RU2774629C2 - Selective egfr mutant inhibitor with insertion in exon 20 - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to a method for the treatment of a patient with malignant tumor expressing EGFR having a mutation with an insertion in exon 20, with a compound selected from a group consisting of: (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dighydropyrimido[5,4-b]indolysin-8-yl)acrylamide; (S)-N-(4-amino-6-methylene-5-(quinolin-3-yl)-7,8-dighydro-6H-pyrimido[5,4-b]pyrrolizin-7-yl)acrylamide; and (S,E)-N-(4-amino-6-methylene-5-(quinolin-3-yl)-7,8-dihydro-6H-pyrimido[5,4-b]pyrrolizin-7-yl)-3-chloracrylamide; and (R)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dighydropyrimido[5,4-b]indolysin-8-yl)-N-methylacrylamide or its salt. The use of the specified compound for the production of an antitumor agent for the treatment of a patient with malignant tumor expressing EGFR having a mutation with an insertion in exon 20 is also proposed.

EFFECT: obtaining a compound for the treatment of malignant tumor.

9 cl, 15 dwg, 8 tbl, 10 ex

Description

Technical field

[0001]

The present invention relates to an anticancer antitumor agent containing a mutant epidermal growth factor receptor with an exon 20 insert (hereinafter referred to as EGFR).

State of the art

[0002]

EGFR is a receptor tyrosine kinase that performs its physiological function in normal tissue by being associated with epidermal growth factor (hereinafter also referred to as EGF), which is a ligand, and promotes the growth and inhibition of apoptosis in epithelial tissues (NPL 1). In addition, a somatic mutation of the EGFR gene is known to be a cancer-causing gene; for example, EGFR in which amino acids 746-750 in the region of exon 19 are deleted (hereinafter also referred to as "exon 19 deletion mutation") and EGFR in which amino acids 858 in the region of exon 21 are mutated from leucine to arginine (hereinafter also referred to as "mutation L858R") permanently induces EGF-independent kinase activity and promotes the growth and survival of cancer cells (NPL 2). These mutations are observed, for example, in 30-50% of cases of non-small cell lung cancer in East Asia. Mutations are also observed in approximately 10% of non-small cell lung cancers in Europe and the USA and are considered as one of the causes of cancer (NPL 3).

[0003]

Thus, research and development of an EGFR inhibitor as an antitumor agent is being actively pursued and implemented in the treatment of EGFR mutant-positive lung cancer. For example, although administration of gefitinib, erlotinib, and afatinib at their therapeutic dose causes gastrointestinal disturbances and skin diseases, which are widely believed to be associated with wild-type EGFR inhibition, as side effects, they have a high antitumor effect against the mutant with deletion in exon 19 and mutant L858R of EGFR-positive lung cancers. The therapeutic effects of these agents are thought to be due to the selective inhibition of mutant EGFR over wild-type EGFR by an EGFR inhibitor (NPL 4).

[0004]

However, recent studies have shown that some cancers have an EGFR mutation in which one or more amino acids are inserted in the region of exon 20 (hereinafter also referred to as "exon 20 insertion mutation"), and that these cancers have low sensitivity to previously known EGFR inhibitors. For example, there are clinical reports showing significantly lower antitumor effects of afatinib against EGFR mutant-positive lung cancer with an exon 20 insertion mutation compared with an exon 19 deletion or L858R (NPL 5) mutation. Because of this, chemotherapy has been used for patients with these cancers. However, since treatment options are limited and sufficient therapeutic effects have not been obtained, an antitumor agent with higher therapeutic effects is required.

[0005]

PTL 1 discloses a compound useful in the treatment of diseases characterized by an EGFR insertion mutant at exon 20. However, the PTL 1 compound differs significantly in structure from the compound of the present invention, and PTL 1 nowhere discloses selectivity based on comparison to wild-type EGFR. , or efficacy in an in vivo model.

[0006]

In addition, although a quinoline substituted compound is disclosed in PTL 2, inhibitory activity against the EGFR mutant with an insertion at exon 20 is nowhere disclosed in PTL 2.

List of referenced documents cited

Patent Literature

[0007]

PTL 1: WO2015/175632A1

PTL 2: WO2015/025936A1

Non-Patent Literature

[0008]

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: Lancet Oncol. Vol. 13, e. 23-31 (2012)

NPL 5: Lancet Oncol. Vol. 16, pp. 830-838 (2015)

Brief summary of the invention

Technical task

[0009]

It is an object of the present invention to provide an antitumor agent with reduced side effects resulting from inhibition of wild-type EGFR, an antitumor agent serving as an inhibitor, which can provide high selectivity for an EGFR mutant with an insert in exon 20, for which the therapeutic effects of previously known EGFR inhibitors are insufficient. .

The solution of the problem

[0010]

The present inventors have conducted extensive studies and found that the EGFR mutant with the exon 20 insert is a suitable target in the treatment of cancer, and that EGFR inhibitors commonly used for treatment have low selectivity between wild-type EGFR and the EGFR mutant with the exon 20 insert. In addition, the inventors also confirmed that the particular compound exhibits exon 20 insertion mutant EGFR selectivity and tumor growth inhibitory effects, and thus is considered superior to afatinib, which is a typical drug for EGFR mutation-positive cancer. Due to this discovery, the inventors have made the present invention.

[0011]

Accordingly, the present invention includes the following embodiments.

[0012]

Paragraph 1.

An anticancer agent for the treatment of a patient with an EGFR-expressing cancer having a mutation with an insertion in exon 20, comprising a compound selected from the group consisting of:

(S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide (hereinafter also called Compound A);

(S)-N-(4-amino-6-methylene-5-(quinolin-3-yl)-7,8-dihydro-6H-pyrimido[5,4-b]pyrrolisin-7-yl)acrylamide (hereinafter herein also referred to as Compound B);

(S,E)-N-(4-amino-6-methylene-5-(quinolin-3-yl)-7,8-dihydro-6H-pyrimido[5,4-b]pyrrolisin-7-yl)- 3-chloroacrylamide (hereinafter also referred to as Compound C); and

(R)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)-N-methylacrylamide (hereinafter in also referred to herein as Compound D),

or its salt.

[0013]

Point 2.

The anticancer agent of claim 1 wherein the compound is (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8 -yl) acrylamide.

[0014]

Point 3.

The antitumor agent according to claim 1 or 2, wherein the patient with an EGFR-expressing cancer having a mutation with an insert in exon 20 is a patient with lung cancer, breast cancer, head and neck cancer, brain tumor, uterine cancer, hematological malignancy, or skin cancer .

[0015]

Item 4.

The anticancer agent of any one of 1 to 3, wherein the patient with an EGFR-expressing cancer having a mutation with an insert in exon 20 is a lung cancer patient.

[0016]

Item 5.

The anticancer agent of any one of 1-4, wherein the exon 20 insertion mutation is a mutation where one or more amino acids are inserted in the exon 20 region.

[0017]

Item 6.

The anticancer agent of any one of 1-5, wherein the exon 20 insertion mutation is a mutation where 1-7 amino acids are inserted in the exon 20 region.

[0018]

Item 7.

The anticancer agent of any one of 1-6, wherein the exon 20 insertion mutation is a mutation where 1-4 amino acids are inserted in the exon 20 region.

[0019]

Item 8.

Противоопухолевое средство по любому из пунктов 1-7, где мутация со вставкой в экзоне 20 представляет собой A763_Y764insFQEA, V769_D770insASV, D770_N771insSVD, D770_N771insNPG, D770_N771insG, D770>GY, N771_P772insN, P772_R773insPR, H773_V774insNPH, H773_V774insPH, H773_V774insAH, H773_V774insH, V774_C774insHV или A761_E762insEAFQ.

[0020]

Item 9.

The anticancer agent of any one of 1-8, wherein the mutation with the insertion in exon 20 is V769_D770insASV, D770_N771insSVD, D770_N771insG, H773_V774insNPH, or H773_V774insPH.

[0021]

Item 10.

A method of treating a patient with a malignant tumor, comprising the step of administering an anticancer agent containing an effective amount of a compound selected from the group consisting of:

(S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide;

(S)-N-(4-amino-6-methylene-5-(quinolin-3-yl)-7,8-dihydro-6H-pyrimido[5,4-b]pyrrolisin-7-yl)acrylamide;

(S,E)-N-(4-amino-6-methylene-5-(quinolin-3-yl)-7,8-dihydro-6H-pyrimido[5,4-b]pyrrolisin-7-yl)- 3-chloroacrylamide; and

(R)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)-N-methylacrylamide,

or a salt thereof to a patient with a cancer expressing an EGFR having an insertion mutation in exon 20.

