[go: up one dir, main page]

WO2023046114A1 - 蝶啶酮衍生物及其应用 - Google Patents

蝶啶酮衍生物及其应用 Download PDF

Info

Publication number
WO2023046114A1
WO2023046114A1 PCT/CN2022/121058 CN2022121058W WO2023046114A1 WO 2023046114 A1 WO2023046114 A1 WO 2023046114A1 CN 2022121058 W CN2022121058 W CN 2022121058W WO 2023046114 A1 WO2023046114 A1 WO 2023046114A1
Authority
WO
WIPO (PCT)
Prior art keywords
substituted
unsubstituted
cancer
compound
egfr
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/CN2022/121058
Other languages
English (en)
French (fr)
Inventor
李洪林
赵振江
陈卓
徐玉芳
王操林
沙文婕
钱旭红
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
East China University of Science and Technology
Original Assignee
East China University of Science and Technology
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 East China University of Science and Technology filed Critical East China University of Science and Technology
Publication of WO2023046114A1 publication Critical patent/WO2023046114A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • 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
    • C07D475/00—Heterocyclic compounds containing pteridine ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic

Definitions

  • the invention belongs to the field of medicinal chemistry; in particular, the invention relates to a compound targeting EGFR mutant protein, a preparation method thereof, and an application in treating EGFR-mediated diseases, such as tumors.
  • EGFR plays an important role in the formation and development of various cancers, so the EGFR tyrosine kinase target has become the preferred target for cancer targeted therapy, especially for non-small cell lung cancer.
  • small molecule inhibitors targeting EGFR have made good clinical progress, such as the first-generation reversible inhibitors gefitinib and erlotinib, the second-generation irreversible afatinib and Daco Tini and the third dioxitinib that has the activity of overcoming the acquired resistance mutation EGFR T790M mutation. But not all patients with EGFR mutations benefit.
  • NSCLC with EGFR exon 20 insertion mutation (EGFR 20ins) has poor or ineffective response to most EGFR inhibitor targeted therapies, and these mutations are often classified as non-classic mutations.
  • These non-canonical mutations mainly include insertion mutations and point mutations of exons 18-21, point mutations and insertion mutations of ERBB2.
  • the object of the present invention is to provide a compound or a pharmaceutically acceptable salt thereof that targets and inhibits non-classical mutant EGFR.
  • the compound should have excellent antitumor activity.
  • the present invention provides a compound represented by formula I or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof:
  • R 1 is independently selected from hydrogen, substituted or unsubstituted C 1 -C 10 alkyl , C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, optionally substituted C 3 -C 8 cycloalkyl, Optionally substituted or unsubstituted aryl, substituted or unsubstituted benzyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aromatic heterocyclic group;
  • R 2 , R 3 , R 4 , R 5 are independently selected from H, halogen, substituted (preferably halogen substituted, more preferably fluorine substituted) or unsubstituted C 1 -C 6 (preferably C 1 -C 3 ) alkoxy or Substituted (preferably halogen substituted, more preferably fluorine substituted) or unsubstituted C 1 -C 6 (preferably C 1 -C 3 ) deuterated alkoxy, substituted or unsubstituted C 1 -C 6 (preferably C 1 -C 3 ) Alkyl, NRcRd ; wherein Rc and Rd are each independently selected from hydrogen , C1-3 alkyl;
  • G is a benzene ring, a five-membered or six-membered heterocyclic ring or a C 3 -C 8 cycloalkyl group or does not exist;
  • R 6 is independently selected from hydrogen, unsubstituted or halogen-substituted C 1 -C 4 alkyl, nitro, amino, halogen, hydroxyl, C 1 -C 6 alkoxy, optionally substituted C 1 -C 6 acyl Oxygen, optionally substituted C 1 -C 6 amido, optionally substituted C 1 -C 6 acyl; wherein, when G is a benzene ring, R 6 is meta-substituted;
  • n is an integer of 0-3;
  • R 7 is independently selected from substituted or unsubstituted NH 2 , substituted or unsubstituted heterocyclic group, substituted or unsubstituted aromatic heterocyclic group, substituted or unsubstituted C 1 -C 10 alkyl;
  • A is selected from the following group or is absent:
  • L is selected from the following group or absent: C 1 -C 10 alkylene, C 1 -C 10 heteroalkylene, -A'-(CH 2 ) m' -W-(CH 2 ) n' -, - (CH 2 ) m' -W-(CH 2 ) n' -O-(CH 2 ) V -and-(CH 2 ) m' -W-[(CH 2 ) n' -O] u -(CH 2 ) v -;
  • A' is selected from the following group or does not exist: 5-membered arylene and 6-membered arylene;
  • W is selected from: phenylene, 5-membered heteroarylene, 6-membered heteroarylene, C 1 -C 10 heterocyclylene and C 1 -C 10 alkylene;
  • n' 0, 1, 2, 3, 4, 5, 6, 7 or 8;
  • n' is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9;
  • each independent u is independently 2, 3 or 4;
  • v 1, 2, 3 or 4
  • B is selected from the following group or absent:
  • R is selected from: hydrogen, methyl and fluorine
  • the compound is a compound shown in the following formula II:
  • R 7 is independently selected from: H, substituted (preferably halogen substituted, more preferably fluorine substituted) or unsubstituted C 1 -C 6 (preferably C 1 -C 3 ) alkyl, substituted (preferably halogen substituted, more preferably fluorine substituted) or unsubstituted C 1 -C 6 (preferably C 1 -C 3 ) deuterated alkyl;
  • R 8 is independently selected from: H, substituted (preferably halogen substituted, more preferably fluorine substituted) or unsubstituted C 1 -C 3 alkyl, substituted (preferably halogen substituted, more preferably fluorine substituted) or unsubstituted C 1 -C 3 deuterated alkyl.
  • R 7 is independently selected from: methyl, deuteromethyl CD 3 , trifluoromethyl, ethyl, deuteroethyl (eg, CD 2 CH 3 , CH 2 CD 3 ), CH 2 CF 3 ;
  • R 8 is independently selected from: hydrogen, methyl, deuteromethyl CD 3 , ethyl, deuteroethyl (eg, CD 2 CH 3 , CH 2 CD 3 ).
  • the compound is selected from:
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the compound described in the first aspect, or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof and optionally pharmaceutically acceptable excipients.
  • the present invention provides the compound described in the first aspect, or its pharmaceutically acceptable salt, solvate, stereoisomer or prodrug in the preparation of prevention or treatment of abnormal expression of EGFR protein activity, ERBB2 overexpression Use in medicine for diseases related to point mutations thereof.
  • said EGFR is mutant EGFR.
  • the mutant EGFR includes at least one of the following mutations: EGFR sensitivity mutation L858R and 19del, EGFR T790M mutation, EGFR 18-21 exon point mutation and insertion mutation, ERBB2 overexpression and point mutation Mutations and insertional mutations.
  • the EGFR 18-21 exon point mutation and insertion mutation contain:
  • Insertion mutations and point mutations in exon 20 include: A763-Y764insFQEA, A763-Y764insFHEA, V769-D770insASV, V769-D770insDNP, D770-N771insNPG, D770-N771insNPH, D770-N771insSVD, D770-N771insP7insASVDN, D771SVG-N7 ⁇ N771-H773dupNPH ⁇ P772-H773insPNP ⁇ P772-H773insPR ⁇ H773-V774insH ⁇ A763-Y764insFQEA ⁇ H773-V774insPH ⁇ H773-V774insNPH ⁇ N771-P772insH ⁇ H771-P772insN ⁇ H773-V774insAH ⁇ D770delinsGY ⁇ V774-C775ins
  • the disease is cancer and is selected from one or more of the following: non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, breast cancer, pancreatic cancer, prostate cancer, ovarian cancer Carcinoma, glioma, head and neck squamous cell carcinoma, cervical cancer, esophageal cancer, liver cancer, kidney cancer, colon cancer, skin cancer, leukemia, lymphoma, stomach cancer or multiple myeloma.
  • the present invention provides a method for preventing or treating diseases related to abnormal expression of EGFR protein activity, ERBB2 overexpression and point mutations thereof, the method comprising treating a therapeutically effective amount of the compound described in the first aspect, or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof, or the pharmaceutical composition described in the second aspect to a subject in need thereof.
  • said subject is a mammal, preferably a human.
  • the EGFR is a mutant EGFR.
  • the mutant EGFR includes at least one of the following mutations: EGFR sensitivity mutation L858R and 19del, EGFR T790M mutation, EGFR 18-21 exon point mutation and insertion mutation, ERBB2 overexpression and point mutation Mutations and insertional mutations.
  • the EGFR 18-21 exon point mutation and insertion mutation contain:
  • Insertion mutations and point mutations in exon 20 include: A763-Y764insFQEA, A763-Y764insFHEA, V769-D770insASV, V769-D770insDNP, D770-N771insNPG, D770-N771insNPH, D770-N771insSVD, D770-N771insP7insASVDN, D771SVG-N7 ⁇ N771-H773dupNPH ⁇ P772-H773insPNP ⁇ P772-H773insPR ⁇ H773-V774insH ⁇ A763-Y764insFQEA ⁇ H773-V774insPH ⁇ H773-V774insNPH ⁇ N771-P772insH ⁇ H771-P772insN ⁇ H773-V774insAH ⁇ D770delinsGY ⁇ V774-C775ins
  • the disease is cancer and is selected from one or more of the following: non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, breast cancer, pancreatic cancer, prostate cancer, ovarian Carcinoma, glioma, head and neck squamous cell carcinoma, cervical cancer, esophageal cancer, liver cancer, kidney cancer, colon cancer, skin cancer, leukemia, lymphoma, stomach cancer or multiple myeloma.
  • Figure 1 shows the in vivo drug efficacy (EGFR L858R/T790M xenograft tumor) activity of compound 2 and compound 3;
  • Figure 2 shows the in vivo drug effect (EGFR L858R/T790M xenograft tumor) activity of compound 5;
  • Figure 3 shows the drug-time curves of compounds 0, 1, 2, 3, 4, 5 in SD rat plasma.
  • alkyl refers to a saturated branched or linear alkyl group with a carbon chain length of 1-10 carbon atoms
  • preferred alkyl groups include 2-8 carbon atoms, 1-6, 1-4 carbon atoms, alkyl groups ranging from 1 to 3 carbon atoms.
  • alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isoisoyl, heptyl, and the like.
  • Alkyl groups may be substituted by one or more substituents, such as 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 with an alkyl group.
  • Preferred alkoxy groups are those with a length of 1 to 6 carbon atoms, more preferably those with a length of 1 to 3 carbon atoms. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, and the like.
  • Alkoxy may be substituted by one or more substituents, such as halogen or haloalkyl.
  • an alkoxy group can be an alkyl group substituted with 1-4 fluorine atoms, or an alkyl group can be an alkyl group substituted with a fluoroalkyl group.
  • alkenyl generally means a monovalent hydrocarbon group having at least one double bond, usually containing 2 to 8 carbon atoms, preferably containing 2 to 6 carbon atoms, and may be straight or branched.
  • alkenyl groups include, but are not limited to, vinyl, propenyl, isopropenyl, butenyl, isobutenyl, hexenyl, and the like.
  • acylamino 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, alkenyl , alkynyl, alkyl substituted by NRcRd, alkenyl substituted by NRcRd and alkynyl substituted by NRcRd, alkyl substituted by halogen, alkenyl substituted by cyano, wherein Rc and Rd can be selected from alkyl group and alkenyl group.
  • aryl refers to a monocyclic, bicyclic or tricyclic aromatic group containing 6 to 14 carbon atoms, including phenyl, naphthyl, phenanthrenyl, anthracenyl, indenyl, fenyl, tetralin base, indanyl, etc.
  • Aryl may be optionally substituted with 1-5 (eg, 1, 2, 3, 4 or 5) substituents selected from the group consisting of halogen, C1-4 aldehyde, C1-6 alkyl, cyano, Nitro, amino, amido, hydroxy, hydroxymethyl, halogen substituted alkyl (eg trifluoromethyl), halogen substituted alkoxy (eg trifluoromethoxy), carboxyl, C1-4 alkoxy , ethoxy formyl, N(CH3) and C1-4 acyl, etc., heterocyclyl or heteroaryl, etc.
  • 1-5 eg, 1, 2, 3, 4 or 5
  • substituents selected from the group consisting of halogen, C1-4 aldehyde, C1-6 alkyl, cyano, Nitro, amino, amido, hydroxy, hydroxymethyl, halogen substituted alkyl (eg trifluoromethyl), halogen substituted alkoxy (eg trifluoromethoxy), carboxyl, C1-4 alkoxy
  • heterocyclic group includes, but is not limited to, 5-membered or 6-membered heterocyclic groups containing 1-3 heteroatoms selected from O, S or N, including but not limited to furyl, thienyl, pyrrolyl , pyrrolidinyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, pyranyl, pyridyl, pyrimidinyl, pyrazinyl, piperidinyl, morpholinyl, etc.
  • aromatic heterocyclic group refers to a group containing 5-14 ring atoms, and 6, 10 or 14 electrons are shared on the ring system. And the contained ring atoms are carbon atoms and optional 1-3 heteroatoms selected from oxygen, nitrogen and sulfur.
  • Useful aromatic heterocyclic groups include piperazinyl, morpholinyl, piperidinyl, pyrrolidinyl, thienyl, furyl, pyryl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, including but not limited to Yu pyrimidinyl and so on.
  • the aromatic heterocyclic group can be optionally substituted by 1-5 (for example, 1, 2, 3, 4 or 5) substituents selected from the group consisting of halogen, C1-4 aldehyde, C1-6 straight chain or branched Alkanyl, cyano, nitro, amino, hydroxy, hydroxymethyl, halogen substituted alkyl (eg trifluoromethyl), halogen substituted alkoxy (eg trifluoromethoxy), carboxyl, C1- 4 alkoxy, ethoxy formyl, N(CH3) and C1-4 acyl.
  • 1-5 for example, 1, 2, 3, 4 or 5
  • substituents selected from the group consisting of halogen, C1-4 aldehyde, C1-6 straight chain or branched Alkanyl, cyano, nitro, amino, hydroxy, hydroxymethyl, halogen substituted alkyl (eg trifluoromethyl), halogen substituted alkoxy (eg trifluoromethoxy), carboxyl, C1- 4 alk
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • substituents selected from the group consisting of halogen, C1 -4 aldehyde, C1-6 straight chain or branched alkyl, cyano, nitro, amino, hydroxyl, hydroxymethyl, halogen substituted alkyl (such as trifluoromethyl), halogen substituted alkoxy ( Examples include trifluoromethoxy), carboxy, C1-4 alkoxy, ethoxyformyl, N(CH3) and C1-4 acyl.
  • 1-5 eg, 1, 2, 3, 4 or 5
  • substituents selected from the group consisting of halogen, C1 -4 aldehyde, C1-6 straight chain or branched alkyl, cyano, nitro, amino, hydroxyl, hydroxymethyl, halogen substituted alkyl (such as trifluoromethyl), halogen substituted alkoxy ( Examples include trifluoromethoxy), carboxy, C1-4 alkoxy, ethoxyformyl, N(CH3) and C1-4 acyl.
  • the compound of the present invention refers to a compound represented by the following general formula I, or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof:
  • the present invention provides a compound represented by formula II, or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof:
  • the present invention provides the following compounds capable of inhibiting EGFR activity, and these compounds have significant inhibitory activity on mutant EGFR (L858R/T790M, 20ins):
  • each group in the compound of the present invention can be further substituted, so as to obtain derivatives that can have the same or similar activity as the compound disclosed in the present invention. things.
  • Each group in the compound of the present invention can be substituted by various conventional substituents in the art, as long as the substitution does not violate the rules of chemical synthesis or valence.
  • substituted refers to the replacement of one or more hydrogen atoms on a specified group with a specified substituent.
  • the specific substituents may be correspondingly described substituents above, specific substituents appearing in each embodiment or conventional substituents in the art. Therefore, in the present invention, the substituents in the general formula can also be independently the corresponding groups in the specific compounds in the examples; Combinations of some of the substituents shown in and other specific substituents appearing in the examples. It is not difficult for those skilled in the art to prepare a compound having such a combination of substituents and to detect whether the obtained compound is active based on conventional technical means in this field.
  • pharmaceutically acceptable salt refers to a salt formed of a compound of the present invention with an acid or a base which is suitable for use as a medicine.
  • Pharmaceutically acceptable salts include inorganic salts and organic salts.
  • a preferred class of salts are the salts of the compounds of the invention with acids.
  • Acids suitable for forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, Maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenemethanesulfonic acid, benzenesulfonic acid and other organic acids; and acidic amino acids such as aspartic acid and glutamic acid.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, Maleic acid, lactic acid, malic acid,
  • the structural formulas described in the present invention are intended to include all isomeric forms (such as enantiomers, diastereoisomers and geometric isomers (or conformational isomers)): for example, containing asymmetric The R, S configuration of the center, the (Z), (E) isomer of the double bond, etc. Accordingly, individual stereochemical isomers of the compounds of the present invention or mixtures thereof as enantiomers, diastereomers or geometric isomers (or conformers) are within the scope of the present invention.
  • tautomer means that structural isomers with different energies can cross a low energy barrier and thereby interconvert.
  • proton tautomers ie, prototropism
  • Valence tautomers include interconversion by recombination of some of the bonding electrons.
  • solvate refers to a complex in which the compound of the present invention coordinates with solvent molecules to form a specific ratio.
  • hydrate refers to a complex formed by coordination between the compound of the present invention and water.
  • the compound of the present invention can obviously be used as a medicine. Therefore, in addition to the various properties that have been examined in the Examples, the compounds of the present invention can also possess various activities inherent as drugs. For example, in vivo activity, bioavailability, druggability, toxicity, differential inhibitory activity, etc.
  • drugs for example, in vivo activity, bioavailability, druggability, toxicity, differential inhibitory activity, etc.
  • those skilled in the art know how to obtain various compounds within the scope of the present invention and detect various activities of various compounds within the scope of the present invention; in other words, based on the teachings of the present invention With conventional technical means in the field, those skilled in the art know how to repeat, verify and implement the present invention.
  • compositions and methods of administration are provided.
  • the compounds of the present invention have targeted inhibition of non-classical mutant EGFR, especially for mutant EGFR
  • the pharmaceutical composition of active ingredients can be used to prevent and/or treat diseases related to abnormal expression of EGFR protein activity, ERBB2 overexpression and point mutations thereof.
  • said EGFR is mutant EGFR.
  • the mutant EGFR includes at least one of the following mutations: EGFR sensitivity mutations L858R and 19del, EGFR T790M mutation, EGFR exon 18-21 point mutation and insertion mutation, ERBB2 point mutation and insertion mutation.
