RU2777595C2 - Drug and composition for treatment of malignant tumors - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: first invention is the use of (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)pyrrolidine-1-yl)prop-2-en-1-one or its pharmaceutically acceptable salt for the treatment of a patient with cancer, receiving one or more additional compounds with an antitumor effect or their pharmaceutically acceptable salt(s), selected from 5-fluoruracil, tegafur/himeracil/potassium oteracil, gemcitabine, paclitaxel, cisplatin, everolimus, MK2206, and trans-3-amino-1-methyl-3-(4-(3-phenyl-5H-imidazo[1,2-c]pyrido[3,4-e][1,3]oxazine-2-yl)phenyl)cyclobutanol, gefitinib. The second invention is a method for the treatment of tumor, including the administration of the specified combinations to a patient who needs it.

EFFECT: group of inventions provides for a synergetic effect of antitumor combinations.

26 cl, 32 dwg, 4 tbl, 12 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to a combined preparation and pharmaceutical composition for the treatment of cancer. More specifically, the present invention relates to a preparation and composition for the treatment of cancer by the combined use of a specific FGFR inhibitor and an antitumor agent.

BACKGROUND OF THE INVENTION

As average life expectancy has increased in recent years, death from cancer, that is, malignant tumors, has become a high percentage of deaths caused by disease. Methods for the treatment of malignant tumors are roughly divided into surgery, radiation therapy and chemotherapy. As drugs for use in chemotherapy, that is, antitumor drugs, antimetabolites, antitumor antibiotics, alkaloid antitumor drugs, and platinum-based drugs, as well as molecularly targeted drugs based on antibodies or small molecular weight compounds, have been developed.

Several aberrant signaling pathways are known to be associated with carcinogenesis, and an association has also been reported between aberrant fibroblast growth factor (FGF)/fibroblast growth factor receptor (FGFR) signaling and various human cancers. It is believed that aberrant activation of FGF/FGFR signaling in human cancers is associated with overexpression and/or amplification of the FGFR gene, gene mutations such as chromosomal translocation, or an autocrine or paracrine mechanism associated with overexpression of its FGF ligands (NPL 1, NPL 2 and NPL 3). In addition, such aberrant signaling is considered to be a partial reason for the resistance of human cancer to treatment with existing chemotherapeutic antitumor agents or other inhibitors of tyrosine kinase receptors (NTP 4).

Accordingly, FGF/FGFR signaling therapeutic agents are expected to be drugs that enhance the therapeutic effects of existing chemotherapeutic anticancer agents or molecularly targeted drugs, including other tyrosine kinase receptor inhibitors, or effective therapeutic agents used alone or in combination with other medicines to treat forms of cancer that are resistant or do not respond to these medicines. Many FGFR inhibitors are currently in clinical development as antitumor agents. For example, AZD4547 is being co-tested with other anticancer agents such as 5-FU, cisplatin, an anti-EGFR antibody, and docetaxel (NPL 5 and NPL 6). In addition, it has been suggested that the concomitant use of the pyrimidine antimetabolite gemcitabine with an FGFR inhibitor is promising (NPL 7).

At the same time, there are reports of the presence of FGFR inhibitory properties in disubstituted alkynyl derivatives of benzene (PL 1). There are also reports that these compounds are effective against cancers characterized by specific FGFR2 mutations (PL 2) and that their bolus administration may be useful as a treatment regimen (PL 3).

CITATED LITERATURE

Patent Literature

PL 1: WO 2013/108809

PL 2: WO 2015/008844

PL 3: WO 2015/008839

Non-patent literature

NPL 1: J. Clin. oncol. 24, 3664-3671 (2006)

NPL 2: Mol. Cancer Res. 3, 655-667 (2005)

NPL 3: Cancer Res., 2085-2094 (2010)

NPL 4: Nature. 26; 487 (7408): 505-9 (2012)

NPL 5: Clin Cancer Res. 2572-83 (2013)

NPL 6: Oncotarget. 2009-22 (2015)

NPL 7: Drug Resistance Updates 9 (2006), 1-18

SUMMARY OF THE INVENTION

Technical problem

Even highly effective antitumor agents must be used with caution, and in some cases cannot be used if these agents have severe side effects or are highly toxic. It is also known that such anticancer agents may differ in their effect in different patients, or their effects may be reduced by long-term administration of the same agent.

The aim of the present invention is to provide a new method for treating cancer using an FGFR inhibitor, which is excellent in antitumor activity and has fewer side effects.

Solution

The inventor of the present invention studied the possibility of using the compound represented by formula (I) and an additional compound having an antitumor effect together, and found that such a combination significantly enhances the antitumor effect without significantly increasing toxicity, compared with the situation when the compound represented by formula (I) or an additional compound having an antitumor effect is used alone.

The present invention proposes the following [1] - [36]:

[1] Combination preparation for the treatment of cancer, comprising a compound represented by formula (I)

where R ₁ is the same or different, and each represents C ₁ -C ₆ alkyl;

X ₁ and X ₂ are independently N or CH;

Y is a group represented by formula (A)

(where the divalent fragment represented by

,

,

is a nitrogen-containing C ₃ -C ₁₀ heterocycloalkylene),

the group represented by formula (B)

(where the divalent fragment represented by

,

,

is C ₃ -C ₁₀ cycloalkylene), or

the group represented by formula (C)

(where the divalent fragment represented by

,

,

is a C ₆ -C ₁₂ arylene);

R ₂ is hydrogen, C ₂ -C ₆ alkynyl, -C(=O)OR _x , -C(=O)N(R _x )(R _y ), hydroxy-C ₁ -C ₆ alkyl, di(C ₁ -C ₆ alkyl)amino-C ₁ -C ₆ alkyl or C ₂ -C ₉ heteroaryl optionally having R ₃ ;

R ₃ is C ₁ -C ₆ alkyl or di(C ₁ -C ₆ alkyl)amino-C ₁ -C ₆ alkyl;

Z is -C(R ₄ )=C(R ₅ )(R ₆ ) or -C≡CR ₇ ;

R ₄ , R ₅ and R ₆ are the same or different, and each represents hydrogen, halogen, C ₁ -C ₆ alkyl optionally having R ₈ , or a group represented by formula (D)

(where the monovalent fragment represented by

,

,

is a nitrogen-containing C ₃ -C ₁₀ heterocycloalkyl),

R ₇ is hydrogen, C ₁ -C ₆ alkyl or hydroxy-C ₁ -C ₆ alkyl;

R ₈ is -OR _x or -N(R _x )(R _y );

R ₉ is C ₁ -C ₆ alkyl, halo or -OR _x ;

R _x and R _y are the same or different and each is hydrogen, C ₁ -C ₆ alkyl, C ₃ -C ₁₀ cycloalkyl, di(C ₁ -C ₆ alkyl)amino-C ₁ -C ₆ alkyl, or C ₁ -C ₆ alkoxy-C ₁ -C ₆ alkyl;

l is an integer from 0 to 3;

m is an integer from 1 to 3; and

n is an integer from 0 to 2; or

its salt; and

one or more additional compounds having an antitumor effect, or a pharmaceutically acceptable salt(s) thereof, selected from an antimetabolite, an alkaloid antitumor agent, a platinum-based drug, a molecularly targeted drug, an antitumor antibiotic, and an alkylating agent.

[2] The combination preparation according to [1], wherein the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is (S)-1-(3-(4-amino-3-((3 ,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof.

[3] The combination preparation according to [1] or [2], wherein the additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof is a pyrimidine antimetabolite, an alkaloid antitumor agent, a platinum-based drug, or a molecularly targeted drug. medicine.

[4] Combination preparation according to [3], characterized in that the pyrimidine antimetabolite is selected from the group consisting of 5-fluorouracil, tegafur/gimeracil/oteracil potassium, capecitabine and gemcitabine.

[5] Combination preparation according to item [3], while (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d ]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof is administered in an amount of 000.1 to 10 moles per mole of pyrimidine antimetabolite.

[6] The combination preparation according to [3], wherein the alkaloid antitumor agent is selected from the group consisting of paclitaxel, docetaxel, irinotecan, and vinblastine.

[7] Combination preparation according to [3], wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d ]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof is administered in an amount of 0.01 to 200 moles per mole of the alkaloid anticancer agent.

[8] The combined preparation according to [3], wherein the platinum-based drug is selected from the group consisting of cisplatin, carboplatin and oxaliplatin.

[9] Combination preparation according to [3], wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d ]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof is administered in an amount of 0.001 to 50 moles per mole of platinum-based drug.

[10] Combination preparation according to [3], characterized in that the molecularly targeted drug is selected from the group consisting of a low molecular weight molecularly targeted drug and an antibody-based molecularly targeted drug.

[11] The combination preparation according to [10], wherein the antibody-based molecularly targeted drug is selected from the group consisting of ramucirumab and bevacizumab.

[12] Combination preparation according to [10], wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d ]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof is administered in an amount of 0.005 to 1 mg per mg of the antibody molecularly targeted drug.

[13] Combination preparation according to [10], characterized in that the low molecular weight molecularly targeted drug is selected from the group consisting of everolimus, MK2206 and trans-3-amino-1-methyl-3-(4-(3- phenyl-5H-imidazo[1,2-c]pyrido[3,4-e][1,3]oxazin-2-yl)phenyl)cyclobutanol.

[14] Combination preparation according to [10], wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d ]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof is administered in an amount of 1 to 1000 moles per mole of the low molecular weight molecularly targeted drug.

[15] The combination preparation according to [1] or [2], wherein the additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof is an antifolate.

[16] The combined preparation according to [15], characterized in that the antifolate is methotrexate.

[17] The combination preparation according to [1] or [2], wherein the additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof is an alkylating agent.

[18] The combination preparation according to any one of [1] to [17], wherein the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and an additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof are administered simultaneously, separately, or sequentially.

[19] The combination preparation according to any one of [1] to [18], wherein the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and an additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof are administered in the same by administration or administered by different routes of administration.

[20] A pharmaceutical composition containing a compound represented by formula (I) or a pharmaceutically acceptable salt thereof and one or more additional compounds having an antitumor effect, or a pharmaceutically acceptable salt(s) thereof, selected from an antimetabolite, an alkaloid antitumor agent, a drug based on platinum, a molecularly targeted drug, an antitumor antibiotic and an alkylating agent.

[21] An antitumor effect enhancer for one or more additional compounds having an antitumor effect, or a pharmaceutically acceptable salt(s) thereof, selected from an antimetabolite, an alkaloid antitumor agent, a platinum-based drug, a molecularly targeted drug, an antitumor antibiotic, and an alkylating agent. an agent containing a compound represented by formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.

[22] An antitumor agent containing a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, used in combination with one or more additional compounds having an antitumor effect, or a pharmaceutically acceptable salt(s) thereof, selected from an antimetabolite, an alkaloid antitumor agent, a platinum-based drug, a molecularly targeted drug, an antitumor antibiotic, and an alkylating agent.

[23] An antitumor agent containing a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, for treating a cancer patient receiving one or more additional compounds having an antitumor effect, or a pharmaceutically acceptable salt(s) thereof, selected from an antimetabolite, an alkaloid anticancer agent, a platinum-based drug, a molecularly targeted drug, an antitumor antibiotic, and an alkylating agent.

[24] The use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of an antitumor effect enhancer for one or more additional compounds having an antitumor effect, or a pharmaceutically acceptable salt(s) thereof, selected from an antimetabolite, an alkaloid antitumor agent, a platinum-based drug, a molecularly targeted drug, an antitumor antibiotic, and an alkylating agent.

[25] The use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof for enhancing the antitumor effect of one or more additional compounds having an antitumor effect, or a pharmaceutically acceptable salt(s) thereof, selected from an antimetabolite, an alkaloid antitumor agent, a drug based on platinum, a molecularly targeted drug, an antitumor antibiotic and an alkylating agent.

[26] A method for treating a tumor, comprising administering a compound represented by formula (I), or a pharmaceutically acceptable salt thereof, and one or more additional compounds having an antitumor effect, or a pharmaceutically acceptable salt(s) thereof, selected from an antimetabolite, an alkaloid antitumor agent, a platinum-based drug, a molecularly targeted drug, an antitumor antibiotic, and an alkylating agent, to a patient in need thereof.

[27] A cancer treatment kit, comprising a compound represented by formula (I), or a pharmaceutically acceptable salt thereof, and one or more additional compounds having an antitumor effect, or a pharmaceutically acceptable salt(s) thereof, selected from an antimetabolite, an alkaloid antitumor agent , a platinum-based drug, a molecularly targeted drug, an antitumor antibiotic, and an alkylating agent.

[28] Combination preparation according to any one of [1] to [19], pharmaceutical composition according to [20], antitumor effect enhancer according to [21], antitumor agent according to [22] or [23], use according to [24] or [25], or the method according to [26], wherein the tumor is selected from the group consisting of lung cancer, esophageal cancer, gastric cancer, duodenal cancer, liver cancer, hepatocellular cancer, bile duct cancer, pancreatic cancer, colorectal cancer, breast cancer, endometrial cancer, ovarian cancer, kidney cancer, bladder cancer, prostate cancer, testicular tumor, thyroid cancer, bone or soft tissue tumor, leukemia, malignant lymphoma , multiple myeloma, head and neck cancer, brain tumors and skin cancer.

[29] Combination preparation according to any of [1] to [19], pharmaceutical composition according to [20], antitumor effect enhancer according to [21], antitumor agent according to [22] or [23], use according to [24] or [25], or the method according to [26], wherein the tumor is selected from the group consisting of gastric cancer, bile duct cancer, uterine cancer, bladder cancer and brain tumor.

[30] Combination preparation according to any one of [1] to [19], pharmaceutical composition according to [20], antitumor effect enhancer according to [21], antitumor agent according to [22] or [23], use according to [24] or [25], or the method according to [26], wherein the tumor is gastric cancer, and the additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof is selected from the group consisting of paclitaxel, docetaxel , tegafur/gimeracil/oteracil potassium, 5-fluorouracil, gemcitabine, capecitabine, oxaliplatin, cisplatin, and ramucirumab.

