EGFR inhibitors resistant to L718 and / or L792 mutations

Abstract

The present invention provides an anti-tumor agent for treating a patient with a malignant tumor expressing EGFR, wherein the EGFR has at least one mutation selected from the group consisting of an L718X mutation in exon 18 and an L792X mutation in exon 20, wherein X represents any amino acid residue, and the anti-tumor agent comprises (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizine-8-yl)acrylamide (compound (A)) or a salt thereof.

Description

Technical Field

[0001] The present invention relates to an antitumor agent against cancer comprising a mutant epidermal growth factor receptor (hereinafter referred to as "EGFR") that is resistant to exon 18 and / or exon 20 treatment. Background Art

[0002] EGFR is a receptor-type tyrosine kinase that exerts its physiological function in normal tissues by binding to epidermal growth factor (hereinafter referred to as EGF) as a ligand, and is involved in growth and apoptosis in epidermal tissues (NPL 1). In addition, somatic mutations of the EGFR gene are considered to be an oncogene; for example, EGFRs that lack codons 746-750 in exon 19 (hereinafter also referred to as "exon 19 deletion mutation") and EGFRs in which leucine encoded by codon 858 in exon 21 is mutated to arginine (hereinafter also referred to as "L858R mutation") constantly induce EGF-independent kinase activity and are involved in the growth and survival of cancer cells (NPL 2). For example, these mutations are observed in 30-50% of non-small cell lung cancers in East Asia. This mutation is also observed in about 10% of non-small cell lung cancers in Europe and the United States, and is considered to be one of the causes of cancer (NPL 3).

[0003] Therefore, the research and development of EGFR inhibitors as antitumor agents has been actively carried out, and it has been introduced into the treatment of various EGFR mutation-positive lung cancers (NPL 2 and NPL 4). Gefitinib, erlotinib and afatinib have been used as therapeutic agents for exon 19 deletion mutation and L858R mutation EGFR-positive lung cancer. Exon 19 deletion mutation and L858R mutation account for 90% of EGFR mutations. In addition, acquired drug resistance occurs during the treatment process using these agents, and 50% of them are caused by drug-resistant mutation EGFR in which codon 790 in exon 20 is changed from threonine to methionine (hereinafter also referred to as "T790M mutation"). For the treatment of lung cancer with this mutation, osimertinib has been used as a therapeutic agent. Therefore, for lung cancer patients with major EGFR mutations, the treatment using EGFR inhibitors is being established.

[0004] Reference file list

[0005] Patent Literature

[0006] PTL 1: WO2015 / 175632A1

[0007] PTL 2: WO2015 / 025936A1

[0008] Non-patent literature

[0009] NPL 1: Nat.Rev.Cancer, vol.6, pp.803-812 (2006)

[0010] NPL 2: Nature Medicine, vol.19, pp.1389-1400(2013)

[0011] NPL 3: Nat.Rev.Cancer, vol.7, pp.169-181(2007)

[0012] NPL 4: Clin.Cancer Res., vol.24, pp.3097-3107(2018) Summary of the invention

[0013] Technical issues

[0014] For lung cancer patients with major EGFR mutations, the treatment using EGFR inhibitors is being established. In contrast, the existence of patients with resistance to osimertinib treatment is also known. It is reported that some of its causes are EGFR gene mutations (NPL 4). For example, it has been confirmed that, except for exon 19 deletion mutations or L858R mutations (both are de novo mutations) and T790M mutations (which are acquired resistance mutations), lung cancer with the following mutations is also resistant to osimertinib treatment: the leucine encoded by codon 718 in exon 18 is replaced by a point mutation of any amino acid (hereinafter also referred to as "L718X mutation"), or the leucine encoded by codon 792 in exon 20 is replaced by a point mutation of any amino acid (hereinafter also referred to as "L792X mutation"). It has been suggested that amino acid substitutions caused by L718X mutation or L792X mutation induce steric hindrance and reduced hydrophobic binding, thereby preventing EGFR from binding to osimertinib (NPL4). Therefore, it is necessary to develop inhibitors with high inhibitory activity against EGFR with compound mutations of de novo active mutations, T790M acquired resistance mutations, and L718X mutation or L792X mutation.

[0015] Therefore, it is believed that the development of a drug with high inhibitory activity against EGFR with a compound mutation containing L718X or L792X can make it show an antitumor effect for lung cancer resistant to osimertinib treatment, and it is expected to help prolong the life and improve the quality of life of mutant EGFR-positive cancer patients for whom no therapy has been established. In addition, a drug with high inhibitory activity against T790M (which is an acquired resistance mutation for treatment with EGFR inhibitors) is expected to reduce the frequency of expression of acquired resistance during treatment with EGFR inhibitors for exon 19 or exon 21 mutant EGFR (which is a de novo mutation); therefore, it is expected to help prolong the life of cancer patients.

[0016] Under such circumstances, an object of the present invention is to provide an inhibitor having high inhibitory activity against L718 and L792 mutant EGFR, which are mutations resistant to osimertinib treatment and against which previously known EGFR inhibitors have insufficient therapeutic effects.

[0017] Solution to the problem

[0018] The inventors of the present invention have conducted in-depth research and found that the conventional EGFR inhibitors introduced for treatment have poor inhibitory activity for compound mutations including L718 and L792 mutations in addition to active mutations and T790M acquired resistance mutations. In addition, the inventors have conducted in-depth research starting from the co-crystal structure of the comparative compound, and have also found that the compounds of the present invention exhibit excellent known activity for EGFR with L718 and L792 mutations, and further show excellent inhibitory activity against the above compound mutant EGFR. Due to this discovery, the inventors have completed the present invention.

[0019] The present invention encompasses the following embodiments.

[0020] Item 1. An antitumor agent for treating patients with malignant tumors expressing EGFR, wherein the EGFR has at least one mutation selected from the following group: L718X mutation in exon 18 and L792X mutation in exon 20, wherein X represents any amino acid residue, and the antitumor agent comprises (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizine-8-yl)acrylamide (compound (A)) or a salt thereof.

[0021] Item 2. An anti-tumor agent according to Item 1, wherein the EGFR also has at least one mutation selected from the group consisting of exon 19 deletion mutation, L858R, L861Q, G719X, E709X, and exon 20 insertion mutation.