  • the EGFR exon 18-21 point mutation and insertion mutation include: exon 18 G719X, E709X, K716A, K728A point mutation and codon 709 deletion mutation; exon 19 insertion mutation I744 -K745insKIPVAI, K745-E746insIPVAIK, K745-E746insVPVAIK, K745-E746insTPVAIK and point mutation D761Y; exon 20 insertion mutation and point mutation include: A763-Y764insFQEA, A763-Y764insFHEA, V769-D770insASV, V710insNPD-ND777 D770-N771insNPH ⁇ D770-N771insSVD ⁇ D770-N771insASVDN ⁇ D770-N771insG ⁇ N771-P772insSVDNP ⁇ N771-H773dupNPH ⁇ P772-
  • the disease is cancer, for example, one or more selected from the following: non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, breast cancer, pancreatic cancer, prostate cancer, Ovarian cancer, glioma, head and neck squamous cell carcinoma, cervical cancer, esophageal cancer, liver cancer, kidney cancer, colon cancer, skin cancer, leukemia, lymphoma, stomach cancer, or multiple myeloma.
  • the pharmaceutical composition of the present invention comprises the compound of the present invention and pharmaceutically acceptable excipients or carriers in a safe and effective amount range.
  • safe and effective dose refers to: the amount of the compound is sufficient to obviously improve the condition without causing severe side effects.
  • the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably 10-200 mg of the compound of the present invention per dose.
  • the "one dose” is a capsule or tablet.
  • “Pharmaceutically acceptable carrier” refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use, and must have sufficient purity and low toxicity. "Compatibility” herein means that the components of the composition can be blended with the compound of the present invention and with each other without significantly reducing the efficacy of the compound.
  • Examples of pharmaceutically acceptable carrier parts include cellulose and derivatives thereof (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid , magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween ), wetting agent (such as sodium lauryl sulfate), coloring agent, flavoring agent, stabilizer, antioxidant, preservative, pyrogen-free water, etc.
  • cellulose and derivatives thereof such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.
  • gelatin such as talc
  • solid lubricants such as stearic acid , magnesium stearate
  • the administration method of the compound or pharmaceutical composition of the present invention is not particularly limited, and representative administration methods include (but not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous).
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active compound is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with (a) fillers or extenders, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow agents, such as paraffin; (f) Absorption accelerators such as quaternary ammonium compounds; (g) wetting agents such as cetyl alcohol and glyceryl monostea, or
  • Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shell materials, such as enteric coatings and others well known in the art. They may contain opacifying agents and, in such compositions, the release of the active compound or compounds may be in a certain part of the alimentary canal in a delayed manner.
  • coatings and shell materials such as enteric coatings and others well known in the art. They may contain opacifying agents and, in such compositions, the release of the active compound or compounds may be in a certain part of the alimentary canal in a delayed manner.
  • Examples of usable embedding components are polymeric substances and waxy substances.
  • the active compounds can also be in microencapsulated form, if desired, with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • liquid dosage forms 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, etc.
  • 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
  • compositions can also contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • Suspensions in addition to the active compounds, may contain suspending agents, for example, ethoxylated isostearyl alcohols, 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 alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous 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 vehicles include water, ethanol, polyols, and suitable mixtures thereof.
  • the compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.
  • the pharmaceutical composition When administered in combination, the pharmaceutical composition also includes one or more (2, 3, 4, or more) other pharmaceutically acceptable compounds.
  • One or more of the other pharmaceutically acceptable compounds may be administered simultaneously, separately or sequentially with the compound of the present invention.
  • 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 is a pharmaceutically effective dosage when administered, for a person with a body weight of 60kg, the daily
  • the dosage is usually 1-2000 mg, preferably 20-500 mg.
  • factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.
  • the compound of the present invention has the activity of inhibiting EGFR protein, especially mutant EGFR (L858R/T790M, 20ins). Therefore, the compound of the present invention can be used for the treatment of diseases related to abnormal expression of EGFR protein, such as various cancers.
  • the compounds of the present invention have excellent pharmacokinetic properties
  • the compound of the present invention has excellent antitumor activity and antitumor activity, so it has great application prospects in the field of medicine.
  • reaction mixture was spin-dried, extracted with ethyl acetate, washed three times with saturated aqueous sodium chloride, the organic phase was dried over anhydrous sodium sulfate, concentrated in vacuo, and recrystallized from petroleum ether to obtain a yellow solid (16.30 g, 87.00%).
  • reaction mixture was spin-dried, extracted with dichloromethane, washed three times with saturated aqueous sodium chloride solution, and the organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo. Methanol was recrystallized to obtain a reddish-brown solid.
  • N-(3-((2-((2-(methoxy-d 3 )-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-nitropyrimidine-4 -yl)amino)phenyl)acrylamide (1.18g, 2.33mmol), iron powder (0.50g, 9.32mmol), ammonium chloride (0.62g, 11.65mmol) and ethanol:water (8mL, 4:1) mixed In a 100mL single-necked bottle, stir at 80°C for 24h.
  • reaction mixture was suction-filtered, the filtrate was adjusted to alkali, extracted with ethyl acetate, washed three times with saturated aqueous sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, and concentrated in vacuo to obtain a purple-black solid (0.45 g, 36.00%).
  • Embodiment 2 N-(3-(2-((2-methoxy-4-(piperazin-1-yl) phenyl) amino)-7-oxo-6-phenylpteridine-8( Synthesis of 7H)-yl)phenyl)acrylamide (intermediate)
  • N-(3-nitrophenyl)acrylamide (5g, 26.1mmol) in a 250mL round bottom flask, add solvent 150mL ethanol to dissolve, 20ml water, then add iron powder (5.8g, 104.4mmol), chlorination Ammonium (6.7g, 130.5mmol) was reacted with stirring at 80°C, followed by TLC, and the reaction was completed after 3h.
  • the reaction solution was filtered with diatomaceous earth, the filtrate was rotary evaporated, and the residue was extracted with EA and 20ml 20% NaOH water, repeated three times. Several layers were combined and evaporated under reduced pressure to obtain 3.5 g of a light green solid with a yield of about 83%. The product was directly used in the next step without purification.
  • reaction solution was filtered with diatomaceous earth, the filtrate was rotary evaporated, and the residue was extracted with EA and 10ml 20% NaOH water, repeated three times. There were several layers combined, and the vacuum rotary After steaming, 0.92 g of a light green solid was obtained, with a yield of about 61%.
  • the product was directly used in the next step without purification.
  • Salts of other compounds were also obtained in a similar manner.
  • reaction mixture was spin-dried, extracted with ethyl acetate, washed three times with saturated aqueous sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, concentrated in vacuo, and recrystallized from petroleum ether to obtain a yellow solid (19.60 g, 87.00%).
  • reaction mixture was suction-filtered, the filtrate was adjusted to alkali, extracted with ethyl acetate, washed three times with saturated aqueous sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, and concentrated in vacuo to obtain a purple-black solid (0.47 g, 36.00%).
  • reaction mixture was spin-dried, extracted with dichloromethane, washed with saturated aqueous sodium chloride, and the organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo to obtain a yellow pure product (2.2 g, 90.2%).
  • tert-butyl 4-(4-nitro-3-(2,2,2-trifluoroethoxy)phenyl)piperazine-1-carboxylate (1.52g, 3.76mmol), 10% palladium on carbon ( 0.39g, 0.38mmol) and ethanol (8.00mL) were mixed in a 100mL three-neck flask, protected by hydrogen, and stirred at 80°C for 24h.
  • reaction mixture was spin-dried, extracted with dichloromethane, washed three times with saturated aqueous sodium chloride solution, and the organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo. An aqueous methanol solution was recrystallized to obtain a reddish-brown solid (0.70 g, 50.72%).
  • reaction mixture was suction-filtered, the filtrate was adjusted to alkali, extracted with ethyl acetate, washed 3 times with saturated aqueous sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, and concentrated in vacuo to obtain a dark brown solid (0.56 g, 75.75%).
  • reaction mixture was spin-dried, extracted with dichloromethane, washed three times with saturated aqueous sodium chloride solution, and the organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo. Recrystallized from methanol: water to obtain a reddish-brown solid (0.70 g, 50.72%).
  • reaction mixture was filtered with suction, the filtrate was adjusted to alkali, extracted with ethyl acetate, washed with saturated aqueous sodium chloride three times, the organic phase was dried over anhydrous sodium sulfate, and concentrated in vacuo to obtain a dark brown solid (0.50 g, 75.75%).
  • EGFR-TK can catalyze the transfer of a phosphate group of adenosine triphosphate (ATP) to the polypeptide substrate poly(Glu,Tyr) 4:1 , and the polypeptide substrate is labeled with two fluorescent groups coumarin ( coumarin) and fluorescein.
  • FRET fluorescence resonance energy transfer
  • EGFR-TK catalyzes the reaction of ATP to cause two fluorescent groups to approach, the donor (coumarin) is excited at 400nM, part of the energy is released, the emission wavelength is 445nM, and the other part of the energy is transferred To fluorescein, the emission wavelength is 520nM.
  • Different compounds have different degrees of inhibition of EGFR-TK, resulting in different degrees of substrate phosphorylation, so the inhibition rate of different compounds is calculated by measuring the ratio of the enzyme-catalyzed substrate phosphorylation percentage.
  • Concentrations of different kinases were determined by optimization experiments and compounds were diluted three-fold from 5.1x10 -9 M to 1x l0 -4 M in DMSO. 4 ⁇ L of compound was dissolved in 96 ⁇ L of water to obtain a 4x compound solution. 40 ⁇ M ATP is dissolved in 1.33x Kinase Buffer, the Kinase/Peptide Mix contains 2x Kinase, 4 ⁇ M Acetine 4-Peptide is ready for use.
  • a 10 ⁇ L kinase reaction consists of 2.5 ⁇ L compound solution, 5 ⁇ L kinase/peptide mix, 2.5 ⁇ L ATP solution.
  • H1975 (EGFR L858R/T790M ) cells were obtained from ATCC. Cell proliferation activity was assessed by MTS assay. The cells were exposed to the treatment conditions for 72 hours, and the number of cells used in each experiment of each cell line was adjusted according to the absorbance value (absorbance value at 490 nm was 1.3-2.2). Six concentration gradients (0.1 nM-10 ⁇ M) were set up for the compounds to be tested, and at least six groups of parallel controls were used for each concentration value.
  • H1975 cells were cultured in the corresponding medium, and the cells were passaged at least twice after recovery, and then used in experiments. Cells in log phase are trypsinized and resuspended in medium. H1975 (1000 cells per well) were seeded in a 96-well plate with a volume of 100 ⁇ L; 6 parallel groups and 7 columns were set up. The orifice plate was placed in an incubator with 5% carbon dioxide at 37°C overnight.
  • the compounds were dissolved in DMSO to prepare a concentration of 10 ⁇ M per liter, and then the compound concentrations were gradually diluted to obtain compound concentrations of 10 ⁇ M, 1 ⁇ M, 0.1 ⁇ M, 0.01 ⁇ M, 0.001 ⁇ M, and 0.0001 ⁇ M per liter, respectively.
  • 2 ⁇ L of compound solution was added to 998 ⁇ L of culture medium, and the mixture was mixed well. 100 ⁇ L of the mixture was added to a 96-well plate. 2 ⁇ L of DMSO was used instead of the compound solution as a 0% inhibition control. After culturing for 68 hours, 20 ⁇ L of MTT (5 mg/mL) was added.
  • the IC 50 (nM) of the deuterated compounds of the present invention for inhibiting EGFR L858R/T790M at the kinase level is between 1.0-1.5, which is similar to that of non-deuterated compounds. It shows that the deuterated derivatives do not affect the interaction between the inhibitor and the kinase. However, compound 4 and compound 5 substituted by trifluoroethoxy group will reduce the kinase inhibitory activity by about 1 times, and it is understandable that the introduction of a larger group will lead to a decrease in the interaction between the two.
  • the inhibitory activity of the compounds of the present invention on H1975 cells is not regular, which is related to the changes in the physical and chemical properties and metabolic properties of the derivatives, and there are many factors.
  • the cellular activity of deuterated compounds was similar to that of non-deuterated compounds.
  • the compounds of the present invention have excellent inhibitory activity on kinases with rare EGFR mutations and 20ins insertion mutations, which is better than the 20ins insertion mutation-positive drug TAK788 that will be launched in 2021.
  • compound 2 performed best and had good application prospects.
  • H1975 cells were transferred from Shanghai Institute of Materia Medica, Chinese Academy of Sciences.
  • the condition of the cell culture medium is 1640+10% FBS (Gibco)
  • the condition of the incubator is a constant temperature carbon dioxide incubator at 37°C
  • the concentration of carbon dioxide is 5%
  • the cells are changed and passaged once every 2-3 days, and the culture is continuously expanded.
  • the cells were digested with trypsin for 4 min, and then the digestion was terminated with medium.
  • Collect the obtained cells centrifuge at 1000r/min for 3min, remove the supernatant, wash twice with serum-free 1640, the cell viability should be maintained above 95% before transplantation, and use serum-free 1640 as a solvent to suspend the cells, the concentration of the suspended cells is 20 million/mL, put the prepared cell suspension on ice for use, in order to maintain cell viability, the cell suspension should be inoculated within 0.5-1h.
  • mice Seventy-two male BALB/c nude mice, 4-5 weeks old, weighing 17-23g, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Cell inoculation was carried out after 2-3 days of adaptation to the animal room environment. Select the rich capillaries on the upper side of the forelimb of nude mice to inject the cell suspension subcutaneously, and the volume of the cell suspension injected per mouse is 0.1 mL.
  • the transplanted tumors can be seen in about 1-2 weeks.
  • the average size of the tumors reaches 100-200mm 3 , they are randomly divided into groups, with 8 mice in each group, so that the tumor volume in each group is basically the same.
  • it can be administered, and the administration is carried out according to the administration compound, administration dose and administration method of each group. After that, it was administered every day, and the tumor volume and mouse body weight were recorded once every 2 days.
  • the same amount of solvent (0.1 mL) was given to the solvent control.
  • the tumor volume and weight of mice need to be recorded before administration. Data were plotted using GraphPad Prism version 4.0. During the whole experiment, the diameter of the transplanted tumor was measured every other day, and the body weight of the mice was weighed at the same time.
  • V0 is the tumor volume measured at the time of cage administration (ie, d0)
  • Vt is the tumor volume at each measurement.
  • Example 11 Inhibitory activity of compounds on cells with high expression of Her2: breast cancer cell SKBR3, lung cancer cell H2170, gastric cancer cell N87
  • Breast cancer cells SKBR3, lung cancer cells H2170, and gastric cancer cells N87 were obtained from the Cell Bank of the Chinese Academy of Sciences. Cell proliferation activity was assessed using a CCK8 assay. Cells were exposed to the treatment conditions for 72 hours, and the number of cells used in each experiment for each cell line was adjusted according to the absorbance value (absorbance value at 450 nm was 1-1.2). Eight concentration gradients (0.1 nM-10 ⁇ M) were set up for the compounds to be tested, and at least six groups of parallel controls were used for each concentration value.
  • the cells were cultured in the corresponding medium, and the cells were subcultured at least twice after thawing, and the cell growth was in the logarithmic growth phase, and then used for the experiment.
  • 3,000 SKBR3 cells/well, 6,000/well H2170 cells, and 9,000/well N87 cells were spread in a 96-well plate with a volume of 90 ⁇ L; 6 parallel groups and 7 columns were set up.
  • the orifice plate was placed in an incubator with 5% carbon dioxide at 37°C overnight.
  • Dissolve the compound in DMSO For the SKBR3 cell line, prepare the stock solution at a concentration of 10 mM per liter, then dilute the compound 100 times with the medium to obtain a maximum concentration of 100 ⁇ M, and then serially dilute the compound concentration by 2 times to gradually dilute 8 concentration gradients; for H2170 For cell lines, the concentration of the mother solution is prepared to be 100 ⁇ M per liter, and then the compound is diluted 100 times with the medium to obtain a maximum concentration of 1 ⁇ M, and then the compound concentration is serially diluted by 3 times to gradually dilute 8 concentration gradients; for the N87 cell line, the concentration of the prepared mother solution is 1 ⁇ M per liter.
  • the compound was diluted 100 times with medium to obtain a maximum concentration of 1 ⁇ M, and then the compound concentration was serially diluted 5 times and gradually diluted to 8 concentration gradients; 10 ⁇ L of the compound solution was added to the laid 96-well plate. Control supplemented with 10ul medium containing 1% DMSO instead of the compound solution was used as 0% inhibition control. After culturing for 72 hours, 10 ⁇ l of CCK8 was added. After 1.5 hours, the plate was read with a microplate reader. Data were calculated using GraphPad Prism version 4.0, and IC50 values were adjusted by non-linear regression models using dose-response curves.
  • compound 1, compound 2 and compound 3 have the best cell activity on lung cancer H2170 and gastric cancer N87 cells.
  • the cellular activity of the deuterated compound was slightly lower compared to Poziotinib.
  • the inhibitory activity IC 50 of the deuterated compound is between 2-3 ⁇ M, which is better than the IC 50 (11 ⁇ M) activity of Poziotinib.
  • the pharmacokinetic properties test of the compound of the present invention is entrusted to Hangzhou Leading Pharmaceutical Technology Co., Ltd. and Medicipia Pharmaceutical Technology (Shanghai) Co., Ltd. to conduct.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Oncology (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

