HK40060754B - L718 and/or l792 mutant treatment-resistant egfr inhibitor - Google Patents

Description

EGFR inhibitors resistant to L718 and/or L792 mutant treatment

Technical Field

This invention relates to antitumor agents targeting cancer, said cancer comprising a mutant epidermal growth factor receptor (hereinafter referred to as "EGFR") that is resistant to treatment with exon 18 and/or exon 20.

Background Technology

EGFR is a receptor-type tyrosine kinase that exerts its physiological functions 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). Furthermore, somatic mutations in the EGFR gene are considered oncogenes; for example, EGFR lacking codons 746-750 in exon 19 (hereinafter also referred to as the "exon 19 deletion mutation") and EGFR with a mutation from leucine to arginine encoded by codon 858 in exon 21 (hereinafter also referred to as the "L858R mutation") continuously induce EGF-independent kinase activity and are involved in cancer cell growth and survival (NPL 2). For example, these mutations are observed in 30-50% of non-small cell lung cancers in East Asia. They are also observed in approximately 10% of non-small cell lung cancers in Europe and the United States, and are considered one of the causes of cancer (NPL 3).

Therefore, research and development of EGFR inhibitors as antitumor agents have been actively undertaken, and they have 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 mutations and L858R mutations in EGFR-positive lung cancer. Exon 19 deletion mutations and L858R mutations account for 90% of EGFR mutations. Furthermore, acquired resistance occurs during treatment with these agents, and 50% of these are caused by resistant EGFR mutations in which codon 790 in exon 20 is changed from threonine to methionine (hereinafter also referred to as the "T790M mutation"). Osimertinib has been used as a therapeutic agent for lung cancer with this mutation. Therefore, the use of EGFR inhibitors is being established for patients with lung cancer having major EGFR mutations.

List of referenced files

Patent documents

PTL 1: WO2015/175632A1

PTL 2: WO2015/025936A1

Non-patent literature

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

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

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

NPL 4: Clin.Cancer Res., vol.24, pp.3097-3107(2018)

Summary of the Invention

Technical issues

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

Therefore, it is believed that developing drugs with high inhibitory activity against EGFR containing compound mutations of L718X or L792X could enable them to exhibit antitumor effects against osimertinib-resistant lung cancer, and is expected to help prolong the lives and improve the quality of life of patients with mutant EGFR-positive cancer for which no therapy has yet been established. Furthermore, drugs with high inhibitory activity against T790M (an acquired resistance mutation to treatment with EGFR inhibitors) are expected to reduce the expression frequency of acquired resistance during treatment with EGFR inhibitors targeting exon 19 or exon 21 mutant EGFR (which are de novo mutations); thus, it is expected to help prolong the lifespan of cancer patients.

In such cases, the object of the present invention is to provide inhibitors with high inhibitory activity against L718 and L792 mutant EGFR mutations, which are mutations resistant to osimertinib treatment and for which previously known EGFR inhibitors are ineffective.

Solution to the problem

The inventors of this invention conducted in-depth research and discovered that conventionally introduced EGFR inhibitors exhibit poor inhibitory activity against complex mutations, including L718 and L792 mutations in addition to active mutations and T790M acquired resistance mutations. Furthermore, the inventors conducted in-depth research starting with comparing the co-crystal structures of the compounds and discovered that the compounds of this invention exhibit excellent known activity against EGFR with L718 and L792 mutations, and further demonstrate excellent inhibitory activity against the aforementioned complex mutant EGFR. Based on these findings, the inventors completed this invention.

The present invention covers the following embodiments.

Project 1. An antitumor agent for treating patients with malignant tumors expressing EGFR, said EGFR having at least one mutation selected from the group consisting of the L718X mutation in exon 18 and the L792X mutation in exon 20, wherein X represents any amino acid residue, said antitumor agent comprising (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimidino[5,4-b]indolazin-8-yl)acrylamide (compound (A)) or a salt thereof.

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

Project 3. The antitumor agent according to Project 2, wherein the EGFR also has a T790M mutation.

Item 4. An antitumor agent according to any one of Items 1-3, wherein the L718X mutation is an L718Q mutation.

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

Project 6. A method for treating a patient with a malignant tumor, comprising the steps of: administering (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimidino[5,4-b]indolazin-8-yl)acrylamide or a salt thereof to a patient with a malignant tumor expressing EGFR, said EGFR having at least one mutation selected from the group consisting of: L718X mutation in exon 18 and L792X mutation in exon 20.

