CH或N；
    X是CH或N；
    Y是CH或N或C卤素；
    Z是CH或N；
    R 1的数量为1-5中任意的整数，任选位于1’、2’、5’、6’或7’位，并且独立选自：H、卤素、C1-C6取代或未取代的烷基、
    Figure PCTCN2020085438-appb-100002
    其中，n为0～4的整数；
    P为1-2个C1-C3烷基或不存在；
    Q为-OH、-SH、-NH 2、-NHCH 3、-COOH、-CONH 2、-NHCONH 2、-NHCONHNH 2、-SO 3H、-SO 2NH 2；
    其中当R 4为
    Figure PCTCN2020085438-appb-100003
    时，在R 4各位置上可以具有一个或多个Q；
    X为O、S、NH；
    R 2选自：氢、C1-C5取代或未取代的烷基甲酰胺基、C2-C5取代或未取代的烯基甲酰胺基；
    R 3选自：氢、卤素、NR 7R 8、取代的N-C1-C3烷基哌嗪基；
    R 7和R 8独立选自：H、C1-C6取代或未取代的烷基、-(CH 2) nNR 9R 10，其中n是0-3的整数；
    R 9和R 10独立选自：H、C1-C3取代或未取代的烷基。
  2. 如权利要求1所述的化合物或其立体异构体或光学异构体或其药学上可接受的盐，其特征在于，
    W是CH；
    X是CH或N；
    Y是CH或N；
    Z是CH；
    R 1的数量为1或2，任选位于1’或5’位，并且独立选自：H、C1-C6取代或未取代的烷基羟基或多羟基、卤素；
    R 2选自：氢、C1-C3取代或未取代的烷基甲酰胺基、C2-C3取代或未取代的烯基甲酰胺基；
    R 3选自：卤素、NR 7R 8；
    R 7和R 8独立选自：H、C1-C3取代或未取代的烷基、-(CH 2) nNR 9R 10，其中n是1或2；
    R 9和R 10独立选自：H、C1-C3取代或未取代的烷基。
  3. 选自以下的化合物或其立体异构体或光学异构体或其药学上可接受的盐：
    Figure PCTCN2020085438-appb-100004
    Figure PCTCN2020085438-appb-100005
  4. 通式II所示的化合物或其立体异构体或光学异构体或其药学上可接受的盐：
    Figure PCTCN2020085438-appb-100006
    式中，
    R 1选自：
    Figure PCTCN2020085438-appb-100007
    其中，n为0～4的整数；
    P为1-2个C1-C3烷基或不存在；
    Q为-OH、-SH、-NH 2、-NHCH 3,、-COOH、-CONH 2、-NHCONH 2、-NHCONHNH 2、-SO 3H、-SO 2NH 2；
    其中当R 4为
    Figure PCTCN2020085438-appb-100008
    时，在R 4各位置上可以具有一个或多个Q；
    X为O、S、NH；
    R 4选自：
    Figure PCTCN2020085438-appb-100009
    A的数量为0-4的任一整数，并且独立选自：卤素、取代或未取代的C1-C3烷氧基；
    V、S、T的数量分别为0-4的任一整数，并且独立选自卤素、取代或未取代的C1-C3烷氧基；
    R 5选自：H、取代或未取代的
    Figure PCTCN2020085438-appb-100010
    R 6选自：H、取代或未取代的C1-C3烷基。
  5. 如权利要求4所述的化合物或其立体异构体或光学异构体或其药学上可接受的盐，其特征在于，
    R 4选自：
    Figure PCTCN2020085438-appb-100011
    A的数量为0-4的任一整数，并且独立选自：卤素、取代或未取代的C1-C3烷氧基；
    V的数量为0-4的任一整数，并且独立选自卤素、取代或未取代的C1-C3烷氧基；
    R 5选自：取代或未取代的
    Figure PCTCN2020085438-appb-100012
    R 6选自：H。
  6. 选自以下的化合物或其立体异构体或光学异构体或其药学上可接受的盐：
    Figure PCTCN2020085438-appb-100013
  7. 一种药物组合物，所述药物组合物含有权利要求1-6中任一项所述的化合物或其立体异构体或光学异构体或其药学上可接受的盐，以及任选的药学上可接受的载体或赋形剂。
  8. 权利要求1-6中任一项所述的化合物或其立体异构体或光学异构体或其药学上可接受的盐，或权利要求7所述的药物组合物在制备治疗或预防EGFR介导的疾病或抑制EGFR的药物中的用途。
  9. 如权利要求9所述的用途，其特征在于，所述EGFR介导的疾病为癌症。
  10. 如权利要求9所述的用途，其特征在于，所述癌症选自下组：非小细胞肺癌、小细胞肺癌、肺腺癌、肺鳞癌、乳腺癌、前列腺癌、神经胶质细胞瘤、卵巢癌、头颈部鳞癌、宫颈癌、食管癌、肝癌、肾癌、胰腺癌、结肠癌、皮肤癌、白血病、淋巴瘤、胃癌、多发性骨髓癌和实体瘤。
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