本发明公开了式I所示的化合物或其药学上可接受的盐。该类化合物表现出了优异的靶向抑制非经典突变EGFR的作用且具有良好的抗肿瘤活性。本发明还公开了所述化合物在制备预防或/和治疗EGFR突变介导的疾病，例如癌症中的应用，以及包含该化合物的药物组合物。

Description

蝶啶酮衍生物及其应用 技术领域
本发明属于药物化学领域；具体地说，本发明涉及靶向EGFR突变蛋白的化合物及其制备方法以及在治疗EGFR介导的疾病，例如肿瘤中的应用。
背景技术
研究发现，在多种癌症的形成和发展中，EGFR都扮演着重要的角色，因此EGFR酪氨酸激酶靶点也就成为癌症靶向治疗的首选作用靶标，特别是在针对非小细胞肺癌的治疗过程中，以EGFR为靶点的小分子抑制剂取得了良好的临床进展，例如第一代可逆抑制剂吉非替尼和埃洛替尼，第二代的不可逆阿法替尼和达可替尼以及具有克服获得性耐药突变EGFR T790M突变活性的第三地奥西替尼。但是并非所有EGFR突变患者都能获益。其中EGFR 20外显子插入突变(EGFR 20ins)的NSCLC对大部分EGFR抑制剂靶向治疗效果差或无效，这些突变常常归为非经典突变。这些非经典突变主要包括18-21外显子的插入突变和点突变，ERBB2的点突变和插入突变。
因此，研究开发靶向非经典突变EGFR抑制剂的药物具有重大的临床意义和应用前景。
发明内容
本发明的目的在于提供一种靶向抑制非经典突变EGFR的化合物或其药学上可接受的盐。所述化合物应具备优异的抗肿瘤活性。
在第一方面，本发明提供式I所示化合物或其药学上可接受的盐、溶剂化物、立体异构体或前药：
Figure PCTCN2022121058-appb-000001
式I中
R 1独立选自氢、取代或未取代的C 1-C l0烷基、C 2-C 6链烯基、C 2-C 6炔基、任选取代的C 3-C 8环烷基、任选取代或未取代的芳基、取代或未取代的苄基、取代或未取代的杂环基、取代或未取代的芳杂环基；
R 2、R 3、R 4、R 5独立选自H、卤素、取代(优选卤素取代，更优选氟取代)或未取代的 C 1-C 6(优选C 1-C 3)烷氧基或取代(优选卤素取代，更优选氟取代)或未取代的C 1-C 6(优选C 1-C 3)氘代烷氧基、取代或未取代的C 1-C 6(优选C 1-C 3)烷基、NR cR d；其中R c和R d各自独立选自氢、C 1-3烷基；
G为苯环、五元或六元杂环或C 3-C 8环烷基或不存在；
R 6独立选自氢、未取代的或卤素取代的C 1-C 4烷基、硝基、氨基、卤素、羟基、C 1-C 6烷氧基、任选取代的C 1-C 6酰氧基、任选取代的C 1-C 6酰氨基、任选取代的C 1-C 6酰基；其中，当G为苯环时，R 6为间位取代；
m为0-3的整数；
R 7独立选自取代或未取代的NH 2、取代或未取代的杂环基、取代或未取代的芳杂环基、取代或未取代的C 1-C 10烷基；
A选自下组或不存在：
Figure PCTCN2022121058-appb-000002
X选自下组或不存在：取代或未取代的C 1-3亚烷基(优选-CH 2-)或氘代亚烷基(优选-CD 2-)、-O-、-C(＝O)-、-C(＝O)NHN＝-；
Y选自下组或不存在：-NHC(＝O)-、-C(＝O)NH-、-＝NNHC(＝O)NH-、-CH 2-、-O-；
L选自下组或不存在：C 1-C 10亚烷基、C 1-C 10亚杂烷基、-A’-(CH 2) m’-W-(CH 2) n’-、-(CH 2) m’-W-(CH 2) n’-O-(CH 2) V-和-(CH 2) m’-W-[(CH 2) n’-O] u-(CH 2) v-；
A’选自下组或不存在：5元亚芳基和6元亚芳基；
W选自：亚苯基、5元亚杂芳基、6元亚杂芳基、C 1-C 10亚杂环基和C 1-C 10亚烷基；
m’是0、1、2、3、4、5、6、7或8；
n’是0、1、2、3、4、5、6、7、8或9；
每个独立的u独立地为2、3或4；
v是1、2、3或4
B选自下组或不存在：
Figure PCTCN2022121058-appb-000003
R选自：氢、甲基和氟；
Q 1选自：-C(R 2a)＝和-N＝；
Q 2选自：-C(R 2b)＝和-N＝；
Q 3选自：-C(R 2c)＝和-N＝；
R 2a、R 2b、R 2c各自独立选自：氢，-C(＝O)-；
Z选自：-CH 2-，-C(＝O)-。
在具体的实施方式中，所述化合物是下式II所示化合物：
Figure PCTCN2022121058-appb-000004
式II中
R 7独立选自：H、取代(优选卤素取代，更优选氟取代)或未取代的C 1-C 6(优选C 1-C 3)烷基、取代(优选卤素取代，更优选氟取代)或未取代的C 1-C 6(优选C 1-C 3)氘代烷基；
R 8独立选自：H、取代(优选卤素取代，更优选氟取代)或未取代的C 1-C 3烷基、取代(优选卤素取代，更优选氟取代)或未取代的C 1-C 3氘代烷基。
在具体的实施方式中，式II中，
R 7独立选自：甲基、氘代甲基CD 3、三氟甲基、乙基、氘代乙基(例如，CD 2CH 3、CH 2CD 3)、CH 2CF 3；
R 8独立选自：氢、甲基、氘代甲基CD 3、乙基、氘代乙基(例如，CD 2CH 3、CH 2CD 3)。
在具体的实施方式中，所述化合物选自：
Figure PCTCN2022121058-appb-000005
在第二方面，本发明提供一种药物组合物，所述药物组合物包含第一方面所述的化合物、或其药学上可接受的盐、溶剂化物、立体异构体或前药和任选的药学上可接受的赋形剂。
在第三方面，本发明提供第一方面所述的化合物、或其药学上可接受的盐、溶剂化物、立体异构体或前药在制备预防或治疗与EGFR蛋白活性异常表达、ERBB2过表达及其点突变相关的疾病的药物中的用途。
在具体的实施方式中，所述EGFR是突变型EGFR。
在具体的实施方式中，所述突变型EGFR包括至少一种以下突变：EGFR敏感性突变L858R和19del，EGFR T790M突变，EGFR 18-21外显子点突变和插入突变，ERBB2过表达及其点突变和插入突变。
在具体的实施方式中，所述EGFR 18-21外显子点突变和插入突变含有：
18外显子G719X、E709X、K716A、K728A点突变与密码子709处缺失突变；
19号外显子插入突变I744-K745insKIPVAI、K745-E746insIPVAIK、K745-E746insVPVAIK、K745-E746insTPVAIK和点突变D761Y；
20号外显子插入突变与点突变包括：A763-Y764insFQEA、A763-Y764insFHEA、V769-D770insASV、V769-D770insDNP、D770-N771insNPG、D770-N771insNPH、D770-N771insSVD、D770-N771insASVDN、D770-N771insG、N771-P772insSVDNP、N771-H773dupNPH、P772-H773insPNP、P772-H773insPR、H773-V774insH、A763-Y764insFQEA、H773-V774insPH、H773-V774insNPH、N771-P772insH、H771-P772insN、H773-V774insAH、D770delinsGY、V774-C775insHV等以及20号外显子点突变S768I；
21号外显子点突变L861Q；
ERBB2的点突变V777L、D769Y、R896C、P1170A和插入突变V777-G778insCG、P780-Y781insGSP等。
在具体的实施方式中，所述疾病为癌症为选自以下的一种或多种：非小细胞肺癌、小细胞肺癌、肺腺癌、肺鳞癌、乳腺癌、胰腺癌、前列腺癌、卵巢癌、神经胶质细胞瘤、头颈部鳞癌、宫颈癌、食管癌、肝癌、肾癌、结肠癌、皮肤癌、白血病、淋巴瘤、胃癌或多发性骨髓癌。
在第四方面，本发明提供一种预防或治疗与EGFR蛋白活性异常表达、ERBB2过表达及其点突变相关的疾病的方法，所述方法包括将治疗有效量的第一方面所述的化合物、或其药学上可接受的盐、溶剂化物、立体异构体或前药或第二方面所述的药物组合物给予有此需要的对象的步骤。
在优选的实施方式中，所述对象是哺乳动物，优选是人。
在优选的实施方式中，所述EGFR是突变型EGFR。
在优选的实施方式中，所述突变型EGFR包括至少一种以下突变：EGFR敏感性突变L858R和19del，EGFR T790M突变，EGFR 18-21外显子点突变和插入突变，ERBB2过表达及其点突变和插入突变。
在优选的实施方式中，所述EGFR 18-21外显子点突变和插入突变含有：
18外显子G719X、E709X、K716A、K728A点突变与密码子709处缺失突变；
19号外显子插入突变I744-K745insKIPVAI、K745-E746insIPVAIK、K745-E746insVPVAIK、K745-E746insTPVAIK和点突变D761Y；
20号外显子插入突变与点突变包括：A763-Y764insFQEA、A763-Y764insFHEA、V769-D770insASV、V769-D770insDNP、D770-N771insNPG、D770-N771insNPH、D770-N771insSVD、D770-N771insASVDN、D770-N771insG、N771-P772insSVDNP、N771-H773dupNPH、P772-H773insPNP、P772-H773insPR、H773-V774insH、A763-Y764insFQEA、H773-V774insPH、H773-V774insNPH、N771-P772insH、H771-P772insN、 H773-V774insAH、D770delinsGY、V774-C775insHV等以及20号外显子点突变S768I；
21号外显子点突变L861Q；
ERBB2的点突变V777L、D769Y、R896C、P1170A和插入突变V777-G778insCG、P780-Y781insGSP等。
在优选的实施方式中，所述疾病为癌症为选自以下的一种或多种：非小细胞肺癌、小细胞肺癌、肺腺癌、肺鳞癌、乳腺癌、胰腺癌、前列腺癌、卵巢癌、神经胶质细胞瘤、头颈部鳞癌、宫颈癌、食管癌、肝癌、肾癌、结肠癌、皮肤癌、白血病、淋巴瘤、胃癌或多发性骨髓癌。
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2、化合物3的动物体内药效(EGFR L858R/T790M移植瘤)活性；
图2显示了化合物5的动物体内药效(EGFR L858R/T790M移植瘤)活性；
图3显示了化合物0、1、2、3、4、5在SD大鼠血浆中的药时曲线。
具体实施方式
发明人经过广泛而深入的研究，发现一批结构全新的能够靶向抑制非经典突变EGFR的小分子化合物。这些化合物具有抑制EGFR蛋白的活性以及良好的抗肿瘤活性，因此这些化合物可用于预防或/和治疗多种癌症，在医药领域具有巨大的应用前景。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个碳原子、1-6个、1-4个碳原子、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、异于基、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1-3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。烷氧基可以被1个或多个取代基取代，例如被卤素或卤代烷基取代。例如，烷氧基可以是被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，“链烯基”通常表示具有至少一个双键的单价烃基，通常含有2-8个碳原子，优 选含有2-6个碳原子，可以是直链或支链。链烯基的例子包括但不限于乙烯基、丙烯基、异丙烯基、丁烯基、异丁烯基、己烯基等等。
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NRcRd取代的烷基、被NRcRd取代的链烯基和NRcRd取代的炔基、被卤素取代的烷基、被氰基取代的链烯基，其中，Rc和Rd可选自烷基和链烯基。
本文中，“芳基”指含有6到14个碳原子的单环、双环或三环芳族基团，包括苯基、萘基、菲基、蒽基、茚基、茀基、四氢化萘基、二氢化茚基等。芳基可任选地被1-5个(例如，1、2、3、4或5个)选自以下的取代基取代：卤素、C1-4醛基、C1-6烷基、氰基、硝基、氨基、酰胺基、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、C1-4烷氧基、乙氧甲酰基、N(CH3)和C1-4酰基等、杂环基或杂芳基等。
本文中，“杂环基”包括但不限于含有1-3个选自O、S或N的杂原子的5元或6元杂环基团，包括但不限于呋喃基、噻吩基、吡咯基、吡咯烷基、吡唑基、咪唑基、三唑基、噁唑基、吡喃基、吡啶基、嘧啶基、吡嗪基、哌啶基、吗啉基等。
本文中，“芳杂环基”是指含有5-14个环原子，并且有6个、10个或14个电子在环体系上共用。而且所含环原子是碳原子和从氧、氮、硫中任选的1-3个杂原子。有用的芳杂环基包括哌嗪基、吗啉基、哌啶基、吡咯烷基、噻吩基、呋喃基、吡喃基、吡咯基、咪唑基、吡唑基、吡啶基、包括但不限制于嘧啶基等。芳杂环基可任选地被1-5个(例如，1、2、3、4或5个)选自以下的取代基取代：卤素、C1-4醛基、C1-6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、C1-4烷氧基、乙氧甲酰基、N(CH3)和C1-4酰基。
本文中，“卤素”指氟、氯、溴或碘。
本文中，“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、C1-4醛基、C1-6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、C1-4烷氧基、乙氧甲酰基、N(CH3)和C1-4酰基。
本发明的化合物
在本文中，本发明的化合物是指以下通式I所示的化合物、或其药学上可接受的盐、溶剂化物、立体异构体或前药：
Figure PCTCN2022121058-appb-000006
式中的取代基如上文所述定义。
进一步地，本发明提供式II所示化合物、或其药学上可接受的盐、溶剂化物、立体异构体或前药：
Figure PCTCN2022121058-appb-000007
式中的取代基如上文所述定义。
在具体的实施方式中，本发明提供了能够具备EGFR抑制活性的以下化合物，这些化合物对突变型EGFR(L858R/T790M、20ins)具有显著的抑制活性：
Figure PCTCN2022121058-appb-000008
Figure PCTCN2022121058-appb-000009
基于本发明的教导以及本领域的公知常识，本领域技术人员会理解，本发明化合物中的各基团可以作进一步的取代，从而得到能够具备与本发明具体公开的化合物活性相同或相似的衍生物。本发明化合物中的各基团可以被本领域常规的各种取代基取代，只要这种取代不违反化学合成规则或者化合价规则。
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或者本领域的常规取代基。因此，在本发明中，通式中的取代基也可以各自独立地为实施例中具体化合物中的相应基团；即，本发明既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。制备具有这样的取代基组合的化合物并检测所得化合物是活性是本领域技术人员基于本领域的惯常技术手段不难做到的。换言之，基于本发明的教导，本领域技术人员能够合成落在本发明的保护范围内的各种化合物，这些化合物并不限于说明书实施例部分公开的具体化合物；本发明的化合物既包括实施例部分公开的具体化合物，也包括这些具体化合物中的某一取代位置的具体取代基与通式中其它取代位置的取代基构成的各种化合物，限于篇幅，不在此一一列举。
