CN112159392B - Substituted pyrimidine compound, pharmaceutical composition thereof and application of compound - Google Patents

Substituted pyrimidine compound, pharmaceutical composition thereof and application of compound Download PDF

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CN112159392B
CN112159392B CN202010966082.0A CN202010966082A CN112159392B CN 112159392 B CN112159392 B CN 112159392B CN 202010966082 A CN202010966082 A CN 202010966082A CN 112159392 B CN112159392 B CN 112159392B
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pharmaceutically acceptable
methyl
compound
cancer
amino
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CN112159392A (en
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范晶晶
唐春雷
范为正
范文华
张立勋
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Nanjing Leizheng Pharmaceutical Technology Co ltd
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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65583—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system each of the hetero rings containing nitrogen as ring hetero atom

Abstract

The invention relates to substituted pyrimidine compounds, pharmaceutical compositions thereof and uses of the compounds. The substituted pyrimidine compounds with the general formula (I) and the pharmaceutically acceptable salts thereof have excellent pharmacodynamic properties, metabolic stability and/or better blood brain barrier permeability.

Description

Substituted pyrimidine compound, pharmaceutical composition thereof and application of compound
Technical Field
The present invention is in the field of chemical medicine and relates to a class of substituted pyrimidine compounds and pharmaceutical compositions of such compounds, and further relates to compounds, pharmaceutical compositions and methods of treating diseases and conditions associated with kinase activity including mutant EGFR and mutant HER2 activity.
Background
Epidermal Growth Factor Receptor (EGFR) is a membrane surface receptor with tyrosine kinase activity that is widely present in human epidermal and stromal cells. In normal cells, EGFR tyrosine kinase (EGFR-TK) activity is precisely controlled, but when the gene site is mutated, it results in a sustained enhancement of its activity, thereby inducing tumors (Chong et al Nature Med.2013; 19(11): 1389-1400).
Lung cancer consists of non-small cell lung cancer (NSCLC), Small Cell Lung Cancer (SCLC) and neuroendocrine tumors. It has been reported that about 10% of NSCLC patients in the united states (10,000 cases/year) and about 35% of NSCLC patients in east asia have been reported to have tumor-associated EGFR mutations. (Lynch et al N Engl J Med.2004; 350(21): 2129-39). The vast majority of NSCLC cases with EGFR mutations also have mutations of another oncogene (e.g., KRAS mutations, ALK rearrangements, etc.). EGFR mutations occur primarily within EGFR exons 18-21, which encode a portion of the EGFR kinase domain. EGFR mutations are often heterozygous, with amplification of mutant allele copy numbers. About 90% of these mutations are exon 19 deletions or exon 21L858R point mutations. These mutations increase the kinase activity of EGFR, leading to overactivation of the downstream pro-survival signaling pathway. (Pao et al Nat Rev Cancer 2010; 10: 760-) 774).
Small deletions, insertions or point mutations in the EGFR kinase domain have been widely reported. The exon 19 mutation resulting in the in-frame deletion of amino acid 747 accounted for 45% of the mutations, the exon 21 mutation resulting in the L858R substitution accounted for 40-45% of the mutations, and the remaining 10% of the mutations involved exons 18 and 20, as reported by Sharma et al (Nat Re. cancer 2007; 7: 169).
Most mutations of the EGFR gene occur in 18-21 exons, most of the mutations are 19 exon non-frameshift deletion mutation and 21 exon L858R point mutation, and patients with the mutations have good effect when using targeted drugs. The third mutation in EGFR is insertion mutation of EGFR 20 exon, accounting for 6% of NSCLC patients with EGFR mutation, and the mutation is mostly occurred in Asia, female, non-smoking and adenocarcinoma. EGFR exon 20 insertions were reported to account for approximately 4-9.2% of all EGFR mutant lung tumors (Arcila et al 2013; 12(2): 220-9; Oxnard et al J Thorac Onco 1.2013; 8(2): 179-84). Most EGFR exon 20 insertions occur in the region of exon 20 encoding amino acids 767 to 774, within the loop following the C-helix of the kinase domain of EGFR (Yasuda et al Lancet Onco 1.2012; 13(1): e 23-31). EGFR exon 20 insertion mutants other than A763_ Y764insFQEA are largely comparable to clinically achievable doses of reversible EGFR TKI erlotinib and gefitinib and irreversible EGFR TKI neratinib, afatinib and dacomitinib in preclinical models (Cancer Res.2007; 67(24): 11924-32; Oncogene 2008:27(34): 4702-11; Cancer Biol ther.2007; 6(5): 661-7)); yasuda et al 2012; yasuda et al Sci Transl Med.2013; 216ra177 (216); the crystal structure of a representative TKI-insensitive mutant (D770-N771 insNPG) revealed that it has an unaltered ATP-binding pocket and, unlike EGFR-sensitizing mutations, it activates EGFR without increasing its affinity for ATP. Patients with tumors that have mutations involving insertion of EGFR exon 20 of amino acids A767, S768, D770, P772 and H773 do not respond to gefitinib or erlotinib (Clin Cancer Res.2008; 14(15): 4877-82; Clin Cancer Res.2011; 17(11): 3812-21). In a retrospective and prospective analysis of patients with NSCLC with a typical EGFR exon 20 insertion, most show progressive disease during treatment with gefitinib or erlotinib or afatinib.
The HER2 mutation was reported to be present in approximately 2-4% of NSCLCs (Int J Cancer 2006; 119: 2586-. The most common mutation is an in-frame insertion within exon 20. In 83% of patients with HER 2-associated NSCLC, the tetra-amino acid YVMA insertion mutation was present at codon 775 in exon 20 of HER 2. (Clin cancer Res 2012; 18: 4910-. In the case of adenocarcinoma histology, the HER2 mutation appears to be more common in never-smokers (Butti tta et al 2006; Shigematsu et al 2005; Stephens et al 2004). However, HER2 mutations can also be found in other NSCLC subgroups (including in previous and current smokers) as well as in other histologies. Exon 20 insertion leads to increased HER2 kinase activity and enhanced signaling through downstream pathways, leading to increased survival, invasiveness, and tumorigenicity (Cancer Cell 2006; 10: 25-38). Tumors with HER2 YVMA mutation were substantially resistant to known EGFR inhibitors.
In recent years, compounds that selectively inhibit both mutant EGFR and mutant HER2 have been the focus of research. However, how to further improve the activity, reduce the toxic and side effects, improve the metabolic stability and/or effectively increase the blood-brain barrier permeability is still a current problem.
Disclosure of Invention
Problems to be solved by the invention
The inventor discovers a novel substituted pyrimidine compound in the process of researching EGFR and HER2 inhibitors, and the substituted pyrimidine compound has excellent inhibitory activity, improved metabolic stability and/or better blood brain barrier permeability on mutant EGFR and mutant HER 2. Further, the invention provides a compound with a general formula (I) or a pharmaceutically acceptable salt thereof, a pharmaceutical composition and application.
Means for solving the problems
The invention provides a compound with a general formula (I) or a pharmaceutically acceptable salt thereof,
Figure BDA0002682367610000021
wherein:
R1selected from hydrogen, C1-C4Alkyl, deuterated methyl, C3-C6Cycloalkyl, -CH2OR4and-CH2OP(＝O)(XR5)YR6，
Wherein R is4Selected from hydrogen, C1-C4Alkyl radical, C3-C6A cycloalkyl group; wherein X or Y are each independently selected from N or O;
wherein R is5And R6Each independently selected from hydrogen and C1-C4Alkyl radical, C6-C12Aryl, heteroaryl, and heteroaryl,
Figure BDA0002682367610000022
Wherein R is7Selected from hydrogen, C1-C8An alkyl group;
R2is selected from
Figure BDA0002682367610000023
Wherein R is8、R9、R10Each independently selected from methyl or methyl substituted with 1-3 deuterium atoms;
R3selected from hydrogen, C1-C4Alkoxy radical, C1-C4Alkyl, halo C1-C4Alkyl, halo C1-C4Alkoxy, halogen or cyano;
z is selected from N or C.
In some embodiments of the invention, R4Selected from hydrogen, methyl, isopropyl;
R5and R6Each independently selected from hydrogen and C1-C4Alkyl, phenyl,
Figure BDA0002682367610000031
Figure BDA0002682367610000032
Further preferably, R1Selected from methyl, deuterated methyl, cyclopropyl, -CH2OH or-CH2OP(＝O)(XR5)YR6Wherein X or Y are each independently selected from N or O, R5And R6Each independently selected from hydrogen and C1-C4An alkyl group.
In some embodiments of the invention, R8、R9、R10Each independently selected from methyl or methyl substituted with 3 deuterium atoms.
In some embodiments of the invention, R3Selected from hydrogen, methoxy, methyl, ethoxy, trifluoroethoxy, halogen or cyano; further preferably R3Selected from methoxy, ethoxy or trifluoroethoxy.
In some embodiments of the invention, the compound of formula (I) is selected from the following compounds:
Figure BDA0002682367610000041
in some embodiments of the invention, the pharmaceutically acceptable salts described herein are inorganic or organic salts, and inorganic salts include hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, bisulfate, nitrate, phosphate, acid phosphate; the organic salt is selected from formate, acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, succinate, glutarate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate, p-toluenesulfonate, ascorbate; further preferably, the pharmaceutically acceptable salt is selected from the hydrochloride, succinate or mesylate salt.
The invention also provides a pharmaceutical composition comprising the compound of the invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, excipient or diluent.
The invention also provides the use of a compound of the invention as hereinbefore described, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of diseases and conditions associated with kinase activity mutated by EGFR and HER2, particularly cancer, in a mammal, particularly a human.
Further wherein the cancer comprises lung cancer, colorectal cancer, pancreatic cancer, head and neck cancer, breast cancer, ovarian cancer, uterine cancer and/or gastric cancer.
The invention also provides the use of a combination of a compound of the invention as hereinbefore described, or a pharmaceutically acceptable salt thereof, and an anti-tumour agent selected from the group consisting of:
(i) antineoplastic drugs acting on the DNA structure;
(ii) antineoplastic agents that affect nucleic acid synthesis;
(iii) anti-tumor drugs that affect nucleic acid transcription;
(iv) tubulin synthesized antineoplastic drugs;
(v) cell signaling pathway inhibitors such as epidermal growth factor receptor inhibitors;
(vi) an anti-tumor monoclonal antibody.
ADVANTAGEOUS EFFECTS OF INVENTION
The present invention provides a novel inhibitor for inhibiting mutant EGFR carrying exon 20 insertion mutation or DT or LT mutation, and for inhibiting mutant HER2, and for methods of treating diseases mediated by any of those mutant EGFR or HER2 proteins, which is effective in ameliorating diseases and disorders associated with mutant EGFR and mutant HER2 activity, and further, which has better pharmacodynamic properties, higher metabolic stability, and is effective in reducing side effects such as skin rash and diarrhea, and in some embodiments, some compounds exhibit better blood brain barrier permeability.
Detailed Description
The inventor discovers a novel substituted pyrimidine compound in the process of researching EGFR and HER2 inhibitors, has excellent inhibitory activity on mutant EGFR and mutant HER2, has lower toxic and side effects, provides a treatment scheme for treating EGFR exon 20 insertion mutation patients, and has longer half-life and better safety. The inhibitor is expected to have good curative effect, is expected to overcome the problems of drug resistance and toxic and side effects, and has good development prospect.
In the present invention C1-C8Alkyl means a straight or branched chain monovalent saturated hydrocarbon group having 1 to 8 carbon atoms, and examples thereof include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, tert-butyl, 1-hexyl, 2-ethylbutyl and the like. C1-C6Alkyl and C1-C4The meaning of alkyl is analogized.
Halogen substituted C1-C4Alkyl means C having one or more hydrogen atoms replaced by halogen atoms1-C4An alkyl group.
C1-C4Alkoxy means a radical of C1-C4Alkyl-substituted oxy radicals, i.e. C1-C4alkyl-O-.
Halogen substituted C1-C4Alkoxy means C having one or more hydrogen atoms replaced by halogen atoms1-C4Alkoxy, for example 2,2, 2-trifluoroethoxy.
C6-C12Aryl means an aromatic hydrocarbon radical having 6 to 12 carbon atoms, wherein the aromatic ring may be substituted by a halogen, C1-C4Alkyl, halo C1-C4Alkyl radical, C1-C4Alkoxy, halo C1-C4Alkoxy, and the like. Examples thereof include, but are not limited to, phenyl, tolyl, xylyl, methoxyphenyl, dimethoxyphenyl, naphthyl, and the like.
C3-C6Cycloalkyl groups include, but are not limited to, cyclopropyl (C3), cyclobutyl (C4), cyclopentyl (C5), cyclohexyl (C6), and the like.
Halogen includes fluorine, chlorine, bromine and iodine.
Figure BDA0002682367610000051
Indicating that the substituent is attached thereto.
By "pharmaceutically acceptable salts" in the context of the present invention is meant those salts which retain the biological effectiveness and properties of the parent compound. The term "salt" refers to any salt of a compound according to the invention prepared from an inorganic or organic acid or base and an internally formed salt. Typically, such salts have a physiologically acceptable anion or cation.
The term "disease" as used herein refers to any condition or disorder that impairs or interferes with the normal function of a cell, organ or tissue.
The term "inhibitor" as used herein refers to a compound or agent that has the ability to inhibit a biological function of a targeted protein or polypeptide, for example by inhibiting the activity or expression of the protein or polypeptide.