[31] Combination preparation according to any one of [1] to [19], pharmaceutical composition according to [20], antitumor effect enhancer according to [21], antitumor agent according to [22] or [23], use according to [24] or [25], or the method according to [26], wherein the tumor is a cancer of the bile ducts, and an additional compound having an antitumor effect, or a pharmaceutically acceptable salt thereof, is selected from the group consisting of gemcitabine and cisplatin.

[32] Combination preparation according to any of [1] to [19], pharmaceutical composition according to [20], antitumor effect enhancer according to [21], antitumor agent according to [22] or [23], use according to [24] or [25], or the method according to [26], wherein the tumor is bladder cancer, and the additional compound having an antitumor effect, or a pharmaceutically acceptable salt thereof, is selected from the group consisting of methotrexate, gemcitabine, paclitaxel, docetaxel, vinblastine, cisplatin, carboplatin, doxorubicin, and atezolizumab.

[33] Combination preparation according to any of [1] to [19], pharmaceutical composition according to [20], antitumor effect enhancer according to [21], antitumor agent according to [22] or [23], use according to [24] or [25], or the method according to [26], wherein the tumor is a brain tumor, and the additional compound having an antitumor effect, or a pharmaceutically acceptable salt thereof, is selected from the group consisting of irinotecan, bevacizumab and temozolomide.

[34] Combination preparation according to any one of [1] to [19], pharmaceutical composition according to [20], antitumor effect enhancer according to [21], antitumor agent according to [22] or [23], use according to [24] or [25], or the method according to [26], wherein the tumor is endometrial cancer, and the additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof is selected from the group consisting of 5-fluorouracil , gemcitabine, cisplatin, carboplatin, paclitaxel, doxorubicin and cyclophosphamide.

[35] Combination preparation according to any one of [1] to [19], pharmaceutical composition according to [20], antitumor effect enhancer according to [21], antitumor agent according to [22] or [23], use according to [24] or [25], or the method according to [26], wherein the FGFR is mutated in the tumor to be treated.

[36] Combination preparation according to any one of [1] to [19], pharmaceutical composition according to [20], antitumor effect enhancer according to [21], antitumor agent according to [22] or [23], use according to [24] or [25], or the method according to [26], wherein the tumor to be treated is resistant to an additional compound having an antitumor effect.

This specification incorporates the contents of Japanese Patent Application No. 2016-042662, on the basis of which priority is claimed for this application.

BENEFICIAL EFFECTS OF THE INVENTION

The present invention relates to a cancer treatment method having strong anti-tumor effects (particularly, a cytoreductive effect and a tumor growth-inhibiting effect (life-prolonging effect)), while at the same time helping to reduce the incidence of side effects of an anti-tumor agent. Thus, the result of the application of this method may be the long-term survival of patients suffering from cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A represents the antitumor effects of compound 1 and tegafur/gimeracil/oteracil potassium (S-1) used alone or together. Relative tumor volumes (RVR) are shown in the drug administration groups and in the control group.

Figure 1B represents the antitumor effects of Compound 1 and S-1 used separately or together. The degree of change in body weight of mice in the drug administration groups and in the control group is shown.

Figure 1C represents the antitumor effects of Compound 1 and S-1 used alone or together. Shown are the effects (P/C (%)) of compound 1 and S-1, used alone or together, on day 15.

Figure 2A presents the antitumor effects of compound 1 and paclitaxel used alone or together. Relative tumor volumes (RVR) are shown in the drug administration groups and in the control group.

Figure 2B presents the antitumor effects of compound 1 and paclitaxel used alone or together. The degree of change in body weight of mice in the drug administration groups and in the control group is shown.

Figure 2C presents the antitumor effects of compound 1 and paclitaxel used alone or together. The effects (P/C (%)) of compound 1 and paclitaxel used alone or together on day 15 are shown.

Figure 3A presents the antitumor effects of compound 1 and cisplatin, used alone or together. Relative tumor volumes (RVR) are shown in the drug administration groups and in the control group.

Figure 3B represents the antitumor effects of compound 1 and cisplatin, used alone or together. The degree of change in body weight of mice in the drug administration groups and in the control group is shown.

Figure 3C represents the antitumor effects of compound 1 and cisplatin used alone or together. The effects (P/C (%)) of compound 1 and cisplatin, used alone or together, are shown on day 11.

Figure 4A presents the antitumor effects of Compound 1 and gemcitabine, used alone or together. Relative tumor volumes (RVR) are shown in the drug administration groups and in the control group.

Figure 4B represents the antitumor effects of Compound 1 and gemcitabine, used alone or together. The degree of change in body weight of mice in the drug administration groups and in the control group is shown.

Figure 4C presents the antitumor effects of Compound 1 and gemcitabine, used alone or together. The effects (P/C (%)) of compound 1 and gemcitabine, used alone or together, are shown on day 15.

Figure 5A presents the antitumor effects of compound 1 and everolimus used alone or together. Relative tumor volumes (RVR) are shown in the drug administration groups and in the control group.

Figure 5B represents the antitumor effects of compound 1 and everolimus used alone or together. The degree of change in body weight of mice in the drug administration groups and in the control group is shown.

Figure 5C represents the antitumor effects of compound 1 and everolimus used alone or together. The effects (P/C (%)) of compound 1 and everolimus used alone or together on day 15 are shown.

Figure 6A shows the effect of the combined use of compound 1 and 5-FU on the growth rate of the SNU-16 cell line (5-FU:compound 1=1250:1).

Figure 6B shows the effect of combined use of compound 1 and 5-FU on the growth rate of the SNU-16 cell line (5-FU:compound 1=1000:1).

Figure 6C shows the effect of the combined use of compound 1 and 5-FU on the growth rate of the SNU-16 cell line (5-FU:compound 1=500:1).

Figure 7A presents the effect of compound 1 used in combination on various carcinoma cells as combination coefficient values. The effect of combining compound 1 with different concentrations of 5-FU is shown.

Figure 7B presents the effect of compound 1 used in combination on various carcinoma cells as combination coefficient values. The effect of combining compound 1 with different concentrations of cisplatin is shown.

Figure 7C presents the effect of compound 1 used in combination on various carcinoma cells as combination coefficient values. The effect of combining compound 1 with different concentrations of paclitaxel is shown.

Figure 7D presents the effect of compound 1 used in combination on various carcinoma cells as combination coefficient values. The effect of combining compound 1 with different concentrations of gemcitabine is shown.

Figure 8A shows the induction of apoptosis when the AKT inhibitor MK2206 and compound 1 are used together. The effect of combining compound 1 with different concentrations of MK2206 is shown.

Figure 8B shows the induction of apoptosis when the AKT inhibitor MK2206 and Compound 1 are used together. The effect of combining different concentrations of Compound 1 with MK2206 is shown.

Figure 9 shows the induction of apoptosis in human AN3CA endometrial cancer cells using the AKT inhibitor MK2206 and compound 1 in combination.

Figure 10A shows the effect of the combined use of compound 1 and everolimus on the survival of human AN3CA endometrial cancer cells.

Figure 10B shows the effect of the combined use of compound 1 and MK2206 on the survival of human AN3CA endometrial cancer cells.

Figure 11 shows the effect of combined use of compound 1 and everolimus or MK2206 on the phosphorylation of AKT, ERK, mTOR and S6 proteins in human AN3CA endometrial cancer cells.

Figure 12A shows the state of EGFR in human non-small cell lung cancer cell lines HCC4006, NCI-H1650 and NCI-H322.

Figure 12B shows the effect of the combined use of compound 1 and gefitinib on the survival of the HCC4006 cell line.

Figure 12C shows the effect of the combined use of compound 1 and gefitinib on the survival of the NCI-H1650 cell line.

Figure 12D shows the effect of the combined use of compound 1 and gefitinib on the survival of the NCI-H322 cell line.

DESCRIPTION OF EMBODIMENTS

The first embodiment of the present invention relates to a combination preparation for the treatment of cancer, comprising a compound represented by formula (I), or a pharmaceutically acceptable salt thereof, and one or more additional compounds having an antitumor effect, or a pharmaceutically acceptable salt(s) thereof, selected from an antimetabolite, an alkaloid antitumor agent, a platinum-based drug, a molecular targeting drug, an antitumor antibiotic, and an alkylating agent, as active ingredients.

In this specification, the term "C ₁ -C ₆ alkyl" means a straight or branched alkyl group containing 1-6 carbon atoms. Specific examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, pentyl, hexyl, and the like. Preferably, C ₁ -C ₆ alkyl is a straight or branched alkyl group containing 1-4 carbon atoms (C ₁ -C ₄ alkyl group), and more preferably methyl, ethyl, isopropyl and tert-butyl.

In this specification, the term "C ₃ -C ₁₀ cycloalkyl" means a monocyclic or polycyclic cycloalkyl group containing 3-10 carbon atoms, and preferably a monocyclic cycloalkyl group containing 3-6 carbon atoms (C ₃ -C ₆ cycloalkyl group). Specific examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, decalyl and the like. Preferred are cyclopropyl and cyclobutyl.

In this specification, the divalent moiety represented by

,

,

group represented by formula (A)

(where R ₂ and l are as above),

is a C ₃ -C ₁₀ divalent heterocycloalkylene group containing at least one nitrogen atom in the ring and additionally containing 0-2 identical or different heteroatoms selected from oxygen and sulfur atoms in the ring (nitrogen-containing C ₃ -C ₁₀ heterocycloalkylene group) , and preferably is a C ₃ -C ₅ heterocycloalkylene group containing 1-3 nitrogen atoms in the ring and additionally containing 0-1 oxygen atom in the ring (nitrogen-containing C ₃ -C ₅ heterocycloalkylene group). Specific examples include azetidinylene, pyrrolidinylene, piperidinylene, piperazinylene, morpholinylene, octahydroquinolinylene, octahydroindolylene, and the like. Of these, azetidinylene, pyrrolidinylene, piperidinylene, piperazinylene and morpholinylene are preferred.

Group represented by formula (A)

,

,

is a divalent nitrogen-containing C ₃ -C ₁₀ heterocycloalkylene group represented by

,

,

where the nitrogen atom has one valency and the other valency is occupied by the substituent (-(CH ₂ ) _l -) and the R ₂ substituent is on the ring.

In this specification, the divalent moiety represented by

,

,

group represented by formula (B)

(where R ₂ and l are as above),

is a monocyclic or polycyclic divalent cycloalkylene group containing 3-10 carbon atoms (C ₃ -C ₁₀ cycloalkylene group), and preferably a monocyclic divalent cycloalkylene group containing 3-6 carbon atoms (C ₃ -C ₆ cycloalkylene group). Specific examples include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, decalylene and the like. Preferred are cyclopropylene and (1,2- or 1,3-)cyclobutylene.

Formula (B)

means a divalent C ₃ -C ₁₀ cycloalkylene group represented by

,

,

where one valence is occupied by the adjacent amino group (NH) and the other valency is occupied by the substituent (-(CH ₂ ) _l -) and the R ₂ substituent is on the ring.

In this specification, the divalent moiety represented by

,

,

group represented by formula (C)

(where R ₂ and l are as above),

is a divalent arylene group containing 6-12 carbon atoms (C ₆ -C ₁₂ arylene group). Specific examples include phenylene, naphthylene, biphenylene and the like. Preferred is phenylene.

Formula (C)

means a divalent C ₆ -C ₁₂ arylene group represented by

,

,

where one valence is occupied by the adjacent amino group (NH) and the other valency is occupied by the substituent (-(CH ₂ ) _l -) and the R ₂ substituent is on the ring.

In this specification, the monovalent moiety represented by

,

,

group represented by formula (D)

(where R ₉ , m and n are as above),

means a C ₃ -C ₁₀ heterocycloalkyl group containing at least one nitrogen atom in the ring and additionally containing 0-2 identical or different heteroatoms selected from oxygen and sulfur atoms in the ring (nitrogen-containing C ₃ -C ₁₀ heterocycloalkylene group), and preferably is a C ₃ -C ₅ heterocycloalkylene group containing 1-3 nitrogen atoms in the ring and additionally containing 0-1 oxygen atom in the ring (nitrogen-containing C ₃ -C ₅ heterocycloalkylene group). Specific examples include azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholino, octahydroquinolinyl, octahydroindolinyl and the like. The azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl and morpholino groups are preferred.

Formula (D)

means a nitrogen-containing C ₃ -C ₁₀ heterocycloalkylene group represented by

,

,

where the nitrogen atom is bonded to a substituent (-(CH ₂ ) _m -) and n substituents (-(R ₉ ) _n ) are on the ring.

In this specification, "C ₂ -C ₉ heteroaryl" means a monocyclic or bicyclic C ₂ -C ₉ heteroaryl group containing 1-3 identical or different heteroatoms selected from nitrogen, oxygen and sulfur atoms; and preferably is a monocyclic C ₂ -C ₅ heteroaryl group containing 1-3 identical or different heteroatoms selected from nitrogen, oxygen and sulfur atoms (C ₂ -C ₅ heteroaryl group). Specific examples include thienyl, furyl, pyrrolyl, triazolyl, imidazolyl, pyrazolyl, oxadiazolyl, isothiazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, isobenzofuryl, indolizinyl, isoindolyl, indolyl, indazolyl, quinolyl, isoquinolyl, phthalazinyl, naphthyridinyl and the like. Preferred is 1,3,4-oxadiazolyl.

In this specification, the term "C ₂ -C ₆ alkynyl" means a straight or branched C ₂ -C ₆ alkynyl group having at least one carbon-carbon triple bond. Specific examples include ethynyl, 2-propynyl, 2-hexynyl and the like. Ethynyl is preferred.

In this specification, the term "hydroxy-C ₁ -C ₆ alkyl" means a linear or branched C ₁ -C ₆ alkyl group containing one hydroxyl group. Specific examples include hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl and the like. Of these, hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl and 2-hydroxybutyl are preferred.

In this specification, the term "di(C ₁ -C ₆ alkyl)amino-C ₁ -C ₆ alkyl group" means a straight or branched C ₁ -C ₆ alkyl group containing an amino group having two straight or branched C ₁ -C ₆ alkyl groups. Preferred is a straight or branched C ₁ -C ₄ alkyl group containing an amino group having two straight or branched C ₁ -C ₄ alkyl groups (di(C ₁ -C ₄ alkyl)amino-C ₁ -C ₄ alkyl group). Specific examples include dimethylaminomethyl, dimethylaminoethyl, dimethylaminopropyl, dimethylaminobutyl, dimethylaminopentyl, dimethylaminohexyl, diethylaminomethyl, diethylaminoethyl, diethylaminopropyl, diethylaminobutyl, diethylaminopentyl, diethylaminohexyl, dipropylaminomethyl, dibutylaminomethyl, dipentylaminomethyl, dihexylaminomethyl, ethyl(methyl)aminomethyl and the like. Preferred are dimethylaminomethyl and diethylaminoethyl.