[0022]

Item 11.

A compound selected from the group consisting of:

(S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide;

(S)-N-(4-amino-6-methylene-5-(quinolin-3-yl)-7,8-dihydro-6H-pyrimido[5,4-b]pyrrolisin-7-yl)acrylamide;

(S,E)-N-(4-amino-6-methylene-5-(quinolin-3-yl)-7,8-dihydro-6H-pyrimido[5,4-b]pyrrolisin-7-yl)- 3-chloroacrylamide; and

(R)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)-N-methylacrylamide,

or a salt thereof for the treatment of a patient with a cancer expressing EGFR having an insertion mutation in exon 20.

[0023]

Item 12.

Application of a compound selected from the group consisting of:

(S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide;

(S)-N-(4-amino-6-methylene-5-(quinolin-3-yl)-7,8-dihydro-6H-pyrimido[5,4-b]pyrrolisin-7-yl)acrylamide; and

(S,E)-N-(4-amino-6-methylene-5-(quinolin-3-yl)-7,8-dihydro-6H-pyrimido[5,4-b]pyrrolisin-7-yl)- 3-chloroacrylamide; and

(R)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)-N-methylacrylamide,

or a salt thereof for the preparation of an antineoplastic agent for the treatment of a patient with a cancer expressing EGFR having an exon 20 insertion mutation.

Useful effects of the invention

[0024]

The antitumor agent of the present invention exhibits high selectivity for an EGFR mutant with an insert in exon 20 without inhibition of wild-type EGFR. Thus, the antitumor agent of the present invention is useful in terms of providing an antitumor agent having reduced side effects resulting from wild-type EGFR inhibition; and having excellent therapeutic effects for a patient with an EGFR-expressing cancer having an insertion mutation at exon 20, for whom the therapeutic effects of previously known EGFR inhibitors are insufficient.

[0025]

Previously known EGFR inhibitors have low selectivity for the exon 20 insertion mutant EGFR compared to wild-type EGFR; thus, the difference between the dosage for providing antitumor effects and the dosage for causing side effects (skin diseases, gastrointestinal disorders, and the like) due to wild-type EGFR inhibition was small. Accordingly, previously known EGFR inhibitors have difficulty in providing sufficient therapeutic effects. On the contrary, since the antitumor agent of the present invention has a high selectivity for the EGFR mutant with an insert in exon 20, the dosage can be increased without causing the side effects caused by wild-type EGFR. Thus, the antitumor agent of the present invention exhibits excellent therapeutic effects for a patient with a cancer expressing an EGFR having an insertion at exon 20.

Brief description of the drawings

[0026]

Figure 1 illustrates the IC ₅₀ ratios of wild-type EGFR to EGFR with insertion mutations at exon 20 calculated from cell growth inhibition tests on wild-type EGFR-expressing cell lines and mutant EGFR-expressing cell lines with Compounds A, B, C, and D, comparative compound, gefitinib, erlotinib and afatinib.

Figure 2 illustrates the GI ₅₀ ratios of wild-type EGFR to EGFR with insertion mutations at exon 20 calculated from cell growth inhibition tests on wild-type-expressing human EGFR cell lines and mutant EGFR-expressing human cell lines by Compounds A, B, C and D, comparative compound, gefitinib, erlotinib and afatinib.

3 illustrates the relative tumor volume (hereinafter also referred to as "RTV") in mouse models transplanted subcutaneously with mutant EGFR-expressing cell lines (NIH3T3-EGFRinsASV cells) to determine the antitumor effect of Compound A.

Fig. 4 illustrates the change in body weight after the formation of groups of mouse models that were subcutaneously transplanted with mutant EGFR-expressing cell lines (NIH3T3-EGFRinsASV cells) to determine the toxicity of compound A.

5 illustrates relative tumor volume in mouse models transplanted subcutaneously with mutant EGFR-expressing cell lines (NIH3T3-EGFRinsSVD cells) to determine the antitumor effect of Compound A.

Fig. 6 illustrates body weight after generation of mouse model groups that were subcutaneously transplanted with mutant EGFR-expressing cell lines (NIH3T3-EGFRinsSVD cells) to determine Compound A toxicity.

7 illustrates relative tumor volume in mouse models transplanted subcutaneously with mutant EGFR-expressing cell lines (H1975-EGFRinsSVD cells) to determine the antitumor effect of Compound A.

Fig. 8 illustrates the post-generation body weight of mouse models transplanted subcutaneously with mutant EGFR-expressing cell lines (H1975-EGFRinsSVD cells) to determine Compound A toxicity.

9 illustrates tumor volume in mouse models transplanted subcutaneously with mutant EGFR-expressing cell lines (NIH3T3-EGFRinsNPH) to determine the antitumor effect of Compound A.

Fig. 10 illustrates body weight after generation of groups of mouse models transplanted subcutaneously with mutant EGFR-expressing cell lines (NIH3T3-EGFRinsNPH) to determine compound A toxicity.

Fig. 11 illustrates tumor volume in rat models transplanted subcutaneously with mutant EGFR-expressing cell lines (H1975-EGFRinsSVD cells) to determine the antitumor effect of Compound A.

Fig. 12 illustrates body weight after generation of groups of rat models that were subcutaneously transplanted with mutant EGFR-expressing cell lines (H1975-EGFRinsSVD cells) to determine Compound A toxicity.

Fig. 13 illustrates tumor volume in mouse models subcutaneously transplanted with a tumor derived from a lung cancer patient who was positive for EGFR with the V769_D770insASV mutation to determine the antitumor effect of Compound A.

Fig. 14 illustrates body weight after grouping of mouse models that were subcutaneously transplanted with a tumor derived from a lung cancer patient who was positive for EGFR with the V769_D770insASV mutation to determine the toxicity of Compound A of the present invention.

In FIG. 15 shows the amino acid sequence of wild-type EGFR (SEQ ID NO:1).

Description of Embodiments

[0027]

Preferred examples of various definitions within the scope of the present invention used in this description are explained in detail below.

[0028]

As used herein, "EGFR" refers to the human epidermal growth factor receptor protein and is also referred to as ErbB-1 or HER1.

[0029]

As used herein, “wild-type EGFR” refers to EGFR free from somatic mutation, which is a protein comprising the amino acid sequence shown in SEQ ID NO:1 (GenBank accession number: NP_005219.2).

[0030]