The term "antineoplastic agent" as used herein refers to any agent useful in the treatment of neoplastic disorders.
The term "pharmaceutically acceptable" as used herein, means a component that is, within the scope of sound medicine, suitable for use in contact with the tissues of humans and other mammals without excessive toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio. "pharmaceutically acceptable salt" refers to any non-toxic salt that, upon administration to a recipient, is capable of providing, directly or indirectly, a compound of the present invention or a prodrug of a compound.
The term "effective amount" or "therapeutically effective amount" as used herein means that the amount of a compound or pharmaceutical composition described herein is sufficient to achieve the intended use, including, but not limited to, the treatment of disease. In some embodiments, the amount is detected to be effective for killing or inhibiting cancer cell growth or spread; the size or number of tumors; or the severity level, stage and progression of the cancer. The therapeutically effective amount may vary depending on the intended application, e.g., in vitro or in vivo, the condition and severity of the disease, the age, weight, or mode of administration of the subject, etc. The term also applies to a particular response in which the dose will induce the target cell, e.g., reduce cell migration. The specific dosage will depend, for example, on the particular compound chosen, the species of the subject and their age/existing health or risk of health, the route of administration, the severity of the disease, administration in combination with other agents, the time of administration, the tissue to which it is administered, and the administration device, among other things.
In the present invention "administering" or "administering" an individual compound means providing a compound of the invention to an individual in need of treatment.
The compounds of the present invention may contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of these compounds are expressly included in the present invention. The compounds of the invention may also exhibit multiple tautomeric forms, in which case the invention expressly includes all tautomeric forms of the compounds described herein. All such isomeric forms of such compounds are included in the present invention. All crystalline forms of the compounds described herein are expressly included in the present invention.
"optional" or "optionally" in the context of the present invention refers to the subsequently described event or circumstanceA condition may or may not occur, and the description includes both the occurrence of the event or circumstance and the non-occurrence of the event or circumstance. For example, ethyl is "optionally" substituted with halo, meaning that ethyl may be unsubstituted (CH)2CH3) Monosubstituted (e.g. CH)2CH2F) Polysubstituted (e.g. CHFCH)2F、CH2CHF2Etc.) or completely substituted (CF)2CF3). It will be appreciated by those skilled in the art that any group containing one or more substituents will not incorporate any substitution or substitution pattern which is sterically impossible and/or cannot be synthesized.
Reference throughout this specification to "an embodiment" or "in another embodiment" or "in certain embodiments" or "in portions of this application" means that a particular reference element, structure or feature described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" or "in another embodiment" or "in certain embodiments" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular elements, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to be open-ended, inclusive meaning that "includes but is not limited to".
It should be understood that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
< Compound or pharmaceutically acceptable salt thereof >
The invention provides a substituted pyrimidine compound or pharmaceutically acceptable salt thereof, a pharmaceutical composition and application thereof. The compound can be used as an EGFR and HER2 inhibitor, and has better pharmacodynamic property and higher metabolic stability.
The present invention provides a compound of the following general formula (I) or a pharmaceutically acceptable salt thereof:
Figure BDA0002682367610000071
wherein:
R1selected from hydrogen, C1-C4Alkyl, deuterated methyl, C3-C6Cycloalkyl, -CH2OR4and-CH2OP(＝O)(XR5)YR6(i.e., the P atom is bound to two oxygens and X and Y),
wherein R is4Selected from hydrogen, C1-C4Alkyl radical, C3-C6A cycloalkyl group; wherein X or Y are each independently selected from N or O;
wherein R is5And R6Each independently selected from hydrogen and C1-C4Alkyl radical, C6-C12Aryl, heteroaryl, and heteroaryl,
Figure BDA0002682367610000072
Wherein R is7Selected from hydrogen, C1-C8An alkyl group;
R2is selected from
Figure BDA0002682367610000073
Wherein R is8、R9,、R10Each independently selected from methyl or methyl substituted with 1-3 deuterium atoms;
R3selected from hydrogen, C1-C4Alkoxy radical, C1-C4Alkyl, halo C1-C4Alkyl, halo C1-C4Alkoxy, halogen or cyano;
z is selected from N or C.
In certain embodiments of the invention, R4Selected from hydrogen, methyl, isopropyl.
In the present inventionIn some embodiments of the invention, R5And R6Each independently selected from hydrogen and C1-C4Alkyl, phenyl,
Figure BDA0002682367610000074
Wherein R is7Selected from hydrogen, C1-C8An alkyl group; further preferably, R5And R6Each independently selected from hydrogen, ethyl, tert-butyl, phenyl,
Figure BDA0002682367610000075
Figure BDA0002682367610000076
In some embodiments of the invention, R is selected to enhance pharmacodynamic activity and metabolic stability1Preferably selected from methyl, deuterated methyl, cyclopropyl, -CH2OH or-CH2OP(＝O)(XR5)YR6Further, R1The compound selected from hydroxymethyl, deuterated methyl and cyclopropyl has high bioactivity on R1Is selected from-CH2OP(＝O)(XR5)YR6The compounds can be converted into compounds with hydroxymethyl groups through in vivo metabolism, and therefore have relatively strong therapeutic potential.
In some embodiments of the invention, X or Y is each independently selected from N or O, R5And R6Each independently selected from hydrogen and C1-C4Alkyl, further preferably, said deuterated methyl is methyl substituted with 3 deuterium atoms, R5And R6Each independently selected from hydrogen, ethyl or tert-butyl. In some embodiments of the invention, X, Y are both O.
In some embodiments of the invention, R2Selected from:
Figure BDA0002682367610000081
wherein R is8、R9,、R10Each independently selected from methyl or methyl substituted with 1-3 deuterium atoms, further preferably, R8、R9、R10Each independently selected from methyl or methyl substituted with 3 deuterium atoms.
In some embodiments of the invention, R3Selected from hydrogen, methoxy, ethoxy, C1-C4Alkyl, halo C1-C4Alkyl, halo C1-C4Alkoxy (especially trifluoroethoxy), halogen or cyano; further preferably, R3Selected from hydrogen, methoxy, methyl, ethoxy, trifluoroethoxy, halogen or cyano; further preferably R3Selected from methoxy, ethoxy or trifluoroethoxy. The substitution of trifluoroethoxy has important positive effects on the pharmacodynamic performance, metabolic stability and blood brain barrier permeability of the compound.
In some embodiments of the invention, the compounds of the invention are selected from:
Figure BDA0002682367610000091
in some embodiments of the invention, the inventors have found that compounds are at R1Or R2Methyl deuteration and/or R at specific position3Is trifluoroethoxy and/or R1The compounds which are cyclopropyl have important positive effects on the pharmacodynamic properties and metabolic stability of the compounds, blood brain barrier permeability, and the relatively long metabolic half-life makes them potentially useful for lowering therapeutic doses and extending the time intervals between administrations.
The compounds of formula (I) include pharmaceutically acceptable salts thereof. The pharmaceutically acceptable salt is inorganic salt or organic salt, and the inorganic salt comprises hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, bisulfate, nitrate, phosphate and acid phosphate; the organic salt is selected from formate, acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, succinate, glutarate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate, p-toluenesulfonate, ascorbate. Still further, the pharmaceutically acceptable salt is selected from the hydrochloride, succinate or mesylate salt.
Further, the compounds of formula (I) or their salts may be isolated in the form of solvates, and thus any such solvate is within the scope of the present invention.
The invention also provides a pharmaceutical composition comprising a compound of the invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, excipient or diluent.
In preparing the pharmaceutical compositions, the compounds of formula (I) or pharmaceutically acceptable salts thereof of the present invention are typically mixed with a pharmaceutically acceptable carrier, excipient or diluent. Wherein, in a unit dosage form (e.g., a tablet or capsule), the compound of formula (I) or a pharmaceutically acceptable salt thereof may be present in an amount of 0.01 to 1000mg, e.g., 0.05 to 800mg, 0.1 to 500mg, 0.01 to 300mg, 0.01 to 200mg, 0.05 to 150mg, 0.05 to 50mg, etc.
The composition of the invention can be prepared into conventional pharmaceutical preparations according to conventional preparation methods. Such as tablets, pills, capsules, powders, granules, emulsions, suspensions, dispersions, solutions, tinctures, syrups, ointments, drops, suppositories, inhalants, sprays and the like.
In certain embodiments, the compounds of the present invention, or pharmaceutically acceptable salts thereof, may be formulated as solid formulations for oral administration, including, but not limited to, capsules, tablets, pills, powders, granules, and the like. In these solid dosage forms, the compounds of formula (I) according to the invention are mixed as active ingredient with at least one conventional inert excipient (or carrier), for example with sodium citrate or dicalcium phosphate, or with one or more of the following ingredients:
(1) fillers or solubilizers, for example, starch, lactose, sucrose, glucose, mannitol, silicic acid, and the like;
(2) binders, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, gum arabic and the like;
(3) humectants, such as glycerol and the like;
(4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate, and the like;
(5) a slow solvent such as paraffin and the like;
(6) absorption accelerators such as quaternary ammonium compounds and the like;
(7) wetting agents such as cetyl alcohol and glyceryl monostearate and the like;
(8) adsorbents, for example, kaolin, and the like;
(9) lubricants, for example, talc, calcium stearate, solid polyethylene glycols, sodium lauryl sulfate, and the like, or mixtures thereof. Capsules, tablets, pills, etc. may also contain buffering agents.
In certain embodiments, the solid dosage forms, e.g., tablets, dragees, capsules, pills, and granules, can be coated or microencapsulated with coating and shell materials such as enteric coatings and other crystalline forms of materials well known in the art. They may contain opacifying agents and the release of the active ingredient in such compositions may be delayed in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active ingredient may also be in microencapsulated form with one or more of the above excipients.
In certain embodiments, the compounds of the present invention, or pharmaceutically acceptable salts thereof, may be formulated in liquid dosage forms for oral administration, including, but not limited to, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, tinctures, and the like. In addition to the compounds of formula (I) or pharmaceutically acceptable salts thereof as active ingredients, the liquid dosage forms may contain inert diluents conventionally employed in the art, such as water and other solvents, solubilizing agents and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, and oils, especially cottonseed oil, peanut oil, corn oil, olive oil, castor oil, sesame oil and the like or mixtures of such materials and the like. In addition to these inert diluents, the liquid dosage forms of the present invention may also include conventional adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, perfuming agents and the like. Such suspending agents include, for example, ethoxylated stearyl alcohol, polyoxyethylene sorbitol, and sorbitan, microcrystalline cellulose, agar, and the like, or mixtures of these materials.
In certain embodiments, the compounds of the present invention and pharmaceutically acceptable salts thereof may be formulated into dosage forms for parenteral injection, including, but not limited to, physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions and dispersions. Suitable carriers, diluents, solvents, excipients include water, ethanol, polyols and suitable mixtures thereof.
In certain embodiments, the compounds of the present invention, or pharmaceutically acceptable salts thereof, may be formulated in dosage forms for topical administration, including, for example, ointments, powders, suppositories, drops, sprays, inhalants and the like. The compounds of the general formula (I) according to the invention or their pharmaceutically acceptable salts as active ingredients are mixed under sterile conditions with physiologically acceptable carriers and optionally preservatives, buffers and, if desired, propellants.
The compounds of the invention or pharmaceutically acceptable salts thereof may be administered alone or in combination with other pharmaceutically acceptable therapeutic agents, particularly in combination with other antineoplastic agents. Such therapeutic agents include, but are not limited to: antineoplastic drugs acting on DNA chemical structures, such as cisplatin, antineoplastic drugs affecting nucleotide synthesis, such as methotrexate, 5-fluorouracil and the like, antineoplastic drugs affecting nucleic acid transcription, such as doxorubicin, epirubicin, aclacinomycin and the like, antineoplastic drugs affecting micro-protein synthesis, such as taxol, vinorelbine and the like, aromatase inhibitors, such as aminoglutethimide, letrozole, rening and the like, cell signaling pathway inhibitors, such as epidermal growth factor receptor inhibitor Imatinib (Imatinib), Gefitinib (Gefitinib), Erlotinib and the like. The components to be combined may be administered simultaneously or sequentially, in a single formulation or in different formulations. Such combinations include not only combinations of one or other active agents of the compounds of the present invention, but also combinations of two or more other active agents of the compounds of the present invention.
< preparation method of Compound >
In another aspect, the present invention also provides a process for the preparation of a compound of general formula (I), said process being carried out by one of the following reaction schemes:
the first reaction scheme is as follows:
Figure BDA0002682367610000111
reaction scheme 1
As shown in a reaction formula 1, taking 2, 4-dichloropyrimidine-5-isopropyl formate as a raw material, and carrying out Friedel-crafts reaction with a compound A under the action of a catalyst to obtain an intermediate a; carrying out substitution reaction on the intermediate a and the compound B to obtain a compound C; wherein R is1Selected from hydrogen, C1-C4Alkyl, deuterated methyl, C3-C6Cycloalkyl radical, R2、R3Z is as defined in formula (I),
in the above reaction, the preparation of the intermediate a is carried out under the action of lewis acid, and the lewis acid can be selected from, but is not limited to, ferric trichloride, aluminum trichloride, zinc chloride, boron trifluoride;
or reaction scheme two:
Figure BDA0002682367610000121
reaction formula 2
As shown in a reaction formula 2, taking 2, 4-dichloropyrimidine-5-isopropyl formate as a raw material, and carrying out Friedel-crafts reaction on the raw material and indole under the action of a catalyst to obtain an intermediate b; carrying out substitution reaction on the intermediate B and the compound B to obtain an intermediate c; finally, the intermediate C is subjected to substitution reaction to obtain a compound C, wherein R2、R3Z is defined by the general formula (I)The meaning is the same.