In this specification, the term "C ₁ -C ₆ alkoxy-C ₁ -C ₆ alkyl" means a straight or branched C ₁ -C ₆ alkyl group having a straight or branched C ₁ -C ₆ alkoxy group. Preferably, it is a straight or branched C ₁ -C ₄ alkyl group having a straight or branched C ₁ -C ₄ alkoxy group (C ₁ -C ₄ alkoxy-C ₁ -C ₄ alkyl group). Specific examples of such groups include methoxymethyl, methoxyethyl, methoxypropyl, methoxybutyl, methoxypentyl, methoxyhexyl, ethoxymethyl, ethoxyethyl, ethoxypropyl, ethoxybutyl, ethoxypentyl, ethoxyhexyl, propoxymethyl, butoxymethyl, pentyloxymethyl, hexyloxymethyl and the like. Of these, 2-methoxyethyl is preferred.

In this specification, examples of "halogen" include chlorine, bromine, fluorine, and iodine. Fluorine is preferred.

In formula (I), the following combinations of X ₁ and X ₂ are preferred. (1) When X ₂ is N, X ₁ is N or CH. (2) When X ₂ is CH, X ₁ is CH.

In formula (I), l is preferably 0 or 1.

In formula (I), Y is preferably a group represented by formula (A)

(wherein R ₂ and l are as above), or a group represented by formula (C)

(where R ₂ and l are as above). More preferably, the divalent moiety represented by

,

,

of the group represented by formula (A) is pyrrolidinylene, azetidinylene or piperidinylene, or a divalent moiety represented by

,

,

the group represented by formula (C) is phenylene.

In formula (I), the following combinations of Y and Z are preferred. When Y is a group represented by formula (A)

(where R ₂ and l are as above),

Z is -C(R ₄ )=C(R ₅ )(R ₆ ) or -C≡CR ₇ . When Y is a group represented by the following formula (B) or (C):

,

,

(where R ₂ and l are as above), Z is -C(R ₄ )=C(R ₅ )(R ₆ ).

In formula (I), R ₁ is preferably C ₁ -C ₄ alkyl, and more preferably methyl or ethyl.

In formula (I), R ₂ is preferably hydrogen, C ₂ -C ₆ alkynyl, -C(=O)OR _x , hydroxy-C ₁ -C ₄ alkyl, or C ₂ -C ₉ heteroaryl optionally having R ₃ , and more preferably ethynyl, methoxycarbonyl, hydroxymethyl or 1,3,4-oxadiazolyl optionally having R ₃ .

In formula (I), R ₃ is preferably C ₁ -C ₄ alkyl or di(C ₁ -C ₄ alkyl)amino-C ₁ -C ₄ alkyl, and more preferably methyl or dimethylaminomethyl.

In formula (I), R ₄ is preferably hydrogen or halogen, more preferably hydrogen or fluorine, and even more preferably hydrogen.

In formula (I), R ₅ and R ₆ are preferably hydrogen, a C ₁ -C ₄ alkyl group optionally having R ₈ , or a group represented by formula (D)

(where R ₉ , m and n are as above), and more preferably hydrogen, methyl having R ₈ , or a group represented by formula (D)

(where R ₉ , m and n are as above).

In formula (I), m is preferably 1.

In formula (I), R ₉ is preferably C ₁ -C ₄ alkyl, fluoro or hydroxy, and more preferably methyl, fluoro or hydroxy.

In formula (I), n is preferably 0 or 1.

In formula (I), R ₇ is preferably hydrogen, C ₁ -C ₄ alkyl or hydroxy-C ₁ -C ₄ alkyl, and more preferably hydrogen, hydroxymethyl, methyl or 2-hydroxy-2-methylethyl.

In formula (I), R ₈ is preferably hydroxy or -N(R _x )(R _y ). In this formula, R _x and R _y are preferably hydrogen, C ₁ -C ₄ alkyl, C ₃ -C ₁₀ cycloalkyl or C ₁ -C ₄ alkoxy-C ₁ -C ₄ alkyl, and more preferably hydrogen, methyl, ethyl, tert-butyl, isopropyl, cyclopropyl, cyclobutyl or 2-methoxyethyl.

Preferred compounds of the present invention are compounds represented by formula (I), where R ₁ represents C ₁ -C ₄ alkyl; X ₁ and X ₂ are independently N or CH; Y is a group represented by the following formula (A) or (C):

;

;

R ₂ is hydrogen, C ₂ -C ₆ alkynyl, -C(=O)OR _x , hydroxy-C ₁ -C ₄ alkyl, or C ₂ -C ₉ heteroaryl optionally having R ₃ ; R ₃ is C ₁ -C ₄ alkyl or di(C ₁ -C ₄ alkyl)amino-C ₁ -C ₄ alkyl; Z is -C(R ₄ )=C(R ₅ )(R ₆ ) or -C≡CR ₇ ; R ₄ is hydrogen or halogen; R ₅ and R ₆ are the same or different, and each is hydrogen, C ₁ -C ₄ alkyl optionally having R ₈ , or a group represented by formula (D)

;

;

R ₇ is hydrogen, C ₁ -C ₄ alkyl or hydroxy-C ₁ -C ₄ alkyl; R ₈ is hydroxy or -N(R _x )(R _y ); R ₉ is C ₁ -C ₄ alkyl, fluoro or hydroxy; R _x and R _y are the same or different and each is hydrogen, C ₁ -C ₄ alkyl, C ₃ -C ₁₀ cycloalkyl or C ₁ -C ₄ alkoxy-C ₁ -C ₄ alkyl; and l is 0 or 1, m is 1, and n is 0 or 1.

More preferred compounds of the present invention are compounds represented by formula (I) wherein R ₁ is C ₁ -C ₄ alkyl, X ₁ and X ₂ are such that (1) when X ₂ is N, X ₁ is N or CH, and (2) when X ₂ is CH, X ₁ is CH; in Y is a bivalent fragment represented by

,

,

group represented by formula (A)

,

,

is pyrrolidinylene, azetidinylene or piperidinylene, or a divalent moiety represented by

,

,

group represented by formula (C)

,

,

is phenylene;

(a) when Y is a group represented by formula (A)

(wherein R ₂ is hydrogen, ethynyl, methoxycarbonyl, hydroxymethyl or 1,3,4-oxadiazolyl optionally having R ₃ ; R ₃ is C ₁ -C ₄ alkyl and l is 0 or 1), Z is -C (R ₄ )=C(R ₅ )(R ₆ ) or -C≡CR ₇ ,

(b) when Y is a group represented by formula (C)

(where R ₂ is hydrogen and l is 0 or 1), Z is -C(R ₄ )=C(R ₅ )(R ₆ ); R ₄ represents hydrogen or fluorine; R ₅ and R ₆ are the same or different, and each is hydrogen, C ₁ -C ₄ alkyl optionally having R ₈ , or a group represented by formula (D)

;

;

R ₇ is hydrogen, hydroxymethyl, methyl or 2-hydroxy-2-methylethyl; R ₈ is -N(R _x )(R _y ); R ₉ is C ₁ -C ₄ alkyl, fluoro or hydroxy; R _x and R _y are the same or different and each is hydrogen, C ₁ C ₄ alkyl, C ₃ -C ₁₀ cycloalkyl or C ₁ -C ₄ alkoxy-C ₁ -C ₄ alkyl, m is 1 and n is 0 or 1.

Even more preferred compounds of the present invention are those represented by formula (I) wherein R ₁ is methyl or ethyl; X ₁ and X ₂ are such that (1) when X ₂ is N, X ₁ is N or CH, and (2) when X ₂ is CH, X ₁ is CH; in Y the bivalent fragment represented by

,

,

is pyrrolidinylene, azetidinylene, piperidinylene, or a divalent moiety represented by

,

,

is phenylene;

(a) when Y is a group represented by formula (A)

(where R ₂ is hydrogen, ethynyl, methoxycarbonyl, hydroxymethyl or 1,3,4-oxadiazolyl, optionally having methyl; and l is 0 or 1), Z is -C(R ₄ )=C(R ₅ )( R ₆ ) or -C≡CR ₇ ,

(b) when Y is a group represented by formula (C)

(where R ₂ is hydrogen; and l is 1), Z is -C(R ₄ )=C(R ₅ )(R ₆ ); R ₄ is hydrogen; R ₅ and R ₆ are the same or different, and each is hydrogen, methyl having R ₈ , or a monovalent moiety represented by

,

,

group represented by formula (D)

,

,

is pyrrolidinyl, piperidinyl, azetidinyl, piperazinyl or morpholinyl; R ₇ is hydrogen, hydroxymethyl, methyl or 2-hydroxy-2-methylethyl; R ₈ is -N(R _x )(R _y ); R ₉ is methyl, fluoro or hydroxy; and R _x and R _y are the same or different and each is hydrogen, methyl, ethyl, t-butyl, isopropyl, cyclopropyl, cyclobutyl, or 2-methoxyethyl; and m is 1 and n is 0 or 1.

Specific examples of preferred compounds as a compound of formula (I) include the following:

(1) (S)-1-(3-(4-Amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1- yl) prop-2-en-1-one,

(2) (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1- il) prop-2-in-1-one,

(3) (S)-1-(3-(4-amino-3-((3,5-diethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1- yl) prop-2-en-1-one,

(4) 1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)azetidin-1-yl)prop- 2-en-1-he,

(5) 1-(3-(4-Amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)azetidin-1-yl)-4 -hydroxybut-2-in-1-one,

(6) 1-(3-(4-Amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)azetidin-1-yl)-4 -(dimethylamino)but-2-en-1-one,

(7) 1-(3-(4-Amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)azetidin-1-yl)-4 -(cyclopropylamino)but-2-en-1-one,

(8) 1-(3-(4-Amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)azetidin-1-yl)-4 -(isopropylamino)but-2-en-1-one,

(9) 1-(3-(4-Amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)azetidin-1-yl)-4 -(ethyl(methyl)amino)but-2-en-1-one,

(10) 1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)azetidin-1-yl)-4 -(cyclobutylamino)but-2-en-1-one,

(11) 1-(3-(4-Amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)azetidin-1-yl)-4 -(diethylamino)but-2-en-1-one,

(12) 1-(3-(4-Amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)azetidin-1-yl)-4 -(tert-butylamino)but-2-en-1-one,

(13) 1-(3-(4-Amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)azetidin-1-yl)-4 -(isopropyl(methyl)amino)but-2-en-1-one,

(14) 1-(3-(4-Amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)azetidin-1-yl)-4 -(piperidin-1-yl)but-2-en-1-one (compound of example 20),

(15) (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)azetidin-1- yl)-4-(3-fluoropyrrolidin-1-yl)but-2-en-1-one,

(16) (R)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)azetidin-1- yl)-4-(3-fluoropyrrolidin-1-yl)but-2-en-1-one,

(17) 1-((2S,4S)-4-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2 -(hydroxymethyl)pyrrolidin-1-yl)prop-2-en-1-one,

(18) 1-(2S,4S)-4-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2- ethinylpyrrolidin-1-yl)prop-2-en-1-one,

(19) (S)-1-(3-(4-amino-5-((3,5-dimethoxyphenyl)ethynyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)pyrrolidin-1- yl)-4-(dimethylamino)but-2-en-1-one,

(20) (S)-1-(3-(4-amino-5-((3,5-dimethoxyphenyl)ethynyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)pyrrolidin-1- yl) prop-2-en-1-one,

(21) (S)-1-(3-(4-amino-5-((3,5-dimethoxyphenyl)ethynyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)pyrrolidin-1- yl)-4-(pyrrolidin-1-yl)but-2-en-1-one,

(22) (S)-1-(3-(4-amino-5-((3,5-dimethoxyphenyl)ethynyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)pyrrolidin-1- yl)-4-(4-hydroxypiperidin-1-yl)but-2-en-1-one,

(23) (S)-1-(3-(4-amino-5-((3,5-dimethoxyphenyl)ethynyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)pyrrolidin-1- silt) but-2-in-1-one,

(24) (S)-1-(3-(4-Amino-5-((3,5-dimethoxyphenyl)ethynyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)pyrrolidin-1- yl)-4-hydroxy-4-methylpent-2-in-1-one,

(25) 1-((S)-3-(4-amino-5-((3,5-dimethoxyphenyl)ethynyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)pyrrolidin-1- yl)-4-((S)-3-fluoropyrrolidin-1-yl)but-2-en-1-one,

(26) (S)-1-(3-(4-amino-5-((3,5-dimethoxyphenyl)ethynyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)pyrrolidin-1- yl)-4-(piperidin-1-yl)but-2-en-1-one,

(27) 1-(3-(4-amino-5-((3,5-dimethoxyphenyl)ethynyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)azetidin-1-yl)prop- 2-en-1-he,

(28) 1-(3-(4-Amino-5-((3,5-dimethoxyphenyl)ethynyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)azetidin-1-yl)-4 -(dimethylamino)but-2-en-1-one,

(29) 1-(3-(4-amino-5-((3,5-dimethoxyphenyl)ethynyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)azetidin-1-yl)-4 -(pyrrolidin-1-yl)but-2-en-1-one,

(30) 1-(3-(4-amino-5-((3,5-dimethoxyphenyl)ethynyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)azetidin-1-yl)-4 -(azetidin-1-yl)but-2-en-1-one,

(31) 1-(3-(4-amino-5-((3,5-dimethoxyphenyl)ethynyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)azetidin-1-yl)-4 -(ethyl(methyl)amino)but-2-en-1-one,

(32) 1-(3-(4-amino-5-((3,5-dimethoxyphenyl)ethynyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)azetidin-1-yl)-4 -(isopropylamino)but-2-en-1-one,

(33) 1-(3-(4-amino-5-((3,5-dimethoxyphenyl)ethynyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)azetidin-1-yl)-4 -(diethylamino)but-2-en-1-one,

(34) 1-(3-(4-amino-5-((3,5-dimethoxyphenyl)ethynyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)azetidin-1-yl)-4 -((2-methoxyethyl)(methyl)amino)but-2-en-1-one,

(35) 1-(3-(4-amino-5-((3,5-dimethoxyphenyl)ethynyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)azetidin-1-yl)-4 -(4-hydroxypiperidin-1-yl)but-2-en-1-one,

(36) (S)-1-(3-(4-amino-5-((3,5-dimethoxyphenyl)ethynyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)azetidin-1- yl)-4-(3-hydroxypyrrolidin-1-yl)but-2-en-1-one,

(37) (R)-1-(3-(4-amino-5-((3,5-dimethoxyphenyl)ethynyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)azetidin-1- yl)-4-(3-hydroxypyrrolidin-1-yl)but-2-en-1-one,

(38) (2S,4S)-methyl-1-acryloyl-4-(4-amino-5-((3,5-dimethoxyphenyl)ethynyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl )pyrrolidine-2-carboxylate,

(39) 1-((2S,4S)-4-(4-amino-5-((3,5-dimethoxyphenyl)ethynyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2 -(1,3,4-oxadiazol-2-yl)pyrrolidin-1-yl)prop-2-en-1-one, and

(40) (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrrolo[3,2-c]pyridin-1-yl)pyrrolidin-1- yl) prop-2-en-1-one.