Project 7. (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimidino[5,4-b]indoleazine-8-yl)acrylamide or a salt thereof for the treatment of patients with malignant tumors expressing EGFR, said EGFR having at least one mutation selected from the group consisting of the L718X mutation in exon 18 and the L792X mutation in exon 20.

Project 8. Use of (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimidino[5,4-b]indoleazine-8-yl)acrylamide or a salt thereof for the treatment of patients with malignant tumors expressing EGFR, said EGFR having at least one mutation selected from the group consisting of the L718X mutation in exon 18 and the L792X mutation in exon 20.

Project 9. Use of (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimidino[5,4-b]indoleazine-8-yl)acrylamide or a salt thereof in the preparation of a medicament for treating patients with malignant tumors expressing EGFR, said EGFR having at least one mutation selected from the group consisting of the L718X mutation in exon 18 and the L792X mutation in exon 20.

Item 10. A pharmaceutical composition comprising: (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimidino[5,4-b]indolazin-8-yl)acrylamide or a salt thereof and a pharmaceutically acceptable carrier, said pharmaceutical composition for treating patients with malignant tumors expressing EGFR, said EGFR having 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.

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

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

(2) When the test results in step (1) reveal that the EGFR gene has at least one mutation selected from the group consisting of the L718X mutation in exon 18 and the L792X mutation in exon 20, where X represents any amino acid residue, the step is to predict that chemotherapy is very likely to show sufficient therapeutic effect on the patient.

Item 12. A method for treating patients with malignant tumors, comprising the steps (1) to (3):

(1) The step of detecting whether there is a mutation in the EGFR gene contained in biological samples obtained from patients;

(2) When the test results in step (1) reveal that the EGFR gene has at least one mutation selected from the group consisting of the L718X mutation in exon 18 and the L792X mutation in exon 20, where X represents any amino acid residue, it is predicted that chemotherapy using a chemotherapeutic agent containing (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimidino[5,4-b]indolazin-8-yl)acrylamide or a salt thereof as the active ingredient is highly likely to show adequate therapeutic effect on the patient.

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

Advantages of the invention

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

The present invention also provides a method for treating patients with malignant tumors expressing EGFR with L718 and L792 mutations.

In the presence of the T790M mutation (an acquired resistance mutation in exon 20) or the L718 and L792 mutations, the EGFR inhibitory activity of conventional EGFR inhibitors is significantly reduced. Therefore, they are difficult to achieve sufficient therapeutic effects. In contrast, the antitumor agent of the present invention exhibits high inhibitory activity against EGFR with L718 and/or L792 mutations, as well as those with active mutations (e.g., Ex19del or L858R mutations); therefore, the antitumor agent of the present invention can exert excellent therapeutic effects on patients with malignant tumors expressing EGFR with these complex mutations. For EGFR with L718 and/or L792 mutations, even if it also has the T790M mutation in addition to the aforementioned mutations, the antitumor agent of the present invention exhibits high inhibitory activity; therefore, the antitumor agent of the present invention can exert excellent therapeutic effects on patients with malignant tumors expressing EGFR with these mutations.

Furthermore, the antitumor agent of the present invention can also be used to reduce the expression frequency of acquired resistance during treatment with EGFR inhibitors targeting exon 18 or exon 21 mutant EGFR (which are de novo mutations), because it has high inhibitory activity against mutant EGFR resistant to exon 18 and exon 20 treatment even in the presence of the T790M mutation (which is an acquired resistance mutation in the exon 20 region).

Attached Figure Description

Figure 1 shows the evaluation results of phosphorylation (inhibitory activity) in the mutant EGFR forced expression system using HEK293 cells.

Detailed Implementation

Preferred examples of the various definitions used within the scope of the invention as described in this specification are explained in detail below.

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

In this specification, "wild-type EGFR" refers to EGFR without somatic mutations, which is a protein containing the amino acid sequence shown in SEQ ID NO: 1 (GenBank accession number: NP_005219.2).

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

In this specification, "mutation resistant to exon 18 treatment" refers to a point mutation or deletion mutation in an amino acid in the exon 18 region of wild-type EGFR (SEQ ID NO: 1). A preferred exon 18 treatment-resistant mutation is a point mutation with a single amino acid substitution in the exon 18 region. More preferably, an exon 18 treatment-resistant mutation is L718X (where X represents any amino acid residue that is not leucine in the amino acids constituting the protein encoded by genetic information), which is a point mutation that replaces leucine encoded by codon 718 of exon 18 with any amino acid. More specifically, preferred examples of L718X include L718Q, a point mutation that replaces leucine encoded by codon 718 of exon 18 with glutamine; and L718V, a point mutation that replaces leucine encoded by codon 718 with valine.