As used herein, "exon 20 insertion mutation" refers to a mutation where one or more amino acids (preferably 1-7, more preferably 1-4) are inserted in the region of exon 20 (amino acid sequence 761 to 823 in SEQ ID NO: 1) EGFR and is preferably a mutation where the amino acid sequence FQEA (phenylalanine, glutamine, glutamic acid and alanine in this order from the N-terminus) is inserted between alanine 763 and tyrosine 764 in the exon 20 region (A763_Y764insFQEA ); a mutation where the ASV amino acid sequence (alanine, serine and valine in that order from the N-terminus) is inserted between valine 769 and aspartic acid 770 in the region of exon 20 (V769_D770insASV); a mutation where the amino acid sequence SVD (serine, valine and aspartic acid in this order from the N-terminus) is inserted between aspartic acid 770 and asparagine 771 in the region of exon 20 (D770_N771insSVD); a mutation where the amino acid sequence NPG (asparagine, proline and glycine in this order from the N-terminus) is inserted between aspartic acid 770 and asparagine 771 in the region of exon 20 (D770_N771insNPG); a mutation where the amino acid G (glycine) is inserted between the 770th aspartic acid and the 771st asparagine (D770_N771insG); a mutation where the 770th aspartic acid in the region of exon 20 is deleted and the amino acid sequence GY (glycine and tyrosine in this order from the N-terminus) is inserted instead (D770>GY); a mutation where the amino acid N (asparagine) is inserted between asparagine 771 and proline 772 in the region of exon 20 (N771_P772insN); a mutation where the amino acid sequence PR (proline and arginine in this order from the N-terminus) is inserted between proline 772 and histidine 773 in the region of exon 20 (P772_R773insPR); a mutation where the amino acid sequence NPH (asparagine, proline and histidine in this order from the N-terminus) is inserted between histidine 773 and valine 774 in the region of exon 20 (H773_V774insNPH); a mutation where the amino acid sequence PH (proline and histidine in this order from the N-terminus) is inserted between histidine 773 and valine 774 in the region of exon 20 (H773_V774insPH); a mutation where the amino acid sequence AH (alanine and histidine in this order from the N-terminus) is inserted between histidine 773 and valine 774 in the region of exon 20 (H773_V774insAH); a mutation where the amino acid H (histidine) is inserted between histidine 773 and valine 774 in the region of exon 20 (H773_V774insH); a mutation where the HV amino acid sequence (histidine and valine in that order from the N-terminus) is inserted between valine 774 and cysteine 775 in the region of exon 20 (V774_C775insHV); a mutation where the amino acid sequence EAFQ (glutamic acid, alanine, phenylalanine, and glutamine in that order from the N-terminus) is inserted between alanine 761 and glutamic acid 762 in the region of exon 20 (A761_E762insEAFQ); etc. More preferred mutations include a mutation where the ASV amino acid sequence (alanine, serine and valine in that order from the N-terminus) is inserted between valine 769 and aspartic acid 770 in the exon 20 region (V769_D770insASV); a mutation where the amino acid sequence SVD (serine, valine and aspartic acid in this order from the N-terminus) is inserted between aspartic acid 770 and asparagine 771 in the region of exon 20 (D770_N771insSVD); a mutation where amino acid G (glycine) is inserted between aspartic acid 770 and asparagine 771 in the region of exon 20 (D770_N771insG); a mutation where the amino acid sequence NPH (asparagine, proline and histidine in this order from the N-terminus) is inserted between histidine 773 and valine 774 in the region of exon 20 (H773_V774insNPH); and a mutation where the amino acid sequence PH (proline and histidine in this order from the N-terminus) is inserted between histidine 773 and valine 774 in the region of exon 20 (H773_V774insPH). More preferred mutations include a mutation where the SVD amino acid sequence (serine, valine and aspartic acid in this order from the N-terminus) is inserted between aspartic acid 770 and asparagine 771 in the exon 20 region (D770_N771insSVD); and a mutation wherein amino acid G (glycine) is inserted between aspartic acid 770 and asparagine 771 in the region of exon 20 (D770_N771insG).

[0031]

As used herein, "an EGFR-expressing cancer patient having an exon 20 insertion mutation" refers to an EGFR-expressing cancer patient having an exon 20 insertion mutation in at least one portion of the EGFR exon 20 region. EGFR may have an insertion mutation in exon 20 in two or more different parts, but preferably in one part. In addition, EGFR may also have a mutation other than an insertion mutation in exon 20 (such as a deletion mutation in exon 19, an L858R mutation, or an L790M mutation).

[0032]

In the present invention, the method for detecting an insertion mutation in exon 20 of EGFR expressed in a cancer patient is not particularly limited as long as the method is capable of detecting a mutation, and any known detection methods can be used. The target of detection when detecting a mutation with an insertion in exon 20 can be any of the genomic sequence of the EGFR gene, the transcription product of the EGFR gene, and the EGFR protein.

[0033]

The sample used to detect a mutation with an insertion in exon 20 is not specifically limited, as long as the sample is a biological sample isolated from a cancer patient, in particular, a sample that is obtained from a cancer patient and contains cancer cells. . Examples of biological samples include biological fluids (eg, blood, urine, and the like), tissues, their extracts, and tissue cultures obtained. The method for isolating the biological sample can be appropriately selected depending on the type of the biological sample.

[0034]

The biological sample is obtained by appropriate processing in accordance with the detection method. In addition, a reagent used for detection (for example, a reagent containing a primer or a probe) can be obtained in a conventional manner according to the detection method.

[0035]

In one embodiment of the present invention, the step of detecting the presence of an insertion mutation in exon 20 of EGFR expressed in a cancer patient may be performed prior to administration of an antitumor agent to the cancer patient.

[0036]

Compounds A to D (Compounds A, B, C, and D) (in the present specification, these compounds are also commonly referred to as "compound of the present invention" or "compound of the present invention") and a method for preparing the same are described below.

[0037]

Compound A ((S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide) is represented by the following chemical formula.

[0038]

[0039]

Compound B ((S)-N-(4-amino-6-methylene-5-(quinolin-3-yl)-7,8-dihydro-6H-pyrimido[5,4-b]pyrrolisin-7-yl) acrylamide) is represented by the following chemical formula.

[0040]

[0041]

Compound C ((S,E)-N-(4-amino-6-methylene-5-(quinolin-3-yl)-7,8-dihydro-6H-pyrimido[5,4-b]pyrrolisin-7- yl)-3-chloroacrylamide) is represented by the following chemical formula.

[0042]

[0043]

Compound D ) is represented by the following chemical formula.

[0044]

[0045]

Compounds A-D can be obtained, for example, using the production method disclosed in WO2015/025936A1, the methods described in the examples, and the like. However, the preparation methods for compounds A to D are not limited to these reaction examples.

[0046]

When compounds A-D of the present invention have isomers such as optical isomers, stereoisomers, rotational isomers and tautomers, any isomers and mixtures thereof are included within the scope of the compound of the present invention unless otherwise indicated. For example, when compounds A-D of the present invention have optical isomers, racemic mixtures and optical isomers isolated from the racemic mixture are also included within the scope of the compound of the present invention, unless otherwise indicated.

[0047]

Salts of compounds A-D refer to any pharmaceutically acceptable salts; examples include base addition salts and acid addition salts.

[0048]

Examples of base addition salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts 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.

[0049]

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, isethionates, benzenesulfonates and p-toluenesulfonates.

[0050]

Compounds A-D and their salts also include their prodrugs. A prodrug refers to a compound that is converted to compounds A to D, or salts thereof, by reaction with an enzyme, gastric acid, or the like under physiological conditions in vivo , i. a compound that is converted into a compound of the present invention or a salt thereof by enzymatic oxidation, reduction, hydrolysis or the like; or a compound that is converted into compounds A to D or a salt thereof by hydrolysis or the like with gastric acid or the like. In addition, the prodrug may be compounds that can be converted to compounds A-D or their salts under physiological conditions, such as described in " Iyakuhin no Kaihatsu [Development of Pharmaceuticals]," Vol. 7, Molecular Design, published in 1990 by Hirokawa Shoten Co., pp. 163-198.

[0051]

Description of diseases

Specific examples of tumors targeted by the present invention include, but are not limited to, head and neck cancer, gastrointestinal cancer (esophageal cancer, gastric cancer, duodenal cancer, liver cancer, biliary tract cancer (e.g., gallbladder cancer). and bile ducts), pancreatic cancer, colorectal cancer (for example, colon cancer and rectal cancer), etc.), lung cancer (for example, non-small cell lung cancer, small cell lung cancer and mesothelioma), breast cancer, cancer genital organs (ovarian cancer, uterine cancer (such as cervical and endometrial cancer), etc.), genitourinary system cancer (such as kidney cancer, bladder cancer, prostate cancer and testicular tumor), hematological malignancy (such as leukemia, malignant lymphoma and multiple myeloma), osteosarcoma, soft tissue sarcoma, skin cancer, brain tumor, and the like. Preferred examples include lung cancer, breast cancer, head and neck cancer, brain tumor, uterine cancer, hematoblastosis, or skin cancer.

[0052]

When compounds A to D or salts thereof are used as a pharmaceutical agent, a pharmaceutical carrier may be added, if necessary, thus forming a suitable dosage form in accordance with the goals of prevention and treatment. Examples of the dosage form include oral preparations, injection forms, suppositories, ointments, patches, and the like. Such dosage forms can be prepared by methods generally known to those skilled in the art.

[0053]

As a pharmaceutical carrier, various solid organic or inorganic carrier materials used as preparation materials can be mixed as an excipient, a binder, a disintegrant, a lubricant, or a coloring agent in the case of solid drug forms; or as a solvent, solubilizing agent, suspending agent, isotonic agent, buffer or emollient in the case of liquid drug forms. In addition, additives such as antiseptics, antioxidants, coloring agents, sweeteners and stabilizers can also be used in pharmaceutical preparations if necessary.

[0054]

Solid oral drugs are prepared as follows. After adding an excipient, optionally with a binder, a disintegrant, a lubricant, a coloring agent, a taste-masking agent or a flavoring agent, etc., to compounds A-D, the resulting mixture is formed into tablets, coated tablets, granules, powders , capsules or the like by conventional methods.