Of these, preferred is (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1 -yl)prop-2-en-1-one.

The compound represented by formula (I) or a pharmaceutically acceptable salt thereof in the above-described combination preparation of the present invention can be synthesized based on the production method described in, for example, WO 2013/108809 (PL 1), although the production method is not specifically limited to this method.

(S)-1-(3-(4-Amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop -2-en-1-one, which is one example of the compound represented by formula (I), is a disubstituted alkynyl benzene derivative having the structure described below. In this specification, for convenience, the connection is referred to as "connection 1". Compound 1 is described as exemplary compound 2 in WO 2013/108809.

In the present invention, the compound represented by formula (I) can be used directly or in the form of a pharmaceutically acceptable salt. The pharmaceutically acceptable salt of the compound represented by formula (I) is not particularly limited, and examples thereof include addition salts with inorganic acids such as hydrochloric acid and sulfuric acid, organic acids such as acetic acid, citric acid, tartaric acid, and maleic acid. ; salts with alkali metals such as potassium and sodium; salts with alkaline earth metals such as calcium and magnesium; and salts with organic bases such as ammonium salts, ethylamine salts and alginate.

The compound represented by formula (I) is an antitumor agent which is excellent in FGFR inhibitory effect and causes fewer side effects, and when used together with various additional compounds having antitumor effects, exhibits the effect of enhancing the antitumor effects of additional compounds having antitumor effects. effect, without a noticeable increase in toxicity.

Accordingly, the combination preparation of the present invention comprises one or more additional compounds having an antitumor effect, or a pharmaceutically acceptable salt(s) thereof, which are different from the compound represented by formula (I). Examples of the number of additional compounds having an antitumor effect, or pharmaceutically acceptable salts thereof, include one or more, preferably one. The additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof is selected from an antimetabolite (purine antimetabolite, pyrimidine antimetabolite, antifolate, etc.), an alkaloid antitumor agent, a platinum-based drug, a molecular target drug (small molecular target drug , an antibody-based molecularly targeted drug, and so on), an antitumor antibiotic, and an alkylating agent.

In this context, the term "additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof" should not include the compound represented by formula (I) because the compound represented by formula (I) is an antitumor agent whose action is based on the FGFR inhibition effect. The additional compound having an antitumor effect is not particularly limited, and examples thereof include antimetabolites (purine antimetabolites, antifolates, and pyrimidine antimetabolites), alkaloid antitumor agents, platinum-based drugs, molecularly targeted drugs (small molecular weight molecularly targeted drugs, antibody-based molecularly targeted drugs and immune checkpoint inhibitors), anticancer antibiotics, alkylating agents, and more specifically include

purine antimetabolites such as fludarabine, cladribine, and nelarabine;

pyrimidine antimetabolites such as 5-fluorouracil (5-FU), tegafur/gimeracil/oteracil potassium (TS-1 or S-1, trade name: "TS-1"), tegafur/uracil (UFT, trade name: "UFT ""), trifluridine/tipiracil hydrochloride (TAS-102, trade name: "LONSURF"), capecitabine, doxyfluridine, 5-fluoro-2'-deoxyuridine (FdUrd), gemcitabine, and cytarabine;

antifolates such as pemetrexed and methotrexate;

alkaloid antineoplastic agents such as paclitaxel (sold under the trade names Taxol, Abraxane, etc.; paclitaxel includes derivatives such as albumin-bound paclitaxel (e.g. ABI-007) and PEG-bound paclitaxel), docetaxel (trade name the name "taxotere" and so on), cabacitaxel, eribulin, irinotecan, noithecan, etoposide, vinorelbine, vincristine and vinblastine;

platinum-based drugs such as cisplatin, carboplatin, oxaliplatin and nedaplatin;

small molecular weight targeted drugs such as imatinib, gefitinib, erlotinib, lapatinib, sunitinib, dasatinib, everolimus, temsirolimus, selumetinib, trametinib, sorafenib, afatinib, regorafenib, dabrafenib, vemurafenib, trans-3-amino-1-methyl-3 -(4-(3-phenyl-5H-imidazo[1,2-c]pyrido[3,4-e][1,3]oxazin-2-yl)phenyl)cyclobutanol (referred to in this specification as "compound 2" ), and their pharmaceutically acceptable salts, as well as 8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-1,2,4-triazolo[3,4-f][1,6]naphthyridine-3( 2H)-one (MK2206) and pharmaceutically acceptable salts thereof;

antibody-based molecularly targeted drugs such as trastuzumab, cetuximab, bevacizumab, panitumumab, veltuzumab, rituximab, and ramucirumab;

immune checkpoint inhibitors such as nivolumab, pembrolizumab, atezolizumab, durvalumab, avelumab, ipilimumab, tremelimumab and abatacept;

antitumor antibiotics such as doxorubicin, daunorubicin, epirubicin, actinomycin D and mitomycin C; and

alkylating agents such as cyclophosphamide, dacarbazine, temozolomide, nimustine, busulfan, procarbazine and melphalan.

The above-described small molecular weight molecularly targeted drugs include those having different mechanisms of action. For example, gefitinib is known to exhibit an antitumor effect by inhibiting EGFR and is referred to as an EGFR inhibitor. The MAPK, PI3K/AKT/mTOR and NF-κB signaling pathways are also known to be aberrantly upregulated in various cancer cells. Inhibitors of these signaling pathways can also be used as antitumor agents of the present invention.

Examples of AKT inhibitors known to exert an antitumor effect by inhibiting AKT may include

trans-3-amino-1-methyl-3-(4-(3-phenyl-5H-imidazo[1,2-c]pyrido[3,4-e][1,3]oxazin-2-yl)phenyl )cyclobutanol (compound 2) and pharmaceutically acceptable salts thereof, and

8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-1,2,4-triazolo[3,4-f][1,6]naphthyridin-3(2H)-one (MK2206) and its pharmaceutical acceptable salts.

Compound 2 above is the compound described in Example 32 of WO 2012/137870 and can be synthesized by the method described in said document.

Examples of mTOR inhibitors known to exert an antitumor effect by inhibiting mTOR may include everolimus.

Examples of pyrimidine antimetabolites include the above compounds. Preferably, the pyrimidine antimetabolite is 5-fluorouracil (5-FU), tegafur/gimeracil/oteracil potassium or gemcitabine, more preferably tegafur/gimeracil/oteracil potassium or gemcitabine, and particularly preferably tegafur/gimeracil/oteracil potassium.

Examples of the alkaloid anticancer agent include the above compounds. Preferably, the alkaloid anticancer agent is paclitaxel.

Examples of the platinum-based drug include the above compounds. Preferably, the platinum-based drug is cisplatin.

Examples of the small molecular weight targeted drug include the above compounds. Preferably, the small molecule targeted drug is an AKT inhibitor, an mTOR inhibitor, or an EGFR inhibitor, and more preferably an AKT inhibitor or an mTOR inhibitor.

Examples of the additional compound having an antitumor effect include the above compounds. The additional compound having an antitumor effect is preferably a pyrimidine antimetabolite, an alkaloid antitumor agent, a platinum-based drug, or a small molecular weight targeting drug. More preferably, the additional compound having an antitumor effect is a pyrimidine antimetabolite, a plant alkaloid antitumor agent, a platinum-based drug, an AKT inhibitor, or an mTOR inhibitor. Even more preferably, the additional compound having an antitumor effect is tegafur/gimeracil/oteracil potassium, gemcitabine, paclitaxel, cisplatin, everolimus, or trans-3-amino-1-methyl-3-(4-(3-phenyl-5H- imidazo[1,2-c]pyrido[3,4-e][1,3]oxazin-2-yl)phenyl)cyclobutanol (compound 2) or a pharmaceutically acceptable salt thereof, 8-[4-(1-aminocyclobutyl) phenyl]-9-phenyl-1,2,4-triazolo[3,4-f][1,6]naphthyridin-3(2H)-one (MK2206) or a pharmaceutically acceptable salt thereof, or everolimus, and particularly preferably, tegafur/gimeracil/oteracil potassium or paclitaxel. Alternatively, an additional compound having an antitumor effect may be provided as a derivative or as a complex, without reducing its pharmacological effect or with the aim of enhancing its pharmacological effect, for example, to improve in vivo pharmacokinetics, improve delivery to a target site, or reduce degradation .

The respective mechanisms of action, doses and therapeutic targets of the above anticancer agents are known and those skilled in the art can obtain these anticancer agents along with the necessary information.

As is known to those skilled in the art, even drugs with excellent antitumor effect may cause additional suffering to patients due to the side effects they cause. The combination preparation of the present invention can help to reduce the dose and frequency of dosing of drugs due to the antitumor effect, and therefore can be effective in reducing side effects.

The malignant tumor that can be treated with the combination preparation of the present invention is not particularly limited, and examples include epithelial cancer (respiratory system cancer, gastrointestinal cancer, genitourinary system cancer, secretory system cancer, breast cancer, and so on), sarcoma , tumor of the hematopoietic system, tumor of the central nervous system and tumor of the peripheral nervous system.

Specific examples of cancers of the respiratory system include lung cancer (non-small cell lung cancer, small cell lung cancer, and so on). Specific examples of gastrointestinal cancers include esophageal cancer, gastric cancer, duodenal cancer, liver cancer, hepatocellular carcinoma, bile duct cancer (gall bladder cancer, cholangiocarcinoma, intrahepatic cholangiocarcinoma, extrahepatic cholangiocarcinoma, and so on), pancreatic cancer, and colorectal cancer (colon cancer, rectal cancer, and so on). Specific examples of genitourinary cancers include ovarian cancer, uterine cancer (cervical cancer, endometrial cancer, and so on), kidney cancer, bladder cancer, prostate cancer, and testicular tumor. Specific examples of cancer of the secretory system include thyroid cancer. Specific examples of a sarcoma include bone or soft tissue tumors. Specific examples of tumors of the hematopoietic system include leukemia, malignant lymphoma, and multiple myeloma. Specific examples of a central nervous system tumor include head and neck cancer and a brain tumor. Specific examples of peripheral nervous system tumors include skin cancer.

Even more preferably, the cancer to be treated according to the present invention is lung cancer, esophageal cancer, stomach cancer, bile duct cancer (gall bladder cancer, cholangiocarcinoma, intrahepatic cholangiocarcinoma or extrahepatic cholangiocarcinoma), endometrial cancer, bladder cancer, breast cancer. gland, osteosarcoma, soft tissue sarcoma, multiple myeloma or brain tumor, and especially preferably stomach cancer, bile duct cancer (gall bladder cancer, cholangiocarcinoma, intrahepatic cholangiocarcinoma or extrahepatic cholangiocarcinoma), endometrial cancer, bladder cancer or brain tumor.

Examples of an additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof, which can preferably be used in the combination preparation of the present invention for treating gastric cancer of the present invention, include pyrimidine antimetabolites, alkaloid antitumor agents, platinum-based drugs, and antitumor antibiotics. Such an additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof is preferably selected from the group consisting of paclitaxel, docetaxel, tegafur/gimeracil/oteracil potassium, 5-fluorouracil, gemcitabine, capecitabine, oxaliplatin, cisplatin and ramucirumab, and more preferably it is tegafur/gimeracil/oteracil potassium or paclitaxel.

Examples of an additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof which can preferably be used in the combination preparation of the present invention for treating bile duct cancer of the present invention include pyrimidine antimetabolites and platinum-based drugs, preferably gemcitabine and cisplatin.

Examples of an additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof, which can preferably be used in the combination preparation of the present invention for treating bladder cancer of the present invention, include pyrimidine antimetabolites, antifolates, alkaloid antitumor agents, platinum-based drugs, anticancer antibiotics and immune checkpoint inhibitors. Such additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof is preferably selected from the group consisting of methotrexate, gemcitabine, paclitaxel, docetaxel, vinblastine, cisplatin, carboplatin, doxorubicin and atezolizumab.

Examples of an additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof, which can preferably be used in the combination preparation of the present invention for treating a brain tumor of the present invention, include antibody-based molecularly targeted drugs, alkaloid antitumor agents, and alkylating agents. . Such an additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof is preferably selected from the group consisting of irinotecan, bevacizumab and temozolomide.

Examples of an additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof, which can preferably be used in the combination preparation of the present invention for treating endometrial cancer of the present invention, include pyrimidine antimetabolites, platinum-based drugs, alkaloid antitumor agents, antitumor antibiotics, and alkylating agents. Such an additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof is preferably selected from the group consisting of 5-fluorouracil, gemcitabine, cisplatin, carboplatin, paclitaxel, doxorubicin and cyclophosphamide.