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

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

In this specification, "therapeutic mutation resistant to exon 20" refers to a point mutation in the amino acid region of exon 20 of wild-type EGFR (SEQ ID NO: 1). A preferred therapeutic mutation resistant to exon 20 is a point mutation involving a single amino acid substitution in the exon 20 region. More preferably, the therapeutic mutation resistant to exon 20 is L792 (where X represents any amino acid residue that is not leucine in 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, a point mutation replacing leucine encoded by codon 792 in exon 20 with histidine; L792F, a point mutation replacing leucine encoded by codon 792 with phenylalanine; L792Y, a point mutation replacing leucine encoded by codon 792 with tyrosine; L792R, a point mutation replacing leucine encoded by codon 792 with arginine; L792V, a point mutation replacing leucine encoded by codon 792 with valine; and L792P, a point mutation replacing leucine encoded by codon 792 with proline. Among these, L792F, L792H, and L792Y are preferred.

In this specification, "exon 20 insertion mutation" refers to the insertion of one or more (preferably 1-7, more preferably 1-4) amino acids into the amino acid sequence of exon 20 region (SEQ ID NO: 1) of EGFR at positions 824-875. Preferred examples include inserting the amino acid sequence FQEA (phenylalanine, glutamine, glutamic acid, and alanine from the N-terminus) into the region of exon 20, between alanine at position 763 and tyrosine at position 764 (A763_Y764insFQEA); inserting the amino acid sequence ASV (alanine, serine, and valine from the N-terminus) into the region of exon 20, between valine at position 769 and aspartic acid at position 770 (V769_D770insASV); inserting the amino acid sequence SVD (serine, valine, and aspartic acid from the N-terminus) into the region of exon 20, between aspartic acid at position 770 and asparagine at position 771 (D770_N771insSVD); and inserting the amino acid sequence SVD (serine, valine, and aspartic acid from the N-terminus) into the region of exon 20, between aspartic acid at position 770 and asparagine at position 771 (D770_N771insSVD). The mutations are as follows: Inserting the acid sequence NPG (asparagine, proline, and glycine from the N-terminus) into the region of exon 20 between asparagine at position 770 and asparagine at position 771 (D770_N771insNPG); inserting amino acid G (glycine) into the region of exon 20 between asparagine at position 770 and asparagine at position 771 (D770_N771insG); deleting asparagine at position 770 in exon 20 and then inserting the amino acid sequence GY (glycine and tyrosine from the N-terminus) into the region of exon 20 (D770>GY); inserting amino acid N (asparagine) into the region of exon 20 between asparagine at position 771 and proline at position 772 (N771_P772insN); and inserting amino acid G (glycine) into the region of exon 20 between asparagine at position 771 and proline at position 772. The following amino acid sequences were inserted into exon 20: PR (proline and arginine from the N-terminus) as a mutation between proline at position 772 and histidine at position 773 (P772_R773insPR); NPH (asparagine, proline, and histidine from the N-terminus) as a mutation between histidine at position 773 and valine at position 774 (H773_V774insNPH); PH (proline and histidine from the N-terminus) as a mutation between histidine at position 773 and valine at position 774 (H773_V774insPH); and AH (alanine and histidine from the N-terminus) as a mutation between exon 20 and position 20. Mutations such as the mutation between histidine at position 773 and valine at position 774 in the exon 20 region (H773_V774insAH), the mutation between histidine at position 773 and valine at position 774 by inserting amino acid H (histidine) into the exon 20 region (H773_V774insH), the mutation between valine at position 774 and cysteine at position 775 by inserting amino acid sequence HV (histidine and valine from the N-terminus) into the exon 20 region (V774_C774insHV), and the mutation between alanine at position 761 and glutamic acid at position 762 by inserting amino acid sequence EAFQ (glutamic acid, alanine, phenylalanine and glutamine from the N-terminus) into the exon 20 region (A761_E762insEAFQ), etc. More preferred examples include a mutation (V769_D770insASV) inserting the amino acid sequence ASV (alanine, serine, and valine from the N-terminus) between valine at position 769 and aspartic acid at position 770 in exon 20; a mutation (D770_N771insSVD) inserting the amino acid sequence SVD (serine, valine, and aspartic acid from the N-terminus) between aspartic acid at position 770 and asparagine at position 771 in exon 20; and a mutation (D770_N771insSVD) inserting amino acid G (glycine) between aspartic acid at position 770 and asparagine at position 771 in exon 20. The D770_N771insG mutation (H773_V774insNPH) involves inserting the amino acid sequence NPH (asparagine, proline, and histidine from the N-terminus) between histidine at position 773 and valine at position 774 in the exon 20 region. The H773_V774insPH mutation (H773_V774insPH) involves inserting the amino acid sequence PH (proline and histidine from the N-terminus) between histidine at position 773 and valine at position 774 in the exon 20 region. Particularly preferred examples include the FQEA amino acid sequence FQEA (phenylalanine, glutamine, glutamic acid, and alanine from the N-terminus). The mutations are: A763_Y764insFQEA (inserting amino acids) between alanine (position 763) and tyrosine (position 764) in exon 20; V769_D770insASV (inserting amino acid sequence ASV, consisting of alanine, serine, and valine from the N-terminus); and D770_N771insSVD (inserting amino acid sequence SVD, consisting of serine, valine, and aspartic acid from the N-terminus) between aspartic acid (position 770) and asparagine (position 771) in exon 20. Mutations such as inserting amino acid G (glycine) into the region of exon 20 between aspartic acid at position 770 and asparagine at position 771 (D770_N771insG), inserting amino acid sequence NPH (asparagine, proline, and histidine from the N-terminus) into the region of exon 20 between histidine at position 773 and valine at position 774 (H773_V774inchsNPH), and inserting amino acid sequence PH (proline and histidine from the N-terminus) into the region of exon 20 between histidine at position 773 and valine at position 774 (H773_V774insPH) are examples of this.