Or reaction scheme three:
Figure BDA0002682367610000122
reaction formula 3
As shown in reaction formula 3, nucleophilic substitution is carried out on the intermediate c and the intermediate D to obtain a compound D;
wherein, X, Y, R5、R6、R2、R3Z is as defined for formula (I).
Or reaction scheme four:
Figure BDA0002682367610000123
reaction formula 4
As shown in the reaction formula 4, the compounds C and R4Q in the presence of a base to give compound D, wherein R4Is as defined in formula (I); the base may be selected from, but is not limited to, triethylamine, N-diethylethylenediamine, sodium carbonate, Q is halogen;
specific reaction conditions of the above reaction can be referred to the conditions in the following examples.
The invention further provides the use of a compound of the invention or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of diseases mediated by EGFR and HER2 mutants, particularly cancer, in mammals, especially humans.
The cancer is further selected from lung cancer, colorectal cancer, pancreatic cancer, head and neck cancer, breast cancer, ovarian cancer, uterine cancer and gastric cancer.
Furthermore, the present invention provides the use of a combination of a compound of the invention, or a pharmaceutically acceptable salt thereof, and an anti-neoplastic agent selected from the group consisting of:
(i) antineoplastic drugs acting on the DNA structure;
(ii) antineoplastic agents that affect nucleic acid synthesis;
(iii) anti-tumor drugs that affect nucleic acid transcription;
(iv) tubulin synthesized antineoplastic drugs;
(v) cell signaling pathway inhibitors such as epidermal growth factor receptor inhibitors;
(vi) an anti-tumor monoclonal antibody.
The compounds of the present invention or pharmaceutically acceptable salts thereof may be administered to mammals including humans, either orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), topically (powders, ointments, drops) or intratumorally.
The compounds of the invention may be administered in a dosage of about 0.01-50mg/kg body weight/day, for example 0.1-45mg/kg body weight/day, 0.5-35mg/kg body weight/day.
Examples
The following examples illustrate but do not limit the synthesis of the compounds of formula (I). The temperatures are given in degrees Celsius. All evaporation was performed under reduced pressure if not otherwise stated. If not otherwise stated, the reagents were purchased from commercial suppliers and used without further purification. The structure of the final products, intermediates and starting materials is confirmed by standard analytical methods, such as elemental analysis, spectroscopic characterization, e.g., MS, NMR. Abbreviations used are those conventional in the art, and some of the intermediates were purchased from Yancheng Zhengchi Biotech, Inc.
The following intermediate substances are involved in the specific examples and can be synthesized by the following process routes:
intermediate 1a N- (5-amino-2- (((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxyphenyl) acrylamide
Figure BDA0002682367610000131
The synthesis method comprises the following steps:
Figure BDA0002682367610000132
step a: preparation of tert-butyl (4-fluoro-2-methoxy-5-nitrophenyl) carbamate
Dissolving 4-fluoro-2-methoxy-5-nitroaniline (3g,16.12mmol) in di-tert-butyl dicarbonate (10mL), heating to 80 ℃, preserving heat for 16h, cooling the reaction liquid to room temperature, separating out solids, filtering, washing a filter cake with n-hexane, and drying to obtain 4.3g of yellow powdery solids.
Step b: preparation of tert-butyl (4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxy-5-nitrophenyl) carbamate
Tert-butyl (4-fluoro-2-methoxy-5-nitrophenyl) carbamate (1.5g,5.24mmol) was dissolved in DMA (15mL), N, N, N' -trimethylethylenediamine (0.8g,7.86mmol) and N, N-diisopropylethylamine (1.01g,7.86mmol) were added sequentially, and the reaction was allowed to warm to 110 ℃ overnight. And adding saturated sodium carbonate aqueous solution to adjust the pH value to 8-9, extracting with ethyl acetate, washing an organic phase with water, drying, and concentrating to obtain 1.8g of light red oily liquid.
Step c: preparation of tert-butyl (5-amino-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) carbamate
Tert-butyl (4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxy-5-nitrophenyl) carbamate (6g,16.3mmol) was dissolved in ethyl acetate (40mL) and reacted overnight at room temperature with the addition of 600mg of Pd/C (5%) under an atmosphere of hydrogen at atmospheric pressure. The reaction solution was filtered, and the filtrate was concentrated to give a crude product of 5g of a black oil, which was directly fed to the next step.
Step d: preparation of tert-butyl (5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) carbamate
Tert-butyl (5-amino-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) carbamate (500mg,1.48mmol) was dissolved in dichloromethane (5mL), N-diisopropylethylamine (228mg,1.77mmol) was added, acryloyl chloride (147mg,1.625mmol) was slowly added dropwise at 5 ℃, after dropping, the temperature was naturally raised to room temperature. After 3 hours, the reaction was washed with water and concentrated to give 500mg of crude product. The next step is directly carried out.
Step e: preparation of N- (5-amino-2- (((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxyphenyl) acrylamide (intermediate 1a) tert-butyl (5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) carbamate (500mg,1.27mmol) was dissolved in HCl-MeOH (1M,10mL) solution and reacted overnight at room temperature, the reaction was concentrated, saturated aqueous sodium bicarbonate solution was added to adjust pH 8-9, dichloromethane was extracted, the organic phase was dried over anhydrous sodium sulfate, concentrated to give a crude product, and purified by column to give 350mg of red liquid.
ESI-MS m/z:293.2[M+H]+,1H-NMR(DMSO-d6,400MHz),8.48(s,1H),7.81(s,1H),6.91(s,1H),6.39-6.30(m,1H),6.24-6.15(m,1H),5.76-5.70(m,1H),3.77(s,3H),2.82(t,J＝8.0Hz,2H),2.66(s,3H),2.25(t,J＝8.0Hz,2H),2.12(s,6H).
Intermediate 2a preparation of N- (5-amino-2- ((2- (dimethylamino) ethyl) (deuterated methyl) amino) -4-methoxyphenyl) acrylamide
Figure BDA0002682367610000141
The synthetic procedure is described for the preparation of intermediate 1a, with the difference that N, N, N' -trimethylethylenediamine is replaced by N1,N1dimethyl-N2Deuterated methylethylenediamine.
ESI-MS m/z:296.2[M+H]+,1H-NMR(DMSO-d6,400MHz),8.48(s,1H),7.81(s,1H),6.91(s,1H),6.39-6.30(m,1H),6.24-6.15(m,1H),5.76-5.70(m,1H),3.77(s,3H),2.82(t,J＝8.0Hz,2H),2.25(t,J＝8.0Hz,2H),2.12(s,6H).
Intermediate 3a N- (5-amino-4-methoxy-2- (methyl (deuterated methyl) amino) ethyl) amino) phenyl) acrylamide
Figure BDA0002682367610000151
The synthetic procedure is described for the preparation of intermediate 1a, with the difference that N, N, N' -trimethylethylenediamine is replaced by N1,N2dimethyl-N1Deuterated methylethylenediamine.
ESI-MS m/z:296.2[M+H]+,1H-NMR(DMSO-d6,400MHz),8.48(s,1H),7.81(s,1H),6.91(s,1H),6.39-6.30(m,1H),6.24-6.15(m,1H),5.76-5.70(m,1H),3.77(s,3H),2.82(t,J＝8.0Hz,2H),2.66(s,3H),2.25(t,J＝8.0Hz,2H),2.12(s,3H).
Intermediate 4a N- (5-amino-2- ((2- (dideuteromethylamino) ethyl) (methyl) amino) -4-methoxyphenyl) acrylamide
Figure BDA0002682367610000152
The synthetic procedure is described for the preparation of intermediate 1a, with the difference that N, N, N' -trimethylethylenediamine is replaced by N1,N1dideuteromethyl-N2-methylethylenediamine.
ESI-MS m/z:299.2[M+H]+,1H-NMR(DMSO-d6,400MHz),8.48(s,1H),7.81(s,1H),6.91(s,1H),6.39-6.30(m,1H),6.24-6.15(m,1H),5.76-5.70(m,1H),3.77(s,3H),2.82(t,J＝8.0Hz,2H),2.66(s,3H),2.25(t,J＝8.0Hz,2H).
Intermediate 5a 5-acrylamide-6-morpholine-2-trifluoroethoxy-pyridine-3-amine
Figure BDA0002682367610000153
The synthesis method comprises the following steps:
Figure BDA0002682367610000154
step f: preparation of 6-chloro-2-trifluoroethoxy-3-nitropyridine
2, 6-dichloro-3-nitropyridine (2g,10.4mmol,1.0eq) was dissolved in toluene (20mL) under nitrogen, cooled to 0 deg.C, added in portions with sodium hydride (0.5g,12.4mmol,1.2eq), stirred at 0 deg.C for 0.5h, then added with trifluoroethanol (1.14g,11.4mmol,1.1eq) and stirred at room temperature for 12 h. And adding water into the reaction solution for quenching, extracting by using ethyl acetate, washing an organic phase by water, and drying. Concentration gave 2.1g, which was reacted in the next step without purification.
Step g: preparation of 6-chloro-2-trifluoroethoxy-pyridin-3-amine
6-chloro-2- (2,2, 2-trifluoroethoxy) -3-nitropyridine (2.2g,8.57mmol,1.0eq) was dissolved in 15mL of ethanol and 5mL of water, and ammonium chloride (2.7g,42.9mmol,5.0eq) and reduced iron powder (2.4g,42.9mmol,5.0eq) were added in this order and stirred at 80 ℃ for 1.5 hours. The reaction solution was filtered through celite while hot, and the residue was extracted with ethyl acetate, and the organic phase was dried, concentrated, and column chromatographed to give a tan solid 1.2 g.
Step h: preparation of N- (6-chloro-2-trifluoroethoxypyridin-3-yl) acetamide
6-chloro-2-trifluoroethoxypyridin-3-amine (2.3g,10.1mmol,1.0eq) was dissolved in dichloromethane (15mL), diisopropylethylamine (1.9g,15.1mmol,1.5eq) was added, acetyl chloride (0.95g,12.1mmol,1.2eq) was slowly added at 0 deg.C, and the reaction was continued for 1 hour. The reaction solution was washed with water, 1N hydrochloric acid and a saturated sodium chloride solution in this order, dried over anhydrous sodium sulfate, concentrated to give a crude product, and subjected to silica gel column chromatography to give 1.5g of a tan solid.
Step i: preparation of N- (6-chloro-2-trifluoroethoxy-5-nitropyridin-3-yl) acetamide
N- (6-chloro-2-trifluoroethoxypyridin-3-yl) acetamide (1.2g,4.47mmol,1.0eq) was dissolved in trifluoroacetic anhydride (10mL), cooled to-l 0 deg.C, fuming nitric acid (0.31g,4.92mmol,1.1eq) was slowly added dropwise, and the reaction was continued for 1.5 hours. The reaction was slowly added to crushed ice to precipitate a solid which was filtered. The resulting crude product was dried at 60 ℃ and slurried with ethyl acetate to give 885mg of a yellow solid. Step j: preparation of N- (6-morpholine-2-trifluoroethoxy-5-nitropyridin-3-yl) acetamide
N- (6-chloro-2-trifluoroethoxy-5-nitropyridin-3-yl) acetamide (400mg,1.27mmol,1.0eq) was dissolved in acetonitrile (5mL), morpholine (133mg,1.53mmol,1.2eq) was added, and the reaction was carried out at 80 ℃ for 3 hours. The reaction mixture was concentrated under reduced pressure to about 1/3 volume, and 0.5mL of ethyl acetate was added to precipitate a solid, which was filtered to obtain 353mg of a yellow solid.
Step k: preparation of N- (6-morpholine-2-trifluoroethoxy-5-aminopyridin-3-yl) acetamide
N- (6-morpholine-2-trifluoroethoxy-5-nitropyridin-3-yl) acetamide (1g) was dissolved in 15mL of methanol, and 100mg of palladium carbon (10%) was added under a hydrogen atmosphere to react for 3 hours. The palladium carbon was removed by filtration, and the filtrate was concentrated to obtain 800mg of a reddish brown oil.
Step l: preparation of 5-acrylamide-6-morpholine-2-trifluoroethoxy-pyridin-3-yl-acetamide
N- (6-morpholine-2-trifluoroethoxy-5-aminopyridin-3-yl) acetamide (334mg,1.0mmol,1.0eq) was dissolved in 10mL of dichloromethane, triethylamine (151mg,1.5mmol,1.5eq) was added, the reaction mixture was cooled to 0 ℃ and acryloyl chloride (108mg,1.2mmol,1.2eq) was slowly added and reacted at 0 ℃ for 2 hours. And washing the reaction solution, drying the organic phase, and concentrating to obtain the product.
Step m: preparation of 5-acrylamide-6-morpholine-2-trifluoroethoxy-pyridin-3-amine
5-acrylamido-6-morpholine-2-trifluoroethoxy-pyridin-3-yl-acetamide (150mg,0.386mmol) was dissolved in methanol (3mL), and 0.15mL of concentrated hydrochloric acid was added and reacted at 60 ℃ for 2.5 hours. Concentrating to remove methanol, dissolving the residue in ethyl acetate, adjusting the pH of the solution to neutral with saturated sodium bicarbonate, washing the organic phase with sodium chloride, drying, concentrating, and performing column chromatography to obtain 118mg of brick red solid.