本文所用的术语，“药学上可接受的盐”是指本发明化合物与酸或碱所形成的适合用作药物的盐。药学上可接受的盐包括无机盐和有机盐。一类优选的盐是本发明化合物与酸形成的盐。适合形成盐的酸包括但并不限于：盐酸、氢溴酸、氢氟酸、硫酸、硝酸、磷酸等无机酸，甲酸、乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、苦味酸、甲磺酸、苯甲磺酸，苯磺酸等有机酸；以及天冬氨酸、谷氨酸等酸性氨基酸。
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。
本文所用的术语，“互变异构体”表示具有不同能量的结构同分异构体可以超过低能垒，从而互相转化。比如，质子互变异构体(即质子移变)包括通过质子迁移进行互变，如1H-吲唑与2H-吲唑。化合价互变异构体包括通过一些成键电子重组而进行互变。
本文所用的术语，“溶剂化物”是指本发明化合物与溶剂分子配位形成特定比例的配合物。
本文所用的术语，“水合物”是指本发明化合物与水进行配位形成的配合物。
进一步地，作为一种具备药学活性的化合物，本发明化合物显然能够作为药物应用。因此，除了实施例中已经检测的各种特性，本发明的化合物还能够具备作为药物而固有的各种活性。例如，体内活性、生物利用度、成药性、毒性、差异抑制活性等等。基于本发明的教导和本领域的常规技术手段，本领域技术人员知晓如何获得本发明范围内的各种化合物以及检测本发明范围内的各种化合物的各种活性；换言之，基于本发明的教导和本领域的常规技术手段，本领域技术人员知晓如何重复、验证以及实现本发明。
药物组合物和施用方法
由于本发明化合物具有靶向抑制非经典突变EGFR，特别是对突变型EGFR
(L858R/T790M、20ins)具有显著的抑制活性，本发明化合物及其各种晶型，药学上可接受的无机或有机盐、前药或溶剂合物或水合物，以及含有本发明化合物为主要活性成分的药物组合物可用于预防和/或治疗与EGFR蛋白活性异常表达、ERBB2过表达及其点突变相关的疾病。
在具体的实施方式中，所述EGFR是突变型EGFR。例如，所述突变型EGFR包括至少一种以下突变：EGFR敏感性突变L858R和19del，EGFR T790M突变，EGFR 18-21外显子点突变和插入突变，ERBB2的点突变和插入突变。
在一优选例中，所述EGFR 18-21外显子点突变和插入突变含有：18外显子G719X、E709X、K716A、K728A点突变与密码子709处缺失突变；19号外显子插入突变I744-K745insKIPVAI、K745-E746insIPVAIK、K745-E746insVPVAIK、K745-E746insTPVAIK和点突变D761Y；20号外显子插入突变与点突变包括：A763-Y764insFQEA、A763-Y764insFHEA、V769-D770insASV、V769-D770insDNP、D770-N771insNPG、D770-N771insNPH、D770-N771insSVD、D770-N771insASVDN、D770-N771insG、N771-P772insSVDNP、N771-H773dupNPH、P772-H773insPNP、P772-H773insPR、H773-V774insH、A763-Y764insFQEA、H773-V774insPH、H773-V774insNPH、N771-P772insH、H771-P772insN、H773-V774insAH、D770delinsGY、V774-C775insHV等以及20号外显子点突变S768I；21号外显子点突变L861Q；ERBB2的点突变V777L、D769Y、R896C、P1170A和插入突变V777-G778insCG、P780-Y781insGSP等。
在优选的实施方式中，所述疾病为癌症，例如选自以下的一种或多种：非小细胞肺癌、小细胞肺癌、肺腺癌、肺鳞癌、乳腺癌、胰腺癌、前列腺癌、卵巢癌、神经胶质细胞瘤、头 颈部鳞癌、宫颈癌、食管癌、肝癌、肾癌、结肠癌、皮肤癌、白血病、淋巴瘤、胃癌或多发性骨髓癌。
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。通常，药物组合物含有1-2000mg本发明化合物/剂，更佳地，含有10-200mg本发明化合物/剂。较佳地，所述的“一剂”为一个胶囊或药片。
“药学上可接受的载体”指的是：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(如吐温
Figure PCTCN2022121058-appb-000010
)、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、肠胃外(静脉内、肌肉内或皮下)。
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、矫味剂和香料。
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。
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。
联合给药时，所述药物组合物还包括与一种或多种(2种，3种，4种，或更多种)其他药学上可接受的化合物。该其他药学上可接受的化合物中的一种或多种可与本发明的化合物同时、分开或顺序地施用。
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，对于60kg体重的人而言，日给药剂量通常为1～2000mg，优选20～500mg。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.本发明的化合物具有抑制EGFR蛋白，特别是突变型EGFR(L858R/T790M、20ins)的活性。因此，本发明的化合物可与用于EGFR蛋白异常表达的相关疾病，如各种癌症的治疗。
2.本发明的化合物具有优异的药代动力学性质；
3.本发明的化合物具有优异的抗肿瘤活性抗肿瘤活性，因此在医药领域具有巨大的应用前景。
以下结合具体实施案例对本发明的技术方案进一步描述，但以下实施例不构成对本发明的限制，所有依据本发明的原理和技术手段采用的各种施用方法，均属于本发明范围。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，否则百分比和份数按重量计算。
实施例1:N-(3-(2-((2-(甲氧基-d 3)-4-(4-甲基哌嗪-1-基)苯基)氨基)-7-氧代-6-苯基蝶呤-8(7H)-基)苯基)丙烯酰胺合成路线(化合物1)：
Figure PCTCN2022121058-appb-000011
4-氟-2-(甲氧基-d 3)-1-硝基苯的合成
Figure PCTCN2022121058-appb-000012
将NaH(0.20g，8.28mmol)、无水四氢呋喃(3mL)混合于50mL两口瓶，N 2保护，冰浴搅拌。将5-氟2-硝基苯酚(1.00g,6.37mmol)溶于无水四氢呋喃(4mL)，缓慢加入反应液，0℃搅拌10min，快速加入CD 3I(2.78g，19.11mmol)。升温至回流，继续反应19h。冷却至室温，将混合物用水淬灭，乙酸乙酯萃取，饱和氯化钠水溶液洗涤3次，有机相用无水硫酸钠干燥，真空浓缩，柱层析，得产物(0.84g,72.00％)。
1-(3-(甲氧基-d 3)-4-硝基苯基)-4-甲基哌嗪的合成
Figure PCTCN2022121058-appb-000013
将4-氟-2-(甲氧基-d 3)-1-硝基苯(11.52g,66.22mmol)、N-甲基哌嗪(6.62g,62.34mmol)、N,N-二异丙基乙胺(12.81g,99.33mmol)和乙腈(20.00mL)混合于100mL的单口瓶中，90℃条件下搅拌12h。反应混合物旋干，乙酸乙酯萃取，饱和氯化钠水溶液洗涤3次，有机相用无水硫酸钠干燥，真空浓缩，石油醚重结晶得黄色固体(16.30g，87.00％)。
2-(甲氧基-d 3)-4-(4-甲基哌嗪-1-基)苯胺的合成
Figure PCTCN2022121058-appb-000014
将4-氟-2-(甲氧基-d 3)-1-硝基苯(1.67g,6.56mmol)、10％钯碳(0.65g,0.65mmol)和乙醇(8.00mL)混合于50mL的两口瓶中，氢气保护，100℃条件下搅拌24h。反应混合物抽滤，然后用二氯甲烷萃取，饱和氯化钠水溶液洗涤3次，有机相用无水硫酸钠干燥，真空浓缩，柱层析(PE-DCM:MeOH30:1)，得褐色油状物。
N-(3-((2-((2-(甲氧基-d 3)-4-(4-甲基哌嗪-1-基)苯基)氨基)-5-硝基嘧啶-4-基)氨基)苯基)丙烯酰胺的合成
Figure PCTCN2022121058-appb-000015
称取2-(甲氧基-d 3)-4-(4-甲基哌嗪-1-基)苯胺(1.22g,5.45mmol)放置于50mL的单口瓶中，加入THF充分搅拌。称取N-(3-((2-氯-5-硝基嘧啶-4-基)氨基)苯基)丙烯酰胺(1.67g,5.45mmol)放置于另一个50mL的单口瓶中，加入THF、DIPEA(1.05g,8.17mmol),缓缓滴入，室温搅拌1h。将反应混合物旋干，用二氯甲烷萃取，饱和氯化钠水溶液洗涤3次，有机相用无水硫酸钠干燥，真空浓缩。甲醇重结晶，得红褐色固体。
N-(3-((5-氨基-2-((2-(甲氧基-d 3)-4-(4-甲基哌嗪-1-基)苯基)氨基)嘧啶-4-基)氨基)苯基)丙烯酰胺的合成
Figure PCTCN2022121058-appb-000016
将N-(3-((2-((2-(甲氧基-d 3)-4-(4-甲基哌嗪-1-基)苯基)氨基)-5-硝基嘧啶-4-基)氨基)苯基)丙烯酰胺(1.18g,2.33mmol)、铁粉(0.50g,9.32mmol)、氯化铵(0.62g,11.65mmol)和乙醇：水(8mL,4:1)混合于100mL的单口瓶中，80℃条件下搅拌24h。反应混合物抽滤，滤液调碱，用乙酸乙酯萃取，饱和氯化钠水溶液洗涤3次，有机相用无水硫酸钠干燥，真空浓缩，得紫黑色固体(0.45g，36.00％)。
N-(3-(2-((2-(甲氧基-d 3)-4-(4-甲基哌嗪-1-基)苯基)氨基)-7-氧代-6-苯基蝶呤-8(7H)-基)苯基)丙烯酰胺的合成
Figure PCTCN2022121058-appb-000017
将N-(3-((5-氨基-2-((2-(甲氧基-d 3)-4-(4-甲基哌嗪-1-基)苯基)氨基)嘧啶-4-基)氨基)苯基)丙烯酰胺(0.36g,0.75mmol)、苯甲酰甲酸乙酯(0.26g,1.12mmol)和乙醇(10mL,4:1)混合于微波反应瓶中，滴加几滴乙酸，微波条件(200PSI,50W,120℃)反应2h。反应混合物调碱，乙酸乙酯萃取，饱和氯化钠水溶液洗涤3次，有机相用无水硫酸钠干燥，真空浓缩，柱层析(DCM:MeOH＝25:1)，得红褐色固体(0.15g，30.00％)。
1H NMR(400MHz,DMSO-d 6)δ10.42(s,1H),8.87(s,1H),8.42(s,1H),8.20(dd,J＝6.8,3.1Hz,2H),7.89(d,J＝7.0Hz,1H),7.75(s,1H),7.54(t,J＝8.0Hz,1H),7.51-7.47(m,3H),7.35(d,J＝8.3Hz,1H),7.15(d,J＝8.0Hz,1H),6.54(d,J＝2.5Hz,1H),6.46(dd,J＝16.9,10.1Hz,1H),6.27(dd,J＝16.9,2.1Hz,1H),6.04(s,1H),5.78(dd,J＝10.0,2.1Hz,1H),3.05(s,4H),2.45(s,4H),2.23(s,3H).HRMS(ESI):[M+H] +calcd for C 33H 26D 6N 8O 3,592.2864；found592.2866.HPLC purity:97.54％,retention time＝6.844min.
实施例2:N-(3-(2-((2-甲氧基-4-(哌嗪-1-基)苯基)氨基)-7-氧代-6-苯基蝶啶-8(7H)-基)苯基)丙烯酰胺的合成(中间体)
Figure PCTCN2022121058-appb-000018
N-(3-硝基苯基)丙烯酰胺的制备
Figure PCTCN2022121058-appb-000019
称3-硝基苯胺(10.00g，72.4mmol)置于1000mL三口烧瓶中，加入250mL二氯甲烷，在0℃搅拌，随后滴加三乙胺(14g，108.6mmol)。另取丙烯酰胺(7.8g，86.87mmol)溶于100mL二氯甲烷，并滴加到上述反应液中，滴加完成后，继续在室温下搅拌1h，TLC跟踪原料基本完全转化。减压旋蒸，残留物加入100ml 20％氢氧化钾溶液有大量白色固体析出，超声后抽滤，随后滤饼用50mL二氯甲烷洗涤，真空干燥，，得到粗产品9.4g，产率约68％。减压回收二氯甲烷。
1H NMR(400MHz,DMSO-d 6):δ8.73(t,J＝2.1Hz,1H),7.98(dd,J＝8.1,1.2Hz,1H),7.91(dd,J＝8.2,1.6Hz,1H),7.62(t,J＝8.2Hz,1H),6.47(dd,J＝17.0,10.1Hz,1H),6.31(dd,J＝17.0,1.9Hz,1H),5.81(dd,J＝10.1,1.9Hz,1H).
N-(3-胺基苯基)丙烯酰胺的制备
Figure PCTCN2022121058-appb-000020
称取N-(3-硝基苯基)丙烯酰胺(5g，26.1mmol)于250mL圆底烧瓶中，加入溶剂150mL乙醇溶解，20ml水，再加入铁粉(5.8g，104.4mmol)，氯化铵(6.7g，130.5mmol)，80℃搅拌反应，TLC跟踪反应，3h后反应结束。反应液用硅藻土过滤，滤液旋蒸，残留物用EA和20ml 20％NaOH水萃取，反复三次。合并有几层，减压旋蒸，得到淡绿色固体3.5g，产率约83％。产品未经纯化，直接用于下步反应。
1H NMR(400MHz,DMSO-d 6): 1H NMR(400MHz,DMSO):δ8.73(t,J＝2.1Hz,1H),7.98(dd,J＝8.1,1.2Hz,1H),7.91(dd,J＝8.2,1.6Hz,1H),7.62(t,J＝8.2Hz,1H),6.47(dd,J＝17.0, 10.1Hz,1H),6.31(dd,J＝17.0,1.9Hz,1H),5.81(dd,J＝10.1,1.9Hz,1H)，5.62(s,J＝13.2,1.9Hz,1H).
N-(3-((2-氯-5-硝基嘧啶-4-基)氨基)苯基)丙烯酰胺的制备
Figure PCTCN2022121058-appb-000021
称取2,4-二氯-5-硝基嘧啶(5g，25.77mmol)、DIPEA(4.99g，38.66mmol)置于100mL圆底三口烧瓶中，加入40mL DMF溶解并置于常温搅拌反应。称取N-(3-氨基苯基)丙烯酰胺(4.1g，25.77mmol)于20ml DMF溶解并将混合液缓慢滴加到圆底烧瓶中，TLC检测反应，5h后反应结束。减压旋蒸除去乙腈，残留物用DCM萃取，反复三次。合并有机层，减压旋蒸。残留物用EA:PE＝1:4析晶，抽滤，滤饼用20ml EA:PE＝1:4混合液冲洗，滤渣真空干燥，得到橙红色固体6.1g，产率约84％。
1H NMR(400MHz,DMSO-d6)δ10.45(s,1H),10.33(s,1H),9.15(s,1H),7.96–7.85(m,1H),7.55(dd,J＝7.9,2.1Hz,1H),7.40(t,J＝8.1Hz,1H),7.26(dd,J＝7.8,2.1Hz,1H),6.48(dd,J＝17.0,10.1Hz,1H),6.28(dd,J＝17.0,2.1Hz,1H),5.78(dd,J＝10.0,2.1Hz,1H).
4-(4-硝基-3-甲氧基苯基)哌嗪-1-羧酸叔丁酯
Figure PCTCN2022121058-appb-000022
称取2-硝基-5-氟苯甲醚(10g，58.47mmol)、DIEA(11.31g，87.7mmol)置于50mL圆底三口烧瓶中，加入100mL DMF溶解，然后再加入哌嗪-1-羧酸叔丁酯(16.31g，87.7mmol)，在90℃条件下搅拌6h后，TLC跟踪至原料完全转化。在室温条搅拌下，向反应液中加入50mL去离子水，有大量黄色固体析出，抽滤，滤饼用50mL去离子水洗涤，真空干燥，得到纯的1-(3-甲氧基-4-硝基苯基)-4-甲基哌嗪黄色固体18g，产率约95％。