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

In this 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 involving a single amino acid substitution in the exon 21 region. More preferably, the exon 21 active mutation is L858X, which is a point mutation replacing leucine encoded by codon 858 in the exon 21 region with any amino acid; or L861X, which is a point mutation replacing leucine encoded by codon 864 in the exon 21 region with any amino acid (X represents any amino acid residue that is not leucine in the amino acids constituting the protein encoded by the genetic information). More specifically, preferred examples include L858R, which is a point mutation replacing leucine encoded by codon 858 in the exon 21 region with arginine; and L861Q, which is a point mutation replacing leucine encoded by codon 864 in the exon 21 region with glutamine.

In this invention, “mutations resistant to treatment of exon 18 and/or exon 20” encompasses “mutations resistant to treatment of exon 18”, “mutations resistant to treatment of exon 20”, and “mutations resistant to treatment of both exon 18 and exon 20”.

In this invention, a "point mutation" refers to a mutation that causes the 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 as nucleic acids.

"EGFR with mutations resistant to treatment of exon 18 and/or exon 20" encompasses "EGFR with mutations resistant to treatment of exon 18", "EGFR with mutations resistant to treatment of exon 20", and "EGFR with mutations resistant to treatment of both exon 18 and exon 21".

In this specification, "EGFR with a mutation resistant to exon 18 therapy" refers to EGFR with at least one 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 a single L718X mutation is preferred. Furthermore, EGFR may have mutations other than those resistant to exon 18 therapy (e.g., exon 19 deletion mutations, L858R mutations, and L790M mutations).

In this specification, "EGFR with a mutation resistant to exon 20 treatment" refers to EGFR with at least one L792X mutation in exon 20 as a mutation resistant to exon 20 treatment. As a mutation resistant to exon 20 treatment, EGFR may have mutations other than the L792X mutation, but preferably a single L718X mutation. Furthermore, EGFR may have mutations other than those resistant to exon 20 treatment (e.g., exon 19 deletion mutations, L858R mutations, and L790M mutations).

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

In this specification, "exon 19 deletion mutation" refers to a mutation that deletes one or more amino acids in the exon 19 region of wild-type EGFR (SEQ ID NO: 1). In addition to deletions in this region, mutations that insert one or more arbitrary amino acids are also included. Examples of exon 19 deletion mutations include a 5-amino acid deletion from glutamate 746 to alanine 750 in the exon 19 region (Del E746-A750), a 7-amino acid deletion followed by a serine insertion after leucine 747 to proline 753 in the exon 19 region (Del 747-P753insS), a 5-amino acid deletion from leucine 747 to threonine 751 in the exon 19 region (Del L747-T751), and a 4-amino acid deletion followed by a proline insertion after leucine 747 to alanine 750 in the exon 19 region (Del 747-A750insP), etc. A preferred example includes a 5-amino acid deletion from glutamate 746 to alanine 750 in the exon 19 region (Del E746-A750).