ESI-MS m/z:347.1[M+H]+,1H-NMR(CDCl3,400MHz):9.30(s,1H),7.71(s,1H),6.46(dd,J＝16.9,1.7Hz,1H),6.39-6.25(m,1H),5.70(dd,J＝10.0,1.8Hz,1H),4.80(q,J＝8.0Hz,2H),3.83(t,J＝4.0Hz,4H),3.30(t,J＝4.0Hz,4H).
Intermediate 6a 5-acrylamide-6-morpholine-2-methoxy-pyridine-3-amine
Figure BDA0002682367610000171
The synthesis was as described for the preparation of intermediate 5a, except that trifluoroethanol was replaced with methanol.
ESI-MS m/z:279.1[M+H]+,1H-NMR(CDCl3,400MHz):9.28(s,1H),7.70(s,1H),6.45(dd,J＝16.9,1.7Hz,1H),6.37-6.24(m,1H),5.69(dd,J＝10.0,1.8Hz,1H),3.85(s,3H),3.82(t,J＝4.0Hz,4H),3.31(t,J＝4.0Hz,4H).
Intermediate 7a 5-acrylamido-6- [ (2-dimethylamino) ethyl) (methyl) amino) ] -2-trifluoroethoxy-pyridin-3-amine
Figure BDA0002682367610000172
The synthesis was as described for the preparation of intermediate 5a, except that morpholine was replaced by N, N' -trimethylethylenediamine.
ESI-MS m/z:362.1[M+H]+,1H-NMR(CDCl3,400MHz):9.32(s,1H),7.73(s,1H),6.46(dd,J＝16.9,1.7Hz,1H),6.39-6.25(m,1H),5.71(dd,J＝10.0,1.8Hz,1H),4.81(q,J＝8.0Hz,2H),2.81(t,J＝8.0Hz,2H),2.67(s,3H),2.23(t,J＝8.0Hz,2H),2.12(s,6H).
Intermediate 8a 5-acrylamido-6- [ (2-dimethylamino) ethyl) (methyl) amino) ] -2-methoxy-pyridin-3-amine
Figure BDA0002682367610000173
The synthesis was as described for the preparation of intermediate 5a, except that morpholine was replaced by N, N' -trimethylethylenediamine and trifluoroethanol was replaced by methanol.
ESI-MS m/z:294.2[M+H]+,1H-NMR(CDCl3,400MHz):9.32(s,1H),7.72(s,1H),6.45(dd,J＝16.9,1.7Hz,1H),6.39-6.23(m,1H),5.72(dd,J＝10.0,1.8Hz,1H),3.78(s,3H),2.82(t,J＝8.0Hz,2H),2.68(s,3H),2.23(t,J＝8.0Hz,2H),2.12(s,6H).
Intermediate 1b 2-chloro-4- (1-methyl-1H-indol-3-yl) pyrimidine-5-carboxylic acid methyl ester
Figure BDA0002682367610000181
The synthesis method comprises the following steps:
Figure BDA0002682367610000182
step n: preparation of 2, 4-dichloropyrimidine-5-carbonyl chloride
Dispersing 3.5g uracil-5-formic acid in phosphorus oxychloride (15mL), adding 12g phosphorus pentachloride in batches at 0 ℃, reacting for 18 hours at 100 ℃ under the protection of nitrogen, removing the phosphorus oxychloride by rotary evaporation, and distilling the concentrated solution under reduced pressure to obtain 3.4g liquid, wherein the yield is as follows: 72.3 percent.
Step o: preparation of isopropyl 2, 4-dichloropyrimidine-5-carboxylate
2, 4-dichloropyrimidine-5-carbonyl chloride (3.4g,16.1mmol) was dissolved in dry tetrahydrofuran (20mL), cooled to-78 deg.C, isopropanol (1.2g,19.3mmol) was added slowly and the mixture was allowed to warm to room temperature overnight. The reaction solution was concentrated and purified by column to obtain 2.4g of a liquid with a yield of 63.5%.
1H-NMR(CDCl3,400MHz),9.00(s,1H),5.34-5.28(m,1H),1.41(d,J＝8.0Hz,6H).
Step p: preparation of isopropyl 2-chloro-4- (1-methyl-1H-indol-3-yl) pyrimidine-5-carboxylate (intermediate E)
2, 4-dichloropyrimidine-5-carboxylic acid isopropyl ester (200mg,0.851mmol) was dissolved in ethylene glycol dimethyl ether (3mL), and anhydrous aluminum trichloride (136mg,1.021mmol) was added in portions at 0 ℃ and stirred at room temperature for 1 hour. Then 1-methyl-indole (122mg,0.936mmol) in ethylene glycol dimethyl ether (0.5mL) was slowly added and stirred at room temperature for 18 hours, the reaction solution was quenched by addition of water, extracted with dichloromethane, the organic phase was dried and concentrated, and the crude product was purified by column to give 100mg of a yellow solid, yield: 35.7 percent
ESI-MS m/z:330.1,332.1[M+H]+,1H-NMR(CDCl3400MHz),8.82(s,1H),8.16(d, J ═ 8.0Hz,1H),8.07(s,1H),7.59(d, J ═ 8.0Hz,1H),7.35 to 7.25(m,1H),5.34 to 5.28(m,1H),3.87(s,3H),1.21(d, J ═ 8.0Hz,6H), intermediate 2b, 2-chloro-4- (1-deuterated methyl-1H-indol-3-yl) pyrimidine-5-carboxylic acid methyl ester preparation
Figure BDA0002682367610000183
The synthetic procedure is as for the preparation of intermediate 1b, except that 1-methyl-indole is replaced by 1-deuterated methyl-indole.
ESI-MS m/z:333.1,335.1[M+H]+,1H-NMR(CDCl3,400MHz),8.82(s,1H),8.16(d,J＝8.0Hz,1H),8.07(s,1H),7.59(d,J＝8.0Hz,1H),7.35-7.25(m,1H),5.34-5.28(m,1H),1.21(d,J＝8.0Hz,6H).
Intermediate 3b preparation of 2-chloro-4- (1-cyclopropyl-1H-indol-3-yl) pyrimidine-5-carboxylic acid methyl ester
Figure BDA0002682367610000191
The synthesis was as described for the preparation of intermediate 1b, except that 1-methyl-indole was replaced by 1-cyclopropyl-indole.
ESI-MS m/z:356.1,358.1[M+H]+,1H-NMR(CDCl3,400MHz),8.82(s,1H),8.16(d,J＝8.0Hz,1H),8.07(s,1H),7.59(d,J＝8.0Hz,1H),7.35-7.25(m,1H),5.34-5.28(m,1H),2.56-2.35(m,1H),1.21(d,J＝8.0Hz,6H),1.07-0.72(m,4H).
Intermediate 4b preparation of 2-chloro-4- (1H-indol-3-yl) pyrimidine-5-carboxylic acid methyl ester
Figure BDA0002682367610000192
The synthesis was as described for the preparation of intermediate 1b, except that 1-methyl-indole was replaced by indole.
ESI-MS m/z:316.1,318.1[M+H]+,1H-NMR(CDCl3,400MHz),8.83(s,1H),8.17(d,J＝8.0Hz,1H),8.06(s,1H),7.58(d,J＝8.0Hz,1H),7.38-7.27(m,1H),5.34-5.28(m,1H),1.21(d,J＝8.0Hz,6H).
Example 1: isopropyl-2- ((5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) -4- (1-deuterated methyl-1H-indol-3-yl) pyrimidine-5-carboxylate
Figure BDA0002682367610000193
Intermediate 2b (24mg,0.073mmol) was dissolved in acetonitrile (2mL), and intermediate 1a (25mg,0.087mmol) and p-toluenesulfonic acid monohydrate (4mg,0.022mmol) were added sequentially, under nitrogen, and heated to 80 ℃ for 18 hours. After the reaction solution was concentrated, the column was purified to obtain 15mg of a gray solid with a yield of 35.3%.
ESI-MS m/z:589.4[M+H]+,1H-NMR(DMSO-d6,400MHz),10.12(s,1H),8.84(s,1H),8.64(d,J＝8.0Hz,2H),8.17(s,1H),7.73(s,1H),7.48(d,J＝8.0Hz,1H),7.19(t,J＝8.0Hz,1H),7.05-7.01(m,2H),6.46-6.40(m,1H),6.29-6.20(m,1H),5.77(d,J＝8.0Hz,1H),5.03-4.96(m,1H),3.87(s,3H),2.89(t,J＝8.0Hz,2H),2.72(s,3H),2.32(t,J＝8.0Hz,2H),2.21(s,6H),1.12(d,J＝8.0Hz,6H).
Example 1A: isopropyl-2- ((5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) -4- (1-deuterated methyl-1H-indol-3-yl) pyrimidine-5-carboxylate succinate
Figure BDA0002682367610000201
Isopropyl-2- ((5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) -4- (1-deuterated methyl-1H-indol-3-yl) pyrimidine-5-carboxylate (400mg,0.68mmol) in tetrahydrofuran (4mL) was slowly added dropwise a solution of succinic acid (84.2mg,0.71mmol) in tetrahydrofuran (1 mL). Stirring for 12 hours under heat preservation. The solid precipitated out, was filtered while hot and dried under vacuum to give 320mg of a pale yellow solid with a yield of 66.7%.
ESI-MS m/z:589.4[M+H]+,1H-NMR(DMSO-d6,400MHz),10.11(s,1H),8.83(s,1H),8.62(d,J＝8.0Hz,2H),8.17(s,1H),7.73(s,1H),7.46(d,J＝8.0Hz,1H),7.18(t,J＝8.0Hz,1H),7.05-6.09(m,2H),6.46-6.41(m,1H),6.29-6.20(m,1H),5.77(d,J＝8.0Hz,1H),5.03-4.96(m,1H),3.87(s,3H),2.89(t,J＝8.0Hz,2H),2.72(s,3H),2.41(s,2H),2.32(t,J＝8.0Hz,2H),2.21(s,6H),1.12(d,J＝8.0Hz,6H).
Example 1B: isopropyl-2- ((5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) -4- (1-deuterated methyl-1H-indol-3-yl) pyrimidine-5-carboxylic acid ester methanesulfonate
Figure BDA0002682367610000202
A solution of methanesulfonic acid (98mg,1.02mmol) in EtOAc (4mL) was slowly added dropwise to a mixed solvent of isopropyl-2- ((5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) -4- (1-deuterated methyl-1H-indol-3-yl) pyrimidine-5-carboxylate (500mg,0.85mmol) in ethanol (10mL) and EtOAc (8mL) at 70 ℃. Stirring for 1.5 hours under heat preservation. The hot residue was filtered and dried under vacuum at 80 ℃ to give 430mg of a pale yellow solid in 74.0% yield.
ESI-MS m/z:589.4[M+H]+,1H-NMR(DMSO-d6,400MHz),10.13(s,1H),8.82(s,1H),8.65(d,J＝8.0Hz,2H),8.16(s,1H),7.74(s,1H),7.48(d,J＝8.0Hz,1H),7.18(t,J＝8.0Hz,1H),7.05-7.01(m,2H),6.46-6.40(m,1H),6.29-6.20(m,1H),5.76(d,J＝8.0Hz,1H),5.04-4.96(m,1H),3.86(s,3H),2.89(t,J＝8.0Hz,2H),2.74(s,3H),2.71(s,3H),2.32(t,J＝8.0Hz,2H),2.21(s,6H),1.12(d,J＝8.0Hz,6H).
Example 2: isopropyl-2- ((5-acrylamido-4- ((2- (dimethylamino) ethyl) (deuterated methyl) amino) -2-methoxyphenyl) amino) -4- (1-methyl-1H-indol-3-yl) pyrimidine-5-carboxylate
Figure BDA0002682367610000203
See the synthesis of example 1, except that intermediate 1a is replaced by intermediate 2a and intermediate 2b is replaced by intermediate 1 b.
ESI-MS m/z:589.4[M+H]+,1H-NMR(DMSO-d6,400MHz),10.12(s,1H),8.84(s,1H),8.64(d,J＝8.0Hz,2H),8.17(s,1H),7.73(s,1H),7.48(d,J＝8.0Hz,1H),7.19(t,J＝8.0Hz,1H),7.05-7.01(m,2H),6.46-6.40(m,1H),6.29-6.20(m,1H),5.77(d,J＝8.0Hz,1H),5.03-4.96(m,1H),3.87(s,3H),3.82(s,3H),2.89(t,J＝8.0Hz,2H),2.32(t,J＝8.0Hz,2H),2.21(s,6H),1.12(d,J＝8.0Hz,6H).