[0055]

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, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylstarch, methylcellulose, ethylcellulose, shellac, calcium phosphate, polyvinylpyrrolidone etc. Examples of disintegrants include dry starch, sodium alginate, agar powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, lactose, and the like. Examples of lubricants include purified talc, sodium stearate, magnesium stearate, borax, polyethylene glycol, and the like. Examples of dyes include titanium oxide, iron oxide, and the like. Examples of taste-masking agents or flavors include sucrose, orange peel, citric acid, tartaric acid, and the like.

[0056]

When preparing a liquid preparation for oral administration, a taste-masking agent, a buffer, a stabilizer, a flavor, and the like can be added to compounds A to D; and the resulting mixture may be formulated as an oral liquid preparation, syrup, elixir, and the like. in accordance with the usual way.

[0057]

Examples of a taste-masking agent or flavor include those mentioned above. Examples of buffers include sodium citrate and the like. Examples of stabilizers include tragacanth, gum arabic, gelatin, and the like. If desired, these oral formulations may be coated according to methods known in the art with an enteric coating or other coating for the purpose of, for example, maintaining the effects. Examples of such coating agents include hydroxypropyl methylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, polyoxyethylene glycol, and Tween 80 (registered trademark).

[0058]

When preparing a solution for injection, a pH adjuster, a buffer, a stabilizer, an isotonic agent, a local anesthetic, and the like can be added to compounds A-D; and the mixture may be prepared as a solution for subcutaneous, intramuscular or intravenous injection according to a conventional method.

[0059]

Examples of the pH adjusting agent and buffer used herein include sodium citrate, sodium acetate, and sodium phosphate. Examples of the stabilizer include pyrosulfite, EDTA, thioglycolic acid and thiolactic acid. Examples of a local anesthetic include procaine hydrochloride and lidocaine hydrochloride. Examples of the tonicity agent include sodium chloride, glucose, D-mannitol, and glycerin.

[0060]

Upon receipt of the suppository, pharmaceutically acceptable carriers known to the person skilled in the art, such as polyethylene glycol, lanolin, cocoa butter and fatty acid triglyceride; and, if necessary, surfactants such as Tween 80 (registered trademark) can be added to compounds A-D and the resulting mixture can be prepared as a suppository according to the usual method.

[0061]

When preparing an ointment, a commonly used base, stabilizer, wetting agent, preservative and the like can be mixed with compounds A to D if necessary; and the resulting mixture may be mixed and prepared as an ointment according to a conventional method.

[0062]

Examples of the base include liquid paraffin, white petrolatum, white beeswax, octyldodecyl alcohol and paraffin.

[0063]

Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, and propyl paraoxybenzoate.

[0064]

Upon receipt of the patch, the above-described ointment, cream, gel, paste or the like can be applied to a conventional substrate in accordance with a conventional method.

[0065]

Examples of substrates include woven fabrics or non-woven fabrics, including cotton, staple fibers or man-made fibers; and films or foam sheets of soft vinyl chloride, polyethylene, polyurethane, and the like. are suitable.

[0066]

The amount of compounds A-D to be included in each of such unit dosage forms depends on the condition of the patient to whom the compound is administered, its dosage forms, and the like. Generally, in the case of an oral agent, the amount of the compound is preferably 0.05-1000 mg per unit dosage form. In the case of injection, the amount of the compound is preferably 0.01-500 mg per unit dosage form; and in the case of a suppository, the amount of the compound is preferably 1-1000 mg per unit dosage form.

[0067]

In addition, the daily dose of the drug in such a dosage form depends on the condition, body weight, age, sex, and the like. patient and cannot be generalized. For example, an adult daily dose (body weight: 50 kg) of compounds A to D as an active ingredient may generally be 0.05-5000 mg, and preferably 0.1-1000 mg; and is preferably administered in a single dose or in two to three divided doses per day.

[0068]

The present invention also relates to a method of treating a cancer patient comprising the step of administering an effective amount of an anticancer agent comprising a compound selected from the group consisting of compounds A to D, or a salt thereof, to a cancer patient expressing EGFR having an exon insertion mutation twenty.

[0069]

The present invention also relates to a compound selected from the group consisting of compounds A to D, or a salt thereof, for the treatment of a patient with a cancer expressing an EGFR having an exon 20 insertion mutation.

[0070]

The present invention also relates to the use of a compound selected from the group consisting of compounds A to D, or a salt thereof, for the treatment of a patient with a cancer expressing an EGFR having an exon 20 insertion mutation.

[0071]

The present invention also relates to the use of a compound selected from the group consisting of compounds A to D or a salt thereof for the preparation of an antitumor agent for the treatment of a patient with a cancer expressing an EGFR having an exon 20 insertion mutation.

[0072]

The present invention is also a method for predicting the therapeutic effects of chemotherapy using an antitumor agent comprising, as an active ingredient, a compound selected from the group consisting of compounds A to D, or a salt thereof, in a patient with a malignant tumor, the method comprising steps (1) and ( 2) below:

(1) a step of detecting the presence or absence of a mutation in the EGFR gene contained in a biological sample obtained from a patient; and

(2) the step of predicting that the patient is highly likely to have sufficient therapeutic effects from chemotherapy when the detection results in step (1) show that the EGFR gene has an insertion mutation in exon 20.

[0073]

The present invention is also a method for treating a cancer patient, comprising steps (1) to (3) below:

(1) a step of detecting the presence or absence of a mutation in the EGFR gene contained in a biological sample obtained from a patient;

(2) a step of predicting that a patient is highly likely to have sufficient therapeutic effects from chemotherapy using an antitumor agent comprising a compound selected from the group consisting of compounds A to D or a salt thereof, when the detection results of step (1) show that the EGFR gene has a mutation with an insertion in exon 20; and

(3) the step of administering an anticancer agent to a patient that is predicted by step (2) to respond sufficiently to chemotherapy with a high probability.

[0074]

The base sequence of the EGFR gene is well known. The GenBank identification number of the cDNA base sequence is NM_005228.4.

[0075]

"Therapeutic effects" can be judged based on tumor shrinking effects, recurrence suppression effects, survival extension effects, and the like. The relapse suppression effects can be represented as the degree of increase in relapse-free life and/or the degree of improvement in the recurrence rate; and the effects of increased life expectancy can be represented as a degree of overall survival time and/or a degree of increase in median disease-free survival or the like. "Sufficient therapeutic effects" of chemotherapy using an antitumor agent comprising Compound A or a salt thereof as an active ingredient means that excellent therapeutic effects are obtained by administering an antitumor agent comprising Compound A or a salt thereof as an active ingredient, such as a significant increase in time survival, significant suppression of recurrence, and the like, compared with treatment without administration.

Examples

[0076]

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

[0077]

Test Example 1

In vitro drug efficacy test

Evaluation of the effect of cell growth inhibition on cell lines expressing wild-type or EGFR mutant EGFR (1)

The inhibitory activity of the compounds against wild-type and mutant EGFR was evaluated using Ba/F3 cells (murine B-lymphocyte progenitor cell lines) introduced with human EGFR genes. Ba/F3 cells were maintained in RPMI-1640 medium (Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin/100 μg/mL streptomycin (Thermo Fisher Scientific) and 1 ng/mL interleukin-3 mice (mlL-3) (CST). PB-CMV-MCS-EF1-GFP+Puro vector or PB-CMV-MCS-EF1-RFP+Puro vector encoded for the human EGFR gene (wild type (WT), V769_D770insASV (insASV), D770_N771insSVD (insSVD), D770_N771insG (insG), H773_V774insNPH (insNPH) or H773_V774insPH (insPH)), were introduced into cells together with the transposase Super PiggyBac expression vector by electroporation using Amaxa (trademark) of the V Cell Line Nucleofector kit (trademark) followed by selection using puromycin (SIGM). Ba/F3 cells expressing wild-type EGFR (hereinafter also referred to as "Ba/F3-EGFR_WT") showed mlL-3 independent growth in the presence of 50 ng/ml EGF (R&D Systems); and Ba/F3 cells expressing a mutant EGFR with an insert at exon 20 (hereinafter also referred to as ʺBa/F3-EGFRinsASV,ʺ ʺBa/F3-EGFRinsSVD,ʺ ʺBa/F3-EGFRinsG,ʺ ʺBa/F3-EGFRinsNPH,ʺ or ʺBa/F3-EGFRinsPHʺ) showed mlL-3 independent growth in the absence of EGF.