[0022] Item 3. An anti-tumor agent according to Item 2, wherein the EGFR also has a T790M mutation.

[0023] Item 4. An antitumor agent according to any one of Items 1 to 3, wherein the L718X mutation is a L718Q mutation.

[0024] Item 5. An antitumor agent according to any one of Items 1 to 4, wherein the L792X mutation is L792H, L792F, or L792Y.

[0025] Item 6. A method for treating patients with malignant tumors, comprising the following steps: administering (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizine-8-yl)acrylamide or a salt thereof to patients with malignant tumors expressing EGFR, wherein the EGFR has at least one mutation selected from the following group: an L718X mutation in exon 18 and an L792X mutation in exon 20.

[0026] Item 7. (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizine-8-yl)acrylamide or its salt is used to treat patients with malignant tumors expressing EGFR, wherein the EGFR has at least one mutation selected from the following group: L718X mutation in exon 18 and L792X mutation in exon 20.

[0027] Item 8. Use of (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizine-8-yl)acrylamide or its salt for treating patients with malignant tumors expressing EGFR, wherein the EGFR has at least one mutation selected from the following group: L718X mutation in exon 18 and L792X mutation in exon 20.

[0028] Item 9. Use of (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizine-8-yl)acrylamide or its salt for the preparation of a medicament for treating patients with malignant tumors expressing EGFR, wherein the EGFR has at least one mutation selected from the following group: L718X mutation in exon 18 and L792X mutation in exon 20.

[0029] Item 10. A pharmaceutical composition comprising: (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizine-8-yl)acrylamide or a salt thereof and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is used to treat patients with malignant tumors expressing EGFR, wherein the EGFR has at least one mutation selected from the following group: L718X mutation in exon 18 and L792X mutation in exon 20, wherein X represents any amino acid residue.

[0030] Item 11. A method for predicting the therapeutic effect of chemotherapy in patients with malignant tumors using an antitumor agent comprising (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof as an active ingredient, the method comprising the following steps (1) and (2):

[0031] (1) a step of detecting whether a mutation exists in the EGFR gene contained in a biological sample obtained from a patient; and

[0032] (2) When the detection result in step (1) finds that the EGFR gene has at least one mutation selected from the group consisting of: L718X mutation in exon 18 and L792X mutation in exon 20, wherein X represents any amino acid residue, a step of predicting that chemotherapy is very likely to show sufficient therapeutic effect on the patient.

[0033] Item 12. A method for treating a patient with a malignant tumor, comprising the following steps (1) to (3):

[0034] (1) a step of detecting whether there is a mutation in the EGFR gene contained in a biological sample obtained from a patient;

[0035] (2) when the detection result in step (1) finds that the EGFR gene has at least one mutation selected from the group consisting of: L718X mutation in exon 18 and L792X mutation in exon 20, wherein X represents any amino acid residue, a step of predicting that chemotherapy using a chemotherapeutic agent comprising (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof as an active ingredient is highly likely to show a sufficient therapeutic effect on the patient;

[0036] (3) A step of administering (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof to a patient with a malignant tumor who has been predicted in step (2) to be highly likely to respond adequately to chemotherapy using an antitumor agent comprising (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof as an active ingredient.

[0037] Advantageous Effects of the Invention

[0038] The antitumor agent of the present invention exerts high inhibitory activity against EGFR having L718 and L792 mutations. Therefore, the antitumor agent of the present invention can be used to provide an antitumor agent having excellent therapeutic effects on patients with malignant tumors expressing EGFR having L718 and L792 mutations.

[0039] The present invention is also intended to provide methods for treating patients with malignancies expressing EGFR having L718 and L792 mutations.

[0040] In the presence of T790M mutation (which is an acquired resistance mutation in the exon 20 region) or in the presence of L718 and L792 mutations, the EGFR inhibitory activity of conventional EGFR inhibitors is significantly reduced. Therefore, it is difficult to exert a sufficient therapeutic effect. In contrast, the anti-tumor agent of the present invention has high inhibitory activity against EGFRs with L718 and / or L792 mutations and with active mutations (e.g., Ex19del or L858R mutations); therefore, the anti-tumor agent of the present invention can exert excellent therapeutic effects on patients with malignant tumors expressing EGFRs with these complex mutations. For EGFRs with L718 and / or L792 mutations, even if they have T790M mutations in addition to the above mutations, the anti-tumor agent of the present invention has high inhibitory activity; therefore, the anti-tumor agent of the present invention can exert excellent therapeutic effects on patients with malignant tumors expressing EGFRs with these mutations.

[0041] In addition, the antitumor agent of the present invention can also be used to reduce the frequency of expression of acquired resistance during treatment with an EGFR inhibitor against exon 18 or exon 21 mutant EGFR, which is a de novo mutation, because it has high inhibitory activity against mutant EGFR resistant to exon 18 and exon 20 treatment even in the presence of T790M mutation, which is an acquired resistance mutation in the exon 20 region. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1Shown are the results of evaluating phosphorylation (inhibitory activity) in a mutant EGFR forced expression system using HEK293 cells. DETAILED DESCRIPTION

[0043] Preferred examples of various definitions within the scope of the invention used in the present specification are explained in detail below.

[0044] In this specification, "EGFR" refers to human epidermal growth factor receptor protein, also known as ErbB-1 or HER1.

[0045] In the present specification, "wild-type EGFR" refers to EGFR without somatic mutation, which is a protein comprising the amino acid sequence shown in SEQ ID NO: 1 (GenBank Accession No.: NP_005219.2).

[0046] In the present specification, "exon 18" refers to the region from position 688 to position 728 in the amino acid sequence of wild-type EGFR (SEQ ID NO: 1).

[0047] In the present specification, "mutation resistant to exon 18 treatment" refers to a point mutation or a deletion mutation in an amino acid in the exon 18 region of wild-type EGFR (SEQ ID NO: 1). A preferred mutation resistant to exon 18 treatment is a point mutation in which one amino acid is substituted in the exon 18 region. More preferably, the mutation resistant to exon 18 treatment is L718X (X represents any amino acid residue other than leucine among the amino acids constituting the protein encoded by genetic information), which is a point mutation in which leucine encoded by codon 718 of exon 18 is replaced by any amino acid. More specifically, preferred examples of L718X include L718Q, which is a point mutation in which leucine encoded by codon 718 of exon 18 is replaced by glutamine; and L718V, which is a point mutation in which leucine encoded by codon 718 is replaced by valine.