Example 3: isopropyl-2- ((5-acrylamido-4- ((2- (N)1-methyl-N1-deuterated methylamino) ethyl) (N)2-methyl) amino) -2-methoxyphenyl) amino) -4- (1-methyl-1H-indol-3-yl) pyrimidine-5-carboxylic acid ester
Figure BDA0002682367610000211
See the synthesis of example 1, except that intermediate 1a is replaced by intermediate 3a and intermediate 2b is replaced by intermediate 1 b. ESI-MS M/z 589.4[ M + H ]]+,1H-NMR(DMSO-d6,400MHz),10.12(s,1H),8.84(s,1H),8.64(d,J＝8.0Hz,2H),8.17(s,1H),7.73(s,1H),7.48(d,J＝8.0Hz,1H),7.19(t,J＝8.0Hz,1H),7.05-7.01(m,2H),6.46-6.40(m,1H),6.29-6.20(m,1H),5.77(d,J＝8.0Hz,1H),5.03-4.96(m,1H),3.87(s,3H),3.82(s,3H),2.89(t,J＝8.0Hz,2H),2.72(s,3H),2.32(t,J＝8.0Hz,2H),2.21(s,3H),1.12(d,J＝8.0Hz,6H).
Example 4: isopropyl-2- ((5-acrylamido-4- ((2- (N)1,N1-dideuteromethylamino) ethyl) (N)2-methyl) amino) -2-methoxyphenyl) amino) -4- (1-methyl-1H-indol-3-yl) pyrimidine-5-carboxylic acid ester
Figure BDA0002682367610000212
See the synthesis of example 1, except that intermediate 1a is replaced by intermediate 4a and intermediate 2b is replaced by intermediate 1 b.
ESI-MS m/z:592.4[M+H]+,1H-NMR(DMSO-d6,400MHz),10.12(s,1H),8.84(s,1H),8.64(d,J＝8.0Hz,2H),8.17(s,1H),7.73(s,1H),7.48(d,J＝8.0Hz,1H),7.19(t,J＝8.0Hz,1H),7.05-7.01(m,2H),6.46-6.40(m,1H),6.29-6.20(m,1H),5.77(d,J＝8.0Hz,1H),5.03-4.96(m,1H),3.87(s,3H),3.82(s,3H),2.89(t,J＝8.0Hz,2H),2.72(s,3H),2.32(t,J＝8.0Hz,2H),1.12(d,J＝8.0Hz,6H).
Example 5: n- {2- (2-Morpholin-6-methoxy) -5- { 5-carboxylic acid isopropyl ester- [4- (1-methyl-lH-indol-3-yl) pyrimidin-2-yl ] amino } -pyridin-3-yl } acrylamide
Figure BDA0002682367610000221
See the synthesis of example 1, except that intermediate 1a is replaced with intermediate 6a and intermediate 2b is replaced with intermediate 1 b.
ESI-MS m/z:572.2[M+H]+,1H-NMR(DMSO-d6,400MHz),10.12(s,1H),8.84(s,1H),8.64(d,J＝8.0Hz,2H),8.17(s,1H),7.73(s,1H),7.48(d,J＝8.0Hz,1H),7.05-7.01(m,2H),6.46-6.40(m,1H),6.29-6.20(m,1H),5.77(d,J＝8.0Hz,1H),5.03-4.96(m,1H),3.87(s,3H),3.85(s,3H),3.82(t,J＝4.0Hz,4H),3.31(t,J＝4.0Hz,4H)1.12(d,J＝8.0Hz,6H).
Example 6
N- {2- (2-Morpholin-6-methoxy) -5- { 5-carboxylic acid isopropyl ester- [4- (1-deuterated methyl-lH-indol-3-yl) pyrimidin-2-yl ] amino } -pyridin-3-yl } acrylamide
Figure BDA0002682367610000222
See the synthesis of example 1, except that intermediate 1a is replaced with intermediate 6 a.
ESI-MS m/z:575.3[M+H]+,1H-NMR(DMSO-d6,400MHz),10.12(s,1H),8.84(s,1H),8.64(d,J＝8.0Hz,2H),8.17(s,1H),7.73(:s,1H),7.48(d,J＝8.0Hz,1H),7.05-7.01(m,2H),6.46-6.40(m,1H),6.29-6.20(m,1H),5.77(d,J＝8.0Hz,1H),5.03-4.96(m,1H),3.85(s,3H),3.82(t,J＝4.0Hz,4H),3.31(t,J＝4.0Hz,4H)1.12(d,J＝8.0Hz,6H).
Example 7: n- {2- (2-Morpholin-6-methoxy) -5- { 5-carboxylic acid isopropyl ester- [4- (1-cyclopropyl-lH-indol-3-yl) pyrimidin-2-yl ] amino } -pyridin-3-yl } acrylamide
Figure BDA0002682367610000223
See the synthesis of example 1, except that intermediate 1a is replaced with intermediate 6a and intermediate 3b is replaced with intermediate 1 b.
ESI-MS m/z:598.3[M+H]+,1H-NMR(DMSO-d6,400MHz),10.13(s,1H),8.83(s,1H),8.64(d,J＝8.0Hz,2H),8.17(s,1H),7.73(s,1H),7.48(d,J＝8.0Hz,1H),7.05-7.01(m,2H),6.46-6.40(m,1H),6.29-6.20(m,1H),5.77(d,J＝8.0Hz,1H),5.03-4.96(m,1H),3.85(s,3H),3.82(t,J＝4.0Hz,4H),3.31(t,J＝4.0Hz,4H),2.54-2.32(m,1H),1.12(d,J＝8.0Hz,6H),1.08-0.72(m,4H).
Example 8: n- {2- [ 2-morpholin-6- (2,2, 2-trifluoroethoxy) ] -5- { 5-carboxylic acid isopropyl ester- [4- (1-methyl-lH-indol-3-yl) pyrimidin-2-yl ] amino } -pyridin-3-yl } acrylamide
Figure BDA0002682367610000231
See the synthesis of example 1, except that intermediate 1a is replaced by intermediate 5a and intermediate 2b is replaced by intermediate 1 b.
ESI-MS m/z:640.2[M+H]+,1H-NMR(DMSO-d6,400MHz),10.11(s,1H),8.84(s,1H),8.63(d,J＝8.0Hz,2H),8.16(s,1H),7.72(s,1H),7.47(d,J＝8.0Hz,1H),7.05-7.01(m,2H),6.45-6.40(m,1H),6.29-6.21(m,1H),5.77(d,J＝8.0Hz,1H),5.03-4.96(m,1H),4.81(q,J＝8.0Hz,2H),3.87(s,3H),3.82(t,J＝4.0Hz,4H),3.31(t,J＝4.0Hz,4H)1.12(d,J＝8.0Hz,6H).
Example 9: n- {2- [ 2-morpholin-6- (2,2, 2-trifluoroethoxy) ] -5- { 5-carboxylic acid isopropyl ester- [4- (1-deuterated methyl-lH-indol-3-yl) pyrimidin-2-yl ] amino } -pyridin-3-yl } acrylamide
Figure BDA0002682367610000232
See the synthesis of example 1, except that intermediate 1a is replaced with intermediate 5 a.
ESI-MS m/z:643.2[M+H]+,1H-NMR(DMSO-d6,400MHz),10.11(s,1H),8.84(s,1H),8.63(d,J＝8.0Hz,2H),8.16(s,1H),7.72(s,1H),7.47(d,J＝8.0Hz,1H),7.05-7.01(m,2H),6.45-6.40(m,1H),6.29-6.21(m,1H),5.77(d,J＝8.0Hz,1H),5.03-4.96(m,1H),4.81(q,J＝8.0Hz,2H),3.82(t,J＝4.0Hz,4H),3.31(t,J＝4.0Hz,4H)1.12(d,J＝8.0Hz,6H).
Example 10: n- {2- [ 2-morpholin-6- (2,2, 2-trifluoroethoxy) ] -5- { 5-carboxylic acid isopropyl ester- [4- (1-cyclopropyl-lH-indol-3-yl) pyrimidin-2-yl ] amino } -pyridin-3-yl } acrylamide
Figure BDA0002682367610000233
The synthesis is as in example 1 except that intermediate 1a is replaced by intermediate 5a and intermediate 2b is replaced by intermediate 3 b.
ESI-MS m/z:666.3[M+H]+,1H-NMR(DMSO-d6,400MHz),10.11(s,1H),8.84(s,1H),8.62(d,J＝8.0Hz,2H),8.17(s,1H),7.72(s,1H),7.47(d,J＝8.0Hz,1H),7.05-7.01(m,2H),6.45-6.40(m,1H),6.29-6.21(m,1H),5.77(d,J＝8.0Hz,1H),5.03-4.96(m,1H),4.81(q,J＝8.0Hz,2H),3.82(t,J＝4.0Hz,4H),3.32(t,J＝4.0Hz,4H),2.57-2.32(m,1H),1.11(d,J＝8.0Hz,6H),1.07-0.71(m,4H).
Example 11: n- {2- { [2- (dimethylamino) ethyl ] (methyl) amino } -6- (2,2, 2-trifluoroethoxy) -5- { 5-carboxylic acid isopropyl ester- [4- (1-methyl-lH-indol-3-yl) pyrimidin-2-yl ] amino } -pyridin-3-yl } acrylamide
Figure BDA0002682367610000241
See the synthesis of example 1, except that intermediate 1a is replaced by intermediate 7a and intermediate 2b is replaced by intermediate 1 b.
ESI-MS m/z:655.3[M+H]+,1H-NMR(DMSO-d6,400MHz),10.11(s,1H),8.84(s,1H),8.63(d,J＝8.0Hz,2H),8.16(s,1H),7.73(s,1H),7.47(d,J＝8.0Hz,1H),7.05-7.01(m,2H),6.45-6.40(m,1H),6.29-6.21(m,1H),5.79(d,J＝8.0Hz,1H),5.07-4.93(m,1H),4.85(q,J＝8.0Hz,2H),3.87(s,3H),2.82(t,J＝8.0Hz,2H),2.67(s,3H),2.23(t,J＝8.0Hz,2H),2.12(s,6H),1.12(d,J＝8.0Hz,6H).
Example 12: n- {2- { [2- (dimethylamino) ethyl ] (methyl) amino } -6- (2,2, 2-trifluoroethoxy) -5- { 5-carboxylic acid isopropyl ester- [4- (1-deuterated methyl-lH-indol-3-yl) pyrimidin-2-yl ] amino } -pyridin-3-yl } acrylamide
Figure BDA0002682367610000242
See the synthesis of example 1, except that intermediate 1a is replaced with intermediate 7 a.
ESI-MS m/z:658.3[M+H]+,1H-NMR(DMSO-d6,400MHz),10.11(s,1H),8.84(s,1H),8.63(d,J＝8.0Hz,2H),8.16(s,1H),7.73(s,1H),7.47(d,J＝8.0Hz,1H),7.05-7.01(m,2H),6.45-6.40(m,1H),6.29-6.21(m,1H),5.79(d,J＝8.0Hz,1H),5.07-4.93(m,1H),4.85(q,J＝8.0Hz,2H),2.82(t,J＝8.0Hz,2H),2.67(s,3H),2.23(t,J＝8.0Hz,2H),2.12(s,6H),1.12(d,J＝8.0Hz,6H).
Example 13: n- {2- { [2- (dimethylamino) ethyl ] (methyl) amino } -6- (2,2, 2-trifluoroethoxy) -5- { 5-carboxylic acid isopropyl ester- [4- (1-cyclopropyl-lH-indol-3-yl) pyrimidin-2-yl ] amino } -pyridin-3-yl } acrylamide
Figure BDA0002682367610000251
The synthesis is as in example 1 except that intermediate 1a is replaced by intermediate 7a and intermediate 2b is replaced by intermediate 3 b.
ESI-MS m/z:681.3[M+H]+,1H-NMR(DMSO-d6,400MHz),10.11(s,1H),8.83(s,1H),8.62(d,J＝8.0Hz,2H),8.15(s,1H),7.75(s,1H),7.46(d,J＝8.0Hz,1H),7.05-7.01(m,2H),6.45-6.40(m,1H),6.29-6.21(m,1H),5.79(d,J＝8.0Hz,1H),5.07-4.93(m,1H),4.85(q,J＝8.0Hz,2H),2.82(t,J＝8.0Hz,2H),2.67(s,3H),2.55-2.31(m,1H),2.23(t,J＝8.0Hz,2H),2.12(s,6H),1.12(d,J＝8.0Hz,6H),1.09-0.71(m,4H).
Example 14: n- {2- { [2- (dimethylamino) ethyl ] (methyl) amino } -6-methoxy-5- { 5-carboxylic acid isopropyl ester- [4- (1-methyl-lH-indol-3-yl) pyrimidin-2-yl ] amino } -pyridin-3-yl } acrylamide
Figure BDA0002682367610000252
See the synthesis of example 1, except that intermediate 1a is replaced with intermediate 8a and intermediate 2b is replaced with intermediate 1 b.
ESI-MS m/z:587.3[M+H]+,1H-NMR(DMSO-d6,400MHz),10.10(s,1H),8.83(s,1H),8.63(d,J＝8.0Hz,2H),8.16(s,1H),7.71(s,1H),7.46(d,J＝8.0Hz,1H),7.05-7.01(m,2H),6.45-6.40(m,1H),6.29-6.21(m,1H),5.79(d,J＝8.0Hz,1H),5.07-4.93(m,1H),3.87(s,3H),3.84(s,1H),2.82(t,J＝8.0Hz,2H),2.67(s,3H),2.23(t,J＝8.0Hz,2H),2.12(s,6H),1.12(d,J＝8.0Hz,6H).