1H NMR(400MHz,DMSO-d6)δ7.91(d,J＝9.3Hz,1H),6.59(dd,J＝9.4,2.5Hz,1H),6.53(d,J＝2.5Hz,1H),3.91(s,3H),1.43(s,8H).
4-(4-氨基-3-甲氧基苯基)哌嗪-1-羧酸叔丁酯
Figure PCTCN2022121058-appb-000023
称取1-(3-甲氧基-4-硝基苯基)-4-甲基哌嗪(2.5g，10mmol)于250mL圆底烧瓶中，加入溶剂150mL乙醇溶解，再加入530mg Pd/C(含10％Pd)，用氢气球通入氢气，在常温下搅拌约3h后，TLC跟踪至原料完全转化。反应液用硅藻土抽滤，50mL乙醇洗涤，然后旋转蒸 发除去溶剂，得到2-甲氧基-4-(4-甲基-1-哌嗪)苯胺固体，纯度约99％。产品未经纯化，直接用于下步反应。
1H NMR(400MHz,DMSO-d6)δ6.52(d,J＝4.5Hz,1H),6.51(d,J＝1.3Hz,1H),6.31(dd,J＝8.3,2.4Hz,1H),4.26(s,2H),3.74(s,3H),3.43(d,J＝10.1Hz,4H),2.87(t,J＝5.1Hz,4H),1.41(s,9H).
N-(3-((2-((2-甲氧基-4-(4-甲基哌嗪-1-基)苯基)氨基)-5-硝基嘧啶-4-基)氨基)苯基酰胺的制备
Figure PCTCN2022121058-appb-000024
称取N-(3-((2-氯-5-硝基嘧啶-4-基)氨基)苯基)丙烯酰胺(2g，7.38mmol)置于100mL圆底烧瓶中，用50mL四氢呋喃溶解，再加入4-(4-氨基-3-甲氧基苯基)哌嗪-1-羧酸叔丁酯(2.18g，7.38mmol)和N,N-二异丙基乙胺(1.06g，8.3mmol)，氩气保护，在常温下搅拌约8h，TLC跟踪至原料完全转化。在室温条搅拌下，向反应液中加入50mL去离子水，有大量红褐色固体析出，抽滤，滤饼用50mL去离子水洗涤，真空干燥，得红褐色固体3.4g，收率约86％。
1H NMR(400MHz,DMSO-d6)δ10.30(s,1H),10.19(s,1H),9.22(s,1H),9.04(s,1H),7.70(s,1H),7.48(d,J＝8.2Hz,1H),7.40(d,J＝8.7Hz,1H),7.33(d,J＝8.2Hz,1H),7.19(t,J＝8.1Hz,1H),6.63(s,1H),6.44(dd,J＝16.9,10.1Hz,1H),6.28(t,J＝13.4Hz,2H),5.77(dd,J＝10.0,2.1Hz,1H),3.76(s,3H),3.46(t,J＝5.0Hz,4H),3.08(s,4H),1.44(s,9H).
4-(4-(((4-((3-丙烯酰胺基苯基)氨基)-5-氨基嘧啶-2-基)氨基)-3-甲氧基苯基)哌嗪-1-甲酸叔丁酯的制备
Figure PCTCN2022121058-appb-000025
称取N-(3-((2-((2-甲氧基-4-(4-甲基哌嗪-1-基)苯基)氨基)-5-硝基嘧啶-4-基)氨基)苯基酰胺(1.5g，2.61mmol)置于100mL圆底烧瓶中，加入溶剂50mL乙醇溶解，10ml水，再加入铁粉(0.58g，10.44mmol)，氯化铵(0.67g，13.05mmol)，80℃搅拌反应，TLC跟踪反应，3h后反应结束。反应液用硅藻土过滤，滤液旋蒸，残留物用EA和10ml 20％NaOH水萃取，反复三次。合并有几层，减压旋蒸，得到淡绿色固体0.92g，产率约61％。产品未经纯化，直接用于下步反应。
1H NMR(400MHz,DMSO-d6)δ10.60(s,1H),8.68(s,1H),8.02–7.95(m,1H),7.90–7.76(m,3H),7.57(t,J＝8.1Hz,1H),7.29(d,J＝8.2Hz,1H),6.63(d,J＝2.5Hz,1H),6.52(dd,J＝16.9,10.1Hz,1H),6.36(dd,J＝8.8,2.5Hz,1H),6.30(dd,J＝17.0,1.9Hz,1H),5.81(dd,J＝10.0,2.0Hz,1H),3.79(s,3H),3.45(t,J＝5.1Hz,4H),3.01(t,J＝5.1Hz,4H),2.44(s,3H),1.42(s,9H).
4-(4-(((8-(3-丙烯酰胺基苯基)-7-氧代-6-苯基-7,8-二氢蝶呤-2-基)氨基)-3-甲氧基苯基)哌嗪-1-羧酸叔丁酯的制备
Figure PCTCN2022121058-appb-000026
称取4-(4-(((4-((3-丙烯酰胺基苯基)氨基)-5-氨基嘧啶-2-基)氨基)-3-甲氧基苯基)哌嗪-1-甲酸叔丁酯(1.5g,2.7mmol)置于50mL圆底烧瓶中，加入1mL冰醋酸、15mL无水乙醇，然后加入苯甲酰甲酸乙酯(515mg,3mmol)，加热至回流搅拌约8h，减压旋蒸除去溶剂，残留物DCM和10ml 20％NaOH水萃取，反复三次，合并有机层，柱层析，得到橙红色固体0.71g，产率40％。
1H NMR(400MHz,DMSO-d6)δ10.41(s,1H),8.87(s,1H),8.44(s,1H),8.20(dd,J＝6.7,3.1Hz,2H),7.89(d,J＝8.0Hz,1H),7.75(d,J＝2.1Hz,1H),7.55(t,J＝8.1Hz,1H),7.51–7.45(m,3H),7.39–7.32(m,1H),7.16(dt,J＝7.9,1.3Hz,1H),6.58(d,J＝2.6Hz,1H),6.47(dd,J＝16.9,10.1Hz,1H),6.27(dd,J＝16.9,2.1Hz,1H),6.04(d,J＝5.4Hz,1H),5.77(dd,J＝10.1,2.1Hz,1H),3.78(s,3H),3.45(t,J＝5.1Hz,4H),3.00(s,4H),1.43(s,9H).
N-(3-(2-((2-甲氧基-4-(哌嗪-1-基)苯基)氨基)-7-氧代-6-苯基蝶啶-8(7H)-基)苯基)丙烯酰胺的制备
Figure PCTCN2022121058-appb-000027
称取4-(4-(((8-(3-丙烯酰胺基苯基)-7-氧代-6-苯基-7,8-二氢蝶呤-2-基)氨基)-3-甲氧基苯基)哌嗪-1-羧酸叔丁酯(50mg,0.07mmol)置于25mL圆底烧瓶中，加入20mL DCM溶解，然后加入三氟乙酸(24mg,0.21mmol)，常温搅拌，TLC跟踪反应2小时后反应结束，减压旋蒸，残留物用DCM和饱和碳酸氢钠水溶液萃取，合并有机层，旋蒸，得到红褐色固体，16mg，产率42％，随后立即投下一步。
1H NMR(400MHz,DMSO-d6)δ10.43(s,1H),8.87(s,1H),8.41(s,1H),8.20(dd,J＝6.5,3.2Hz,2H),7.79–7.70(m,2H),7.49(h,J＝4.1Hz,4H),7.35(dd,J＝22.3,15.9,7.2Hz,3H),7.15(d,J＝8.2Hz,1H),6.51(d,J＝7.8Hz,1H),6.49–6.41(m,1H),6.31–6.22(m,1H),6.04(d,J＝5.4Hz,1H),5.78(dd,J＝10.1,2.1Hz,1H).
实施例3:N-(3-(2-((2-甲氧基-4-(4-(甲基-d 3)哌嗪-1-基)苯基)氨基)-7-氧代-6-苯基蝶呤-8(7H)-基)苯基)丙烯酰胺合成(化合物2)：
Figure PCTCN2022121058-appb-000028
将NaH(0.20g，8.28mmol)、无水四氢呋喃(3mL)混合于50mL两口瓶，N 2保护，冰浴搅拌。将N-(3-(2-((2-甲氧基-4-(哌嗪-1-基)苯基)氨基)-7-氧代-6-苯基蝶呤-8(7H)-基)苯基)丙烯酰胺(3.66g,6.37mmol)溶于无水四氢呋喃(4mL)，缓慢加入反应液，0℃搅拌10min，快速加入CD 3I(2.78g，19.11mmol)。升温至回流，继续反应19h。冷却至室温，将混合物用水淬灭，乙酸乙酯萃取，饱和氯化钠水溶液洗涤3次，有机相用无水硫酸钠干燥，真空浓缩，柱层析，得产物(1.21g,32.00％)。
1H NMR(400MHz,DMSO-d 6)δ10.41(s,1H),8.86(s,1H),8.42(s,1H),8.19(s,2H),7.88(d,J＝7.7Hz,1H),7.75(s,1H),7.54(t,J＝8.1Hz,1H),7.51-7.46(m,3H),7.35(d,J＝8.3Hz,1H),7.15(d,J＝8.2Hz,1H),6.54(s,1H),6.46(dd,J＝16.9,10.1Hz,1H),6.27(d,J＝18.1Hz,1H),6.06(s,1H),5.78(d,J＝10.4Hz,1H),3.77(s,3H),3.05(s,4H),2.45(s,4H).HRMS(ESI):[M+H] +calcd for C 33H 26D 6N 8O 3,592.2864；found 592.2862.HPLC purity:98.77％,retention time＝6.839min.
化合物2的甲磺酸盐合成：
Figure PCTCN2022121058-appb-000029
称取化合物化合物2(100mg,17mmol)于100mL的圆底烧瓶中，加入10mL二氯甲烷形成橙红色浑浊，在30℃下搅拌10min，加入甲磺酸(16mg,17mmol)，混合物逐渐变成橙黄色澄清溶液，3min后缓慢变浑浊，继续室温搅拌1h后停止，室温静置过夜，少量的二氯甲烷挥发，壁上有黄色固体析出，超声后抽滤并用少量二氯甲烷洗涤，得到112mg黄色固体，产率96.5％。
其它化合物盐也采用类似方法获得。
实施例4:N-(3-(2-((2-(甲氧基-d 3)-4-(4-(甲基-d 3)哌嗪-1-基)苯基)氨基)-7-氧代-6-苯基蝶呤-8(7H))-基)苯基)丙烯酰胺合成路线(化合物3)：
Figure PCTCN2022121058-appb-000030
4-(3-(甲氧基-d 3)-4-硝基苯基)哌嗪-1-羧酸叔丁酯的合成
Figure PCTCN2022121058-appb-000031
将4-氟-2-(甲氧基-d 3)-1-硝基苯(14.90g,66.22mmol)、叔丁氧羰基哌嗪(11.60g,62.34mmol)、N,N-二异丙基乙胺(12.81g,99.33mmol)和乙腈(20.00mL)混合于100mL的单口瓶中，90℃条件下搅拌12h。反应混合物旋干，乙酸乙酯萃取，饱和氯化钠水溶液洗涤3次，有机相用无水硫酸钠干燥，真空浓缩，石油醚重结晶得黄色固体(19.60g，87.00％)。
4-(4-氨基-3-(甲氧基-d 3)苯基)哌嗪-1-羧酸叔丁酯的合成
Figure PCTCN2022121058-appb-000032
将4-(3-(甲氧基-d 3)-4-硝基苯基)哌嗪-1-羧酸叔丁酯(2.21g,6.56mmol)、10％钯碳(0.65g,0.65mmol)和乙醇(8.00mL)混合于50mL的两口瓶中，氢气保护，100℃条件下搅拌24h。反应混合物抽滤，然后用二氯甲烷萃取，饱和氯化钠水溶液洗涤3次，有机相用无水硫酸钠干燥，真空浓缩，柱层析(PE-DCM:MeOH30:1)，得褐色油状物。
4-(4-((4-((3-丙烯酰胺苯基)氨基)-5-硝基嘧啶-2-基)氨基)-3-(甲氧基-d 3)苯基)哌嗪-1-羧酸叔丁酯的合成
Figure PCTCN2022121058-appb-000033
(1)称取4-(4-氨基-3-(甲氧基-d 3)苯基)哌嗪-1-羧酸叔丁酯(1.74g,5.45mmol)放置于50mL的单口瓶中，加入THF充分搅拌，然后滴入反应体系(2)中。
(2)称取N-(3-((2-氯-5-硝基嘧啶-4-基)氨基)苯基)丙烯酰胺(1.67g,5.45mmol)放置于50mL的单口瓶中，加入THF、DIPEA(1.05g,8.17mmol),将反应体系(1)缓缓滴入，室温搅拌1h。将反应混合物旋干，用二氯甲烷萃取，饱和氯化钠水溶液洗涤3次，有机相用无水硫酸钠干燥，真空浓缩。甲醇重结晶，得红褐色固体。
4-(4-((4-((3-丙烯酰胺苯基)氨基)-5-氨基嘧啶-2-基)氨基)-3-(甲氧基-d 3)苯基)哌嗪-1-羧酸叔丁酯的合成
Figure PCTCN2022121058-appb-000034
将4-(4-((4-((3-丙烯酰胺苯基)氨基)-5-硝基嘧啶-2-基)氨基)-3-(甲氧基-d 3)苯基)哌嗪-1-羧酸叔丁酯(1.38g,2.33mmol)、铁粉(0.50g,9.32mmol)、氯化铵(0.62g,11.65mmol)和乙醇：水(8mL,4:1)混合于100mL的单口瓶中，80℃条件下搅拌24h。反应混合物抽滤，滤液调碱，用乙酸乙酯萃取，饱和氯化钠水溶液洗涤3次，有机相用无水硫酸钠干燥，真空浓缩，得紫黑色固体(0.47g，36.00％)。
叔丁基4-(4-((8-(3-丙烯酰胺苯基)-7-氧代-6-苯基-7,8-二氢蝶呤-2-基)氨基)-3-(甲氧基-d 3)苯基)哌嗪-1-羧酸酯的合成
Figure PCTCN2022121058-appb-000035
将4-(4-((4-((3-丙烯酰胺苯基)氨基)-5-氨基嘧啶-2-基)氨基)-3-(甲氧基-d 3)苯基)哌嗪-1-羧酸叔丁酯(0.42g,0.75mmol)、苯甲酰甲酸乙酯(0.26g,1.12mmol)和乙醇(10mL,4:1)混合于微波反应瓶中，滴加几滴乙酸，微波条件(200PSI,50W,120℃)反应2h。反应混合物调碱，乙酸乙酯萃取，饱和氯化钠水溶液洗涤3次，有机相用无水硫酸钠干燥，真空浓缩，柱层析(DCM:MeOH＝25:1),得红褐色固体(0.15g，30.00％)。
N-(3-(2-((2-(甲氧基-d 3)-4-(哌嗪-1-基)苯基)氨基)-7-氧代-6-苯基蝶呤-8(7H)-基)苯基)丙烯酰胺的合成
Figure PCTCN2022121058-appb-000036
将叔丁基4-(4-((8-(3-丙烯酰胺苯基)-7-氧代-6-苯基-7,8-二氢蝶呤-2-基)氨基)-3-(甲氧基-d 3)苯基)哌嗪-1-羧酸酯(0.42g,0.75mmol)、二氯甲烷(3mL)混合于25mL反应瓶中，冰浴下滴加几滴三氟乙酸，常温反应。反应混合物调碱，乙酸乙酯萃取，饱和氯化钠水溶液洗涤3次，有机相用无水硫酸钠干燥，真空浓缩，柱层析(DCM:MeOH＝25:1),得红褐色固体(0.30g，70.00％)。
N-(3-(2-((2-(甲氧基-d 3)-4-(4-(甲基-d 3)哌嗪-1-基)苯基)氨基)-7-氧代-6-苯基蝶呤-8(7H))-基)苯基)丙烯酰胺的合成(化合物3)
Figure PCTCN2022121058-appb-000037
将NaH(0.20g，8.28mmol)、无水四氢呋喃(3mL)混合于50mL两口瓶，N 2保护，冰浴搅拌。将N-(3-(2-((2-(甲氧基-d 3)-4-(哌嗪-1-基)苯基)氨基)-7-氧代-6-苯基蝶呤-8(7H)-基)苯基)丙烯酰胺(3.66g,6.37mmol)溶于无水四氢呋喃(4mL)，缓慢加入反应液，0℃搅拌10min，快速加入CD 3I(2.78g，19.11mmol)。升温至回流，继续反应19h。冷却至室温，将混合物用水淬灭，乙酸乙酯萃取，饱和氯化钠水溶液洗涤3次，有机相用无水硫酸钠干燥，真空浓缩，柱层析，得产物(1.21g,32.00％)。
1H NMR(600MHz,DMSO-d 6)δ10.42(s,1H),8.87(s,1H),8.41(s,1H),8.19(s,2H),7.89(s,1H),7.75(s,1H),7.54(t,J＝8.1Hz,1H),7.51-7.47(m,3H),7.35(s,1H),7.15(d,J＝7.8Hz,1H),6.53(d,J＝2.6Hz,1H),6.46(dd,J＝16.9,10.2Hz,1H),6.27(dd,J＝16.9,2.0Hz,1H),6.02(s,1H),5.78(dd,J＝10.1,2.0Hz,1H),3.05(s,4H),2.46(s,4H).HRMS(ESI):[M+H] +calcd for C 33H 26D 6N 8O 3,595.3052；found 595.3057.HPLC purity:98.82％,retention time＝6.825min.