[0078]

To evaluate the effect of cell growth inhibition, Ba/F3-EGFR_WT cells were suspended in RPMI-1640 medium containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 ng/ml EGF; and the cell suspension was seeded in each well of a 96-well flat-bottomed microplate so that the number of cells per well was 30,000. Ba/F3 cells expressing EGFR with an exon 20 insertion mutation were suspended in RPMI-1640 medium containing 10% FBS , 100 u/ml penicillin and 100 mcg/ml streptomycin; and the cell suspension was seeded in each well of a 96-well flat-bottomed microplate so that the number of cells per well was 15,000. Then, (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl) -8,9-dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide (compound A), (S)-N-(4-amino-6-methylene-5-(quinolin-3-yl)- 7,8-dihydro-6H-pyrimido[5,4-b]pyrrolisin-7-yl)acrylamide (compound B), (S,E)-N-(4-amino-6-methylene-5-(quinoline- 3-yl)-7,8-dihydro-6H-pyrimido[5,4-b]pyrrolisin-7-yl)-3-chloroacrylamide (compound C), and (R)-N-(4-amino-6- methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)-N-methylacrylamide (compound D) prepared according to the preparation method disclosed in PTL 2 , and (S)-N-(4-amino-5-(quinolin-3-yl)-6,7,8,9-tetrahydropyrimido[5,4-b]indolizin-8-yl)acrylamide prepared according to with the method of preparation disclosed in WO2013/125709A1 (the compound of example 1 in WO2013/125709A1, which is also referred to as "comparative compound" below) was dissolved in DMSO, and diluted with DMSO or medium milk used to suspend the cells. These compounds were individually added to each well of the cell culture plate and incubated in a 5% CO ₂ gas-containing incubator at 37° C. for 3 days. The number of cells after incubation was measured using CellTiter-Glo (trademark) for fluorescent cell viability assay (Promega Corporation) according to the manufacturer's recommended protocol. The growth rate was calculated using the following formula, and the concentration of each test compound for 50% inhibition (IC ₅₀ (μM)) was determined.

[0079]

Growth rate (%)=T/C×100

T: luminescence intensity of the well to which the test compound was added.

C: luminescence intensity of a well to which no test compound has been added.

[0080]

In addition, the ratio of IC ₅₀ between wild-type EGFR and EGFR with an insertion mutation at exon 20 was determined using the following formula. In FIG. 1 shows the results.

[0081]

ratio IC ₅₀ =IC ₅₀ (WT)/IC ₅₀ (ex20ins)

IC_fifty(WT): IC_fiftyfor EGFR wild type

IC ₅₀ (ex20ins): IC ₅₀ for EGFR with insertion mutation in exon 20

[0082]

As can be seen from FIG. 1, compounds A-D show inhibitory effects on cell growth on cell lines expressing EGFR with an exon 20 insert; and their mutational selectivity was higher than that of the comparative compound, gefitinib, erlotinib and afatinib.

[0083]

Test Example 2

Effect of cell growth inhibition on wild-type or mutant EGFR-expressing human cell lines (2)

The following cells were used to evaluate the inhibitory activity of the compounds against wild-type EGFR and mutant EGFR: NCI-H1975 cells, which are human lung adenocarcinoma cell lines expressing EGFR with the D770_N771insSVD mutation by gene modification (which is also referred to as “H1975- EGFRinsSVD"); and A431 cells, which are human epithelial cancer cell lines expressing wild-type EGFR. H1975-EGFRinsSVD cells were generated as follows. The PB-CMV-MCS-EF1-RFP+Puro vector encoded for D770_N771insSVD (insSVD) was introduced into NCI-H1975 cells, along with the Super PiggyBac expression vector Transposase, by electroporation using an Amaxa (trademark) R Cell Line kit Nucleofector (trademark), followed by selection using puromycin (SIGMA). XTN (trademark) TALEN site-specific nucleases (Transposagen) were introduced into cells by electroporation using the Amaxa (trademark) set of R cell lines Nucleofector (trademark), and endogenous-EGFR (T790M/L858R)-knockout cells were selected by sequencing.

[0084]

To evaluate the effect of cell growth inhibition, individual cell types were suspended in the medium recommended by ATCC. Cell suspensions were seeded in each well of respective 96-well flat bottom plates so that the number of cells per well was 3000 and incubated in a 5% CO ₂ -containing incubator at 37°C for 1 day. Compound A, reference compound, gefitinib, erlotinib, and afatinib were individually dissolved in DMSO, and diluted with DMSO such that the concentration of these test compounds was 200 times the final concentration. These DMSO solutions of the test compounds were diluted with the medium used for cell suspension and added to each well of the cell culture plates so that the final concentration of DMSO was 0.5%, and the cells were incubated in a 5% CO ₂ -containing incubator at 37°C in within 3 days. The number of cells at the start of incubation (Day 0) and the number of cells after incubation (Day 3) were measured using CellTiter-Glo (trademark) for fluorescent cell viability assay (Promega Corporation) according to the manufacturer's recommended protocol. The growth rate was calculated using the following formula, and the concentration of each test compound for 50% inhibition (GI ₅₀ (μM)) was determined. Table 1 shows the results.

[0085]

1) If T on day 3 ≥ C on day 0:

Growth rate (%)=(T on day 3 - C on day 0)/(C on day 3 - C on day 0)×100

T: luminescence intensity of the well to which the test compound was added.

C: luminescence intensity of a well to which no test compound has been added.

Day 0: The day the test compound was added.

Day 3: The day the assessment was made.

[0086]

2) If T on day 3 < C on day 0:

Growth rate (%)=(T on day 3 - C on day 0)/(C on day 0)×100

T: luminescence intensity of the well to which the test compound was added.

C: luminescence intensity of a well to which no test compound has been added.

Day 0: The day the test compound was added.

Day 3: The day the assessment was made.

[0087]

Table 1

GI ₅₀ (µM)

A431 H1975EGFRinsSVD Connection A 0.396 0.031 Comparative connection 0.543 0.364 gefitinib 0.310 1.903 erlotinib 0.612 2.775 afatinib 0.023 0.189 osimertinib 0.321 0.194

[0088]

In addition, the ratio of GI ₅₀ between wild-type EGFR and EGFR with an insertion mutation at exon 20 was determined using the following formula. Table 2 shows the results.

[0089]

Ratio GI ₅₀ =GI ₅₀ (A431)/GI ₅₀ (H1975 EGFRinsSVD)

GI_fifty(A431): GI_fiftyfor EGFR wild type

GI ₅₀ (H1975 EGFRinsSVD): GI ₅₀ for EGFR with insertion mutation at exon 20

[0090]

As can be seen from Table 1 and Fig. 2, Compound A exhibited a cell growth inhibitory effect on cell lines expressing a mutant EGFR with an insert at exon 20, their mutation selectivity was higher than that of the comparative compound, gefitinib, erlotinib, afatinib, and osimertinib.

[0091]

Test Example 3

Evaluation of the inhibitory activity of phosphorylated EGFR against wild-type or mutant EGFR-expressing human cell lines

A431 cells, which are human epithelial cancer cell lines overexpressing wild-type EGFR, and H1975-EGFRinsSVD cells, which are human lung adenocarcinoma cell lines expressing EGFR with a D770_N771insSVD mutation by gene modification, were suspended in appropriate media. These cell suspensions were individually seeded in a 60 mm dish and incubated in a 5% CO ₂ -containing incubator at 37°C for 1 day. Compound A was dissolved in DMSO, and diluted with DMSO so that the concentration of the test compound was 1000 times the final concentration. The DMSO solution of the test compound was diluted with each medium used for cell suspension, and each diluted solution was added to the respective cell culture dishes so that the final concentration of DMSO was 0.1%, followed by incubation in a 5% CO ₂ -containing incubator at 37 °C for 6 hours. After incubation, the cells were collected and stored at -80°C in the form of pellets until use. RIPA buffer (Thermo Fisher Scientific) containing a mixture of protease inhibitors (Thermo Fisher Scientific) was added to the pellets and intracellular proteins were extracted. Protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific) and each sample was adjusted to have a protein concentration suitable for measuring phosphorylated EGFR expression. Phosphorylated EGFR expression was measured using the Simple Western (Trademark) Assay System (ProteinSimple) according to the manufacturer's recommended protocol. The primary antibody used in the measurement was phospho-EGF receptor (Tyr1068) #3777 (CST) diluted to 1/50.