Example 15: n- {2- { [2- (dimethylamino) ethyl ] (methyl) amino } -6-methoxy-5- { 5-carboxylic acid isopropyl ester- [4- (1-deuterated methyl-lH-indol-3-yl) pyrimidin-2-yl ] amino } -pyridin-3-yl } acrylamide
Figure BDA0002682367610000253
See the synthesis of example 1, except that intermediate 1a is replaced with intermediate 8 a.
ESI-MS m/z:590.3[M+H]+,1H-NMR(DMSO-d6,400MHz),10.10(s,1H),8.83(s,1H),8.63(d,J＝8.0Hz,2H),8.16(s,1H),7.71(s,1H),7.46(d,J＝8.0Hz,1H),7.05-7.01(m,2H),6.45-6.40(m,1H),6.29-6.21(m,1H),5.79(d,J＝8.0Hz,1H),5.07-4.93(m,1H),3.84(s,1H),2.82(t,J＝8.0Hz,2H),2.67(s,3H),2.23(t,J＝8.0Hz,2H),2.12(s,6H),1.12(d,J＝8.0Hz,6H).
Example 16: n- {2- { [2- (dimethylamino) ethyl ] (methyl) amino } -6-methoxy-5- { 5-carboxylic acid isopropyl ester- [4- (1-cyclopropyl-lH-indol-3-yl) pyrimidin-2-yl ] amino } -pyridin-3-yl } acrylamide
Figure BDA0002682367610000261
See the synthesis of example 1, except that intermediate 1a is replaced by intermediate 8a and intermediate 2b is replaced by intermediate 3 b.
ESI-MS m/z:613.3[M+H]+,1H-NMR(DMSO-d6,400MHz),10.10(s,1H),8.83(s,1H),8.63(d,J＝8.0Hz,2H),8.16(s,1H),7.72(s,1H),7.45(d,J＝8.0Hz,1H),7.05-7.01(m,2H),6.45-6.40(m,1H),6.30-6.21(m,1H),5.78(d,J＝8.0Hz,1H),5.07-4.93(m,1H),3.84(s,1H),2.82(t,J＝8.0Hz,2H),2.67(s,3H),2.56-2.31(m,1H),2.23(t,J＝8.0Hz,2H),2.12(s,6H),1.11(d,J＝8.0Hz,6H),1.08-0.70(m,4H).
Example 17: isopropyl-2- ((5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) -4- (1-hydroxymethyl-1H-indol-3-yl) pyrimidine-5-carboxylate
Figure BDA0002682367610000262
Preparation of isopropyl-2- ((5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) -4- (1H-indol-3-yl) pyrimidine-5-carboxylate
See the synthesis of example 1, except that intermediate 2b is replaced with intermediate 4 b. ESI-MS M/z 572.3[ M + H ]]+Preparation of isopropyl-2- ((5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) -4- (1-hydroxymethyl-1H-indol-3-yl) pyrimidine-5-carboxylate
Isopropyl-2- ((5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) -4- (1H-indol-3-yl) pyrimidine-5-carboxylate (100mg,0.175mmol) was suspended in water (1mL), dichloromethane (1mL), methanol (1mL), and aqueous formaldehyde (37%, 0.4mL) and tetrabutylammonium fluoride (1M,0.08mL) were added. The reaction mixture was extracted with dichloromethane overnight at room temperature, the organic phase was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and column purified to give 80mg of a solid in 76% yield.
ESI-MS m/z:602.3[M+H]+,1H-NMR(DMSO-d6,400MHz),10.12(s,1H),8.85(s,1H),8.65(d,J＝8.0Hz,2H),8.16(s,1H),7.73(s,1H),7.48(d,J＝8.0Hz,1H),7.21(t,J＝8.0Hz,1H),7.07-7.01(m,2H),6.46-6.41(m,1H),6.39(d,J＝4.0Hz,2H),6.29-6.20(m,1H),5.77(d,J＝8.0Hz,1H),5.03-4.96(m,1H),3.87(s,3H),2.89(t,J＝8.0Hz,2H),2.72(s,3H),2.32(t,J＝8.0Hz,2H),2.21(s,6H),1.12(d,J＝8.0Hz,6H).
Example 18: n- (2- { 2-dimethylaminoethyl-methylamino } -4-methoxy-5- { [4- (5-isopropylcarboxylate-1-methylphosphonic acid di-tert-butyl ester-indol-3-yl) pyrimidin-2-yl ] amino } phenyl) acrylamide
Figure BDA0002682367610000271
Isopropyl-2- ((5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) -4- (1H-indol-3-yl) pyrimidine-5-carboxylate (100mg,0.175mmol) was dissolved in 2mL DMF, NaH (8.4mg,0.210mmol) was added under ice bath, reaction was performed at 0 ℃ for 0.5H after the addition was completed, then di-tert-butyl chloromethyl phosphate (58.5mg,0.210mmol) was dissolved in DMF (0.5mL) and added to the reaction solution, reaction was performed at room temperature for 2H, the reaction solution was poured into water, DCM was extracted, the organic phase was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, column purification gave 30mg of a solid, yield: 21.6%.
ESI-MS m/z:794.4[M+H]+,1H-NMR(DMSO-d6,400MHz),10.14(s,1H),8.84(s,1H),8.63(d,J＝8.0Hz,2H),8.17(s,1H),7.74(s,1H),7.48(d,J＝8.0Hz,1H),7.23(t,J＝8.0Hz,1H),7.07-7.00(m,2H),6.46-6.41(m,1H),6.39(d,J＝4.0Hz,2H),6.29-6.20(m,1H),5.77(d,J＝8.0Hz,1H),5.03-4.96(m,1H),4.03(q,J＝8.0Hz,4H)，3.87(s,3H),2.89(t,J＝8.0Hz,2H),2.72(s,3H),2.32(t,J＝8.0Hz,2H),2.21(s,6H),1.41(t,J＝8.0Hz,6H),1.32(s,18H),1.12(d,J＝8.0Hz,6H).
Example 19: n- (2- { 2-dimethylaminoethyl-methylamino } -4-methoxy-5- { [4- (5-isopropylcarboxylate-1-methylphosphonic acid diethyl ester-indol-3-yl) pyrimidin-2-yl ] amino } phenyl) acrylamide
Figure BDA0002682367610000272
See example 18 for synthesis except that di-tert-butyl chloromethyl phosphate was replaced with diethyl chloromethyl phosphate in 18.6% yield.
ESI-MS m/z:738.3[M+H]+,1H-NMR(DMSO-d6,400MHz),10.14(s,1H),8.84(s,1H),8.63(d,J＝8.0Hz,2H),8.17(s,1H),7.74(s,1H),7.48(d,J＝8.0Hz,1H),7.23(t,J＝8.0Hz,1H),7.07-7.00(m,2H),6.46-6.41(m,1H),6.39(d,J＝4.0Hz,2H),6.29-6.20(m,1H),5.77(d,J＝8.0Hz,1H),5.03-4.96(m,1H),4.03(q,J＝8.0Hz,4H),3.87(s,3H),2.89(t,J＝8.0Hz,2H),2.72(s,3H),2.32(t,J＝8.0Hz,2H),2.21(s,6H),1.41(t,J＝8.0Hz,6H),1.12(d,J＝8.0Hz,6H).
Example 20: n- (2- { 2-dimethylaminoethyl-methylamino } -4-methoxy-5- { [4- (5-isopropylcarboxylate-1-methylphosphonic acid-indol-3-yl) pyrimidin-2-yl ] amino } phenyl) acrylamide
Figure BDA0002682367610000281
N- (2- { 2-dimethylaminoethyl-methylamino } -4-methoxy-5- { [4- (5-isopropylcarboxylate-1-methylphosphonic acid di-tert-butyl-indol-3-yl) pyrimidin-2-yl ] amino } phenyl) propan-2-eneamide (100mg) was dissolved in 1mL of dilute hydrochloric acid (1M) and 1mL of tetrahydrofuran, stirred at room temperature for 1 hour, adjusted to pH 6-7, extracted with dichloromethane, dried and then spun to give 30mg of a solid in 35% yield.
ESI-MS m/z:682.3[M+H]+,1H-NMR(DMSO-d6,400MHz),10.14(s,1H),8.84(s,1H),8.63(d,J＝8.0Hz,2H),8.17(s,1H),7.74(s,1H),7.48(d,J＝8.0Hz,1H),7.23(t,J＝8.0Hz,1H),7.07-7.00(m,2H),6.46-6.41(m,1H),6.39(d,J＝4.0Hz,2H),6.29-6.20(m,1H),5.77(d,J＝8.0Hz,1H),5.03-4.96(m,1H),3.87(s,3H),2.89(t,J＝8.0Hz,2H),2.72(s,3H),2.32(t,J＝8.0Hz,2H),2.21(s,6H),1.12(d,J＝8.0Hz,6H).
Example 21: n- {2- { [2- (dimethylamino) ethyl ] (methyl) amino } -6- (2,2, 2-trifluoroethoxy) -5- { 5-carboxylic acid isopropyl ester- [4- (1-hydroxymethyl-lH-indol-3-yl) pyrimidin-2-yl ] amino } -pyridin-3-yl } acrylamide
Figure BDA0002682367610000282
See example 17 for a synthetic method except that intermediate 1a was replaced with intermediate 7a in 45.6% yield.
ESI-MS m/z:671.3[M+H]+,1H-NMR(DMSO-d6,400MHz),10.11(s,1H),8.85(s,1H),8.64(d,J＝8.0Hz,2H),8.17(s,1H),7.73(s,1H),7.48(d,J＝8.0Hz,1H),7.05-7.01(m,2H),6.45-6.40(m,1H),6.39(d,J＝4.0Hz,2H),6.29-6.21(m,1H),5.79(d,J＝8.0Hz,1H),5.07-4.93(m,1H),4.85(q,J＝8.0Hz,2H),2.82(t,J＝8.0Hz,2H),2.67(s,3H),2.23(t,J＝8.0Hz,2H),2.12(s,6H),1.12(d,J＝8.0Hz,6H).
Example 22: n- {2- { [2- (dimethylamino) ethyl ] (methyl) amino } -6- (2,2, 2-trifluoroethoxy) -5- { 5-carboxylic acid isopropyl ester- [4- (1-methylphosphonic acid di-tert-butyl-lH-indol-3-yl) pyrimidin-2-yl ] amino } -pyridin-3-yl } acrylamide
Figure BDA0002682367610000283
See example 18 for a synthetic method except that intermediate 1a is replaced with intermediate 7a in 15.6% yield.
ESI-MS m/z:863.4[M+H]+,1H-NMR(DMSO-d6,400MHz),10.11(s,1H),8.86(s,1H),8.64(d,J＝8.0Hz,2H),8.17(s,1H),7.73(s,1H),7.48(d,J＝8.0Hz,1H),7.07-7.01(m,2H),6.45-6.41(m,1H),6.43(d,J＝4.0Hz,2H),6.29-6.20(m,1H),5.78(d,J＝8.0Hz,1H),5.07-4.93(m,1H),4.88(q,J＝8.0Hz,2H),2.82(t,J＝8.0Hz,2H),2.67(s,3H),2.23(t,J＝8.0Hz,2H),2.12(s,6H),1.32(s,18H),1.12(d,J＝8.0Hz,6H).
Example 23: n- {2- { [2- (dimethylamino) ethyl ] (methyl) amino } -6- (2,2, 2-trifluoroethoxy) -5- { 5-carboxylic acid isopropyl ester- [4- (1-methylphosphonic acid diethyl ester-lH-indol-3-yl) pyrimidin-2-yl ] amino } -pyridin-3-yl } acrylamide
Figure BDA0002682367610000291
See example 18 for a synthetic method, except that di-tert-butyl chloromethyl phosphate is replaced by diethyl chloromethyl phosphate and intermediate 1a is replaced by intermediate 7 a. The yield was 17.3%.
ESI-MS m/z:807.3[M+H]+,1H-NMR(DMSO-d6,400MHz),10.12(s,1H),8.88(s,1H),8.64(d,J＝8.0Hz,2H),8.17(s,1H),7.73(s,1H),7.48(d,J＝8.0Hz,1H),7.07-7.02(m,2H),6.45-6.41(m,1H),6.41(d,J＝4.0Hz,2H),6.29-6.21(m,1H),5.78(d,J＝8.0Hz,1H),5.07-4.92(m,1H),4.87(q,J＝8.0Hz,2H),4.03(q,J＝8.0Hz,4H),2.82(t,J＝8.0Hz,2H),2.67(s,3H),2.23(t,J＝8.0Hz,2H),2.12(s,6H),1.41(t,J＝8.0Hz,6H),1.12(d,J＝8.0Hz,6H).
Example 24: n- {2- { [2- (dimethylamino) ethyl ] (methyl) amino } -6- (2,2, 2-trifluoroethoxy) -5- { 5-carboxylic acid isopropyl ester- [4- (1-methylphosphonic acid-lH-indol-3-yl) pyrimidin-2-yl ] amino } -pyridin-3-yl } acrylamide
Figure BDA0002682367610000292
See example 20 for a synthetic method except that intermediate 1a was replaced with intermediate 7a in 25.4% yield.
ESI-MS m/z:751.3[M+H]+,1H-NMR(DMSO-d6,400MHz),10.11(s,1H),8.86(s,1H),8.64(d,J＝8.0Hz,2H),8.17(s,1H),7.73(s,1H),7.48(d,J＝8.0Hz,1H),7.07-7.01(m,2H),6.45-6.41(m,1H),6.41(d,J＝4.0Hz,2H),6.29-6.20(m,1H),5.78(d,J＝8.0Hz,1H),5.07-4.93(m,1H),4.88(q,J＝8.0Hz,2H),2.82(t,J＝8.0Hz,2H),2.67(s,3H),2.23(t,J＝8.0Hz,2H),2.12(s,6H),1.12(d,J＝8.0Hz,6H).