In the combination preparation of the present invention, a compound represented by formula (I) or a pharmaceutically acceptable salt thereof and one or more additional compounds having an antitumor effect or a pharmaceutically acceptable salt(s) thereof, selected from a pyrimidine antimetabolite, an alkaloid antitumor agent, a drug based on platinum and a molecularly targeted drug, may be formulated separately in multiple formulations, or may be co-formulated in a single formulation. In addition, the combination preparation of the present invention may further contain an active ingredient other than the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and an additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof, but is preferably a combination preparation, containing only the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and an additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof as active ingredients.

When a compound represented by formula (I) or a pharmaceutically acceptable salt thereof and an additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof are contained as active ingredients in preparations, a pharmaceutical carrier may be added to each active ingredient, if necessary, to form various suitable dosage forms for prophylactic and therapeutic purposes. Examples of the dosage form include oral preparations, injections, suppositories, ointments and patches. Oral preparations are preferred. Oral preparations may be in such form as tablets, capsules, granules, powders and syrups, and are not particularly limited. Such dosage forms can be manufactured by methods well known to those skilled in the art. The formulations or pharmaceutical compositions may further contain a suitable carrier such as an excipient, diluent, excipient or disintegrant appropriate to the dosage forms, or as required.

The daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof may vary depending on the state of health, body weight, age and sex of the patient, and so on, and cannot be generalized. Generally, the daily dose for an adult (body weight: 60 kg) is about 1 to 1000 mg, preferably about 10 to 500 mg, and more preferably about 20 to 300 mg of the compound represented by formula (I), or pharmaceutically acceptable salt.

In the case of daily administration of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is administered in an amount of about 1-200 mg per day, preferably 2-100 mg per day. day, more preferably 4-50 mg per day, and even more preferably 10-40 mg per day.

In the case of administration every other day of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is administered in an amount of about 2-1000 mg per day, preferably 10-500 mg per day, more preferably 20-200 mg per day, and even more preferably 50-160 mg per day.

When the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and an additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof are formulated separately as two or more different preparations, the preparation containing the compound represented by formula (I) or its a pharmaceutically acceptable salt, and a preparation containing an additional compound having an antitumor effect, or a pharmaceutically acceptable salt thereof, can be administered simultaneously, separately or sequentially. The dosing interval for separate administration is not particularly limited and can be selected so that the respective effects of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and an additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof are optimally achieved, as well as the effect of their joint use. In the case of sequential administration, a preparation containing a compound represented by formula (I) or a pharmaceutically acceptable salt thereof and a preparation containing an additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof may be administered in any order.

When using the combination preparation of the present invention, a preparation containing a compound represented by formula (I) or a pharmaceutically acceptable salt thereof and a preparation containing an additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof can be administered by the same or different routes. introductions. For example, both of a preparation containing a compound represented by formula (I) or a pharmaceutically acceptable salt thereof and a preparation containing an additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof can be administered orally. Alternatively, for example, a preparation containing a compound represented by formula (I) or a pharmaceutically acceptable salt thereof may be administered orally, while a preparation containing an additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof may be administered by intravenous injection. The route of administration can be appropriately determined depending on the active ingredients to be administered, and taking into account the progression of the cancer in the patient, the general health of the patient, and so on.

The combination preparation of the present invention may be administered to a patient before or after surgery, and may also be administered to an inoperable patient. The combination preparation of the present invention may further include a drug to enhance the antitumor effect, and may also include a drug to reduce side effects.

In one aspect of the present invention, for the treatment of gastric cancer, for example, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and the tegafur/gimeracil/oteracil potassium pyrimidine antimetabolite can be used together, and both drugs can be administered orally as active ingredients in combination preparation or as active ingredients in a pharmaceutical composition described below.

In another aspect of the present invention, for the treatment of gastric cancer, for example, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and the alkaloid antitumor agent paclitaxel may be used together, and the compound represented by formula (I) or a pharmaceutically acceptable salt thereof may be administered. orally, while paclitaxel can be administered intravenously.

In one aspect of the present invention, for the treatment of bile duct cancer, for example, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and the alkaloid antitumor agent gemcitabine can be used together, and the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be administered orally, while gemcitabine can be administered intravenously.

In another aspect of the present invention, for the treatment of bile duct cancer, for example, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and the platinum-based drug cisplatin can be used together, and the compound represented by formula (I) or a pharmaceutically acceptable salt thereof. salt can be administered orally, while cisplatin can be administered intravenously.

In one aspect of the present invention, for the treatment of bladder cancer, for example, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and the platinum-based drug cisplatin can be used together and the compound represented by formula (I) or a pharmaceutically acceptable salt thereof. salt can be administered orally, while cisplatin can be administered intravenously.

In one aspect of the present invention, for the treatment of a brain tumor, for example, a compound represented by formula (I) or a pharmaceutically acceptable salt thereof and an alkylating agent temozolomide may be used together, and a compound represented by formula (I) or a pharmaceutically acceptable salt thereof may be administered. orally, while temozolomide can be administered intravenously or orally.

In one aspect of the present invention, for the treatment of endometrial cancer, for example, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and the platinum-based drug cisplatin, the pyrimidine antimetabolite gemcitabine, or the molecularly targeted drug everolimus can be used together, wherein the route administration and dosing frequency of each drug can be selected accordingly.

The proportions for administering or mixing the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and an additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof are not particularly limited, as long as the proportions are in the range that provides an enhancing effect. for antitumor effect. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount of about 0.001-1000 moles, preferably about 0.01-100 moles, based on the free form of the compound, per mole of an additional compound having an antitumor effect.

When the additional compound having an antitumor effect is a pyrimidine antimetabolite, for example, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, can be used in the range of preferably 0.001-10 moles, and more preferably 0.01-1 moles, per mole of pyrimidine antimetabolite.

When the additional compound having an antitumor effect is an alkaloid antitumor agent, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.01-200 moles, and more preferably 0.1-100 moles, per mole of alkaloid antitumor agent.

When the additional compound having an antitumor effect is a platinum-based drug, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.001-50 moles, and more preferably 0.01-10 moles, per mole of platinum-based drug.

When the additional compound having an antitumor effect is a small molecular weight drug, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 1-1000 moles, and more preferably 10-100 moles, per mole of a low molecular weight molecularly targeted drug.

When the additional compound having an antitumor effect is an mTOR inhibitor, for example, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 1-1000 moles, and more preferably 10-100 moles, per mole of inhibitor mTOR.

When the additional compound having an antitumor effect is an antibody-based molecularly targeted drug, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.005-1 mg, and more preferably 0.01- 0.5 mg, per mg of antibody-based molecularly targeted drug.

When the additional compound having an antitumor effect is tegafur/gimeracil/oteracil potassium (pyrimidine antimetabolite), for example, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount of 0.01-10 moles, preferably 0, 1-2 moles per mole of tegafur. In the case of daily administration of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.01-1 mol, and more preferably 0 05-0.5 mol, per mol of tegafur. In the case of administration every other day of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.01-10 moles, and more preferably 0.1-3 moles per mole of tegafur.

When the additional compound having an antitumor effect is 5-fluorouracil (pyrimidine antimetabolite), the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in the amount of 0.0001-1 mol, preferably 0.0005-0.5 mole, per mole of 5-fluorouracil. In the case of daily administration of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.0001-0.1 mol or more. preferably 0.005-0.05 moles per mole of 5-fluorouracil. In the case of administration every other day of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.0001-1 mol, and more preferably 0.001-0.5 mol, per mol of 5-fluorouracil.

When the additional compound having an antitumor effect is capecitabine (pyrimidine antimetabolite), the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount of 0.001-1 mol, preferably 0.005-0.1 mol, per mol of capecitabine. In the case of daily administration of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.001-0.1 mol, and more preferably 0.005 -0.05 mol, per mol of capecitabine. In the case of administration every other day of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.005-0.5 mol, and more preferably 0.01-0.1 mol, per mol of capecitabine.

When the additional compound having an antitumor effect is gemcitabine (pyrimidine antimetabolite), the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount of 0.001-1 mol, preferably 0.005-0.1 mol, per mol of gemcitabine. In the case of daily administration of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.001-0.1 mol, and more preferably 0.005 -0.05 mol, per mol of gemcitabine. In the case of administration every other day of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in the range of amounts, preferably 0.005-1 mol, and more preferably 0. 01-0.1 mol, per mol of gemcitabine.

When the additional compound having an antitumor effect is paclitaxel (an alkaloid antitumor agent), the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount of 0.01 to 10 moles, preferably 0.05 to 5 moles, per mole of paclitaxel. In the case of daily administration of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.01-1 mol, and more preferably 0 05-0.5 mol, per mol of paclitaxel. In the case of administration every other day of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.1-10 moles, and more preferably 0.2-5 moles per mole of paclitaxel.

When the additional compound having an antitumor effect is docetaxel (an alkaloid antitumor agent), the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount of 0.05 to 10 moles, preferably 0.1 to 5 moles, per mole of docetaxel. In the case of daily administration of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.05-1 mol, and more preferably 0 .1-0.5 mol, per mol of docetaxel. In the case of administration every other day of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.1-10 moles, and more preferably 0.5-5 moles per mole of docetaxel.

When the additional compound having an antitumor effect is irinotecan (an alkaloid antitumor agent), the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount of 0.01-2 moles, preferably 0.02-1 moles, per mole of irinotecan. In the case of daily administration of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.01-1 mol, and more preferably 0 .02-0.1 mol, per mol of irinotecan. In the case of administration every other day of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.05-2 moles, and more preferably 0.1-1 mole, per mole of irinotecan.

When the additional compound having an antitumor effect is vinblastine (an alkaloid antitumor agent), the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount of 1-200 moles, preferably 5-100 moles, per mole of vinblastine. In the case of daily administration of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 1-30 moles, and more preferably 5-20 moth, per mole of vinblastine. In the case of administration every other day of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 5-200 moles, and more preferably 10- 100 moles per mole of vinblastine.

When the additional compound having an antitumor effect is cisplatin (a platinum-based drug), the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount of 0.01-50 moles, preferably 0.05-10 moles , per mole of cisplatin. In the case of daily administration of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.01-10 moles, and more preferably 0 05-2 moles per mole of cisplatin. In the case of administration every other day of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.1-10 moles, and more preferably 0.2-6 moles per mole of cisplatin.

When the additional compound having an antitumor effect is oxaliplatin (a platinum-based drug), the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount of 0.01-5 moles, preferably 0.05-2 moles , per mole of oxaliplatin. In the case of daily administration of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.01-1 mol, and more preferably 0 05-0.5 mol, per mol of oxaliplatin. In the case of administration every other day of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.05-5 moles, and more preferably 0.1-2 moles per mole of oxaliplatin.

When the additional compound having an antitumor effect is carboplatin (a platinum-based drug), the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount of 0.001-1 mol, preferably 0.01-0.5 mol , per mole of carboplatin. In the case of daily administration of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.001-0.1 mol, and more preferably 0 .01-0.1 mol, per mol of carboplatin. In the case of administration every other day of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.01-1 mol, and more preferably 0.05-0.5 mol, per mol of carboplatin.

When the additional compound having an antitumor effect is everolimus (a small molecular weight targeted drug), the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount of 1-1000 moles, preferably 10-100 moles, per mole everolimus.

When the additional compound having an antitumor effect is ramucirumab (antibody molecularly targeted drug), the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount of 0.01-1 mg, preferably 0.02 -0.5 mg, per mg of ramucirumab. In the case of daily administration of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.01-0.2 mg or more. preferably 0.02-0.1 mg per mg of ramucirumab. In the case of administration every other day of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.02-1 mg, and more preferably 0.05-0.5 mg, per mole of ramucirumab.

When the additional compound having an antitumor effect is bevacizumab (antibody molecularly targeted drug), the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount of 0.005-1 mg, preferably 0.01-0 .5 mg per mg of bevacizumab. In the case of daily administration of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.005-0.1 mg, and more preferably 0 01-0.1 mg per mg of bevacizumab. In the case of administration every other day of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.01-1 mg, and more preferably 0.05-0.5 mg per mg of bevacizumab.

When the additional compound having an antitumor effect is atezolizumab (an immune checkpoint inhibitor), the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount of 0.001-1mg, preferably 0.01-0.5mg, per mg of atezolizumab. In the case of daily administration of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.001-0.1 mg, and more preferably 0 0.01-0.05 mg per mg of atezolizumab. In the case of administration every other day of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.01-1 mg, and more preferably 0.03-0.5 mg, per mg of atezolizumab.

When the additional compound having an antitumor effect is methotrexate (antifolate), the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount of 0.1 to 20 moles, preferably 0.2 to 5 moles, per mole of methotrexate . In the case of daily administration of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.1-2 moles, and more preferably 0 ,2-1 mol, per mol of methotrexate. In the case of administration every other day of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.5-10 moles, and more preferably 1-5 moles per mole of methotrexate.

When the additional compound having an antitumor effect is doxorubicin (an antitumor antibiotic), the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount of 0.05-20 mol, preferably 0.1-10 mol, per mol doxorubicin. In the case of daily administration of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.05-2 moles, and more preferably 0 ,1-1 mol, per mol of doxorubicin. In the case of administration every other day of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.1-20 moles, and more preferably 0.5-10 moles per mole of doxorubicin.

When the additional compound having an antitumor effect is temozolomide (an alkylating agent), the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount of 0.001-1 mol, preferably 0.01-0.5 mol, per mol temozolomide. In the case of daily administration of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.001-0.1 mol, and more preferably 0 .01-0.05 mol, per mol of temozolomide. In the case of administration every other day of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.01-1 mol, and more preferably 0.05-0.5 mol, per mol of temozolomide.

When the additional compound having an antitumor effect is cyclophosphamide (an alkylating agent), the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount of 0.001-1 mol, preferably 0.01-0.3 mol, per mol cyclophosphamide. In the case of daily administration of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in an amount range of preferably 0.001-0.1 mol, and more preferably 0 .01-0.05 mol, per mol of cyclophosphamide. In the case of administration every other day of a daily dose of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be used in the range of amounts, preferably 0.005-1 mol, and more preferably 0. 01-0.2 mol, per mol of cyclophosphamide.

In a second embodiment, the present invention also relates to a pharmaceutical composition comprising a compound represented by formula (I), or a pharmaceutically acceptable salt thereof, and one or more additional compounds having an antitumor effect, or a pharmaceutically acceptable salt(s) thereof, selected from an antimetabolite, an alkaloid antitumor agent, a platinum-based drug, a molecularly targeted drug, an antitumor antibiotic, and an alkylating agent.