[0048] In the present specification, "exon 18 active mutation" refers to a point mutation or a deletion mutation in an amino acid in the exon 18 region of the wild-type EGFR (SEQ ID NO: 1). A preferred exon 18 active mutation is a point mutation in which one amino acid is substituted in the exon 18 region. More preferably, the exon 18 active mutation is E709X, which is a point mutation that replaces glutamic acid encoded by codon 709 of exon 18 with any amino acid; or G719X, which is a point mutation that replaces glycine encoded by codon 719 of exon 18 with any amino acid. More specifically, preferred examples of E709X include E709K, which is a point mutation that replaces glutamic acid encoded by codon 709 in the exon 18 region with lysine; and E709A, which is a point mutation that replaces glycine encoded by codon 709 in the exon 18 region with alanine. Preferred examples of G719X include G719A, which is a point mutation that replaces glycine encoded by codon 719 in the exon 18 region with alanine; G719S, which is a point mutation that replaces glycine encoded by codon 719 in the exon 18 region with serine; and G719C, which is a point mutation that replaces glycine encoded by codon 719 in the exon 18 region with cysteine.

[0049] In the present invention, "exon 20" refers to the region from position 824 to position 875 in the amino acid sequence of wild-type EGFR (SEQ ID NO: 1).

[0050] In the present specification, "mutation resistant to exon 20 treatment" refers to a point mutation in the amino acid in the exon 20 region of wild-type EGFR (SEQ ID NO: 1). A preferred mutation resistant to exon 20 treatment is a point mutation with one amino acid substitution in the exon 20 region. More preferably, the mutation resistant to exon 20 treatment is L792 (X represents any amino acid residue other than leucine among the amino acids constituting the protein encoded by genetic information), which is a point mutation that replaces leucine encoded by codon 792 in exon 20 with any amino acid. Specific examples include L792H, which is a point mutation that replaces leucine encoded by codon 792 in the exon 20 region with histidine; L792F, which is a point mutation that replaces leucine encoded by codon 792 with phenylalanine; L792Y, which is a point mutation that replaces leucine encoded by codon 792 with tyrosine; L792R, which is a point mutation that replaces leucine encoded by codon 792 with arginine; L792V, which is a point mutation that replaces leucine encoded by codon 792 with valine; and L792P, which is a point mutation that replaces leucine encoded by codon 792 with proline. Among these, L792F, L792H and L792Y are preferred.

[0051] In the present specification, "exon 20 insertion mutation" refers to the insertion of one or more (preferably 1-7, more preferably 1-4) amino acids into the 824-875 region of the amino acid sequence of the exon 20 region of EGFR (SEQ ID NO: 1). Preferred examples include a mutation in which the amino acid sequence FQEA (phenylalanine, glutamine, glutamic acid and alanine, in order from the N-terminal side) is inserted between the 763rd alanine and the 764th tyrosine in the exon 20 region (A763_Y764insFQEA), a mutation in which the amino acid sequence ASV (alanine, serine and valine, in order from the N-terminal side) is inserted between the 769th valine and the 770th aspartic acid in the exon 20 region (V769_D770insASV), a mutation in which the amino acid sequence SVD (serine, valine and aspartic acid ... The mutations included insertion of amino acid (A) into aspartic acid 770 and asparagine 771 in the exon 20 region (D770_N771insSVD), insertion of amino acid sequence NPG (asparagine, proline, and glycine, in order from the N-terminal side) into aspartic acid 770 and asparagine 771 in the exon 20 region (D770_N771insNPG), insertion of amino acid G (glycine) into aspartic acid 770 and asparagine 771 in the exon 20 region (D770_N771insG), deletion of amino acid (A) into aspartic acid 770 and asparagine 771 in the exon 20 region (D770_N771insSVD), insertion of amino acid sequence NPG (asparagine, proline, and glycine, in order from the N-terminal side) into aspartic acid 770 and asparagine 771 in the exon 20 region (D770_N771insNPG), insertion of amino acid G (glycine) into aspartic acid 770 and asparagine 771 in the exon 20 region (D770_N771ins The mutations included insertion of the amino acid sequence GY (glycine and tyrosine in order from the N-terminal side) after aspartic acid at position 770 (D770>GY), insertion of the amino acid N (asparagine) between asparagine at position 771 and proline at position 772 in the exon 20 region (N771_P772insN), insertion of the amino acid sequence PR (proline and arginine in order from the N-terminal side) between proline at position 772 and histidine at position 773 in the exon 20 region (P772_R773insPR), insertion of the amino acid sequence NPH (asparag ... , proline and histidine) are inserted between the 773rd histidine and the 774th valine in the exon 20 region (H773_V774insNPH), the amino acid sequence PH (proline and histidine in sequence from the N-terminal side) is inserted between the 773rd histidine and the 774th valine in the exon 20 region (H773_V774insPH), the amino acid sequence AH (alanine and histidine in sequence from the N-terminal side) is inserted between the 773rd histidine and the 774th valine in the exon 20 region (H773_V774insAH),The amino acid H (histidine) is inserted into the mutation between the 773rd histidine and the 774th valine in the exon 20 region (H773_V774insH), the amino acid sequence HV (histidine and valine in sequence from the N-terminal side) is inserted into the mutation between the 774th valine and the 775th cysteine ​​in the exon 20 region (V774_C774insHV), the amino acid sequence EAFQ (glutamic acid, alanine, phenylalanine and glutamine in sequence from the N-terminal side) is inserted into the mutation between the 761st alanine and the 762nd glutamate in the exon 20 region (A761_E762insEAFQ), etc. More preferred examples include a mutation in which the amino acid sequence ASV (alanine, serine, and valine in order from the N-terminal side) is inserted between the 769th valine and the 770th aspartic acid in the exon 20 region (V769_D770insASV), a mutation in which the amino acid sequence SVD (serine, valine, and aspartic acid in order from the N-terminal side) is inserted between the 770th aspartic acid and the 771st asparagine in the exon 20 region (D770_N771insSVD), and a mutation in which the amino acid G The mutations were as follows: a mutation in which the amino acid sequence NPH (asparagine, proline, and histidine, in that order from the N-terminal side) was inserted between aspartic acid 770 and asparagine 771 in the exon 20 region (D770_N771insG), a mutation in which the amino acid sequence NPH (asparagine, proline, and histidine, in that order from the N-terminal side) was inserted between histidine 773 and valine 774 in the exon 20 region (H773_V774insNPH), and a mutation in which the amino acid sequence PH (proline and histidine, in that order from the N-terminal side) was inserted between aspartic acid 770 and asparagine 771 in the exon 20 region (D770_N771insG). Particularly preferred examples include a mutation in which the amino acid sequence FQEA (phenylalanine, glutamine, glutamic acid, and alanine, in order from the N-terminal side) is inserted between alanine at position 763 and tyrosine at position 764 in the exon 20 region (A763_Y764insFQEA), an amino acid sequence ASV (alanine, serine, and valine, in order from the N-terminal side) is inserted between alanine at position 763 and tyrosine at position 764 in the exon 20 region (A763_Y764insFQEA), and an amino acid sequence ASV (alanine, serine, and valine, in order from the N-terminal side) is inserted between alanine at position 769 and tyrosine at position 764 in the exon 20 region (A763_Y764insFQEA). a mutation in which the amino acid sequence SVD (serine, valine, and aspartic acid in order from the N-terminal side) is inserted between aspartic acid at position 770 and asparagine at position 771 in the exon 20 region (D770_N771insSVD); a mutation in which the amino acid G (glycine) is inserted between aspartic acid at position 770 and asparagine at position 771 in the exon 20 region (D770_N771insG);The amino acid sequence NPH (asparagine, proline, and histidine in order from the N-terminal side) is inserted into the mutation between the 773rd histidine and the 774th valine in the exon 20 region (H773_V774inchsNPH), and the amino acid sequence PH (proline and histidine in order from the N-terminal side) is inserted into the mutation between the 773rd histidine and the 774th valine in the exon 20 region (H773_V774insPH), etc.,