Example 25: isopropyl-2- ((5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) -4- (1-methoxymethyl-indol-3-yl) pyrimidine-5-carboxylic acid ester
Figure BDA0002682367610000301
Isopropyl-2- ((5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) -4- (1-hydroxymethyl-1H-indol-3-yl) pyrimidine-5-carboxylate (i.e. the compound of example 17) (100mg,0.166mmol) was dissolved in 2mL of dry DMF, NaH (8mg,0.2mmol) was added under ice bath, after which time 0.5H reaction was carried out at 0 ℃, iodomethane (28mg,0.2mmol) was then dissolved in DMF and added to the reaction mixture, reaction was carried out at room temperature for 2H, the reaction mixture was poured into water, DCM was extracted, the organic phase was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, column purification gave 25mg of solid, yield: 24.5%.
ESI-MS m/z:616.3[M+H]+,1H-NMR(DMSO-d6,400MHz),10.12(s,1H),8.85(s,1H),8.65(d,J＝8.0Hz,2H),8.16(s,1H),7.73(s,1H),7.48(d,J＝8.0Hz,1H),7.21(t,J＝8.0Hz,1H),7.07-7.01(m,2H),6.46-6.41(m,1H),6.36(d,J＝4.0Hz,2H),6.29-6.20(m,1H),5.77(d,J＝8.0Hz,1H),5.03-4.96(m,1H),3.87(s,3H),3.38(s,3H),2.89(t,J＝8.0Hz,2H),2.72(s,3H),2.32(t,J＝8.0Hz,2H),2.21(s,6H),1.12(d,J＝8.0Hz,6H).
Example 26: isopropyl-2- ((5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-isopropylphenyl) amino) -4- (1-methoxymethyl-indol-3-yl) pyrimidine-5-carboxylic acid ester
Figure BDA0002682367610000302
See example 25 for a synthetic method, except that methyl iodide was replaced with bromoisopropane in 21.8% yield.
ESI-MS m/z:616.3[M+H]+,1H-NMR(DMSO-d6,400MHz),10.12(s,1H),8.85(s,1H),8.65(d,J＝8.0Hz,2H),8.16(s,1H),7.73(s,1H),7.48(d,J＝8.0Hz,1H),7.21(t,J＝8.0Hz,1H),7.07-7.01(m,2H),6.46-6.41(m,1H),6.36(d,J＝4.0Hz,2H),6.29-6.20(m,1H),5.77(d,J＝8.0Hz,1H),5.03-4.96(m,1H),3.87(s,3H),3.65-3.52(m,1H),3.38(s,3H)2.89(t,J＝8.0Hz,2H),2.72(s,3H),2.32(t,J＝8.0Hz,2H),2.21(s,6H),1.18(d,J＝8.0Hz,6H),1.14(d,J＝8.0Hz,6H).
Example 27: n- {2- { [2- (dimethylamino) ethyl ] (methyl) amino } -6- (2,2, 2-trifluoroethoxy) -5- { 5-carboxylic acid isopropyl ester- [4- (1-methoxymethyl-lH-indol-3-yl) pyrimidin-2-yl ] amino } -pyridin-3-yl } acrylamide
Figure BDA0002682367610000311
See example 25 for synthesis except that the compound of example 17 was replaced with the compound of example 21, yield: 18.8 percent.
:ESI-MS m/z:685.3[M+H]+,1H-NMR(DMSO-d6,400MHz),10.11(s,1H),8.85(s,1H),8.64(d,J＝8.0Hz,2H),8.17(s,1H),7.73(s,1H),7.48(d,J＝8.0Hz,1H),7.05-7.01(m,2H),6.45-6.40(m,1H),6.37(d,J＝4.0Hz,2H),6.29-6.21(m,1H),5.79(d,J＝8.0Hz,1H),5.07-4.93(m,1H),4.85(q,J＝8.0Hz,2H),3.38(s,3H),2.82(t,J＝8.0Hz,2H),2.67(s,3H),2.23(t,J＝8.0Hz,2H),2.12(s,6H),1.12(d,J＝8.0Hz,6H).
Example 28: n- {2- { [2- (dimethylamino) ethyl ] (methyl) amino } -6- (2,2, 2-trifluoroethoxy) -5- { 5-carboxylic acid isopropyl ester- [4- (1-isopropoxymethyl-lH-indol-3-yl) pyrimidin-2-yl ] amino } -pyridin-3-yl } acrylamide
Figure BDA0002682367610000312
See example 25 for synthesis with the exception that the compound of example 17 was replaced with the compound of example 21 and methyl iodide was replaced with bromoisopropane, yield 20.3%.
ESI-MS m/z:713.3[M+H]+,1H-NMR(DMSO-d6,400MHz),10.11(s,1H),8.85(s,1H),8.64(d,J＝8.0Hz,2H),8.17(s,1H),7.73(s,1H),7.48(d,J＝8.0Hz,1H),7.05-7.01(m,2H),6.45-6.40(m,1H),6.35(s,2H),6.29-6.21(m,1H),5.79(d,J＝8.0Hz,1H),5.07-4.93(m,1H),4.85(q,J＝8.0Hz,2H),3.65-3.52(m,1H),2.82(t,J＝8.0Hz,2H),2.67(s,3H),2.23(t,J＝8.0Hz,2H),2.12(s,6H),1.18(d,J＝8.0Hz,6H),1.13(d,J＝8.0Hz,6H).
The corresponding succinate and mesylate salts can be prepared in a similar manner as in examples 1A and 1B above for examples 2 to 28.
Biological examples:
application example 1: ASV and NPG EGFR exon 20 insertion mutations
The activity of compounds in selectively inhibiting EGFR exon 20 insertion mutations can be assessed using the murine progenitor B cell line Ba/F3 cells that have been transduced with EGFR exon 20 insertion. Expression vector pLVX-IRES puro (Clontech) encoding human EGFR exon 20 insertion into NPG (H773-V774insNPG) or ASV (V769-D770insASV) was transfected into HEK293 cells by the trans-lentivirus ORF assembly system (Thermo Scientific) to generate a virus encoding EGFR exon 20 insertion. Infection by EGFR exon 20 virus was maintained in a medium supplemented with 10% fetal bovine serum, 200. mu. M L-glutamine/200. mu.g/mL penicillin/200. mu.g/mL streptomycin (Life Technology), and 10ng/mL IL-3 (R)&D system) in RPMI1640 medium and subsequently selected by puromycin (Life Technology) selection and IL-3 depletion. Ba/F3 cells expressing EGFR exon 20 insertions (designated Ba/F3-EGFR-exon 20-NPG or Ba/F3-EGFR-exon 20-ASV) can be propagated in the absence of IL-3. Method for determining the antiproliferative activity of a compound: BaF3-EGFR exon 20 cells (NPG or ASV) (2500 cells/well) seeded in 96-well plates were treated with test compounds (dissolved in DMSO) at a range of concentrations (4-fold dilution, maximum concentration: 10,000 nM). Incubate at 37 ℃ in 5% CO2Plates were incubated for 72 hours and passed
Figure BDA0002682367610000322
Aqueous single solution cell proliferation assay (Promega) to indirectly measure the number of viable cells in each well. This assay is a colorimetric method for determining the number of viable cells by measuring the metabolic activity of viable cells by detecting the enzymatic conversion of tetrazolium salt to blue formazan derivatives. Reagents (20 μ L) were added to each well and the plates were returned to the incubator for 2 hours. The absorbance in each well was then measured at 490nm using an Envision plate reader (PerkinElmer). IC was calculated by determining the concentration of compound required to reduce the MTS signal by 50% compared to DMSO control in a best-fit curve using Microsoft XLFit software or Accelrys Pipeline Pilot50The value is obtained.
Application example 2: EGFR exon 19 deletion and exon 20T790M parallel mutations
The activity of compounds in selectively inhibiting EGFR exon 19 deletion and T790M concurrent mutations can be assessed using the murine progenitor B cell line Ba/F3 cells that have been transduced with EGFR exon 19 deletion and T790M mutations. The expression vector pLVX-IRES puro (Clontech) encoding the deletion of EGFR E746-A750 and the mutation of T790M was transfected into HEK293 cells by the trans lentiviral ORF assembly system (Thermo Scientific) to generate a virus encoding the deletion of EGFR exon 19 and the mutation of T790M. Infection by EGFR E746-A750 deletion and T790M mutant virus was maintained in a supplemented medium of 10% fetal bovine serum, 200. mu. M L-glutamine/200. mu.g/mL penicillin/200. mu.g/mL streptomycin (Life Technology), and 10ng/mL IL-3 (R/L, E.sub.&D system) in RPMI1640 medium and subsequently selected by puromycin (Life Technology) selection and IL-3 depletion. Ba/F3 cells expressing an EGFR E746-A750 deletion and a T790M mutation (designated Ba/F3-EGFR-Del/T790M) can proliferate in the absence of IL-3. Method for determining the antiproliferative activity of a compound: BaF3-EGFR-Del/T790M cells (2500 cells/well) seeded in 96-well plates were treated with test compounds (dissolved in DMSO) at a range of concentrations (4-fold dilution, maximum concentration: 10,000 nM). Incubate at 37 ℃ in 5% CO2Plates were incubated for 72 hours, and passed through Cell
Figure BDA0002682367610000321
The number of viable cells in each well was measured indirectly by an aquous single solution cell proliferation assay (Promega; this assay is a colorimetric method for determining the number of viable cells by measuring the metabolic activity of viable cells by detecting the enzymatic conversion of tetrazolium salt to blue formazan derivative). Reagents (20 μ L) were added to each well and the plates were returned to the incubator for 2 hours. The absorbance in each well was then measured at 490nm using an Envision plate reader (Perkin Elmer). IC was calculated by determining the concentration of compound required to reduce the MTS signal by 50% compared to DMSO control in a best-fit curve using Microsoft XLFit software or Accelrys Pipeline Pilot50The value is obtained.
Application example 3: EGFR exon 21L858R and exon 20T790M parallel mutations
The activity of compounds in selectively inhibiting EGFR L858R and T790M concurrent mutations can be assessed using the murine progenitor B cell line Ba/F3 cells that have been transduced with EGFR L858R and T790M double mutations. The expression vector pLVX-IRES puro (Clontech) encoding the double mutation of human EGFR L858R and T790M was transfected into HEK293 cells by the trans-lentiviral ORF assembly system (Thermo Scientific) to generate a virus encoding the double mutation of EGFR L858R and T790M. Infection by EGFR L858R and T790M double mutant viruses was maintained in a supplemented medium of 10% fetal bovine serum, 200. mu. M L-glutamine/200. mu.g/mL penicillin/200. mu.g/mL streptomycin (Li fe Technology), and 10ng/mL IL-3 (R.sub.&D system) in RPMI1640 medium, and subsequently selected by glance sideways at lingomycin (Life Technology) selection and IL-3 depletion. Ba/F3 cells expressing double mutations of EGFR L858R and T790M (named Ba/F3-EGFR L858R/T790M) can proliferate in the absence of IL-3. Method for determining the antiproliferative activity of a compound: BaF3-EGFR L858R/T790M cells (2500 cells/well) seeded in 96-well plates were treated with test compounds (dissolved in DMSO) at a range of concentrations (4-fold dilution, maximum concentration: 10,000 nM). Incubate at 37 ℃ in 5% CO2Plates were incubated for 72 hours next and passed through Ce11Ti
Figure BDA0002682367610000323
The number of viable cells in each well was measured indirectly by an aquous single solution cell proliferation assay (Promega; this assay is a colorimetric method for determining the number of viable cells by measuring the metabolic activity of viable cells by detecting the enzymatic conversion of tetrazolium salt to blue formazan derivative). Reagents (20 μ L) were added to each well and the plates were returned to the incubator for 2 hours. The absorbance in each well was then measured at 490nm using an Envision plate reader (Perkin Elmer). IC was calculated by determining the concentration of compound required to reduce the MTS signal by 50% compared to DMSO control in a best-fit curve using Microsoft XLFit software or Accelrys Pipeline Pilot50The value is obtained.
Application example 4: HER2 exon 20YVMA insertion mutation
The activity of compounds in selectively inhibiting HER2 exon 20YVMA insertion mutations can be assessed using the murine progenitor B cell line Ba/F3 cell that has been transduced with ER2 exon 20YVMA insertion. Expression vector pLVX-IRES puro (Clontech) encoding human EGFR exon 20 insertion into YVMA (A775-G776ins YVMA) was transfected into HEK293 cells by trans-lentiviral ORF assembly system (Thermo Scientific) to generate virus encoding EGFR exon 20 insertion. Infection by EGFR exon 20 virus was maintained in a medium supplemented with 10% fetal bovine serum, 200. mu. M L-glutamine/200. mu.g/mL penicillin/200. mu.g/mL streptomycin (Life Technology), and 10ng/mL IL-3 (R)&D system) in RPMI1640 medium and subsequently selected by puromycin (Life Technology) selection and IL-3 depletion. Ba/F3 cells expressing Her2 exon 20YVMA insertion (designated Ba/F3-Her2 exon 20YVMA) can proliferate in the absence of IL-3. Method for determining the antiproliferative activity of a compound: BaF3-Her2 exon 20YVMA cells (2500 cells/well) seeded in 96-well plates were treated with test compounds (dissolved in DMSO) at a range of concentrations (4-fold dilution, maximum concentration: 10,000 nM). Incubate at 37 ℃ in 5% CO2Plates were incubated for 72 hours and passed
Figure BDA0002682367610000332
Aqueous single solution cell expansionThe number of viable cells in each well is indirectly measured by a colonization assay (Promega; this assay is a colorimetric method for determining the number of viable cells by measuring the metabolic activity of viable cells by detecting the enzymatic conversion of tetrazolium salt to blue formazan derivative). Reagents (20 μ L) were added to each well and the plates were returned to the incubator for 2 hours. The absorbance in each well was then measured at 490nm using an Envision plate reader (Perkin Elmer). IC was calculated by determining the concentration of compound required to reduce the MTS signal by 50% compared to DMSO control in a best-fit curve using Microsoft XLFit software or Accelrys Pipeline Pilot50The value is obtained.