The pharmaceutical composition of the present invention contains a compound represented by formula (I), or a pharmaceutically acceptable salt thereof, and one or more additional compounds having an antitumor effect, or a pharmaceutically acceptable salt(s) thereof, as active ingredients in the same composition, in while the combination preparation described above includes these active ingredients in separate preparations. The proportions for mixing a compound represented by formula (I) or a pharmaceutically acceptable salt thereof and one or more additional compounds having an antitumor effect or a pharmaceutically acceptable salt(s) thereof in the composition may be within the ranges described above.

In a third embodiment, the present invention also relates to an antitumor effect enhancer for one or more additional compounds having an antitumor effect, or a pharmaceutically acceptable salt(s) thereof, selected from an antimetabolite, an alkaloid antitumor agent, a platinum-based drug, a molecularly targeted drug. an agent, an antitumor antibiotic, and an alkylating agent containing a compound represented by formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.

In a fourth embodiment, the present invention also relates to an antitumor agent containing a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein the antitumor agent is used in combination with one or more additional compounds having an antitumor effect, or a pharmaceutically acceptable salt thereof. (salts) selected from an antimetabolite, an alkaloid anticancer agent, a platinum-based drug, a molecularly targeted drug, an antitumor antibiotic, and an alkylating agent.

In a fifth embodiment, the present invention also relates to a cancer treatment kit comprising a compound represented by formula (I) or a pharmaceutically acceptable salt thereof and one or more additional compounds having an antitumor effect, or a pharmaceutically acceptable salt(s) thereof, selected of an antimetabolite, an alkaloid antitumor agent, a platinum-based drug, a molecularly targeted drug, an antitumor antibiotic, and an alkylating agent.

In a sixth embodiment, the present invention also relates to an antitumor agent containing a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, for treating a cancer patient receiving one or more additional compounds having an antitumor effect, or a pharmaceutically acceptable salt thereof. (i) selected from an antimetabolite, an alkaloid anticancer agent, a platinum-based drug, a molecularly targeted drug, an antitumor antibiotic, and an alkylating agent.

In a seventh embodiment, the present invention also relates to the use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of an antitumor effect enhancer for one or more additional compounds having an antitumor effect, or a pharmaceutically acceptable salt(s) thereof, selected from an antimetabolite, an alkaloid anticancer agent, a platinum-based drug, a molecularly targeted drug, an antitumor antibiotic, and an alkylating agent.

In an eighth embodiment, the present invention also relates to the use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof for enhancing the antitumor effect of one or more additional compounds having an antitumor effect, or a pharmaceutically acceptable salt(s) thereof, selected from an antimetabolite, an alkaloid antitumor agent, a platinum-based drug, a molecularly targeted drug, an antitumor antibiotic, and an alkylating agent.

In a ninth embodiment, the present invention also relates to a method for treating a tumor, comprising administering a compound represented by formula (I) or a pharmaceutically acceptable salt thereof and one or more additional compounds having an antitumor effect, or a pharmaceutically acceptable salt(s) thereof, selected from an antimetabolite, an alkaloid antitumor agent, a platinum-based drug, a molecularly targeted drug, an antitumor antibiotic, and an alkylating agent, to a patient in need thereof.

It has been confirmed that embodiments of the present invention are highly effective not only against a tumor having a wild-type FGFR, but also against a tumor having an amplified or mutated FGFR. Accordingly, the treatment target of the present invention also includes, but is not specifically limited to, tumors having a wild-type FGFR, or an amplified or mutated FGFR. Although the target for treatment of the present invention is not limited to a tumor having a particular wild-type FGFR, it is preferably a tumor having a wild-type FGFR3. In addition, although the target for treatment of the present invention is not limited to a tumor with amplification of a particular FGFR, it is preferably a tumor with amplification of FGFR1 or FGFR2. In addition, although the target for treatment of the present invention is not limited to a tumor having a particular FGFR mutation, it is preferably a tumor having an FGFR2 mutation.

The compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be effectively used even in the case of a tumor having resistance to an additional compound having an antitumor effect. For example, the effects of the EGFR inhibitor gefitinib are known to decrease with long-term use. However, its combined use with a compound represented by formula (I) or a pharmaceutically acceptable salt thereof can lead to a significant decrease in the survival of tumor cells. In addition, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof can be effectively used even in a tumor having resistance to an FGFR inhibitor other than the compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

EXAMPLES

Hereinafter, the present invention is described in detail with reference to examples; however, the scope of the present invention is not limited to these examples. While the present invention is fully explained by way of examples, it should be understood that various changes or modifications may be made by those skilled in the art. Accordingly, such changes or modifications are part of the present invention without departing from the scope of the present invention. The various reagents used in the examples are commercially available unless otherwise noted.

Example 1 Measurement of the Antitumor Effect of Compounds 1 and S-1 Used in Combination on Tumor from SNU-16 Human Gastric Cancer Cells Subcutaneously Implanted in Nude Mice

Using the technique described in Clin Cancer Res. 2013; 19 (9): 2572-83, a suspension of SNU-16 human gastric cancer cells (obtained from the American Type Culture Collection) was subcutaneously implanted in 6-week-old male BALB/cAJcl-nu/nu mice (CLEA Japan, Inc.) in an amount of 8 × 10 ⁶ cells/mouse. To separate mice into groups (n=10/group), after implantation of the cell suspension, tumor volume (VR) was calculated by the formula below, and mice having VR from 100 to 300 mm ³ were selected and divided into groups so that the average VR was the same in all groups. The day on which the animals were assigned to groups was defined as day 0.

OO (mm ³ )=(long axis × short axis ² )/2 (the units for long axis and short axis were mm).

Compound 1 at a dose of 5 or 15 mg/kg/day and tegafur/gimeracil/oteracil potassium (S-1; molar ratio of the mixture: 1:0.4:1) at a dose of 8.3 mg/kg/day (based on tegafur) was orally administered once a day for 14 days.

The dose of Compound 1 used was 15 mg/kg, which is the effective dose for this subcutaneously implanted mouse model, and a low dose of 5 mg/kg was used to confirm the effect of using the drug in combination. The dose of S-1 used was determined based on the maximum tolerated dose in nude mice, and so on.

Antitumor effects were evaluated based on the differences between the mean values of the relative tumor volume (RVR) in the two groups compared on the day of the assessment, in the form of an index. The OO was calculated using the formula below based on the OO values on the day of measurement and on the day of grouping. In addition, P/C (%) was calculated based on the mean values of LLC in the groups of drug administration and in the control group.

OOO = (OD on the day of measurement) / (OD on the day of division into groups)

P/C (%)=(mean PFO in each treatment group on assessment day)/(mean PFO in control group on assessment day) × 100

As a result, each of Compound 1 treatment (5 and 15 mg/kg) and S-1 treatment (8.3 mg/kg) alone resulted in growth inhibition of the subcutaneously implanted SNU-16 tumor, with corresponding P/C values (% ) on the day of assessment, amounting to 83.6%, 69.0% and 84.3%. In contrast, co-treatment with a combination of 5 or 15 mg/kg Compound 1 and 8.3 mg/kg S-1 resulted in tumor growth inhibition to a greater extent than treatment with either drug alone, with corresponding P/C values (%), constituting 54.4% and 43.9%.

The tumor growth inhibitory effect of compound 1 and S-1 used in combination was superior to the computer program predicted effect of both drugs combined (P/C=70.5% and 58.2%) based on the effect of each drug used alone, as an index, indicating that sharing results in synergistic effects (P<0.05; Bliss method, Student's t-test). These results are presented in Figures 1A and 1C. On the other hand, the average rate of change in body weight in the compound 1/S-1 combination group was not significantly different from the rate of change in body weight in the administration group of each drug (Figure 1B).

Example 2 Measurement of the antitumor effect of compound 1 and paclitaxel used in combination on tumor from human gastric cancer cell line SNU-16 subcutaneously implanted in nude mice

As in Example 1, a suspension of SNU-16 human gastric cancer cells was subcutaneously implanted in 6-week-old male BALB/cAJcl-nu/nu mice (CLEA Japan, Inc.) at 8 x 10 ⁶ cells/mouse. To separate mice into groups (n=10/group), after implantation of the cell suspension, mice having a tumor volume (VR) from 100 to 300 mm ³ were selected and divided into groups so that the average VR was the same in all groups.

Compound 1 was orally administered daily at a dose of 5 or 15 mg/kg/day for 14 days once a day. Paclitaxel was administered at a single dose of 60 mg/kg/day, based on the maximum soluble amount for the injectable solution, by injection into the tail vein on day 1.

Antitumor effects were evaluated based on the differences between the mean values of the relative tumor volume (RVR) in the two groups compared on the day of the assessment, in the form of an index. In addition, P/C (%) was calculated based on the mean values of LLC in the groups of drug administration and in the control group.

As a result, each of compound 1 treatment (5 and 15 mg/kg) and paclitaxel (60 mg/kg) treatment alone resulted in growth inhibition of the subcutaneously implanted SNU-16 tumor, with corresponding P/C (%) values on the day of assessment , constituting 83.6%, 69.0% and 12.9%. In contrast, co-treatment with a combination of 5 or 15 mg/kg of Compound 1 and 60 mg/kg of paclitaxel resulted in tumor growth inhibition to a greater extent than treatment with either drug alone, with corresponding P/C (%) values of 3.1 % and 1.5%.

The tumor growth inhibitory effect of compound 1 and paclitaxel used in combination was superior to the computer program predicted effect of both drugs combined (P/R=10.8% and 8.9%) based on the effect of each drug used alone as index, indicating that sharing leads to synergistic effects (P<0.05; Bliss method, Student's t-test). These results are presented in Figures 2A and 2C. On the other hand, a transient decrease in body weight was observed in mice after administration of paclitaxel, indicating a side effect of paclitaxel. The average rate of change in body weight in the compound 1/paclitaxel combination group was not significantly different from the rate of change in body weight in the administration group for each drug (Figure 2B).

Example 3 Measurement of the Antitumor Effect of Compound 1 and Cisplatin in Combination on Tumor from Human Endometrial Cancer Cell Line AN3CA Subcutaneously Implanted in Nude Mice

Using the technique described in Gynecol Oncol. 2014; 132 (2): 468-73, a suspension of AN3CA human endometrial cancer cells (obtained from the American Type Culture Collection) was subcutaneously implanted in 6-week-old female BALB/cAJcl-nu/nu mice (CLEA Japan, Inc.) at 1×10 ⁷ cells/mouse. The grafted tumor was removed from mice, minced into 2 square mm fragments, and then subcutaneously implanted in 6-week-old female BALB/cAJcl-nu/nu mice. To separate mice into groups (n=6/group) after tumor implantation, mice having a tumor volume (TOV) of 100 to 300 mm ³ were selected and divided into groups so that the average OV was the same in all groups.

Compound 1 was orally administered daily at a dose of 15 mg/kg/day for 14 days once a day. Cisplatin was administered at a dose of 7 mg/kg/day via the tail vein on day 1. The dose of 7 mg/kg cisplatin was the maximum tolerated dose in a single dose in nude mice.

Antitumor effects were evaluated based on the differences between the mean values of the relative tumor volume (RVR) in the two groups compared on the day of the assessment, in the form of an index. In addition, P/C (%) was calculated based on the mean values of LLC in the groups of drug administration and in the control group.

As a result, each of treatment with Compound 1 (15 mg/kg) and treatment with cisplatin (7 mg/kg) alone resulted in growth inhibition of the subcutaneously implanted AN3CA tumor, with corresponding P/C (%) values at assessment day of 46, 0% and 53.0%. In contrast, co-treatment with a combination of 15 mg/kg Compound 1 and 7 mg/kg cisplatin resulted in tumor growth inhibition to a greater extent than treatment with either drug alone, with a corresponding P/C (%) of 14.9%.

The tumor growth inhibitory effect of compound 1 and cisplatin used in combination was statistically significantly enhanced compared to the effect of each drug used alone (P<0.05; Aspin-Welch test). In addition, the tumor growth inhibitory effect of compound 1 and cisplatin used in combination was superior to the computer program predicted effect of both drugs combined (P/R=24.4%) based on the effect of each drug used alone as an index ( Bliss method). These results are presented in Figures 3A and 3C. On the other hand, a transient decrease in body weight was observed in mice after administration of cisplatin, indicating a side effect of cisplatin. The change in body weight of mice over time did not increase with the combined use of compound 1 and cisplatin, compared with the change in body weight of mice treated with cisplatin alone (Figure 3B).

Example 4 Measurement of the Antitumor Effect of Compound 1 and Gemcitabine in Combination on Tumor from Human Endometrial Cancer Cell Line AN3CA Subcutaneously Implanted in Nude Mice

As in Example 3, a suspension of AN3CA human endometrial cancer cells was subcutaneously implanted in 6-week-old female BALB/cAJcl-nu/nu mice (CLEA Japan, Inc.) at 1 x 10 ⁷ cells/mouse. The grafted tumor was removed from mice, minced into 2 square mm fragments, and then subcutaneously implanted in 6-week-old female BALB/cAJcl-nu/nu mice. To separate mice into groups (n=6/group) after tumor implantation, mice having a tumor volume (TOV) of 100 to 300 mm ³ were selected and divided into groups so that the average OV was the same in all groups.

Compound 1 was orally administered daily at a dose of 15 mg/kg/day for 14 days. Gemcitabine was administered at a dose of 100 mg/kg/day by tail vein on day 1 and day 8. Gemcitabine was used at a dose of 100 mg/kg at which drug effect could be expected, based on an article in J Pharmacol. Exp Ther., 2008; 325:484-490, and so on.

Antitumor effects were evaluated based on the differences between the mean values of the relative tumor volume (RVR) in the two groups compared on the day of the assessment, in the form of an index. In addition, P/C (%) was calculated based on the mean values of LLC in the groups of drug administration and in the control group.

Treatment with Compound 1 (15 mg/kg) and treatment with gemcitabine (100 mg/kg) alone resulted in growth inhibition of the subcutaneously implanted AN3CA tumor, with corresponding P/C (%) values at assessment day of 42.1% and 75.5%. In contrast, co-treatment with a combination of 15 mg/kg of Compound 1 and 100 mg/kg of gemcitabine resulted in tumor growth inhibition to a greater extent than treatment with either drug alone, with a corresponding P/C (%) of 24.7%.