[0052] In the present invention, "exon 21" refers to the region from position 824 to position 875 in the amino acid sequence of wild-type EGFR (SEQ ID NO: 1).

[0053] In the present specification, "exon 21 active mutation" refers to a point mutation in an amino acid in exon 21 of wild-type EGFR (SEQ ID NO: 1). A preferred exon 21 active mutation is a point mutation in which one amino acid is substituted in the exon 21 region. More preferably, the exon 21 active mutation is L858X, which is a point mutation in which leucine encoded by codon 858 in the exon 21 region is substituted with any amino acid; or L861X, which is a point mutation in which leucine encoded by codon 864 in the exon 21 region is substituted with any amino acid (X represents any amino acid residue other than leucine in the amino acids constituting the protein encoded by the genetic information). More specifically, preferred examples include L858R, which is a point mutation in which leucine encoded by codon 858 in the exon 21 region is mutated to arginine; and L861Q, which is a point mutation in which leucine encoded by codon 864 in the exon 21 region is substituted with glutamine.

[0054] In the present invention, “mutations resistant to treatment with exon 18 and / or exon 20” encompass “mutations resistant to treatment with exon 18”, “mutations resistant to treatment with exon 20” and “mutations resistant to treatment with exon 18 and exon 20”.

[0055] In the present invention, "point mutation" refers to a mutation that causes substitution, insertion or deletion of one or more (e.g., about 1-10, preferably about 1-5, more preferably about 1, 2 or 3) amino acid residues; and may include in-frame insertions and / or deletions of nucleic acids.

[0056] “EGFR having a mutation that confers resistance to treatment with exon 18 and / or exon 20” encompasses “EGFR having a mutation that confers resistance to treatment with exon 18,” “EGFR having a mutation that confers resistance to treatment with exon 20,” and “EGFR having a mutation that confers resistance to treatment with exon 18 and exon 21.”

[0057] In the present specification, "EGFR having a mutation resistant to exon 18 therapy" refers to an EGFR having at least the L718X mutation in exon 18 as a mutation resistant to exon 18 therapy. As a mutation resistant to exon 18 therapy, EGFR may also have exon 18 mutations other than L718X, but preferably has a single L718X mutation. In addition, EGFR may have mutations other than mutations resistant to exon 18 therapy (e.g., exon 19 deletion mutations, L858R mutations, and L790M mutations).

[0058] In the present specification, "EGFR having a mutation resistant to exon 20 therapy" refers to an EGFR having at least the L792X mutation in exon 20 as a mutation resistant to exon 20 therapy. As a mutation resistant to exon 20 therapy, EGFR may have mutations other than the L792X mutation, but preferably has a single L718X mutation. In addition, EGFR may have mutations other than mutations resistant to exon 20 therapy (e.g., exon 19 deletion mutation, L858R mutation, and L790M mutation).

[0059] In the present specification, "exon 19" refers to the region from positions 729 to 823 in the amino acid sequence of wild-type EGFR (SEQ ID NO: 1).

[0060] In the present specification, "exon 19 deletion mutation" refers to a mutation in which one or more amino acids are deleted in the exon 19 region of the wild-type EGFR (SEQ ID NO: 1). In addition to deletions in this region, mutations in which one or more arbitrary amino acids are inserted are also included. Examples of exon 19 deletion mutations include a mutation in which 5 amino acids from glutamic acid at position 746 to alanine at position 750 are deleted in the exon 19 region (Del E746-A750), a mutation in which serine is inserted after 7 amino acids from leucine at position 747 to proline at position 753 are deleted in the exon 19 region (Del 747-P753insS), a mutation in which 5 amino acids from leucine at position 747 to threonine at position 751 are deleted in the exon 19 region (Del L747-T751), a mutation in which proline is inserted after 4 amino acids from leucine at position 747 to alanine at position 750 are deleted in the exon 19 region (Del 747-A750insP), and the like. Preferred examples include a mutation that deletes 5 amino acids from glutamic acid at position 746 to alanine at position 750 in the exon 19 region (Del E746-A750).