Table 1 shows the results of application examples 1 to 4. That is, Table 1 shows ASV and NPG insertion mutant exon 20EGFR IC of example compounds and reference compounds (commercially available compound TAK-788)50Data on IC of DT mutations50Data, and YVMA insertional mutation exon 20HER2 IC50And (4) data.
TABLE 1 data (nM) of the activity assays of the examples of the invention and of the reference compounds
Figure BDA0002682367610000331
Figure BDA0002682367610000341
Figure BDA0002682367610000351
Where n is the number of experimental repetitions
The results show that the compounds with methyl replaced by hydroxymethyl and methyl replaced by deuteromethyl improve the bioactivity of the examples, and show stronger therapeutic potential due to the comparative example TAK-788. Although examples 18-20, 22-28 did not demonstrate superior results to TAK-788 in the cell viability assay, examples 18-20, 22-28 could be metabolized in vivo to give example 17 or example 21, with similar therapeutic potential.
Application example 5: evaluation of Compound stability Using human liver microsomes
The liver microsomal enzyme stability of the example compounds was compared to the reference compound TAK-788.
Measurement System: the metabolic stability of the compound of the present invention was tested using 1mM NADPH for liver microparticles mixed in men and women. The samples were analyzed using a mass spectrometer. HRMS was used to determine peak area response ratios (peak area corresponding to test compound or control divided by peak area of the analytical internal standard) without running a standard curve. In order to detect all possible metabolites, HRMS scans were performed at the appropriate m/z range.
The measurement conditions were as follows: the assay was performed with one incubation (N ═ 1). Test compounds were incubated at 37 ℃ in buffer containing 0.5 mg/ml liver microsomal protein. Reactions were initiated by addition of cofactors and samples taken at 0, 2,4, 8, 16, 24, 36, 48 hours, positive controls (5 μ M testosterone) were incubated in parallel and samples taken at 0, 2,4, 8, 16, 24, 36, 48 hours.
And (3) measuring quality control: the control compound testosterone was performed in parallel to confirm the enzymatic activity of the (liver) microsomes. After the final time point, the addition of NADPH to the reaction mixture was confirmed using fluorimetry. The T1/2 of the control met acceptable internal standards.
The analysis method comprises the following steps: liquid chromatography column: thermo BDS Hypersil c 1830 x2.0mm, 3 μm, with guard column m.p., buffer: 25mM formic acid buffered solution, pH 3.5; aqueous phase (a): 90% water, 10% buffer; organic phase (B): 90% acetonitrile, 10% buffer; flow rate: 300 microliters/minute; automatic sample injector: the injection volume was 10 microliters.
See table 2 for gradient program.
TABLE 2 gradient program
Time (minutes) ％A ％B
0.0 100 0
1.5 0 100
2.0 0 100
2.1 100 0
3.5 100 0
The results of the metabolic half-life measurement of the examples as described in the present invention by using human liver microsomes are shown in Table 3.
TABLE 3 metabolic half-lives of the example compounds and the reference compounds
Compound (I) Half-life (hours)
Example 1 24
Example 2 18
Example 3 19
Example 4 26
Example 5 19
Example 6 26
Example 7 22
Example 8 19
Example 9 23
Example 10 25
Example 11 20
Example 12 24
Example 13 23
Example 14 15
Example 15 23
Example 16 22
Example 17 17
Example 21 16
TAK-788 16
The results show that methyl deuterated, trifluoroethoxy substituted, and or cyclopropyl substituted compounds at specific positions improve the metabolic stability of the examples, and that the relatively long metabolic half-lives allow them the potential to reduce the therapeutic dose and extend the dosing interval.
Application example 6: blood brain barrier penetration assay
According to the literature (Journal of Medicinal Chemistry,2013,56(1):2-12) Kp, uu brainAnd Kp，uu CSFBoth are the main parameters measured and optimized during CNS drug discovery. Relationship K between the concentration of unbound drug in brain and bloodp，uuBrain prediction of drug on brain Leptomeningeal Metastasis (LM) caused by metastatic spread of cancer to leptomeningeal, metastatic tumor caused central nervous system dysfunction. Kp，uu CSFIndicating the drug distribution in CSF compared to the distribution of the drug in blood, which drives the drug response during leptomeningeal transfer therapy.
In vitro blood and brain binding assays were performed on HT dialysis plates with semipermeable membranes. Diluted blood (with DPBS 1:1, pH7.4) and brain homogenate (with DPBS 1:3, pH7.4) were spiked with 5 μ M test compound (in triplicate) and dialyzed in 150 μ L of an equal volume of 100mM PBS buffer (pH7.4) at 37 ℃ for 4 hours in a slowly rotating plate. At the end of the incubation, 50 μ L aliquots from the receiver side and 5 μ L aliquots from the donor compartment were taken. mu.L of the sample was further diluted with 45. mu.L of blank blood or brain homogenate. Paired samples were matrix matched with buffer or blank blood/brain homogenates and mixed for 2min and then precipitated with 150 μ L cold acetonitrile with 100ng/mL tolbutamide as an internal standard. After centrifugation at 4000rpm for 20min, the supernatant was diluted with 0.1% aqueous formic acid and analyzed for LC/MS. The unbound fraction (fu) of the test compound in the brain homogenate and diluted blood was calculated by the ratio of the buffer side reaction to the brain homogenate/blood side reaction, and the following equation f was usedu，bl(fu，br)＝(1/D)/[(1/fu-1)+1/D)]Calculating the unbound fraction of test compound in undiluted blood and tissue from the measured fu in the homogenate and diluted blood (f)u，blAnd fu，br). D is the dilution factor.
The short term oral absorption (SOA) model is an in vivo screening model for identifying brain penetration of a compound. Six male wistar rats (university of Yangzhou, comparative medicine center) were dosed orally with 2mg/kg of compound in 1% methylcellulose. After 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours and 7 hours of administration, cerebrospinal fluid (CSF) was collected from the cisterna magna and blood samples (> 60 μ L/time point/each site) were collected via cardiac puncture into individual EDTA coagulation tubes and then immediately diluted with 3 volumes of water. Brain tissue was harvested and homogenized in 3 volumes of phosphate buffered saline (pH 7.4). All samples were stored at about-70 ℃ prior to LC/MS analysis.
Standards were prepared by labeling blank blood, brain homogenate, and artificial CSF from 0.2ng/mL to 500 ng/mL. Homogenized brain tissue along with blood samples were precipitated by adding 3 volumes of cold acetonitrile containing an internal standard (40ng/mL dexamethasone and 40ng/mL diclofenac), and 10 μ L of CSF sample was precipitated with 100 μ L of cold acetonitrile containing an internal standard. After vortexing for 2min and centrifugation at 14,000rpm for 5min, the supernatant was analyzed by LC/MS/MS. Two sets of standard curves were run at the beginning and end of each batch from the analysis of the blood samples. Standard curves were made for brain and CSF samples, along with the test samples.
Following oral administration, the AUC brain/AUC blood measurements in rodents are expressed as the brain/blood ratio (K)pBrain) total brain levels. The free fraction of the test compound in the biological matrix is determined by an in vitro blood and brain binding assay. K is calculated by the following equationp, uu brainAnd Kp，uu CSF∶Kp, uu brain＝AUCBrain/AUCBlood, blood-enriching agent and method for producing the same×(fu, brain/fu. blood) And Kp，uu CSF＝AUCCSF/(AUCBlood, blood-enriching agent and method for producing the same×fu. blood)。
The data determined for some of the examples of the invention and obtained for the reference compound TAK-788 (free base form) are shown in the table below:
TABLE 4 experimental data on brain barrier permeability of examples of the invention and reference compounds
Compound (I) Kp, uu brain Kp，uu CSF
Example 1 0.17 0.38
Example 2 0.16 0.35
Example 3 0.15 0.36
Example 4 0.16 0.40
Example 8 0.17 0.39
Example 9 0.19 0.48
Example 10 0.20 0.49
Example 11 0.17 0.45
Example 12 0.18 0.46
TAK-788 0.14 0.35
When compared with TAK-788, the compound has better brain barrier permeability characteristics and better potential for treating non-small cell lung cancer brain metastasis.
While the invention has been illustrated by the foregoing specific embodiments, it is not to be construed as being limited thereby; but that the present invention encompass the generic aspects previously disclosed. Various modifications and embodiments can be made without departing from the spirit and scope of the invention.

Claims (12)

1. A compound having the general formula (I) or a pharmaceutically acceptable salt thereof,
Figure FDA0003288260980000011
wherein:
R1selected from deuterated methyl, -CH2OR4and-CH2OP(＝O)(XR5)YR6，
Wherein R is4Selected from hydrogen, C1-C4An alkyl group;
wherein X or Y are each independently selected from N or O;
wherein R is5And R6Each independently selected from hydrogen and C1-C4Alkyl radical, C6-C12Aryl, heteroaryl, and heteroaryl,
Figure FDA0003288260980000012
Wherein R is7Selected from hydrogen, C1-C8An alkyl group;
R2is selected from
Figure FDA0003288260980000013
Wherein R is8、R9、R10Each is independently selected from methyl;
R3is selected from C1-C4Alkoxy, halo C1-C4An alkoxy group;
z is selected from N or C;
when R is1Selected from deuterated methyl, the compound of the general formula (I) is selected from the following compounds:
Figure FDA0003288260980000014
when R is1Is selected from-CH2OR4or-CH2OP(＝O)(XR5)YR6When R is in the above-mentioned range2Is selected from
Figure FDA0003288260980000015
2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof,
R4selected from hydrogen, methyl, isopropyl;
R5and R6Each independently selected from hydrogen and C1-C4Alkyl, phenyl,
Figure FDA0003288260980000021
Figure FDA0003288260980000022
3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof,
R3selected from methoxy, ethoxy or trifluoroethoxy.
4. A compound according to any one of claims 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the compound of general formula (I) is selected from the following compounds:
Figure FDA0003288260980000023
5. the compound according to any one of claims 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is an inorganic salt or an organic salt, and the inorganic salt includes hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, bisulfate, nitrate, phosphate, acid phosphate; the organic salt is selected from formate, acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, succinate, glutarate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate, p-toluenesulfonate, ascorbate.
6. A compound according to claim 5, or a pharmaceutically acceptable salt thereof, selected from the hydrochloride, succinate or mesylate salt.
7. A pharmaceutical composition comprising a compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient or diluent.
8. Use of a compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of diseases and conditions associated with kinase activity that is mutated to EGFR and HER2 in a mammal.
9. Use of a compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a cancer associated with kinase activity of the EGFR mutation and the HER2 mutation in a human.
10. The use of claim 9, wherein the cancer comprises lung cancer, colorectal cancer, pancreatic cancer, head and neck cancer, breast cancer, ovarian cancer, uterine cancer, and/or gastric cancer.
11. Use of a compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, in combination with an anti-neoplastic agent selected from the group consisting of:
(i) antineoplastic drugs acting on the DNA structure;
(ii) antineoplastic agents that affect nucleic acid synthesis;
(iii) anti-tumor drugs that affect nucleic acid transcription;
(iv) tubulin synthesized antineoplastic drugs;
(v) inhibitors of cellular signaling pathways;
(vi) an anti-tumor monoclonal antibody.
12. The use of claim 11, wherein the cell signaling pathway inhibitor is an epidermal growth factor receptor inhibitor.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016029839A1 (en) * 2014-08-25 2016-03-03 四川海思科制药有限公司 (substituted phenyl) (substituted pyrimidine) amino derivative, preparation method therefor, and medication use
CN106559991A (en) * 2014-06-19 2017-04-05 阿里亚德医药股份有限公司 For the heteroaryl compound of kinase inhibition
CN110790749A (en) * 2018-08-03 2020-02-14 北京普祺医药科技有限公司 Nitrogen-containing heterocyclic compound, pharmaceutical composition and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106559991A (en) * 2014-06-19 2017-04-05 阿里亚德医药股份有限公司 For the heteroaryl compound of kinase inhibition
WO2016029839A1 (en) * 2014-08-25 2016-03-03 四川海思科制药有限公司 (substituted phenyl) (substituted pyrimidine) amino derivative, preparation method therefor, and medication use
CN110790749A (en) * 2018-08-03 2020-02-14 北京普祺医药科技有限公司 Nitrogen-containing heterocyclic compound, pharmaceutical composition and application thereof

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