The tumor growth inhibitory effect of compound 1 and gemcitabine used in combination tended to be greater than the effect of compound 1 used alone (P=0.0594; Aspin-Welch test) and was also statistically significantly greater than the effect of gemcitabine used alone (P<0, 05; Aspin-Welch test). The tumor growth inhibitory effect of Compound 1 and gemcitabine used in combination was superior to the computer program predicted effect of both drugs combined (P/R=31.8%) based on the effect of each drug used alone as an index (Bliss method) . These results are presented in Figures 4A and 4C. On the other hand, the change in body weight of mice over time was not markedly increased by the combined use of compound 1 and gemcitabine, compared with the change in body weight of mice treated with gemcitabine alone (Figure 4B).

Example 5 Measurement of the Antitumor Effect of Compound 1 and Everolimus Used in Combination on Tumor from Human Endometrial Cancer Cell Line AN3CA Subcutaneously Implanted in Nude Mice

As in Example 3, a suspension of AN3CA human endometrial cancer cells was subcutaneously implanted in 6-week-old female BALB/cAJcl-nu/nu mice (CLEA Japan, Inc.) at 1 x 10 ⁷ cells/mouse. The grafted tumor was removed from mice, minced into 2 square mm fragments, and then subcutaneously implanted in 6-week-old female BALB/cAJcl-nu/nu mice. To separate mice into groups (n=6/group) after tumor implantation, mice having a tumor volume (TOV) of 100 to 300 mm ³ were selected and divided into groups so that the average OV was the same in all groups.

Compound 1 was orally administered daily at 15 and 50 mg/kg/day for 14 days. Everolimus was orally administered daily at a dose of 2 mg/kg/day for 14 days. Everolimus was used at a dose of 2mg/kg at which drug effect could be expected, based on an article in Neoplasia, 2013; 15:1391-1399, and so on.

Antitumor effects were evaluated based on the differences between the mean values of the relative tumor volume (RVR) in the two groups compared on the day of the assessment, in the form of an index. In addition, P/C (%) was calculated based on the mean values of LLC in the groups of drug administration and in the control group.

Compound 1 treatment (15 and 50 mg/kg) and everolimus treatment (2 mg/kg) alone each resulted in growth inhibition of the subcutaneously implanted AN3CA tumor, with corresponding P/C (%) values on assessment day of 33.3 %, 12.6% and 27.1%. In contrast, co-treatment with a combination of 15 or 50 mg/kg of Compound 1 and 2 mg/kg of everolimus resulted in tumor growth inhibition to a greater extent than treatment with either drug alone, with corresponding P/C (%) values of 5.1 % and 1.9%.

The tumor growth inhibitory effect of compound 1 and everolimus used in combination was statistically significantly enhanced compared to the effect of each drug used alone (P<0.05; Aspin-Welch test). In addition, the tumor growth inhibitory effect of Compound 1 and everolimus used in combination was superior to the computer program predicted effect of both drugs combined (P/R=9.0% and 3.4%) based on the effect of each drug used alone. , as an index (Bliss method). These results are presented in Figures 5A and 5C. On the other hand, the change in body weight of mice over time was not markedly increased by the combined use of compound 1 and everolimus, compared with the change in body weight of mice treated with everolimus alone (Figure 5B).

Example 6 Evaluation of the Tumor Inhibiting Effect of Compounds 1 and 5-FU Used in Combination in vitro

A. Materials and methods

Using the technique described in Clin Cancer Res. 2013; 19 (9): 2572-83, SNU-16 human gastric cancer cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum. Cells were maintained at 37°C in an atmosphere with 5% CO ₂ and subcultured in a ratio of from 1:5 to 1:20 1-2 passages per week.

Cell survival analysis

Cell survival measurements were performed using CellTiter-Glo (manufactured by Promega Corp.). Cells were harvested in the usual manner, then suspended in RPMI-1640 medium containing 10% fetal bovine serum and plated in a 96-well plate. The number of cells added to the well was 2000 cells/80 μl. After incubation at 37°C for 24 hours in an atmosphere with 5% CO ₂ 10 μl of medium containing compound 1 and 5-FU or solvent (DMSO) was added to each well. Used 10 concentrations of compound 1, obtained by serial 3-fold dilution, with the highest final concentration of 100 μm. 10 concentrations of 5-FU obtained from each of 3 serial 3-fold dilutions were used, with the highest final concentrations being 80, 100 and 200 μM. In addition to the groups for each drug alone, groups of two drugs added at the same time were prepared in parallel. After adding drugs to the wells, the cells were incubated at 37°C for 72 hours in an atmosphere with 5% CO ₂ . To determine cell viability, 100 μl of CellTiter-Glo solution was added to each well, the cells were incubated at room temperature for 10 minutes, and then the intensity of chemiluminescence in each well was measured on a plate reader (ARVO). Drug-added cell survival rates were calculated as a percentage relative to the control group (100%) according to the following formula:

Cell survival (%)=(Chemiluminescence intensity when drug added)/(Chemiluminescence intensity in the control group) × 100

In addition, IF (affected fraction) values of cells were calculated by subtracting 1/100 of the cell survival rate from 1.

The concentration at which each drug caused a 50% inhibition of cell growth (IC ₅₀ ) was determined using the CalcuSyn 2.0 median effect analysis program (CalcuSyn, Inc.). The combination index (IC) value was then determined for each combined drug concentration. The presence or absence of a combinatorial effect of two drugs was evaluated as antagonistic, additive and synergistic effect when the SI value was greater than 1, was equal to 1 and was less than 1, respectively (Table 1; Pharmacol Rev. 2006; 58 (3): 621- 81, BMC Complement Altern Med. 2013; 13: 212, and Anticancer Res. 2005; 25 (3B): 1909-17).

Table 1

IP range (upper limit) Description 0.1 Very strong synergy 0.3 Strong synergy 0.7 Synergy 0.85 Moderate synergy 0.9 Weak synergy one Almost additivity 1.2 Weak Antagonism 1.45 moderate antagonism 3.3 Antagonism ten Strong antagonism >10 Very strong antagonism

Concentration combinations of Compound 1 and 5-FU that resulted in an MF value of 0.5 (corresponding to ED ₅₀ ), 0.75 (corresponding to ED ₇₅ ) or 0.9 (corresponding to ED ₉₀ ) were derived from MF values for cells SNU-16 calculated for combined concentrations of Compound 1 and 5-FU and used to fit a linear curve using CalcuSyn software (HULINKS Inc.) to obtain IP.

B. Results

The combined use of Compound 1 and 5-FU resulted in synergistic effects for all combinations with the 3 concentration ratios used (Figures 6A - 6C). In particular, synergistic effects as strong as IS=0.3-0.4 were found in the concentration range of 10-200 nM added Compound 1. Table 2 below lists the values of each coupling index obtained at a ratio of 5-FU :compound 1 at 1250:1, 1000:1 or 500:1 at each time point when the cell growth inhibitory effect of drug sharing was 50%, 75% and 90% (ED ₅₀ , ED ₇₅ and ED ₉₀ ).

Based on the results described above, it was concluded that the preferred sharing effect is achieved when Compound 1 or a pharmaceutically acceptable salt thereof is used in the range of 0.0008 to 0.002 moles per mole of 5-FU.

table 2

cell line 5-FU: Compound 1 Combination index value _ED50 ED _{75 ED90} SNU-16 1250:1 0.69 0.53 0.43 1000:1 0.99 0.56 0.32 500:1 0.51 0.40 0.32

Example 7: Evaluation in vitro effect of compound 1 and 5-FU, paclitaxel, cisplatin or gemcitabine used in combination

A. Materials and methods

Human gastric cancer cell line SNU-16 (obtained from the American Type Culture Collection), human breast cancer cell line MFM223 (obtained from the European Cell Culture Collection), human lung cancer cell line H1581 (obtained from the American Type Culture Collection), DMS114 ( obtained from the American Type Culture Collection) and LK2 (obtained from the Cell Bank Research Facility of the Department of Health), AN3CA human endometrial cancer cells (obtained from the American Type Culture Collection) and MFE280 (obtained from DS Pharma Biomedical Co., Ltd.) , as well as human bladder cancer cells line RT112/84 (obtained from the European collection of cell cultures) were cultured using any of the media RPMI-1640, DMEM and MEM containing 10% fetal bovine serum. Cells of each line were maintained at 37°C in an atmosphere with 5% CO ₂ and subcultured in a ratio of from 1:5 to 1:20 1-2 passages per week. The names of the cells used, the carcinomas that are their source, and information on FGFR mutations are given below.

Table 3

cell line Source, carcinoma Information about changes in the FGFR gene SNU-16 Stomach cancer FGFR2 amplification MFM223 Mammary cancer Amplification of FGFR1,2 H1581 Lung cancer FGFR1 amplification DMS114 Lung cancer FGFR1 amplification LK2 Lung cancer FGFR1 amplification AN3CA endometrial cancer FGFR2 K310R N549K MFE280 endometrial cancer FGFR2 S252W RT112/84 bladder cancer FGFR3 DT overexpression, TACC3 translocation

Cell survival analysis

Cell survival measurements were performed using CellTiter-Glo. Cells were harvested in the usual manner, suspended in the above medium containing 10% fetal bovine serum, and then plated in a 96-well plate. The number of cells added to the well was 2000 cells/80 μl. Cells were incubated at 37°C for 24 hours in an atmosphere with 5% CO ₂ and then 10 μl of medium containing compound 1 and 5-FU, paclitaxel, cisplatin or gemcitabine, or solvent, was added to each well.

Given the sensitivity of cells to each drug,

compound 1 was used at four concentrations obtained from 3-fold serial dilutions starting at 10, 100 and 1000 nM and zero concentration (DMSO);

5-FU was used at five concentrations obtained from 3-fold serial dilutions starting at 10 and 100 μM and zero concentration (DMSO);

paclitaxel was used at five concentrations obtained from 3-fold serial dilutions starting at 100 nM and zero concentration (DMSO);

cisplatin was used at five concentrations obtained from 3-fold serial dilutions starting at 60 and 166 μM and zero concentration (DMSO);

gemcitabine was used at five concentrations obtained from 3-fold serial dilutions starting at 30, 100, 300, and 10,000 nM and zero concentration (DMSO), and

tested each combination.

After the addition of the drug, the cells were additionally incubated at 37°C for 72 hours in an atmosphere with 5% CO ₂ . To determine cell viability, 100 μl of CellTiter-Glo solution was added to each well, the cells were incubated at room temperature for 10 minutes, and then the intensity of chemiluminescence in each well was measured on an ARVO plate reader. Cell viability upon drug addition was calculated as in Example 6.

In addition, IF (affected fraction) values of cells were calculated by subtracting 1/100 of the cell survival rate from 1.

The combination index (IC) value was then determined for each combined drug concentration. The presence or absence of the effect of the joint use of two drugs was evaluated as an antagonistic, additive and synergistic effect, when the SI value was more than 1, was equal to 1 and was less than 1, respectively.

The SI value for the combined use of Compound 1 and each chemotherapeutic agent was calculated for 8 cell lines derived from 5 different cancer tissues. The IC values for each condition are shown in Figure 7. The abscissa of Figure 7 shows the concentration of Compound 1 (nM) and the concentrations of the anticancer agents are shown in the drawing captions.

B. Results

When compound 1 was used in combination with 5-FU, paclitaxel, cisplatin, or gemcitabine, synergistic co-administration effects (IS<1) occurred over a wide range of chemotherapeutic agent concentrations. With respect to the concentration of compound 1 itself, synergistic effects of sharing were also established for the range from 1.1 to 111 nM. Moreover, a synergistic enhancement of the effects of chemotherapeutic agents by compound 1 was confirmed, regardless of the mechanisms of action of chemotherapeutic agents. In addition, different cancer cells in which the synergistic effect of sharing was demonstrated had different FGFR aberrations (amplification, point mutation, and translocation), indicating that synergistic effects occur regardless of the types of FGFR aberrations.

More specifically, as shown in Figure 7A, when using Compound 1 in combination with 5-FU, there were synergistic effects (IC < 1) in the concentration range from 11 to 100 nM added Compound 1, and the synergistic effects shown were strong (IC < 0 ,5) in the case of human lung cancer cell line DMS114.

As shown in Figure 7B, when Compound 1 was used in combination with cisplatin, there were synergistic effects (IS < 1) over a concentration range of 3.3 to 111 nM added Compound 1.

As shown in Figure 7C, when using Compound 1 in combination with paclitaxel, there were synergistic effects (IS<1) in the concentration range from 10 to 33 nM of added Compound 1.

As shown in Figure 7D, when Compound 1 was used in combination with gemcitabine, there were synergistic effects (IC < 1) over the concentration range of 3.7 to 111 nM added Compound 1, and the synergistic effects shown were strong (IC < 0.5) in the case of human endometrial cancer cells of the AN3CA line.

Example 8 Evaluation of the effect of compound 1 and an AKT inhibitor used in combination

A. Materials and Methods

AN3CA human endometrial cancer cells were cultured in MEM medium containing 10% fetal bovine serum. Cells were maintained at 37°C in an atmosphere with 5% CO ₂ and subcultured in a ratio of from 1:5 to 1:20 1-2 passages per week.

Cell survival analysis

Cell survival measurements were performed using CellTiter-Glo. Cells were harvested in the usual manner, suspended in the above medium containing 10% fetal bovine serum, and then plated in a 96-well plate. The number of cells added to the well was 2000 cells/80 μl. Cells were incubated at 37°C for 24 hours in an atmosphere with 5% CO ₂ and then 10 μl of medium containing compound 1 and AKT inhibitor 8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-1,2 ,4-triazolo[3,4-f][1,6]naphthyridin-3(2H)-one (MK2206) dihydrochloride, or diluent, was added to each well. Used 5 concentrations of compound 1, obtained by serial 3-fold dilution, with the highest final concentration of 0.03 μm. 4 concentrations of MK2206 dihydrochloride, obtained by serial 3-fold dilution, were used, with the highest concentration being 1 μM (calculated as free form). The DMSO addition group was used as a control, and Compound 1 alone, MK2206 dihydrochloride alone, and two drug combinations were studied at each concentration. Cells were additionally incubated at 37°C for 72 hours in an atmosphere with 5% CO ₂ (day 3). 100 μl of CellTiter-Glo solution was added to each well and cells were incubated at room temperature for 10 minutes, followed by measurement of chemiluminescence intensity on an ARVO plate reader. Based on the obtained data, the cell growth inhibitory effect was calculated relative to the results for the vehicle group.