In addition, EGFRs with mutations resistant to exon 18 and/or exon 20 may also have at least one mutation selected from the group consisting of: exon 19 deletion mutations, L858R, L861Q, G719X, E709X, and exon 20 insertion mutations. Specific examples include EGFRs with an L718X mutation in the exon 18 region and an exon 19 deletion mutation, EGFRs with an L792X mutation in the exon 20 region and an exon 19 deletion mutation, EGFRs with an L718X mutation in the exon 18 region and an L858R mutation, EGFRs with an L792X mutation in the exon 20 region and an L858R mutation, EGFRs with an L718X mutation in the exon 18 region and an L861Q mutation, and EGFRs with an L792X mutation in the exon 20 region and an L861Q mutation. FR, EGFR with L718X mutation in exon 18 and G719X, EGFR with L792X mutation in exon 20 and G719X, EGFR with L718X mutation in exon 18 and E709X, EGFR with L792X mutation in exon 20 and E709X, EGFR with L718X mutation in exon 18 and exon 20 insertion mutation, and EGFR with L792X mutation in exon 20 and exon 20 insertion mutation. Among these, the preferred EGFRs are those with an L718X mutation in exon 18 and an exon 19 deletion mutation, those with an L792X mutation in exon 20 and an exon 19 deletion mutation, those with an L718X mutation in exon 18 and an L858R mutation, and those with an L792X mutation in exon 20 and an L858R mutation.

In addition to the exon 19 deletion mutations, L858R, L861Q, G719X, E709X, and exon 20 insertion mutations mentioned above, EGFR mutations resistant to exon 18 and/or exon 20 treatment can also carry the T790M mutation. T790M is an acquired resistance mutation in the exon 20 region. It is known that T790M arises from the use of existing EGFR inhibitors. The acquisition of T790M often reduces the effectiveness of existing drugs in patients with malignant tumors.

In this invention, examples include: EGFRs having an L718X mutation in exon 18, an exon 19 deletion mutation, and a T790M mutation; EGFRs having an L792X mutation in exon 20, an exon 19 deletion mutation, and a T790M mutation; EGFRs having an L718X mutation in exon 18, an L858R mutation, and a T790M mutation; EGFRs having an L792X mutation in exon 20, an L858R mutation, and a T790M mutation; EGFRs having an L718X mutation in exon 18, an L861Q mutation, and a T790M mutation; and EGFRs having an L792X mutation in exon 20, an L861Q mutation, and a T790M mutation. EGFR with L718X mutation in exon 18, EGFR with G719X mutation and T790M mutation, EGFR with L792X mutation in exon 20, EGFR with E709X mutation and T790M mutation, EGFR with L718X mutation in exon 18, EGFR with E709X mutation and T790M mutation, EGFR with L718X mutation in exon 18, EGFR with exon 20 insertion mutation and T790M mutation, and EGFR with L792X mutation in exon 20, EGFR with exon 20 insertion mutation and T790M mutation. Among these, the preferred EGFRs are those with the L718X mutation in exon 18, the exon 19 deletion mutation, and the T790M mutation; those with the L792X mutation in exon 20, the exon 19 deletion mutation, and the T790M mutation; those with the L718X mutation in exon 18, the L858R mutation, and the T790M mutation; and those with the L792X mutation in exon 20, the L858R mutation, and the T790M mutation.

Among the EGFRs with the above compound mutations, EGFRs with the L718Q mutation in the region of exon 18, EGFRs with the L792F mutation in the region of exon 20, EGFRs with the L792H mutation, and EGFRs with the L792Y mutation are particularly preferred.

In this invention, the method for detecting mutations in EGFR expressed by patients with malignant tumors is not particularly limited, as long as the method can detect the mutations, and any known detection method can be used.

There are no particular restrictions on the samples used for detecting EGFR mutations, as long as the sample is a biological sample isolated from a patient with malignant tumors, particularly a sample obtained from a patient with malignant tumors and containing malignant tumor cells. Examples of biological samples include bodily fluids (e.g., blood, urine, etc.), tissues, their extracts, and cultures of the resulting tissues. Depending on the type of biological sample, the method for obtaining the biological sample may be appropriately selected.