[0061] In addition, the EGFR having a mutation resistant to exon 18 and / or exon 20 treatment may also have at least one mutation selected from the group consisting of an exon 19 deletion mutation, L858R, L861Q, G719X, E709X, and an exon 20 insertion mutation. Specific examples include an EGFR having an L718X mutation in the exon 18 region and also having an exon 19 deletion mutation, an EGFR having an L792X mutation in the exon 20 region and also having an exon 19 deletion mutation, an EGFR having an L718X mutation in the exon 18 region and also having an L858R, an EGFR having an L792X mutation in the exon 20 region and also having an L858R, an EGFR having an L718X mutation in the exon 18 region and also having an L861Q, an EGFR having an L792X mutation in the exon 20 region and also having an L861Q. FR, EGFR having an L718X mutation in the exon 18 region and also having a G719X, EGFR having an L792X mutation in the exon 20 region and also having a G719X, EGFR having an L718X mutation in the exon 18 region and also having an E709X, EGFR having an L792X mutation in the exon 20 region and also having an E709X, EGFR having an L718X mutation in the exon 18 region and also having an exon 20 insertion mutation, and EGFR having an L792X mutation in the exon 20 region and also having an exon 20 insertion mutation. Preferred among these are EGFR having an L718X mutation in the exon 18 region and also having an exon 19 deletion mutation, EGFR having an L792X mutation in the exon 20 region and also having an exon 19 deletion mutation, EGFR having an L718X mutation in the exon 18 region and also having L858R, and EGFR having an L792X mutation in the exon 20 region and also having L858R.

[0062] In addition to the above-mentioned exon 19 deletion mutation, L858R, L861Q, G719X, E709X and exon 20 insertion mutation, the EGFR with a mutation that is resistant to exon 18 and / or exon 20 treatment may also have a T790M mutation. T790M is an acquired drug resistance mutation in the exon 20 region. It is known that T790M is produced by the use of existing EGFR inhibitors. The acquisition of T790M often reduces the effect of existing drugs on patients with malignant tumors.

[0063] In the present invention, examples include EGFR having an L718X mutation in the exon 18 region, an exon 19 deletion mutation, and a T790M mutation, EGFR having an L792X mutation in the exon 20 region, an exon 19 deletion mutation, and a T790M mutation, EGFR having an L718X mutation in the exon 18 region, an L858R mutation, and a T790M mutation, EGFR having an L792X mutation in the exon 20 region, an L858R mutation, and a T790M mutation, EGFR having an L718X mutation in the exon 18 region, an L861Q mutation, and a T790M mutation, EGFR having an L792X mutation in the exon 20 region, an L861Q mutation, and a T790M mutation EGFR with an L718X mutation in the exon 18 region, a G719X mutation, and a T790M mutation, EGFR with an L792X mutation in the exon 20 region, a G719X mutation, and a T790M mutation, EGFR with an L718X mutation in the exon 18 region, an E709X mutation, and a T790M mutation, EGFR with an L792X mutation in the exon 20 region, an E709X mutation, and a T790M mutation, EGFR with an L718X mutation in the exon 18 region, an exon 20 insertion mutation, and a T790M mutation, and EGFR with an L792X mutation in the exon 20 region, an exon 20 insertion mutation, and a T790M mutation. Preferred among these are EGFR having an L718X mutation in the exon 18 region, an exon 19 deletion mutation, and a T790M mutation, EGFR having an L792X mutation in the exon 20 region, an exon 19 deletion mutation, and a T790M mutation, EGFR having an L718X mutation in the exon 18 region, an L858R, and a T790M mutation, and EGFR having an L792X mutation in the exon 20 region, an L858R, and a T790M mutation.

[0064] Among the EGFRs having the above compound mutations, EGFR having L718Q mutation in the exon 18 region, EGFR having L792F mutation in the exon 20 region, EGFR having L792H mutation, and EGFR having L792Y mutation are particularly preferred.

[0065] In the present invention, the method for detecting a mutation in EGFR expressed by a malignant tumor patient is not particularly limited as long as the method is capable of detecting a mutation, and any known detection method may be used.

[0066] The sample for detecting EGFR mutation is not particularly limited, as long as the sample is a biological sample isolated from a malignant tumor patient, in particular a sample obtained from a malignant tumor patient and containing malignant tumor cells. Examples of biological samples include body fluids (e.g., blood, urine, etc.), tissues, extracts thereof, and cultures of the resulting tissues. Depending on the type of biological sample, the method for obtaining the biological sample can be appropriately selected.

[0067] The biological sample is prepared by appropriately treating the biological sample according to the measurement method. In addition, a reagent containing a primer or a probe for detection can be prepared by a conventional method according to the measurement method thereof.

[0068] In one embodiment of the present invention, the step of detecting the presence of the mutation of the present invention in EGFR expressed by a malignant tumor patient may be performed before administering an anti-tumor agent to the malignant tumor patient.

[0069] Malignant tumors can include two or more different types of malignant tumor cells. In addition, two or more malignant tumors can occur in a single patient. Therefore, a single patient can have different EGFR mutations at the same time (for example, exon 18 mutations are L718Q and L718V exon 18 mutations; exon 20 mutations are L792F, L792H, L792Y, L792R, L792V and L792P exon 20 mutations; however, it is not limited).

[0070] The antitumor agent of the present invention comprises (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide (compound (A)) or a salt thereof as an active ingredient. Compound (A) is represented by the following chemical formula.

[0071] [Chemical formula 1]

[0072]

[0073] Hereinafter, the process for preparing the compound of the present invention is explained.

[0074] Compound A of the present invention can be produced by the production method disclosed in WO2015 / 025936A1, the method described in Examples, etc. However, the production method of the compound of the present invention is not limited to these reaction examples.

[0075] When the compound A of the present invention has isomers such as optical isomers, stereoisomers and tautomers, unless otherwise specified, any isomers and mixtures thereof are included in the scope of the compound of the present invention. For example, when the compound of the present invention has optical isomers, unless otherwise specified, racemic mixtures and optical isomers separated from the racemic mixtures are also included in the scope of the compound of the present invention.