实施例5:N-(3-(7-氧代-6-苯基-2-((4-(哌嗪-1-基)-2-(2,2,2-三氟乙氧基)苯基)氨基)蝶啶-8(7H))-基)苯基)丙烯酰胺的合成(中间体2)
Figure PCTCN2022121058-appb-000038
4-(4-硝基-3-(2,2,2-三氟乙氧基)苯基)哌嗪-1-羧酸叔丁酯的合成
Figure PCTCN2022121058-appb-000039
将4-氟-1-硝基-2-(2,2,2-三氟乙氧基)苯(1.00g,4.184mmol)、哌嗪-1-羧酸叔丁酯(1.17g,6.276mmol)、N,N-二异丙基乙胺(0.648g,5.021mmol)和DMF(15.00mL)混合于100mL的单口瓶中，90℃条件下搅拌12h。反应混合物旋干，二氯甲烷萃取，饱和氯化钠水溶液洗涤，有机相用无水硫酸钠干燥，真空浓缩，得黄色纯产物(2.2g，90.2％)。
4-(4-氨基-3-(2,2,2-三氟乙氧基)苯基)哌嗪-1-羧酸叔丁酯的合成
Figure PCTCN2022121058-appb-000040
将4-(4-硝基-3-(2,2,2-三氟乙氧基)苯基)哌嗪-1-羧酸叔丁酯(1.52g,3.76mmol)、10％钯碳(0.39g,0.38mmol)和乙醇(8.00mL)混合于100mL的三口瓶中，氢气保护，80℃条件下搅拌24h。反应混合物抽滤，然后用二氯甲烷萃取，饱和氯化钠水溶液洗涤3次，有机相用无水硫酸钠干燥，真空浓缩，柱层析(DCM:MeOH＝50:1)，得产物(1.02g,78％)。
4-(4-((4-((3-丙烯酰胺苯基)氨基)-5-硝基嘧啶-2-基)氨基)-3-(2,2,2-三氟乙氧基)苯基)哌嗪-1-羧酸叔丁酯的合成
Figure PCTCN2022121058-appb-000041
称取4-(4-氨基-3-(2,2,2-三氟乙氧基)苯基)哌嗪-1-羧酸叔丁酯(0.90g,2.42mmol)放置于50mL的单口瓶中，加入THF充分搅拌，然后滴入反应体系中。称取N-(3-((2-氯-5-硝基嘧啶-4-基)氨基)苯基)丙烯酰胺(0.78g,2.42mmol)放置于100mL的单口瓶中，加入THF、DIPEA(0.47g,3.63mmol),将反应体系缓缓滴入，室温搅拌1h。将反应混合物旋干，用二氯甲烷萃取，饱和氯化钠水溶液洗涤3次，有机相用无水硫酸钠干燥，真空浓缩。甲醇的水溶液重结晶，得红褐色固体(0.70g,50.72％)。
4-(4-((4-((3-丙烯酰胺苯基)氨基)-5-氨基嘧啶-2-基)氨基)-3-(2,2,2-三氟乙氧基)苯基)哌嗪-1-羧酸叔丁酯的合成
Figure PCTCN2022121058-appb-000042
将N-(3-((2-((4-(4-甲基哌嗪-1-基)-2-(2,2,2三氟乙氧基)苯基)氨基)-5-硝基嘧啶-4-基)氨基)苯基)丙烯酰胺(0.80g,1.22mmol)、铁粉(0.27g,4.90mmol)、氯化铵(0.33g,6.12mmol)和乙醇：水(25mL,4:1)混合于100mL的单口瓶中，80℃条件下搅拌24h。反应混合物抽滤，滤液调碱，用乙酸乙酯萃取，饱和氯化钠水溶液洗涤3次，有机相用无水硫酸钠干燥，真空浓缩，得深褐色固体(0.56g,75.75％)。
N-(3-(7-氧代-6-苯基-2-((4-(哌嗪-1-基)-2-(2,2,2-三氟乙氧基)苯基)氨基)蝶啶-8(7H))-基)苯基)丙烯酰胺的合成
Figure PCTCN2022121058-appb-000043
将N-(3-((2-((4-(4-甲基哌嗪-1-基)-2-(2,2,2三氟乙氧基)苯基)氨基)-5-胺基嘧啶-4-基)氨基)苯基)丙烯酰胺(0.33g,0.52mmol)、苯甲酰甲酸乙酯(0.19g,1.05mmol)和乙醇混合于微波反应瓶中，滴加几滴乙酸，微波条件(200PSI,50W,100℃)反应2h。反应混合物调碱，二氯甲烷萃取，饱和氯化钠水溶液洗涤3次，有机相用无水硫酸钠干燥，真空浓缩，柱层析(DCM:MeOH＝25:1),得红褐色固体(0.3g,78.78％)。
1H NMR(400MHz,DMSO-d 6)δ10.43(s,1H),8.90–8.80(m,1H),8.40(s,1H),8.18(dd,J＝6.1,2.6Hz,2H),7.84(d,J＝8.1Hz,1H),7.74(s,1H),7.54(d,J＝8.0Hz,1H),7.51–7.47(m,3H),7.33(s,1H),7.13(d,J＝7.6Hz,1H),6.65(s,1H),6.45(dd,J＝17.0,10.1Hz,1H),6.30–6.23(m,1H),6.17(s,1H),5.78(d,J＝10.0Hz,1H),4.71(q,J＝8.9Hz,2H),3.06(s,4H),2.44(s,4H).
实施例6:N-(3-(2-((4-(4-(甲基-d3)哌嗪-1-基)-2-(2,2,2-三氟乙氧基)苯基)氨基)-7-氧代-6-苯基蝶啶)-8(7H)-基)苯基)丙烯酰胺的合成(化合物4)
Figure PCTCN2022121058-appb-000044
将NaH(0.20g，8.28mmol)、无水四氢呋喃(3mL)混合于50mL两口瓶，N 2保护，冰浴搅拌。将N-(3-(7-氧代-6-苯基-2-((4-(哌嗪-1-基)-2-(2,2,2三氟乙氧基)苯基)氨基)蝶啶-8(7H)-基))苯基)丙烯酰胺(4.10g,7.12mmol)溶于无水四氢呋喃(4mL)，缓慢加入反应液，0℃搅拌10min，快速加入CD 3I(2.78g，19.11mmol)。升温至回流，继续反应19h。冷却至室温，将混合物用水淬灭，乙酸乙酯萃取，饱和氯化钠水溶液洗涤3次，有机相用无水硫酸钠干燥，真空浓缩，柱层析，得产物(1.21g,32.00％)。
LC-MS:m/z＝660[M+H] +.1H NMR(600MHz,DMSO-d6)δ10.43(s,1H),8.88(s,1H),8.37(s,1H),8.21(dd,J＝6.7,3.1Hz,2H),7.87(d,J＝8.2Hz,1H),7.76(d,J＝2.2Hz,1H),7.54(t,J＝8.1Hz,1H),7.52–7.45(m,3H),7.43–7.27(m,1H),7.15(dd,J＝7.8,1.9Hz,1H),6.69(s, 1H),6.48(dd,J＝16.9,10.2Hz,1H),6.28(dd,J＝16.9,2.0Hz,1H),6.23–6.09(m,1H),5.79(dd,J＝10.1,2.0Hz,1H),4.75(q,J＝8.9Hz,2H),3.09(s,4H),2.49(s,4H).
实施例7:N-(3-(2-((4-(4-甲基哌嗪-1-基)-2-(2,2,2-三氟乙氧基)苯基)氨基)-7-氧代-6-苯基蝶啶-8(7H))-基)苯基)丙烯酰胺的合成(化合物5)
Figure PCTCN2022121058-appb-000045
4-(4-硝基-3-(2,2,2-三氟乙氧基)苯基)哌嗪-1-羧酸叔丁酯的合成
Figure PCTCN2022121058-appb-000046
将4-氟-1-硝基-2-(2,2,2-三氟乙氧基)苯(1.00g,4.184mmol)、N-甲基哌嗪(0.63g,6.276mmol)、N,N-二异丙基乙胺(0.648g,5.021mmol)和DMF(15.00mL)混合于100mL的单口瓶中，90℃条件下搅拌12h。反应混合物旋干，二氯甲烷萃取，饱和氯化钠水溶液洗涤3次，有机相用无水硫酸钠干燥，真空浓缩，得黄色纯产物(1.20g，90.2％)。
LC-MS:m/z＝320[M+H] +. 1H NMR(400MHz,DMSO-d 6)δ7.91(d,J＝9.4Hz,1H),6.69(dd,J＝9.4,2.4Hz,1H),6.63(d,J＝2.3Hz,1H),4.92(q,J＝8.8Hz,2H),3.47–3.42(m,4H),2.44–2.40(m,4H),2.22(s,3H).
4-(4-甲基哌嗪-1-基)-2-(2,2,2-三氟乙氧基)苯胺的合成
Figure PCTCN2022121058-appb-000047
将1-甲基-4-(4-硝基-3-(2,2,2-三氟乙氧基)苯基)哌嗪(1.20g,3.76mmol)、10％钯碳(0.39g,0.38mmol)和乙醇(8.00mL)混合于100mL的三口瓶中，氢气保护，80℃条件下搅拌24h。反应混合物抽滤，然后用二氯甲烷萃取，饱和氯化钠水溶液洗涤3次，有机相用无水硫酸钠干燥，真空浓缩，柱层析(DCM:MeOH＝50:1)，得产物(0.85g,78％)。
N-(3-((2-((4-(4-甲基哌嗪-1-基)-2-(2,2,2-三氟乙氧基)苯基)氨基)-5-硝基嘧啶-4-基)氨基)苯 基)丙烯酰胺的合成
Figure PCTCN2022121058-appb-000048
称取4-(4-甲基哌嗪-1-基)-2-(2,2,2-三氟乙氧基)苯胺(0.70g,2.42mmol)放置于50mL的单口瓶中，加入THF充分搅拌。称取N-(3-((2-氯-5-硝基嘧啶-4-基)氨基)苯基)丙烯酰胺(0.78g,2.42mmol)放置于100mL的单口瓶中，加入THF、DIPEA(0.47g,3.63mmol)，将反应液混合，室温搅拌反应1h。将反应混合物旋干，用二氯甲烷萃取，饱和氯化钠水溶液洗涤3次，有机相用无水硫酸钠干燥，真空浓缩。甲醇:水重结晶，得红褐色固体(0.70g,50.72％)。
N-(3-((2-((4-(4-甲基哌嗪-1-基)-2-(2,2,2三氟乙氧基)苯基)氨基)-5-胺基嘧啶-4-基)氨基)苯基)丙烯酰胺
Figure PCTCN2022121058-appb-000049
将N-(3-((2-((4-(4-甲基哌嗪-1-基)-2-(2,2,2三氟乙氧基)苯基)氨基)-5-硝基嘧啶-4-基)氨基)苯基)丙烯酰胺(0.70g,1.22mmol)、铁粉(0.27g,4.90mmol)、氯化铵(0.33g,6.12mmol)和乙醇：水(25mL,4:1)混合于100mL的单口瓶中，80℃条件下搅拌24h。反应混合物抽滤，滤液调碱，用乙酸乙酯萃取，饱和氯化钠水溶液洗涤3次，有机相用无水硫酸钠干燥，真空浓缩，得深褐色固体(0.50g,75.75％)。
N-(3-(2-((4-(4-甲基哌嗪-1-基)-2-(2,2,2-三氟乙氧基)苯基)氨基)-7-氧代-6-苯基蝶啶-8(7H))-基)苯基)丙烯酰胺
Figure PCTCN2022121058-appb-000050
将N-(3-((2-((4-(4-甲基哌嗪-1-基)-2-(2,2,2三氟乙氧基)苯基)氨基)-5-胺基嘧啶-4-基)氨基)苯基)丙烯酰胺(0.28g,0.52mmol)、苯甲酰甲酸乙酯(0.19g,1.05mmol)和乙醇混合于微波反应瓶中，滴加几滴乙酸，微波条件(200PSI,50W,100℃)反应2h。反应混合物调碱，二氯甲烷萃取，饱和氯化钠水溶液洗涤3次，有机相用无水硫酸钠干燥，真空浓缩，柱层析 (DCM:MeOH＝25:1),得红褐色固体(0.26g,78.78％)。
LC-MS:m/z＝657[M+H] +. 1H NMR(400MHz,DMSO-d 6)δ10.43(s,1H),8.86(s,1H),8.41(s,1H),8.18(dd,J＝6.8,3.0Hz,2H),7.84(d,J＝8.3Hz,1H),7.74(t,J＝2.0Hz,1H),7.54(d,J＝8.0Hz,1H),7.51–7.46(m,3H),7.33(s,1H),7.13(d,J＝8.2Hz,1H),6.65(s,1H),6.45(dd,J＝17.0,10.2Hz,1H),6.26(dd,J＝16.9,2.0Hz,1H),6.15(s,1H),5.78(dd,J＝10.0,2.1Hz,1H),4.71(q,J＝8.9Hz,2H),3.06(s,4H),2.44(t,J＝5.0Hz,4H),2.22(s,3H),1.22(d,J＝2.6Hz,3H).
实施例8.激酶和细胞活性测试
测试方法
a)激酶活性测试方法
EGFR-TK能催化三磷酸腺昔(ATP)的一个磷酸基团转移到多肤底物poly(Glu,Tyr) 4:1上，而多肤底物标记有两个荧光基团香豆素(coumarin)和荧光素(fluorescein)。基于荧光能量共振转移(FRET)的方法，EGFR-TK催化ATP发生反应导致两个荧光基团接近，供体(coumarin)在400nM处被激发，部分能量释放，发射波长为445nM，另一部分能量转移到fluorescein，发射波长为520nM。不同化合物对EGFR-TK的抑制程度不同，导致底物磷酸化的程度不同，从而通过测定酶催化底物磷酸化百分比的比值，计算不同化合物的抑制率。
在384孔板中加入2.5μL Test Compounds，5μL Kinase/Peptide Substrate Mixture，2.5μL ATP Solution，10μL反应体系振荡30s混匀，室温孵育1h；加入匀5μL Development Solution，15μL反应体系振荡30s混匀，室温孵育1h；加入5μL Stop Reagent，总体积20μL反应体系振荡30s混匀，使用酶标仪进行荧光信号的检测，激发波长为400nm，发射波长分别为445nm和520nm。测定化合物在7个浓度梯度下的抑制率，通过Origin8.0拟合曲线计算各个化合物的IC 50值。实验过程进行阳性对照确证反应体系可行性，每次实验三个平行。
体外酶活性分析：野生型及突变型(L858/T790M，Del19)EGFR均购自于Invitrogen。为所有的待测试化合物设置了从5.1×10 -11mo1/L到1.0×10 -6mo1/L的10个浓度梯度。
不同激酶的浓度由优化实验决定，化合物在DMSO中从5.1x10 -9M到1x l0 -4M稀释三倍。4μL化合物溶于96μL水，得到4x的化合物溶液。40μM ATP溶于1.33x激酶缓冲液，激酶/肽混合物包含2x激酶、4μM醋氨酸4肽准备好待用。10μL激酶反应包括2.5μL化合物溶液，5μL激酶/肽混合物，2.5μL ATP溶液。5μL磷酸化肽溶液代替激酶/肤混合物用作100％磷酸化对照。2.5μL 1.33x激酶缓冲液代替ATP溶液用作100％抑制对照，2.5μL 4％DMSO代替化合物溶液用作0％抑制对照。板内溶液充分混合后在室温下培养1.5小时。每孔加入5μL Development Solution后继续在室温下培养1小时，非磷酸化肤在此时间内被裂解。最后，加入5μL Stop Reagent结束反应。孔板用EnVisionMultilabel Reader(Perkin Elmer)进行测量。实验数据使用GraphPad Prismversion 4.0进行计算。每次实验均重复3次以上。
b)细胞增殖抑制活性测试方法
细胞增殖及生长抑制分析：H1975(EGFR L858R/T790M)细胞均从ATCC获得。细胞增殖活性采用MTS分析法进行评估。细胞暴露在处理条件下72小时，各细胞系每次实验所使用的细胞数根据吸光度值(490nm处的吸光度值为1.3-2.2)进行调整。为待测试化合物设置了6个浓 度梯度(0.1nM-10μM)，每个浓度值至少使用6组平行对照。
H1975细胞在相应的培养基中培养，细胞在复苏后至少传代两次，然后用于实验使用。对数期的细胞受膜蛋白酶作用并在培养基中再悬浮。H1975(每孔1000细胞)播种于96孔板中，体积100μL；设置6组平行及7列。孔板放于37℃5％二氧化碳的培养箱中过夜。将化合物溶于DMSO，配制浓度为每升10μM，随后将化合物浓度逐步稀释得到的化合物浓度分别为每升10μM、1μM、0.1μM、0.01μM、0.001μM、0.0001μM。2μL化合物溶液加到998μL的培养基中，混合物经充分混合。100μL的混合物加入96孔板中。2μL DMSO代替化合物溶液用作0％抑制对照。培养68小时之后，加入20μL MTT(5mg/mL)。4小时后，抛弃上清液并加入150μL DMSO。摇振10分钟之后，孔板用Synergy HT(Bio TeK)(OD490)读取数据。数据使用GraphPad Prism version 4.0进行计算，IC 50值通过使用剂量反应曲线的非线性回归模型调整得到。
结果如表1所示：
表1.化合物对EGFR(L858R/T790M)激酶和H1975细胞的抑制活性