Biological samples are prepared by appropriately processing them according to the measurement method. Furthermore, reagents containing primers or probes for detection can be prepared using conventional methods, depending on the measurement method used.

In one embodiment of the invention, the step of detecting the presence of the mutation of the invention in EGFR expressed by the malignant tumor patient may be performed before administering an antitumor agent to the patient with a malignant tumor.

Malignant tumors can include two or more different types of malignant tumor cells. Furthermore, two or more types of malignant tumors can occur in a single patient. Therefore, a single patient can simultaneously have different EGFR mutations (e.g., 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, this is not a limitation).

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

[Chemical Formula 1]

The methods for preparing the compounds of the present invention are explained below.

Compound A of the present invention can be prepared by the preparation methods disclosed in WO2015/025936A1, the methods described in the examples, etc. However, the preparation methods of the compounds of the present invention are not limited to these reaction examples.

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

A salt of compound A is any pharmaceutically acceptable salt; examples include base addition salts and acid addition salts.

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

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, ascorbic acid salts, and trifluoroacetates; and sulfonates, such as methanesulfonates, isothiocyanates, benzenesulfonates, and p-toluenesulfonates.

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

Disease description

Specific examples of tumors targeted in this invention include, but are not particularly limited to, head and neck cancer, gastrointestinal cancers (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), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, and mesothelioma), breast cancer, reproductive tract cancers (ovarian cancer, uterine cancer (e.g., cervical cancer and endometrial cancer), urinary system cancers (e.g., kidney cancer, bladder cancer, prostate cancer, and testicular tumors), hematopoietic system tumors (e.g., leukemia, malignant lymphoma, and multiple myeloma), osteosarcoma, soft tissue sarcoma, skin cancer, and brain tumors. Preferred examples include lung cancer, breast cancer, head and neck cancer, brain tumors, uterine cancer, gastrointestinal cancer, hematopoietic tumors, and skin cancer. Lung cancer is particularly preferred.

When the compounds of the present invention or their salts are used as pharmaceutical agents, a drug carrier may be added if necessary to form a suitable dosage form for preventive and therapeutic purposes. Examples of dosage forms include oral preparations, injections, suppositories, ointments, patches, etc. Oral preparations are preferred. Such dosage forms can be formed by methods conventionally known to those skilled in the art.

As pharmaceutically acceptable carriers, various conventional organic or inorganic carrier materials used as preparation materials can be incorporated into solid dosage forms as excipients, binders, disintegrants, lubricants, or colorants; or into liquid dosage forms as solvents, solubilizers, suspending agents, isotonic agents, buffers, or soothing agents. Furthermore, pharmaceutical additives, such as preservatives, antioxidants, colorants, sweeteners, and stabilizers, may be used if desired.

Oral solid dosage forms are prepared as follows: after adding excipients, optionally along with other excipients, binders, disintegrants, lubricants, colorants, taste maskers, or flavorings, to the compounds of the present invention, the resulting mixture is formulated into tablets, coated tablets, granules, powders, capsules, etc., by conventional methods.

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, methylcellulose, ethylcellulose, shellac, calcium phosphate, and polyvinylpyrrolidone. Examples of disintegrants include dry starch, sodium alginate, powdered agar, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, glyceryl monostearate, and lactose. Examples of lubricants include purified talc, sodium stearate, magnesium stearate, borax, and polyethylene glycol. Examples of colorants include titanium dioxide and iron oxide. Examples of taste masking agents or flavoring agents include sucrose, bitter orange peel, citric acid, and tartaric acid.

When preparing oral liquid formulations, masking agents, buffers, stabilizers, flavoring agents, etc., can be added to the compounds of the present invention; the resulting mixture can be formulated into oral liquid formulations, syrups, elixirs, etc., according to common methods.

When preparing injectable formulations, pH adjusters, buffers, stabilizers, isotonic agents, local anesthetics, etc., can be added to the compounds of the present invention; the mixture can be formulated into subcutaneous, intramuscular, or intravenous injection formulations according to common methods.

Examples of pH adjusters and buffers used in this article include sodium citrate, sodium acetate, and sodium phosphate. Examples of stabilizers include sodium metabisulfite, 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.