[0092]

For each cell type, a calibration curve of protein concentration (x-axis) and phosphorylated EGFR expression level (y-axis) was obtained, and the expression level of phosphorylated EGFR of each sample was converted to protein concentration based on the calibration curve. The rate of phosphorylated EGFR was calculated using the following formula to determine the test compound concentration at which phosphorylated EGFR is inhibited by 50% (IC ₅₀ (μM)).

[0093]

Phosphorylated EGFR rate (%)=T/C×100

T: equivalent amount for the protein concentration of the sample to which the test compound was added.

C: equivalent amount for the protein concentration of the sample to which no test compound has been added.

[0094]

In addition, selectivity for wild-type EGFR and EGFR with an insertion mutation at exon 20 was calculated using the following formula. Table 2 shows the results.

[0095]

Ratio IC ₅₀₌ IC ₅₀ (A431)/IC ₅₀ (H1975 EGFRinsSVD)

IC_fifty(A431): IC_fiftyfor EGFR wild type

IC ₅₀ (H1975 EGFRinsSVD): EGFR IC ₅₀ with insertion mutation at exon 20

[0096]

table 2

IC ₅₀ (µM) A431 H1975EGFRinsSVD IC ₅₀ (µM) 0.535 0.023 _IC50 ratio - 23.3

[0097]

As shown in Table 2, Compound A exhibited selective inhibitory activity against EGFR with an insertion mutation in exon 20.

[0098]

Test Example 4

Evaluation of the inhibitory activity of phosphorylated EGFR against wild-type or mutant EGFR-expressing human cell lines (2)

The autophosphorylation inhibitory activity of the compound against wild type and EGFR mutant EGFR was evaluated using NIH-3T3 cells, which are mouse fibroblast cell lines introduced with the human EGFR gene. NIH-3T3 cells were maintained in D-MEM (high glucose) medium (Wako Pure Chemical Industries, Ltd.) containing 10% neonatal calf serum (NBCS), 1500 mg/l sodium bicarbonate and 100 U/ml penicillin/100 µg/ml streptomycin (Thermo Fisher Scientific). The PB-CMV-MCS-EF1-RFP+Puro vector encoding the human EGFR gene (WT, insASV, insSVD, insG, insNPH, or insPH) was introduced into cells, together with the Super PiggyBac expression vector Transposase, by electroporation using Amaxa (trademark) of the R Cell Line Nucleofector kit (trademark), followed by selection using puromycin (SIGMA). NIH-3T3 cells expressing wild-type EGFR (hereinafter also referred to as 'NIH3T3-EGFR_WT') grew in the presence of 50 ng/ml EGF (R&D Systems) under 1% NBCS. NIH-3T3 cells expressing EGFR with an exon 20 insertion mutation (hereinafter also referred to as ʺNIH3T3-EGFRinsASV,ʺ ʺNIH3T3-EGFRinsSVD,ʺ ʺNIH3T3-EGFRinsG,ʺ ʺNIH3T3-EGFRinsNPHʺ, or ʺNIH3T3-EGFRinsPHʺ) grew in the absence of EGF under conditions 1 % NBCS.

[0099]

To evaluate EGFR autophosphorylation inhibitory activity, NIH3T3 cells injected with human EGFR were suspended in appropriate media. These cell suspensions were individually seeded in a 60 mm dish or 6-well flat bottom plate and incubated in a 5% CO ₂ -containing incubator at 37° C. for 1 day. Compound A was dissolved in DMSO, and diluted with DMSO so that the concentration of the test compound was 400 times the final concentration. Test compound DMSO solutions were diluted with cell suspension medium and added to cell culture dishes such that the final concentration of DMSO was 0.25%. In addition, EGF was added to the NIH3T3-EGFR_WT cell culture dish to give a final concentration of 50 ng/mL. All culture dishes were subjected to incubation. All culture dishes were subjected to incubation in a 5% CO ₂ -containing incubator at 37°C for 6 hours. After incubation, the cells were collected and stored at -80°C in the form of pellets until use. RIPA buffer (Thermo Fisher Scientific) containing a mixture of protease inhibitors (Thermo Fisher Scientific) was added to the pellets and intracellular proteins were extracted. Protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific) and each sample was adjusted to a protein concentration suitable for measuring phosphorylated EGFR expression. Phosphorylated EGFR expression was measured using the Simple Western (trademark) assay system (ProteinSimple) according to the manufacturer's recommended protocol. The primary antibody used in the measurement was phospho-EGF receptor (Tyr1068) #3777 (CST) diluted to 1/50.

[0100]

For each cell type, a calibration curve of protein concentration (x-axis) and phosphorylated EGFR expression level (y-axis) was obtained, and the expression level of phosphorylated EGFR of each sample was converted to protein concentration based on the calibration curve. The inhibition rate of phosphorylated EGFR was calculated using the following formula to determine the test compound concentration at which phosphorylated EGFR is inhibited by 50% (IC ₅₀ (μM)).

[0101]

Phosphorylated EGFR Inhibition Rate (%)=T/C×100

T: equivalent amount for the protein concentration of the sample to which the test compound was added.

C: equivalent amount for the protein concentration of the sample to which no test compound has been added.

[0102]

In addition, selectivity for wild-type EGFR and EGFR with an insertion mutation at exon 20 was calculated using the following formula. Table 3 shows the results.

[0103]

Ratio IC ₅₀ =IC ₅₀ (WT)/IC ₅₀ (EGFR mutation with insert in exon 20)

[0104]

Table 3

wt insASV insSVD insG insNPH insPH IC ₅₀ (µM) 0.571 0.197 0.033 0.073 0.083 0.159 _IC50 ratio - 2.9 17.1 7.9 6.9 3.6

[0105]

As shown in Table 3, Compound A exhibited selective inhibitory activity against EGFR with insertion mutations in exon 20.

[0106]

As can be seen from the results of Test Examples 1-4, Compounds A-D exhibited a cell growth inhibitory effect accompanied by an EGFR inhibitory effect on cell lines expressing EGFR with an exon 20 insertion mutation; and the effect and their mutational selectivity were higher than those of the comparative compound, gefitinib, erlotinib, afatinib and osimertinib.

[0107]

Test Example 5

In Vivo Drug Efficacy Test

Evaluation of the antitumor effect in a model with subcutaneously transplanted EGFR-expressing mutant cell lines

Nude mice were subcutaneously transplanted with NIH3T3-EGFRinsASV cells, NIH3T3-EGFRinsSVD cells, or H1975-EGFRinsSVD cells into which mutant human EGFR had been introduced. At the point when the tumor volume of the grafted tumor in nude mice grew to about 100-200 mm ³ , the mice were divided into groups, 5-6 mice per group, by stratified randomization such that the average tumor volume between groups was the same. Mice were then treated orally with Compound A or afatinib once a day for 14 consecutive days.

The dose of afatinib was 20 mg/kg/day, which is the maximum tolerated dose (the maximum dose at which weight loss during the dosing period is less than 20%) for 14 days, the dosing period in this test; and the compound A dose was 200 mg/kg/day (maximum tolerated dose). The maximum tolerated dose was determined according to the "Guidelines Involving Experimental Neoplasia Proposals in Mice and Rats" of the National Cancer Institute (NCI), from a humanitarian point of view.

[0108]

To compare changes in tumor growth over time due to administration of individual test compounds, the relative tumor volume (hereinafter also referred to as 'RTV') was calculated based on the tumor volume at the time the mice were grouped (which is taken as 1 for the tumor growth index) using the following formula . For the toxicity index, body weight was measured over time, and the average change in body weight (hereinafter also referred to as "BWC (%)") from the day the mice were divided into groups was calculated by the following formula. In FIG. 3-8 show changes in mean RTV and mean BWC in mice.

[0109]

RTV=(Tumor volume on day of tumor volume measurement)/(Tumor volume on day mice were divided into groups)

BWC (%)=(body weight measured on the day of body weight measurement)/(body weight on the day the mice were divided into groups)

[0110]

When the mean RTV in the Compound A group on the last day of assessment was less than the mean RTV in the afatinib group, and at the same time there was a statistically significant difference (Student's t-test, p<0, 05), Compound A was found to be significantly more effective than afatinib. Such a case is indicated by the symbol "*" in the figures. T/C (%) on the last day of evaluation was calculated according to the following formula. Table 4 shows the results.