Figure PCTCN2022121058-appb-000051
Figure PCTCN2022121058-appb-000052
表中数据可以看出，本发明的氘代化合物在激酶水平对EGFR L858R/T790M抑制IC 50(nM)在1.0-1.5之间，与非氘代化合物相似。说明氘代衍生物不影响抑制剂和激酶相互作用。而三氟乙氧基取代后的化合物4和化合物5会导致激酶抑制活性下降1倍左右，较大基团引入导致二者相互作用降低是可以理解的。
本发明的化合物对H1975细胞的抑制活性表现规律性不强，这与衍生物的理化性质和代谢性质改变有关，因素较多。氘代化合物与非氘代化合物的细胞活性相似。
实施例9.EGFR 20ins及罕见突变激酶活性测试结果
EGFR 20ins及罕见突变激酶活性测试(委托国外测试公司：Reaction Biology检测，国内代理公司：科瑞斯生物)
测试方法：
RBC--EGFR罕见突变激酶检测方案(Protocol)
a)在新制备的反应缓冲液中制备底物
b)将任何所需的辅因子输送到上述底物溶液中
c)将激酶输送到底物溶液中并轻轻混合
d)通过Acoustic将100％DMSO中的化合物输送到激酶反应混合物中技术(Echo550；纳升范围)，在室温下孵育20分钟
e)将33P-ATP输送到反应混合物中以启动反应
f)室温孵育2小时
g)P81过滤结合法检测激酶活性
Figure PCTCN2022121058-appb-000053
Figure PCTCN2022121058-appb-000054
从上表的数据可以看出，本发明的化合物对EGFR罕见突变和20ins插入突变的激酶的抑制活性表现优异，比2021年上市的20ins插入突变阳性药TAK788要好。其中化合物2表现最好，有良好应用前景。
实施例10.动物体内药效(EGFR L858R/T790M移植瘤)活性测试结果
动物体内活性评价方法(化合物对H1975细胞移植瘤的体内药效评价)：
实验目的：连续14天口服(PO)给予H1975移植瘤BALB/c裸鼠受试物，随时间记录观察裸鼠体重和肿瘤大小变化，并观察裸鼠给药后的生理活动变化，用以考察受试物抗肿瘤药效。
H1975细胞准备
H1975细胞为上海中科院药物所协议转让。细胞培养基条件为1640+10％FBS(Gibco)，培养箱条件为37℃恒温二氧化碳培养箱，二氧化碳浓度为5％，细胞2-3天换液传代一次并不断扩大培养。待传至所需细胞量进行细胞移植，用胰酶消化细胞4min，然后使用培养基终止消化。收集所得的细胞，1000r/min，3min离心，去除上清，用无血清的1640洗涤2次，移植前细胞活力应保持在95％以上，并用无血清1640做溶媒悬浮细胞，悬液细胞浓度为2000万/mL，将配好的细胞悬液放在冰上待用，为保持细胞活力，细胞悬液应在0.5-1h内接种完成。
H1975移植瘤建模
雄性BALB/c裸鼠72只，4-5周月龄，体重17-23g，由江苏集萃药康生物科技股份有限公司购买，适应动物房环境2-3天后便进行细胞接种。选择裸鼠前肢上侧毛细血管丰富处进行皮下注射细胞悬液，每只注射细胞悬浮液量为0.1mL。
动物房实验条件：实验室温度:23±3℃；
实验室湿度：30-60％；
各12小时开灯/关灯设置；
1-2周左右可见移植瘤成型，待肿瘤平均大小达100-200mm 3时进行随机分组，每组8只小鼠，使得每组肿瘤体积基本一致。然后可以给药，按照各组别的给药化合物、给药剂量，给药方式进行给药。之后每天给药，2天1次记录肿瘤体积和小鼠体重，溶剂对照给等量溶剂(0.1mL)。给药前需要记录小鼠的肿瘤体积，小鼠重量。数据使用GraphPad Prism version 4.0画图。整个实验过程中，隔天测量移植瘤直径，同时称量小鼠体重。肿瘤体积(tumor volume,TV)的计算公式为：TV＝1/2×L×W2，其中L、W分别表示长、宽，单位mm。其中V0为分笼给药时(即d0)测量所得肿瘤体积，Vt为每一次测量时的肿瘤体积。抗肿瘤活性的评价指标为肿瘤体积增长抑制率TGI，计算公式如下：％TGI＝{[1-(TVt/TV0)/(CVt/CV0)]/(1-CV0/CVt)}×100，TVt＝治疗组在t时的肿瘤体积均值,TV0＝治疗组在加药前的肿瘤体积均值，CVt＝溶剂对照组在t时的肿瘤体积均值，CV0＝溶剂对照组在加药前的肿瘤体积均值。
Figure PCTCN2022121058-appb-000055
结果见图1和图2。从图1看出，化合物2、化合物3动物体内药效(EGFR L858R/T790M移植瘤)活性优异，化合物3(10mg/Kg)活性TGI就达到了96.2％，接近100％；化合物2(10mg/Kg)活性TGI就达到了106.2％。化合物2(5mg/Kg)活性TGI也达到了近80％，效果很明显。从图2看出，化合物5动物体内药效(EGFR L858R/T790M移植瘤)活性表现一般。
实施例11.化合物对Her2高表达细胞：乳腺癌细胞SKBR3、肺癌细胞H2170、胃癌细胞N87的抑制活性
细胞增殖抑制活性测试方法
细胞增殖及生长抑制分析：乳腺癌细胞SKBR3、肺癌细胞H2170、胃癌细胞N87均从中科院细胞库获得。细胞增殖活性采用CCK8分析法进行评估。细胞暴露在处理条件下72小时，各细胞系每次实验所使用的细胞数根据吸光度值(450nm处的吸光度值为1-1.2)进行调整。为待测试化合物设置了8个浓度梯度(0.1nM-10μM)，每个浓度值至少使用6组平行对照。
细胞在相应的培养基中培养，细胞在复苏后至少传代两次，细胞生长处在对数生长期，然后用于实验使用。SKBR3细胞3000个/孔，H2170细胞6000/孔，N87细胞9000/孔，铺在96孔板中，体积90μL；设置6组平行及7列。孔板放于37℃5％二氧化碳的培养箱中过夜。
将化合物溶于DMSO，关于SKBR3细胞株，配制母液浓度为每升10mM，随后用培养 基将化合物稀释100倍，得到最大浓度100μM，然后化合物浓度2倍梯度稀释逐步稀释8个浓度梯度；关于H2170细胞株，配制母液浓度为每升100μM，随后用培养基将化合物稀释100倍，得到最大浓度1μM，然后化合物浓度3倍梯度稀释逐步稀释8个浓度梯度；关于N87细胞株，配制母液浓度为每升100μM，随后用培养基将化合物稀释100倍，得到最大浓度1μM，然后化合物浓度5倍梯度稀释逐步稀释8个浓度梯度；10μL化合物溶液加到铺好的96孔板中。对照补10ul含1％DMSO的培养基代替化合物溶液用作0％抑制对照。培养72小时之后，加入10μl CCK8。1.5小时后，孔板用酶标仪读取数据。数据使用GraphPad Prism version 4.0进行计算，IC 50值通过使用剂量反应曲线的非线性回归模型调整得到。
化合物 SKBR3(μM) H2170(nM) N87(nM)
Poziotinib 11.16±3.27 0.74±0.086 0.069±0.035
化合物0 2.68±0.91 17.51±3.09 3.39±1.26
化合物1 2.37±0.79 24.67±4.16 2.72±0.70
化合物2 2.14±0.60 20.29±3.64 2.95±1.36
化合物3 1.92±0.41 21.77±6.91 2.92±1.65
化合物4 2.79±0.76 25.68±4.45 8.02±2.43
化合物5 3.32±0.91 35.73±2.34 8.29±3.22
从上表的数据可以看出，在肺癌H2170、胃癌N87细胞上，化合物1、化合物2、化合物3的细胞活性最优。相比Poziotinib，氘代化合物的细胞活性略低。在乳腺癌SKBR3细胞上，氘代化合物的抑制活性IC 50在2-3μM之间，相比于Poziotinib的IC 50(11μM)活性更好。
实施例12.化合物的药代动力学性质测试
本发明化合物的药代动力学性质测试委托杭州先导医药科技有限公司和美迪西普亚医药科技(上海)有限公司进行。
Figure PCTCN2022121058-appb-000056
Figure PCTCN2022121058-appb-000057
如上所述，化合物1-化合物6的药代性质都有明显改善。以在双突变EGFR L858R/T790M表现良好，在EGFR 20ins主要插入突变中表现最好的化合物2为例，其生物利用度达到29.4％；Cmax为223.96ng/mL；AUC (0-∞)为2643.35ng/mL*h。作为化合物2的盐形式，其甲磺酸盐的药代动力学性质会更好。这些性质对该类型抑制剂在后期临床应用中具有极大优势，且在降低有效剂量，改善安全性方面优势更为明显。
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. 式I所示化合物或其药学上可接受的盐、溶剂化物、立体异构体或前药：
    Figure PCTCN2022121058-appb-100001
    式I中
    R 1独立选自氢、取代或未取代的C 1-C l0烷基、C 2-C 6链烯基、C 2-C 6炔基、任选取代的C 3-C 8环烷基、任选取代或未取代的芳基、取代或未取代的苄基、取代或未取代的杂环基、取代或未取代的芳杂环基；
    R 2、R 3、R 4、R 5独立选自H、卤素、取代(优选卤素取代，更优选氟取代)或未取代的C 1-C 6(优选C 1-C 3)烷氧基或取代(优选卤素取代，更优选氟取代)或未取代的C 1-C 6(优选C 1-C 3)氘代烷氧基、取代或未取代的C 1-C 6(优选C 1-C 3)烷基、NR cR d；其中R c和R d各自独立选自氢、C 1-3烷基；
    G为苯环、五元或六元杂环或C 3-C 8环烷基或不存在；
    R 6独立选自氢、未取代的或卤素取代的C 1-C 4烷基、硝基、氨基、卤素、羟基、C 1-C 6烷氧基、任选取代的C 1-C 6酰氧基、任选取代的C 1-C 6酰氨基、任选取代的C 1-C 6酰基；其中，当G为苯环时，R 6为间位取代；
    m为0-3的整数；
    R 7独立选自取代或未取代的NH 2、取代或未取代的杂环基、取代或未取代的芳杂环基、取代或未取代的C 1-C 10烷基；
    A选自下组或不存在：
    Figure PCTCN2022121058-appb-100002
    X选自下组或不存在：取代或未取代的C 1-3亚烷基(优选-CH 2-)或氘代亚烷基(优选-CD 2-)、-O-、-C(＝O)-、-C(＝O)NHN＝-；
    Y选自下组或不存在：-NHC(＝O)-、-C(＝O)NH-、-＝NNHC(＝O)NH-、-CH 2-、-O-；
    L选自下组或不存在：C 1-C 10亚烷基、C 1-C 10亚杂烷基、-A’-(CH 2) m’-W-(CH 2) n’-、-(CH 2) m’-W-(CH 2) n’-O-(CH 2) V-和-(CH 2) m’-W-[(CH 2) n’-O] u-(CH 2) v-；
    A’选自下组或不存在：5元亚芳基和6元亚芳基；
    W选自：亚苯基、5元亚杂芳基、6元亚杂芳基、C 1-C 10亚杂环基和C 1-C 10亚烷基；
    m’是0、1、2、3、4、5、6、7或8；
    n’是0、1、2、3、4、5、6、7、8或9；
    每个独立的u独立地为2、3或4；
    v是1、2、3或4
    B选自下组或不存在：
    Figure PCTCN2022121058-appb-100003
    R选自：氢、甲基和氟；
    Q 1选自：-C(R 2a)＝和-N＝；
    Q 2选自：-C(R 2b)＝和-N＝；
    Q 3选自：-C(R 2c)＝和-N＝；
    R 2a、R 2b、R 2c各自独立选自：氢，-C(＝O)-；
    Z选自：-CH 2-，-C(＝O)-。
  2. 如权利要求1所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或前药，其特征在于，所述化合物是下式II所示化合物：
    Figure PCTCN2022121058-appb-100004
    式II中
    R 7独立选自：H、取代(优选卤素取代，更优选氟取代)或未取代的C 1-C 6(优选C 1-C 3)烷基、取代(优选卤素取代，更优选氟取代)或未取代的C 1-C 6(优选C 1-C 3)氘代烷基；
    R 8独立选自：H、取代(优选卤素取代，更优选氟取代)或未取代的C 1-C 3烷基、取代(优选卤素取代，更优选氟取代)或未取代的C 1-C 3氘代烷基。
  3. 如权利要求2所述的化合物或其药学上可接受的盐、溶剂化物、立体异构体或前药，其特征在于，式II中，
    R 7独立选自：甲基、氘代甲基CD 3、三氟甲基、乙基、氘代乙基(例如，CD 2CH 3、CH 2CD 3)、CH 2CF 3；
    R 8独立选自：氢、甲基、氘代甲基CD 3、乙基、氘代乙基(例如，CD 2CH 3、CH 2CD 3)。
  4. 下组所示的化合物，或其药学上可接受的盐、溶剂化物、立体异构体或前药，其特征在于，所述化合物选自：
    Figure PCTCN2022121058-appb-100005
    Figure PCTCN2022121058-appb-100006
  5. 一种药物组合物，所述药物组合物包含权利要求1-4中任一项所述的化合物、或其药学上可接受的盐、溶剂化物、立体异构体或前药和任选的药学上可接受的赋形剂。
  6. 如权利要求1-4中任一项所述的化合物、或其药学上可接受的盐、溶剂化物、立体异构体或前药在制备预防或治疗与EGFR蛋白活性异常表达、ERBB2过表达及其点突变相关的疾病的药物中的用途。
  7. 如权利要求6所述的用途，其特征在于，所述EGFR是突变型EGFR。
  8. 如权利要求7所述的用途，其特征在于，所述突变型EGFR包括至少一种以下突变：EGFR敏感性突变L858R和19del，EGFR T790M突变，EGFR 18-21外显子点突变和插入突变，ERBB2过表达及其点突变和插入突变。
  9. 如权利要求8所述的用途，其特征在于，所述EGFR 18-21外显子点突变和插入突变含有：
    18外显子G719X、E709X、K716A、K728A点突变与密码子709处缺失突变；
    19号外显子插入突变I744-K745insKIPVAI、K745-E746insIPVAIK、K745-E746insVPVAIK、K745-E746insTPVAIK和点突变D761Y；
    20号外显子插入突变与点突变包括：A763-Y764insFQEA、A763-Y764insFHEA、V769-D770insASV、V769-D770insDNP、D770-N771insNPG、D770-N771insNPH、D770-N771insSVD、D770-N771insASVDN、D770-N771insG、N771-P772insSVDNP、N771-H773dupNPH、P772-H773insPNP、P772-H773insPR、H773-V774insH、A763-Y764insFQEA、H773-V774insPH、H773-V774insNPH、N771-P772insH、H771-P772insN、H773-V774insAH、D770delinsGY、V774-C775insHV等以及20号外显子点突变S768I；
    21号外显子点突变L861Q；
    ERBB2的点突变V777L、D769Y、R896C、P1170A和插入突变V777-G778insCG、P780-Y781insGSP等。
  10. 如权利要求6-9中任一项所述的用途，其特征在于，所述疾病为癌症为选自以下的一种或多种：非小细胞肺癌、小细胞肺癌、肺腺癌、肺鳞癌、乳腺癌、胰腺癌、前列腺癌、卵巢癌、神经胶质细胞瘤、头颈部鳞癌、宫颈癌、食管癌、肝癌、肾癌、结肠癌、皮肤癌、白血病、淋巴瘤、胃癌或多发性骨髓癌。
PCT/CN2022/121058 2021-09-24 2022-09-23 蝶啶酮衍生物及其应用 Ceased WO2023046114A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111123442 2021-09-24
CN202111123442.1 2021-09-24