[0076] The salt of compound A refers to any pharmaceutically acceptable salt; examples include base addition salts and acid addition salts.

[0077] Examples of base addition salts include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; ammonium salts; and organic amine salts such as trimethylamine salt, triethylamine salt, dicyclohexylamine salt, ethanolamine salt, diethanolamine salt, triethanolamine salt, procaine salt and N,N-dibenzylethylenediamine salt.

[0078] Examples of acid addition salts include inorganic acid salts such as hydrochlorides, sulfates, nitrates, phosphates and perchlorates; organic acid salts such as acetates, formates, maleates, fumarates, tartrates, citrates, ascorbates and trifluoroacetates; and sulfonates such as methanesulfonates, isothiocyanates, benzenesulfonates and p-toluenesulfonates.

[0079] The compounds of the present invention or their salts also include their prodrugs. Prodrugs refer to compounds that can be converted into compounds of the present invention or their salts by reaction with enzymes, gastric acid, etc. under physiological conditions in vivo, that is, compounds that can be converted into compounds of the present invention or their salts by enzymatic oxidation, hydrolysis, reduction, etc.; or compounds that can be converted into compounds of the present invention or their salts by hydrolysis with gastric acid, etc. or similar methods. In addition, prodrugs can be "compounds that can be converted into compounds of the present invention or their salts under physiological conditions", such as those described in: "Iyakuhin no Kaihatsu [Development of Pharmaceuticals]," Vol. 7, Molecular Design, published in 1990 by Hirokawa Shoten Co., pp. 163-198.

[0080] Disease Description

[0081] Specific examples of tumors targeted in the present invention include, but are not particularly limited to, head and neck cancer, gastrointestinal cancer (esophageal cancer, gastric cancer, duodenal cancer, liver cancer, bile duct cancer (e.g., gallbladder cancer and bile duct cancer), pancreatic cancer, colorectal cancer (e.g., colon cancer and rectal cancer), etc.), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer and mesothelioma), breast cancer, reproductive tract cancer (ovarian cancer, uterine cancer (e.g., cervical cancer and endometrial cancer), etc.), urinary system cancer (e.g., kidney cancer, bladder cancer, prostate cancer and testicular tumor), hematopoietic system tumor (e.g., leukemia, malignant lymphoma and multiple myeloma), osteosarcoma, soft tissue sarcoma, skin cancer, brain tumor, etc. Preferred examples include lung cancer, breast cancer, head and neck cancer, brain tumor, uterine cancer, gastrointestinal cancer, hematopoietic tumor and skin cancer. Lung cancer is particularly preferred.

[0082] When the compound of the present invention or its salt is used as a pharmaceutical agent, a pharmaceutical carrier may be added if necessary to form a suitable dosage form according to the purpose of prevention and treatment. Examples of dosage forms include oral preparations, injections, suppositories, ointments, patches, etc. Oral preparations are preferred. Such dosage forms can be formed by conventional methods known to those skilled in the art.

[0083] As pharmaceutically acceptable carriers, various conventional organic or inorganic carrier materials used as preparation materials can be blended into solid preparations as excipients, binders, disintegrants, lubricants or colorants; or blended into liquid preparations as solvents, solubilizers, suspending agents, isotonic agents, buffers or soothing agents. In addition, if necessary, pharmaceutical preparation additives such as preservatives, antioxidants, colorants, sweeteners and stabilizers can also be used.

[0084] Oral solid preparations are prepared as follows: After adding excipients, optionally together with excipients, binders, disintegrants, lubricants, colorants, taste-masking agents or flavoring agents, etc. to the compound of the present invention, the resulting mixture is formulated into tablets, coated tablets, granules, powders, capsules, etc. by a common method.

[0085] Examples of excipients include lactose, sucrose, D-mannitol, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose and silicic anhydride. Examples of binders include water, ethanol, 1-propanol, 2-propanol, simple syrup, liquid glucose, liquid α-starch, liquid gelatin, D-mannitol, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shellac, calcium phosphate, polyvinyl pyrrolidone, etc. Examples of disintegrants include dry starch, sodium alginate, powdered agar, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, lactose, etc. Examples of lubricants include purified talc, sodium stearate, magnesium stearate, borax, polyethylene glycol, etc. Examples of colorants include titanium oxide, iron oxide, etc. Examples of taste masking agents or flavoring agents include sucrose, bitter orange peel, citric acid, tartaric acid, etc.

[0086] When preparing liquid preparations for oral administration, masking agents, buffers, stabilizers, flavoring agents, etc. may be added to the compounds of the present invention; the resulting mixture may be formulated into oral liquid preparations, syrups, elixirs, etc. according to conventional methods.

[0087] When preparing injections, pH adjusters, buffers, stabilizers, isotonic agents, local anesthetics, etc. may be added to the compounds of the present invention; the mixture may be formulated into subcutaneous, intramuscular or intravenous injections according to common methods.

[0088] Examples of pH regulators and buffers used herein include sodium citrate, sodium acetate, and sodium phosphate. Examples of stabilizers include sodium pyrosulfite, EDTA, thioglycolic acid, and thiolactic acid. Examples of local anesthetics include procaine hydrochloride and lidocaine hydrochloride. Examples of isotonic agents include sodium chloride, glucose, D-mannitol, and glycerol.

[0089] When preparing suppositories, pharmaceutically acceptable carriers known in the relevant art, such as polyethylene glycol, lanolin, cocoa butter and fatty acid triglycerides, may be added to Compound A, and if necessary, a surfactant such as Tween 80 (registered trademark) may be added to Compound A, and the resulting mixture may be formulated into suppositories according to a common method.

[0090] When preparing an ointment, a commonly used base, stabilizer, wetting agent, preservative, etc. may be incorporated into compound A if necessary; and the resulting mixture is mixed and formulated into an ointment according to a commonly used method.

[0091] Examples of the base include liquid paraffin, white petrolatum, white beeswax, octyldodecanol and paraffin.

[0092] Examples of preservatives include methylparaben, ethylparaben, and propylparaben.

[0093] When a patch is prepared, the ointment, cream, gel, paste or the like as described above can be applied to a conventional base material according to a conventional method.