When preparing suppositories, pharmaceutically acceptable carriers known in the relevant field, such as polyethylene glycol, lanolin, cocoa butter, and fatty acid triglycerides, can be added to compound A, and surfactants such as Tween 80 (registered trademark) can be added to compound A if necessary, and the resulting mixture can be formulated into suppositories according to commonly used methods.

When preparing ointments, commonly used alkalis, stabilizers, wetting agents, preservatives, etc., can be incorporated into compound A if necessary; the resulting mixture is then prepared into an ointment using common methods.

Examples of alkalis include liquid paraffin, white petrolatum, white beeswax, octyldodecyl alcohol, and paraffin wax.

Examples of preservatives include methyl paraben, ethyl paraben, and propyl paraben.

When preparing patches, ointments, creams, gels, pastes, etc., as described above can be coated onto common substrates using common methods.

Examples of substrates include woven or nonwoven fabrics containing cotton, short fibers, or chemical fibers; films or foam sheets made of soft chloroethylene, polyethylene, polyurethane, etc., may also be used.

The amount of compound A to be added to each of these unit dosage forms depends on the patient's condition, the dosage form, etc., when administering the compound. Generally, in the case of oral dosage forms, the amount of compound is preferably 0.05 mg to 1000 mg per unit dosage form. In the case of injectable dosage forms, the amount of compound is preferably 0.01 mg to 500 mg per unit dosage form; and in the case of suppositories, the amount of compound is preferably 1 to 1000 mg per unit dosage form.

Furthermore, the daily dosage of this type of drug depends on the patient's condition, weight, age, sex, etc., and cannot be generalized. Generally, the daily dose of the compound of the present invention for adults (weight: 50 kg) is usually 0.05 to 5000 mg, preferably 0.1 to 1000 mg; and preferably administered once daily, or in two or three separate doses.

The present invention also provides a method for treating patients with malignant tumors, comprising the steps of administering compound A or a salt thereof to a patient with a malignant tumor who expresses EGFR with a mutation resistant to treatment of exon 18 and/or exon 20.

The present invention also provides compound A or a salt thereof for treating patients with malignant tumors who express EGFR with mutations resistant to treatment of exon 18 and/or exon 20.

The present invention also provides the use of compound A or a salt thereof for treating patients with malignant tumors who express EGFR with mutations resistant to treatment of exon 18 and/or exon 20.

The present invention also provides the use of compound A or a salt thereof for the preparation of a medicament for treating patients with malignant tumors who express EGFR with mutations resistant to treatment of exon 18 and/or exon 20.

The present invention also provides a method for predicting the therapeutic effect of chemotherapy in patients with malignant tumors using an antitumor agent containing compound A or a salt thereof as an active ingredient, the method comprising the steps of (1) and (2):

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

(2) When the test results in step (1) reveal that the EGFR gene has a mutation that is resistant to treatment of exon 18 and/or exon 20, the step is to predict that the chemotherapy is very likely to show sufficient therapeutic effect on the patient.

The present invention also provides a method for treating patients with malignant tumors, comprising the steps (1) to (3):

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

(2) When the test results in step (1) reveal that the EGFR gene has a mutation that is resistant to treatment of exon 18 and/or exon 20, it is predicted that chemotherapy with an antitumor agent containing (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimidino[5,4-b]indoleazine-8-yl)acrylamide or a salt thereof as the active ingredient is very likely to show sufficient therapeutic effect on the patient.

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

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

"Therapeutic efficacy" can be evaluated through tumor shrinkage effect, recurrence inhibition effect, and life-prolonging effect. The recurrence inhibition effect can be expressed as the degree of extension of the recurrence-free period or the degree of improvement in the recurrence rate; and the life-prolonging effect can be expressed as the degree of extension of overall survival or the median extension of progression-free survival. "Sufficient therapeutic efficacy" of chemotherapy using an antitumor agent containing compound A or its salt as the active ingredient means obtaining superior therapeutic effects by administering an antitumor agent containing compound A or its salt as the active ingredient, such as extended survival time or recurrence inhibition compared to no administration.

Example

The invention is described in more detail below with reference to the following test examples. However, the invention is not limited to these embodiments (test examples).