Publications (1)

Publication Number Publication Date
WO2023046114A1 true WO2023046114A1 (zh) 2023-03-30

Family

ID=85661142

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/121058 Ceased WO2023046114A1 (zh) 2021-09-24 2022-09-23 蝶啶酮衍生物及其应用

Country Status (2)

Country Link
CN (2) CN115850281A (zh)
WO (1) WO2023046114A1 (zh)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1373763A (zh) * 1999-09-15 2002-10-09 沃尼尔·朗伯公司 作为激酶抑制剂的蝶啶酮
WO2006002367A1 (en) * 2004-06-23 2006-01-05 Irm Llc Compounds and compositions as protein kinase inhibitors
WO2013170671A1 (zh) * 2012-05-14 2013-11-21 华东理工大学 蝶啶酮衍生物及其作为egfr、blk、flt3抑制剂的应用
WO2016192609A1 (zh) * 2015-05-29 2016-12-08 华东理工大学 蝶啶酮衍生物作为egfr抑制剂的应用
CN108721298A (zh) * 2017-04-19 2018-11-02 华东理工大学 作为布鲁顿酪氨酸激酶抑制剂的嘧啶并杂环化合物及其应用
WO2020142612A1 (en) * 2019-01-03 2020-07-09 Genentech, Inc. Pyrido-pyrimidinone and pteridinone compounds as inhibitors of endoribonuclease inositol requiring enzyme i (ire i alpha) for the treatment of cancer diseases.
WO2022042755A1 (zh) * 2020-08-28 2022-03-03 华东理工大学 抑制突变型egfr的化合物及其应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1373763A (zh) * 1999-09-15 2002-10-09 沃尼尔·朗伯公司 作为激酶抑制剂的蝶啶酮
WO2006002367A1 (en) * 2004-06-23 2006-01-05 Irm Llc Compounds and compositions as protein kinase inhibitors
WO2013170671A1 (zh) * 2012-05-14 2013-11-21 华东理工大学 蝶啶酮衍生物及其作为egfr、blk、flt3抑制剂的应用
WO2016192609A1 (zh) * 2015-05-29 2016-12-08 华东理工大学 蝶啶酮衍生物作为egfr抑制剂的应用
CN108721298A (zh) * 2017-04-19 2018-11-02 华东理工大学 作为布鲁顿酪氨酸激酶抑制剂的嘧啶并杂环化合物及其应用
WO2020142612A1 (en) * 2019-01-03 2020-07-09 Genentech, Inc. Pyrido-pyrimidinone and pteridinone compounds as inhibitors of endoribonuclease inositol requiring enzyme i (ire i alpha) for the treatment of cancer diseases.
WO2022042755A1 (zh) * 2020-08-28 2022-03-03 华东理工大学 抑制突变型egfr的化合物及其应用

Also Published As

Publication number Publication date
CN115850282A (zh) 2023-03-28
CN115850281A (zh) 2023-03-28

Similar Documents

Publication Publication Date Title
CN105832740B (zh) 芳胺基嘌呤衍生物及其制备方法和在医药上的用途
RU2705579C2 (ru) Новое пиразоло[3, 4-d]пиримидиновое соединение или его соль
CN112839937B (zh) 异吲哚啉-1-酮衍生物、其制备方法及其用途
CN107428763B (zh) 作为egfr抑制剂的嘧啶并嘧啶二酮衍生物及其应用
CN106883213A (zh) 一种新型egfr和alk激酶的双重抑制剂
RO117849B1 (ro) Derivati de chinazolina, procedeu de preparare a acestora, compozitie farmaceutica si metoda de producere a unui efect antiproliferativ
KR20190021345A (ko) Egfr 저해제로 제공되는 아닐린 피리미딘 화합물의 결정
EP4324833A1 (en) Alkynylphenylbenzamide compound and use thereof
EP3476846A1 (en) Novel heterocyclic derivative compound and use thereof
TW201934546A (zh) 嘧啶類化合物、其製備方法及其醫藥用途
CN103421010A (zh) 作为egfr抑制剂的蝶啶酮衍生物及其应用
WO2017144025A1 (zh) 作为EGFR抑制剂的嘧啶并[4,5-d][1,3]噁嗪-2-酮衍生物及其应用
CN108047204A (zh) 2,4-二芳氨基嘧啶衍生物及其制备方法和应用
CN113135898B (zh) 一种抗癌喹喔啉嘧啶胺杂环化合物及其制备方法和应用
CN111116585B (zh) 具有c-MET激酶抑制活性化合物、制备方法、组合物及用途
WO2017101862A1 (zh) 作为egfr抑制剂的5,8-二氢蝶啶-6,7-二酮衍生物及其应用
WO2023046114A1 (zh) 蝶啶酮衍生物及其应用
CN110467616A (zh) 含杂芳基取代哒嗪酮结构的三唑并吡嗪类化合物的制备及应用
CN110407839A (zh) 含杂芳基酰胺结构的三唑并杂环类化合物的制备及应用
KR20210122192A (ko) 벤조티아졸 유도체 화합물
CN107056754B (zh) 内嵌脲类结构的wnt通路抑制剂
CN106565682B (zh) 取代吲哚满酮衍生物及其用途
CN119552166B (zh) 一种parp1/cdk6双靶点抑制剂及其制备方法与应用
CN113493436B (zh) 胺基取代吡啶衍生物及其制法和药物组合物与用途
US10040803B2 (en) 4-((2-acrylamidophenyl)amino)thieno[3,2-D]pyrimidine-7-carboxamide derivatives as protein kinase inhibitors

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: 22872158

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: 22872158

Country of ref document: EP

Kind code of ref document: A1