[0094] Examples of the substrate include woven fabrics or nonwoven fabrics containing cotton, staple fibers or chemical fibers; films or foam sheets of soft vinyl chloride, polyethylene, polyurethane or the like may also be used.

[0095] The amount of compound A to be added to each such unit dosage form depends on the condition of the patient to whom the compound is administered, its dosage form, etc. In general, in the case of an oral agent, the amount of the compound is preferably 0.05 mg to 1000 mg per unit dosage form. In the case of an injection, the amount of the compound is preferably 0.01 mg to 500 mg per unit dosage form; in the case of a suppository, the amount of the compound is preferably 1 to 1000 mg per unit dosage form.

[0096] In addition, the daily dose of the drug of such dosage form depends on the patient's condition, body weight, age, sex, etc., and cannot be generalized. Usually, the daily dose of the compound of the present invention for an adult (body weight: 50 Kg) can usually be 0.05 to 5000 mg, preferably 0.1 to 1000 mg; and preferably one dose per day, or two to three divided doses.

[0097] The present invention also provides a method for treating a patient with a malignant tumor, comprising the step of administering Compound A or a salt thereof to a patient with a malignant tumor expressing EGFR having a mutation that is resistant to treatment with Exon 18 and / or Exon 20.

[0098] The present invention also provides Compound A or a salt thereof for use in treating a patient with a malignant tumor expressing EGFR having a mutation that is resistant to treatment with exon 18 and / or exon 20.

[0099] The present invention also provides use of Compound A or a salt thereof for treating a patient with a malignant tumor expressing EGFR having a mutation that is resistant to treatment with exon 18 and / or exon 20.

[0100] The present invention also provides the use of compound A or a salt thereof for preparing a pharmaceutical agent for treating a patient with a malignant tumor expressing EGFR having a mutation that is resistant to treatment with exon 18 and / or exon 20.

[0101] The present invention also provides a method for predicting the therapeutic effect of chemotherapy using an anti-tumor agent comprising Compound A or a salt thereof as an active ingredient in a patient with a malignant tumor, the method comprising the following steps (1) and (2):

[0102] (1) a step of detecting whether a mutation exists in the EGFR gene contained in a biological sample obtained from a patient; and

[0103] (2) when the test results in step (1) reveal that the EGFR gene has a mutation that is resistant to treatment with exon 18 and / or exon 20, a step of predicting that the chemotherapy is very likely to show a sufficient therapeutic effect on the patient;

[0104] The present invention also provides a method for treating a patient with a malignant tumor, comprising the following steps (1) to (3):

[0105] (1) a step of detecting whether a mutation exists in the EGFR gene contained in a biological sample obtained from a patient; and

[0106] (2) when the detection result in step (1) finds that the EGFR gene has a mutation that confers resistance to treatment with exon 18 and / or exon 20, a step of predicting that chemotherapy using an antitumor agent comprising (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof as an active ingredient is highly likely to show a sufficient therapeutic effect on the patient;

[0107] (3) A step of administering (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof to a patient with a malignant tumor who has been predicted in step (2) to be highly likely to respond adequately to chemotherapy using an antitumor agent comprising (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof as an active ingredient.

[0108] The basic sequence of the EGFR gene is publicly known. The GenBank accession number of the basic sequence of the cDNA is NM_005228.4.

[0109] "Therapeutic effect" can be evaluated by tumor shrinkage effect, recurrence inhibition effect, life extension effect, etc. The effect of inhibiting recurrence can be expressed as the degree of extension of the non-recurrence period or the degree of improvement of the recurrence rate; and the life extension effect can be expressed as the degree of extension of the entire survival time or the median of progression-free survival, etc. "Sufficient therapeutic effect" of chemotherapy using an antitumor agent containing compound A or a salt thereof as an active ingredient means that a superior therapeutic effect is obtained by administering an antitumor agent containing compound A or a salt thereof as an active ingredient, such as prolonging survival time, inhibiting recurrence, etc. compared with no administration.

[0110] Example

[0111] The present invention is hereinafter described in more detail with reference to the following test examples. However, the present invention is not limited to this embodiment (test example).

[0112] Test Example 1

[0113] In vitro efficacy testing

[0114] Evaluation results of intracellular phosphorylation in a mutant EGFR forced expression system using HEK293 cells (inhibition (Activity)

[0115] The intracellular target inhibitory activity of the compound was evaluated based on the following indicators: intracellular EGFR phosphorylation in a mutant EGFR forced expression system using Jump-in (trademark) Grip (trademark) HEK293 cells (Thermo Fisher Scientific Inc.) (hereinafter also referred to as "HEK293 cells"). HEK293 cells were maintained in D-MEM (high glucose) (Thermo Fisher Scientific Inc.) containing GlutaMAX (trademark)-I, which contains 10% dialyzed FBS. HEK293 cells were seeded in each well of a 96-well flat-bottom microplate, with a cell count of 10,000 in each well, and incubated overnight at 37°C in an incubator containing 5% CO2 gas. Subsequently, pcDNA3.0 encoding human EGFR gene (Del E746-A750 (hereinafter also referred to as "Ex19del"), Ex19del+T790M (symbol "+" indicates that both mutations are included), Ex19del+T790M+L718Q, Ex19del+T790M+L792H, Ex19del+T790M+L792F, Ex19del+T790M+L792Y, L858R, L858R+T790M, L858R+T790M+L718Q, L858R+T790M+L792H, L858R+T790M+L792F or L858R+T790M+L792Y) was added. TM6.2 / V5-DEST vector was introduced with ViaFect (trademark) transfection reagent (Promega Corporation) together with Opti-MEM (trademark) I (Thermo Fisher Scientific Inc.). The cells were incubated overnight at 37°C in an incubator containing 5% CO2 gas again. The next day, compound A, erlotinib, afatinib and osimertinib (erlotinib, afatinib and osimertinib can be referred to as "comparison compounds" hereinafter) were dissolved in DMSO independently and diluted with DMSO or culture medium. The solution was then added to each well of the cell culture plate independently, and the cells were incubated at 37°C for 6 hours in an incubator containing 5% CO2 gas. After incubation, the cells were fixed with 20% neutral buffered formalin (Wako Pure Chemical Industries, Ltd.) and blocked with Odyssey (trademark) blocking buffer (PBS) (M&S Techno Systems Inc.). The cells were then reacted with primary antibodies (EGFR antibody mixture #AHR5062 (Thermo Fisher Scientific Inc.) and phosphorylated EGFR receptor (Tyr1068) antibody #2234L (CST)), diluted to 1 / 200 with Odyssey (trademark) blocking buffer (PBS), and the cells were allowed to stand at 4°C overnight. On the next day, the cells were reacted with secondary antibodies (IRDye800CW Goat aRabbit #926-32211 and IRDye 680RD Goat aMouse #926-68070 (M&S TechnoSystems Inc.), diluted to 1 / 800 with Odyssey (trademark) blocking buffer (PBS), and the cells were allowed to stand at room temperature for 1 hour. Fluorescence intensity (hereinafter also referred to as "FI") was detected at fluorescence wavelengths of 800 nm and 700 nm using the Odyssey (trademark) CLx infrared imaging system (LI-CORBioscience).