[0111]

Table 4

transplanted tumor Compound 20 mg/kg 200 mg/kg NIH3T3
EGFRinsASV Compound A of the present invention N.D. 3 Afatinib 46 N.D. NIH3T3
EGFRinsSVD Compound A of the present invention N.D. 5 Afatinib 39 N.D. H1975
EGFRinsSVD Compound A of the present invention N.D. 6 Afatinib 79 N.D.

N.D.: no data available.

[0112]

As can be seen from the results of Figures 3-8 and Table 4, Compound A showed a significant antitumor effect on cell lines expressing EGFR with an exon 20 insertion mutation subcutaneously transplanted into nude mice. The effect was also superior to that of afatinib without symptoms such as severe weight loss, abnormal feces, or abnormal skin in mice.

[0113]

Test Example 6

In Vivo Drug Efficacy Test

Evaluation of the antitumor effect in a model with subcutaneously transplanted EGFR-expressing mutant cell lines

Naked mice were subcutaneously transplanted with NIH3T3-EGFRinsNPH cells into which mutant human EGFR was introduced. At the point when the volume of the inoculated tumor in nude mice grew to about 100-200 mm ³ , the mice were divided into groups, 6 mice per group, by stratified randomization so that the average tumor volume between groups was the same. Mice were then treated orally with Compound A or afatinib once a day for 10 consecutive days.

[0114]

The dose of afatinib was 20 mg/kg/day, which is the maximum tolerated dose (the maximum dose at which weight loss during the dosing period is less than 20%); and the dose of Compound A was 100 and 200 mg/kg/day. The maximum tolerated dose was determined according to the "Guidelines Involving Experimental Neoplasia Proposals in Mice and Rats" of the National Cancer Institute (NCI), from a humanitarian point of view.

[0115]

To compare changes in tumor growth over time due to the administration of individual test compounds, the tumor volume (hereinafter also referred to as 'TV') of each mouse was calculated using the following formula. For the toxicity index, body weight was measured over time, and the change in body weight (hereinafter also referred to as "BWC (%)") from the day the mice were divided into groups was calculated by the following formula. In FIG. 9 and 10 show the changes in mean TV and mean BWC of mice.

[0116]

TV (mm ³ )=(major axis × short axis ² )/2

BWC (%)=(body weight measured on the day of body weight measurement)/(body weight on the day the mice were divided into groups)

[0117]

When the mean TV in the compound A group on the day after the final administration was less than the mean TV in the control group, at the same time a statistically significant difference was observed (Dunnett's test, p<0.05), it was determined that compound A is effective in antitumor effect. Such a case is indicated by the symbol "*" in the figures. T/C (%) on the last day of evaluation was calculated according to the following formula. Table 5 shows the results.

[0118]

T/C (%)=(tumor volume of test compound administered group)/(tumor volume of control group)

[0119]

Table 5

transplanted tumor Compound 20 mg/kg 200 mg/kg NIH3T3
EGFRinsNPH Connection A N.D. 2 Afatinib 59 N.D.

N.D.: no data available.

[0120]

As can be seen from FIG. 9 and 10 and Table 5, Compound A of the present invention demonstrated a significant antitumor effect on cell lines expressing EGFR with an insertion mutation at exon 20 subcutaneously transplanted into nude mice, accompanied by tumor growth inhibition or tumor regression. In the evaluation, the mice also did not show severe weight loss.

[0121]

Test Example 7

Evaluation of the antitumor effect in a rat model with subcutaneously transplanted EGFR-mutant-expressing cell lines

Nude rats were subcutaneously transplanted with H1975-EGFRinsSVD cells into which mutant human EGFR was introduced. At the point when the tumor volume of the grafted tumor in nude rats grew to about 200-500 mm ³ , the rats were divided into groups, 6 rats per group, by stratified randomization such that the average tumor volume between groups was the same. The rats were then orally administered Compound A once a day for 14 consecutive days.

[0122]

The dose was 20 or 40 mg/kg/day, which is less than the maximum tolerated dose (the maximum dose at which weight loss during the dosing period is less than 20%) for 14 days, the dosing period in this test. The maximum tolerated dose was determined according to the "Guidelines Involving Experimental Neoplasia Proposals in Mice and Rats" of the National Cancer Institute (NCI), from a humanitarian point of view.

To compare changes in tumor growth over time due to the administration of test compounds, the tumor volume (hereinafter also referred to as 'TV') of each rat was calculated using the following formula. For the toxicity index, body weight was measured over time, and the change in body weight (hereinafter also referred to as "BWC (%)") from the day the rats were divided into groups was calculated by the following formula. In FIG. 11 and 12 show the changes in mean TV and mean BWC of rats.

[0123]

TV (mm ³ )=(major axis × short axis ² )/2

BWC (%)=(body weight measured on the day of body weight measurement)/(body weight on the day the rats were divided into groups)

[0124]

When the mean TV in the compound A group on the last day of evaluation was less than the mean TV in the control group, while a statistically significant difference was observed (Dunnett's test, p<0.05), it was determined that compound A is effective in antitumor effect. Such a case is indicated by the symbol "*" in the figures. T/C (%) on the last day of evaluation was calculated according to the following formula. Table 6 shows the results.

[0125]

T/C (%)=(tumor volume of test compound administered group)/(tumor volume of control group)

[0126]

Table 6

transplanted tumor Compound 20 mg/kg 40 mg/kg H1975
EGFRinsSVD Connection A 7 3

[0127]

As can be seen from FIG. 11 and 12 and Table 6, compound A demonstrated a significant antitumor effect on cell lines expressing EGFR with an insertion mutation at exon 20 subcutaneously transplanted into nude rats, accompanied by tumor growth inhibition or tumor regression. In the evaluation, the rats did not show severe weight loss.

[0128]

Test Example 8

Evaluation of the antitumor effect in a mouse model with a subcutaneously transplanted tumor obtained from a patient with mutant EGFR-positive lung cancer

Nude mice were subcutaneously transplanted with LXF 2478, which is a tumor derived from a human lung cancer patient who was positive for EGFR with the V769_D770insASV mutation. At the point when the tumor volume of the grafted tumor in nude mice grew to about 100-200 mm ³ , the mice were divided into groups, 8 mice per group, by stratified randomization such that the average tumor volume between groups was the same. Mice were then treated orally with Compound A or afatinib once a day for 28 consecutive days, and a two-week follow-up period was established.

[0129]

The dose of afatinib was 20 mg/kg/day, which is the maximum tolerated dose (the maximum dose at which weight loss during the dosing period is less than 20%); and the dose of Compound A was 100 and 200 mg/kg/day. The maximum tolerated dose was determined according to the "Guidelines Involving Experimental Neoplasia Proposals in Mice and Rats" of the National Cancer Institute (NCI), from a humanitarian point of view.

[0130]

To compare changes in tumor growth over time due to administration of individual test compounds, the relative tumor volume (hereinafter also referred to as 'RTV') was calculated based on the tumor volume at the time the mice were grouped (which is taken as 1 for the tumor growth index) using the following formula . For the toxicity index, body weight was measured over time, and the change in body weight (hereinafter also referred to as "BWC (%)") from the day the mice were divided into groups was calculated by the following formula. In FIG. 13 and 14 show changes in mean RTV and mean BWC in mice.

[0131]

RTV=(tumor volume on the day of tumor volume measurement)/(tumor volume on the day the mice were divided into groups)

BWC (%)=(body weight measured on the day of body weight measurement)/(body weight on the day the mice were divided into groups)

[0132]

When the mean RTV in the Compound A group on the day after the last dose (Day 28) was less than the mean RTV in the control group, there was also a statistically significant difference (Dunnett's test, p<0.05 ), Compound A was determined to be effective. Such a case is indicated by the symbol "*" in the figures. T/C (%) on the next day after the last administration (day 28) was calculated according to the following formula. Table 7 shows the results.

[0133]

T/C (%)=(RTV of test compound administered group)/(RTV of control group)

[0134]

Table 7

transplanted tumor Compound 20 mg/kg 100 mg/kg 200 mg/kg LXF2478 Connection A N.D. 2.0 0.1 Afatinib 16.8 N.D. N.D.