Test Example 1

In vitro efficacy testing

Evaluation results of intracellular phosphorylation in a mutant EGFR forced expression system using HEK293 cells (inhibition) (Active)

The intracellular target inhibitory activity of the compounds was assessed based on intracellular EGFR phosphorylation in a mutant EGFR forced expression system using Jump-in (trademarked) Grip (trademarked) HEK293 cells (Thermo Fisher Scientific Inc.) (hereinafter also referred to as "HEK293 cells"). HEK293 cells were stored in D-MEM (high glucose) (Thermo Fisher Scientific Inc.) containing GlutaMAX (trademarked)-I and 10% dialysis FBS. HEK293 cells were seeded into each well of a 96-well flat-bottomed microplate to a cell count of 10,000 per well and incubated overnight at 37°C in an incubator containing 5% _CO2 gas. Subsequently, the pcDNA™ 6.2/V5-DEST vector encoding the human EGFR gene (Del E746-A750 (hereinafter also referred to as "Ex19del"), Ex19del+T790M (the 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 combined with Opti-MEM (trademark) I (Thermo Fisher Scientific). Scientific Inc. used ViaFect transfection reagent (Promega Corporation) to introduce the cells. The cells were incubated again overnight at 37°C in an incubator with 5% _CO2 gas. The next day, Compound A, erlotinib, afatinib, and osimertinib (erlotinib, afatinib, and osimertinib can each be referred to as "comparative compounds" below) were independently dissolved in DMSO and diluted with DMSO or culture medium. The solutions were then independently added to each well of the cell culture plate, and the cells were incubated at 37°C for 6 hours in an incubator with 5% _CO2 gas. After incubation, the cells were fixed with 20% neutral buffered formalin (Wako PureChemical Industries, Ltd.) and blocked with Odyssey blocking buffer (PBS) (M&S TechnoSystems Inc.). 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 blocking buffer (PBS) and incubated overnight at 4°C. The next day, cells were reacted with secondary antibodies (IRDye800CW Goat aRabbit #926-32211 and IRDye 680RD Goat aMouse #926-68070 (M&S Techno Systems Inc.) diluted to 1/800 with Odyssey blocking buffer (PBS) and incubated 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 an Odyssey CLx infrared imaging system (LI-CORBioscience).

The value obtained by subtracting the FI of wells without primary antibody from the FI detected at fluorescence wavelengths of 800 nm or 700 nm is called FI(800, EGFR)-blank (for 800 nm) and FI(700, p-EGFR)-blank (for 700 nm). FI(p-EGFR/EGFR) is determined by dividing the FI(700, p-EGFR)-blank of each well by the FI(800, EGFR)-blank. The EGFR phosphorylation rate was calculated using the following formula to determine the concentration of the test compound at which EGFR was phosphorylated by 50% ( _IC50 ) (μM). Table 1 illustrates the results.

EGFR phosphorylation rate (%) = T/C × 100

T: FI (p-EGFR/EGFR) added to the pores of the test compound.

C: FI (p-EGFR/EGFR) of wells without test compound.

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

[Table 1]

sequence list

<110> TAIHO PHARMACEUTICAL CO., LTD.

<120> EGFR inhibitors resistant to L718 and/or L792 mutant treatments

<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 Ser 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 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 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 (7)

1. Use of (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimidino[5,4-b]indoleazine-8-yl)acrylamide or a salt thereof in the preparation of a medicament for treating patients with malignant tumors expressing EGFR, said EGFR having at least one mutation selected from the group consisting of the L718X mutation in exon 18 and the L792X mutation in exon 20, wherein said 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 comprises 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 1-3, wherein the L718X mutation is an L718Q mutation. 5. The use according to any one of claims 1-3, wherein the L792X mutation is L792H, L792F or L792Y. 6. The use according to any one of claims 1-3, wherein the agent is a pharmaceutical composition comprising: (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimidino[5,4-b]indolazin-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-dihydropyrimidino[5,4-b]indoleazine-8-yl)acrylamide or a salt thereof in the preparation of a medicament for treating patients with malignant tumors expressing EGFR, said EGFR having at least one mutation selected from the group consisting of: L718X mutation in exon 18 and L792X mutation in exon 20, said use comprising the steps (1) and (2): (1) The step of detecting whether there is a mutation in the EGFR gene contained in a biological sample obtained from the patient; and (2) When the test results in step (1) reveal that the EGFR gene has at least one mutation selected from the group consisting of the L718X mutation in exon 18 and the L792X mutation in exon 20, where X represents any amino acid residue, the patient is given a drug containing (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimidino[5,4-b]indolazin-8-yl)acrylamide or a salt thereof as the active ingredient.