[0116] The values ​​obtained by subtracting the FI of the wells without primary antibody from the FI detected at a fluorescence wavelength of 800 nm or 700 nm are referred to as FI(800, EGFR)-blank (for 800 nm) and FI(700, p-EGFR)-blank (for 700 nm). The value obtained by dividing the FI(700, p-EGFR)-blank of each well by the FI(800, EGFR)-blank is determined as FI(p-EGFR / EGFR). The phosphorylated EGFR rate is calculated using the following formula to determine the test compound concentration at which EGFR is phosphorylated by 50% (IC 50 Table 1 shows the results.

[0117] Phosphorylation EGFR rate (%) = T / C × 100

[0118] T: FI (p-EGFR / EGFR) of the wells to which the test compound was added.

[0119] C: FI (p-EGFR / EGFR) of wells to which no test compound was added.

[0120] As shown in Table 1, compound A has high inhibitory activity against intracellular phosphorylation of compound mutant EGFR containing L718Q and L792X; its activity is higher than that of erlotinib, afatinib and osimertinib.

[0121] [Table 1]

[0122] Sequence Listing <110> TAIHO PHARMACEUTICAL CO., LTD. <120> EGFR inhibitors resistant to L718 and / or L792 mutations <130> P19-261WO <150> JP 2018-247131 <151> 2018-12-28 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 1210 <212> PRT <213> Homo sapiens <400> 1 Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala 1 5 10 15 Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln 20 25 30 Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe 35 40 45 Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn 50 55 60 Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys 65 70 75 80 Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val 85 90 95 Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr 100 105 110 Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn 115 120 125 Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu 130 135 140 His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu 145 150 155 160 Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met 165 170 175 Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro 180 185 190 Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln 195 200 205 Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg 210 215 220 Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys 225 230 235 240 Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp 245 250 255 Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro 260 265 270 Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly 275 280 285 Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His 290 295 300 Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu 305 310 315 320 Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val 325 330 335 Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn 340 345 350 Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp 355 360 365 Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr 370 375 380 Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu 385 390 395 400 Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp 405 410 415 Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln 420 425 430 His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu 435 440 445 Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser 450 455 460 Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu 465 470 475 480 Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu 485 490 495 Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro 500 505 510 Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn 515 520 525 Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly 530 535 540 Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro 545 550 555 560 Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro 565 570 575 Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val 580 585 590 Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp 595 600 605 Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys 610 615 620 Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly 625 630 635 640 Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu 645 650 655 Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His 660 665 670 Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu 675 680 685 Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu 690 695 700 Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser 705 710 715 720 Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu 725 730 735 Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser 740 745 750 Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser 755 760 765 Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser 770 775 780 Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp 785 790 795 800 Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn 805 810 815 Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg 820 825 830 Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro 835 840 845 Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala 850 855 860 Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp 865 870 875 880 Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp 885 890 895 Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser 900 905 910 Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu 915 920 925 Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr 930 935 940 Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys 945 950 955 960 Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln 965 970 975 Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro 980 985 990 Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp 995 1000 1005 Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe 1010 1015 1020 Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu 1025 1030 1035 Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn 1040 1045 1050 Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg 1055 1060 1065 Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp 1070 1075 1080 Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro 1085 1090 1095 Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln 1100 1105 1110 Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro 1115 1120 1125 His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln 1130 1135 1140 Pro Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala 1145 1150 1155 Gln Lys Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln 1160 1165 1170 Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe Lys 1175 1180 1185 Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gln 1190 1195 1200 Ser Ser Glu Phe Ile Gly Ala 1205 1210

Claims

1. Use of (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizine-8-yl)acrylamide or a salt thereof in the preparation of a medicament for treating patients with malignant tumors expressing EGFR, wherein the EGFR has at least one mutation selected from the group consisting of an L718X mutation in exon 18 and an L792X mutation in exon 20, wherein the mutation position corresponds to the amino acid sequence shown in SEQ ID NO:

1.

2. The use according to claim 1, wherein the EGFR further has at least one mutation selected from the group consisting of exon 19 deletion mutation, L858R, L861Q, G719X, E709X, and exon 20 insertion mutation.

3. The use according to claim 2, wherein the EGFR further has a T790M mutation.

4. The use according to any one of claims 1 to 3, wherein the L718X mutation is a L718Q mutation.

5. The use according to any one of claims 1 to 3, wherein the L792X mutation is L792H, L792F or L792Y.

6. The use according to any one of claims 1 to 3, wherein the medicament is a pharmaceutical composition comprising: (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof and a pharmaceutically acceptable carrier.

7. Use of (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizine-8-yl)acrylamide or a salt thereof in the preparation of a medicament for treating a patient with a malignant tumor expressing EGFR, wherein the EGFR has at least one mutation selected from the group consisting of an L718X mutation in exon 18 and an L792X mutation in exon 20, the use comprising the following steps (1) and (2): (1) a step of detecting whether a mutation exists in the EGFR gene contained in a biological sample obtained from a patient; and (2) When the detection result in step (1) finds that the EGFR gene has at least one mutation selected from the group consisting of: L718X mutation in exon 18 and L792X mutation in exon 20, wherein X represents any amino acid residue, administering to the patient a pharmaceutical agent comprising (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide or a salt thereof as an active ingredient.

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