[0135]

As can be seen from FIG. 13 and 14 and Table 7, Compound A demonstrated a significant antitumor effect on a tumor obtained from a lung cancer patient positive for exon 20 insertion mutant EGFR subcutaneously transplanted into nude mice, accompanied by tumor regression. The effect was maintained during the observation period, and no serious weight loss was observed in mice.

[0136]

Test Example 9

Evaluation of the life-prolonging effect in a model with transplanted mutant EGFR-expressing cell lines in the lung

The H1975-EGFRinsSVD-Luc strain was generated by introducing luciferase into H1975-EGFRinsSVD, which is a human mutant EGFR-introduced cell line. pJTI (trademark) Fast DEST vector, which was obtained by encoding luciferase in NCI-H1975-EGFRinsSVD cells, was introduced into H1975-EGFRinsSVD-Luc cells, together with pJTI (trademark) PhiC31 integrase expression vector, by electroporation using Amaxa ( trademark) of the R Cell Line Nucleofector kit (trademark), followed by selection using hygromycin B (Nacalai Tesque Inc.).

[0137]

In evaluating the life-prolonging effect, an equivalent amount of Matrigel was added to a suspension of cultured H1975-EGFRinsSVD-Luc cells to obtain a cell suspension, and the cell suspension was transplanted into the right lung of nude mice. On the 6th day after transplantation, all live mice were injected with luciferin via the tail vein and divided into groups of 9 mice per group by stratified randomization, so that the average luminescence intensity between groups was the same. Mice were then treated orally with Compound A or afatinib once daily on consecutive days. The dose of afatinib was 20 mg/kg/day, which is the maximum tolerated dose (the maximum dose at which weight loss during the dosing period is less than 20%); and the dose of Compound A was 100 and 200 mg/kg/day. The maximum tolerated dose was determined according to the "Guidelines Involving Experimental Neoplasia Proposals in Mice and Rats" of the National Cancer Institute (NCI), from a humanitarian point of view.

[0138]

To evaluate the life extension effect, the survival period after transplantation was observed and the survival time of each mouse was determined. From the survival time, the mean survival time (hereinafter also referred to as "MST") for each group was calculated, and the effect of prolonging the survival period (that is, the increase in lifespan, which is also referred to as 'I.L.S. (%)' in the following) was calculated based on the MST of the control group and group to which the test compound was administered using the following formula. For the toxicity index, body weight was measured over time, and the change in body weight (hereinafter also referred to as "BWC (%)") from the day the mice were divided into groups was calculated by the following formula.

[0139]

I.L.S. (%)=(T/C-1)×100

T: MST of the group administered with the test compound

C: MST of the control group

[0140]

BWC (%)=(body weight measured on the day of body weight measurement)/(body weight on the day the mice were divided into groups)

[0141]

When the MST of the Compound A treated group was greater than the MST of the control group, while showing a statistically significant difference (Wilcoxon test, p < 0.05), Compound A was determined to be effective in prolonging life. Table 8 shows the results.

[0142]

Table 8

transplanted tumor Compound MST I.L.S. (%) p value H1975
EGFR
insSVD Solvent Injection Control 44 N.A. N.A. Connection A
100 mg/kg 70 59 <0.01 Connection A
200 mg/kg 89 102 <0.01 Afatinib
20 mg/kg 54 23 N.S.

N.A.: Analysis not applied.

N.S.: No significant differences were observed.

[0143]

As shown in Table 8, Compound A showed a significant life-prolonging effect in nude mouse models transplanted in the same part of their lung with cell lines expressing EGFR with an exon 20 insertion mutation. However, afatinib did not show such a life-prolonging effect on mouse models. Mice treated with Compound A also showed no significant weight loss.

[0144]

Test Example 10

Evaluation of Phosphorylated-EGFR Inhibitory Activity in Tumor Graft and Mouse Skin Tissue

Naked mice were subcutaneously transplanted with NIH3T3-EGFRinsSVD cells into which mutant human EGFR was introduced. At the point when the tumor volume of the tumor inoculated in nude mice had grown to about 250-500 mm ³ , the mice were divided into groups, 3 mice per group, by stratified randomization such that the average tumor volume between groups was the same. The mice were then orally administered Compound A or afatinib once. One hour and three hours after administration, that is, approximately at the time when the maximum concentration of compound A and afatinib in the blood is reached, the tumor and skin tissues were collected. The harvested tissue was flash-frozen with liquid nitrogen and stored at -80°C until use. The tumor and skin tissue were homogenized with the addition of RIPA buffer (Thermo Fisher Scientific) containing a mixture of protease inhibitors (Thermo Fisher Scientific), and intracellular proteins were extracted. Protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific) and each sample was adjusted to a protein concentration suitable for measuring phosphorylated EGFR expression. Proteins were separated by SDS-PAGE and transferred to a PVDF membrane. After blocking, the phospho-EGF receptor (Tyr1068)#2234 (CST), which is the primary antibody, was diluted with 0.1% TBS-T buffer to 1/1000 and allowed to interact at 4°C overnight. Thereafter, HRP-labeled anti-rabbit antibody #NA9340V (GE Healthcare), which is a secondary antibody, was diluted to 1/2500 with 5% skim milk solution, with 0.1% TBS-T buffer, and allowed to react at room temperature for 40 minutes. After interaction with ECL-Prime (GE Healthcare), detection was performed using a LAS-3000 image analyzer (GE Healthcare).

[0145]

The test results show that Compound A selectively inhibits mutant EGFR in tumors compared to wild-type EGFR in skin.

Sequence list

P17-145WO_PCT_exon 20insertion mutation _20171013_112510_12.txt

--->

SEQUENCE LIST

<110> TAIHO PHARMACEUTICAL CO., LTD.

<120> SELECTIVE INHIBITOR OF EGFR MUTANT WITH EXON 20 INSERTION

<130> P17-145WO

<150> JP 2016-213072

<151> 2016-10-31

<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 (19)

1. A method of treating a patient with a malignant tumor, comprising the step of administering an effective amount of a compound selected from the group consisting of: (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide; (S)-N-(4-amino-6-methylene-5-(quinolin-3-yl)-7,8-dihydro-6H-pyrimido[5,4-b]pyrrolisin-7-yl)acrylamide; (S,E)-N-(4-amino-6-methylene-5-(quinolin-3-yl)-7,8-dihydro-6H-pyrimido[5,4-b]pyrrolisin-7-yl)- 3-chloroacrylamide; and (R)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)-N-methylacrylamide, or a salt thereof to a patient with a cancer expressing an EGFR having an insertion mutation in exon 20. 2. The method of claim 1 wherein the compound is (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizine -8-yl)acrylamide. 3. The method of claim 1 or 2, wherein the patient with the EGFR-expressing cancer having the exon 20 insertion mutation is a patient with lung cancer, breast cancer, head and neck cancer, brain tumor, uterine cancer, hematological malignancy, or skin cancer. 4. The method according to any one of paragraphs. 1-3, wherein the patient with an EGFR-expressing cancer having an insertion mutation in exon 20 is a lung cancer patient. 5. The method according to any one of paragraphs. 1-4, where an insertion mutation in exon 20 is a mutation where one or more amino acids are inserted in the region of exon 20. 6. The method according to any one of paragraphs. 1-5, where the exon 20 insertion mutation is a mutation where 1-7 amino acids are inserted in the exon 20 region. 7. The method according to any one of paragraphs. 1-6, where the exon 20 insertion mutation is a mutation where 1-4 amino acids are inserted in the exon 20 region. 8. The method according to any one of paragraphs. 1-7, where the exon 20 insertion mutation is V769_D770insASV, D770_N771insSVD, D770_N771insG, H773_V774insNPH, or H773_V774insPH. 9. Use of a compound selected from the group consisting of: (S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide; (S)-N-(4-amino-6-methylene-5-(quinolin-3-yl)-7,8-dihydro-6H-pyrimido[5,4-b]pyrrolisin-7-yl)acrylamide; and (S,E)-N-(4-amino-6-methylene-5-(quinolin-3-yl)-7,8-dihydro-6H-pyrimido[5,4-b]pyrrolisin-7-yl)- 3-chloroacrylamide; and (R)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)-N-methylacrylamide, or a salt thereof for the preparation of an anticancer agent for treating a patient with a cancer expressing EGFR having a mutation with an insertion in exon 20.

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