The degree of cell growth inhibition for each drug concentration was calculated as described below based on the ratio between the chemiluminescence intensity in the control group on day 3 (S) and the chemiluminescence intensity in the control group on the day of drug addition (B) and plotted.

In case the chemiluminescence intensity on day 3 (S) was > chemiluminescence intensity on the day of drug addition (B),

Cell Growth Rate=(Chemiluminescence intensity on day 3, resulting from exposure to drug A at concentration X-B)/(S-B).

In case the chemiluminescence intensity on day 3 (S) was < the chemiluminescence intensity on the day of drug addition (B),

Cell Growth Rate=(Chemiluminescence intensity on day 3 resulting from drug A exposure at concentration X - B)/B

where drug A is Compound 1 or MK2206 dihydrochloride and concentration X is any concentration in the dilution series.

B. Results

It was found that in the case of AN3CA endometrial cancer cells having a FGFR2 mutation, the combined use of compound 1 and MK2206 dihydrochloride resulted in an increase in the cell growth inhibitory effect depending on the concentration of each drug, compared with the cell growth inhibitory effect of each drug used. separately (Figures 8A and 8B). In particular, it was found that when Compound 1 and MK2206 dihydrochloride were used together at high concentrations (for example, a combination of 0.01 μM or higher concentration of Compound 1 and 0.1 μM or higher concentration of MK2206) there was a cell killing effect, the amount which decreased compared to the number of cells during the addition of the drug, indicating the induction of apoptosis.

Based on the results described above, it was concluded that a preferred sharing effect is achieved when Compound 1 or a pharmaceutically acceptable salt thereof is used in the range of 0.001 to 1 mole per mole of MK2206.

Example 9 Evaluation of the effect of compound 1 and an AKT inhibitor used in combination (confirmation of apoptosis induction)

A. Materials and methods

AN3CA human endometrial cancer cells were cultured in MEM medium containing 10% fetal bovine serum. Cells were maintained at 37°C in an atmosphere with 5% CO ₂ and subcultured in a ratio of from 1:5 to 1:20 1-2 passages per week.

Cell survival analysis

The presence or absence of induction of apoptosis by drug sharing was analyzed using CellEvent™ Green Caspase-3/7 Detection Reagent (Invitrogen Corp.). Cells were collected in the usual manner, suspended in the above medium containing 10% fetal bovine serum, and then seeded in a 12-well plate. The number of cells added to the well was 1 x 10 ⁶ cells/800 µl. Cells were incubated at 37°C for 24 hours in an atmosphere with 5% CO ₂ and then 100 μl of medium containing compound 1 and MK2206 dihydrochloride, or solvent, was added to each well. The final concentration of Compound 1 added was 10 nM. The concentration of MK2206 dihydrochloride was 300 nM (free form). The effects of these drugs were compared, used alone (DMSO was added as a control) or in combination. After adding the drug, the cells were incubated at 37°C for 24 hours in an atmosphere of 5% CO ₂ . Apoptosis was detected by adding 1 μl of CellEvent™ Green Caspase-3/7 Detection Reagent solution (Invitrogen Corp.) to each cell well, incubating the cells at 37°C for 30 minutes, followed by cell imaging and measurement of fluorescent signals in bright field under a fluorescent microscope. The observed fluorescent signals indicated the induction of caspase 3/7, and the amplitude of the signals indicated the presence or absence of induction of cell death due to apoptosis.

B. Results

As shown in Figure 9, the induction of cell death could be hardly detected in the group without the addition of drugs, as well as in cells to which Compound 1 or MK2206 dihydrochloride was added alone. On the other hand, an image with a strong fluorescent color indicative of cell death induction was found in the compound 1 and MK2206 dihydrochloride addition group, indicating a strong apoptosis-inducing effect of the two drugs used in combination.

Example 10 Evaluation of the Antitumor Effect of Compound 1 Used in Combination with an AKT Inhibitor or an mTOR Inhibitor

A. Materials and methods

AN3CA human endometrial cancer cells were cultured in MEM medium containing 10% fetal bovine serum. Cells were maintained at 37°C in an atmosphere with 5% CO ₂ and subcultured in a ratio of from 1:5 to 1:20 1-2 passages per week.

Cell survival analysis

Cell survival measurements were performed using CellTiter-Glo. Cells were harvested in the usual manner, suspended in the above medium containing 10% fetal bovine serum, and then plated in a 96-well plate. The number of cells added to the well was 2000 cells/80 μl. Cells were incubated at 37° C. for 24 hours in a 5% CO ₂ atmosphere, and then 10 μl of medium containing compound 1 and MK2206 dihydrochloride or the mTOR inhibitor everolimus or solvent was added to the cells in each well. Used 10 concentrations of compound 1 obtained by serial 2-fold dilution, with the highest final concentration of 0.16 μm. 10 concentrations of MK2206 dihydrochloride, obtained by serial 2-fold dilution, were used, with the highest final concentration being 6.4 μM (calculated as free form). Used 10 concentrations of everolimus obtained by serial 2-fold dilution, with the highest final concentration of 0.032 μm. DMSO was added as a control, and the effect of using each drug alone or in combination with compound 1 was studied. After the drug was added to the cells, the cells were further incubated at 37° C. for 72 hours in a 5% CO ₂ atmosphere. 100 μl of CellTiter-Glo solution was added to each well and cells were incubated at room temperature for 10 minutes, followed by chemiluminescence measurement on an ARVO plate reader. Cell growth inhibitory effects relative to the solvent group were calculated from the data obtained in a similar manner as in Examples 7 and 8.

The concentration at which each drug caused 50% inhibition of cell growth (IC ₅₀ ) was determined using the CalcuSyn 2.0 median effect analysis program (CalcuSyn, Inc.) similarly to the procedure of Example 6. The combination index (IC) value for each combination was then determined. drug concentrations.

The concentration combinations of Compound 1 and MK2206 dihydrochloride or everolimus that resulted in MF values of 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 were derived from the MF values calculated for the combined concentrations of compound 1 and MK2206 dihydrochloride or everolimus in the case of AN3CA cells, and used to fit a linear curve using the CalcuSyn program to obtain IS.

B. Results

IC values for combinations of compound 1 and MK2206 dihydrochloride or everolimus concentrations that resulted in an MF of 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 were calculated and shown in Table 4 In the table, molar ratios between drugs are shown in brackets. It was found that the combined use of compound 1 and everolimus or MK2206 dihydrochloride leads to strong synergistic effects (IC <0.3) in all tested ranges of drug concentrations (Figures 10A and 10B and Table 4).

Based on the results described above, it was concluded that the preferred sharing effect is achieved when Compound 1 or a pharmaceutically acceptable salt thereof is used in an amount of 5 moles per mole of everolimus.

It was also concluded that a preferred sharing effect is achieved when Compound 1 or a pharmaceutically acceptable salt thereof is used in an amount of 0.025 moles per mole of MK2206.

Table 4

Medicine Everolimus: Compound 1 (1:5) PF 0.4 0.270 0.5 0.264 0.6 0.260 0.7 0.257 0.8 0.255 0.9 0.258 Medicine MK2206: Compound 1 (1:0.025) PF 0.4 0.256 0.5 0.195 0.6 0.149 0.7 0.111 0.8 0.078 0.9 0.046

Example 11 Effect of Compound 1 and an AKT Inhibitor or an mTOR Inhibitor Used in Combination on the MAPK/ERK Pathway and the PI3K/AKT Pathway

A. Materials and methods

Human AN3CA endometrial cancer cells were grown in MEM medium containing 10% fetal bovine serum. Cells were maintained at 37°C in an atmosphere with 5% CO ₂ and subcultured in a ratio of from 1:5 to 1:20 1-2 passages per week.

Western blotting

Levels of intracellular phosphorylation of ERK, AKT, mTOR, and S6 were determined by Western blotting. Cells were collected in the usual manner, suspended in the above medium containing 10% fetal bovine serum, and then seeded in a 12-well plate. The number of cells added to the well was 1 x 10 ⁶ cells/800 µl. Cells were incubated at 37°C for 24 hours in an atmosphere with 5% CO ₂ and then 100 μl of medium containing compound 1 and MK2206 dihydrochloride or everolimus or solvent was added to each well. Compound 1 was added to cells at final concentrations of 10 and 100 nM. Similarly, MK2206 dihydrochloride was added at a final concentration of 5 μM (calculated as free form) and everolimus was added at a final concentration of 3 nM. Studied the effects of each drug individually and each combination. DMSO was added as a control at a final concentration of 0.2%.

Cells were incubated at 37°C for 2 hours in an atmosphere with 5% CO ₂ . Cell lysates were then prepared in RIPA buffer [composition: 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% (w/w) Nonidet P-40, 0.1% (w/w) deoxycholate sodium and 0.1% (by weight) SDS]. The amounts of phosphorylated AKT, phosphorylated ERK, phosphorylated mTOR, phosphorylated S6 and GAPDH were determined by conventional Western blotting.

B. Results

As shown in Figure 11, compound 1 at both concentrations of 10 and 100 nM strongly inhibited ERK phosphorylation in AN3CA cells treated with compound 1 for 24 hours. On the other hand, treatment of MK2206 alone with dihydrochloride or everolimus resulted in inhibition of AKT phosphorylation or mTOR phosphorylation, respectively. The combined use of Compound 1 and MK2206 dihydrochloride resulted in inhibition of both ERK phosphorylation and AKT phosphorylation. In particular, strong inhibition of S6 protein phosphorylation occurred only with the combination of drugs. With regard to the combined use of compound 1 and everolimus, the combined inhibition of ERK phosphorylation and S6 protein phosphorylation was observed only when both drugs were used together.

Based on the results described above, it was concluded that a preferred co-use effect is achieved when Compound 1 or a pharmaceutically acceptable salt thereof is used in the range of 0.002 to 0.02 moles per mole of everolimus.

It was also concluded that a preferred sharing effect is achieved when Compound 1 or a pharmaceutically acceptable salt thereof is used in the range of 0.002 to 0.02 moles per mole of MK2206.

Example 12: Activation of the FGF/FGFR Pathway Responsible for EGFR Inhibitor Resistance and the Effect of Compound 1 Addition

A. Materials and methods

Human lung cancer cell lines HCC4006 (obtained from American Type Culture Collection), NCI-H1650 (obtained from American Type Culture Collection), and NCI-H322 (obtained from DS Pharma Biomedical Co., Ltd.) were cultured in RPMI medium containing 10% fetal bovine serum. Cells of each line were maintained at 37°C in an atmosphere with 5% CO ₂ and subcultured in a ratio of from 1:5 to 1:20 1-2 passages per week.

Cell survival analysis

Cell survival measurements were performed using CellTiter-Glo. Cells were harvested in the usual manner, suspended in the above medium containing 10% fetal bovine serum, and plated in a 96-well plate. The number of cells added per well was 2000 cells/70 μl. Cells were incubated at 37°C for 24 hours in an atmosphere with 5% CO ₂ and then 10 μl of medium containing compound 1, EGFR inhibitor gefitinib, solvent (DMSO) or 2 drugs: compound 1 and gefitinib were added to each well . To study the effects on the activation of the FGF/FGFR pathway in cells, the FGF2 or FGF7 ligand was added to the cells and compared to a control without addition. 10 concentrations of compound 1 and gefitinib, obtained from serial 3-fold dilutions, were used, with the highest final concentrations being 100 nM and 10 μM, respectively. DMSO was added to the control well and 10 ng/ml FGF2 or 100 ng/ml FGF7 was added to the medium.

Cells were incubated at 37°C for 72 hours in an atmosphere with 5% CO ₂ . Then 100 μl of CellTiter-Glo solution was added to each well, and the cells were incubated at room temperature for 10 minutes, followed by measurement of chemiluminescence intensity on a plate reader (ARVO). Based on the obtained data, the cell growth inhibitory effects were calculated relative to the solvent group.

B. Results

The HCC4006 and NCI-H1650 lung cancer cell lines are deficient in EGFR inhibitor-responsive EGFR exon 9 (Figure 12A). The addition of the FGFR FGF7 ligand attenuates the drug effect of gefitinib (Figures 12B and 12C). When FGFR activation upon addition of FGF was inhibited by the addition of compound 1 to it, the effect of gefitinib was restored to the same level as in the absence of the ligand, or further enhanced. Similar effects of Compound 1 were found in FGFR-expressing NCI-H322 cells lacking the EGFR mutation (Figure 12D). These results demonstrate that the combined use of Compound 1 and an EGFR inhibitor is effective in the treatment of lung cancer in which FGF/FGFR signals cause resistance or low responsiveness to an EGFR inhibitor.

Based on the results described above, it was concluded that a preferred co-use effect is achieved when Compound 1 or a pharmaceutically acceptable salt thereof is used in the range of 0.01 to 1 mole per mole of gefitinib.

INDUSTRIAL APPLICABILITY

The present invention can greatly enhance the anti-tumor effect compared to the situation where conventional anti-tumor agents are administered separately, and the effect can also take place in the case of drug-resistant tumors; thus, the possibilities of chemotherapy in the treatment of malignant tumors can be significantly expanded.

All publications, patents and patent applications cited in this document are incorporated herein by reference in their entirety.

Claims (26)

1. Application of (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1- yl) prop-2-en-1-one or a pharmaceutically acceptable salt thereof for the treatment of a cancer patient receiving one or more additional compounds having an antitumor effect, or a pharmaceutically acceptable salt(s) thereof, selected from 5-fluorouracil, tegafur/ gimeracil/oteracil potassium, gemcitabine, paclitaxel, cisplatin, everolimus, MK2206 and trans-3-amino-1-methyl-3-(4-(3-phenyl-5H-imidazo[1,2-c]pyrido[3,4 -e][1,3]oxazin-2-yl)phenyl)cyclobutanol, gefitinib. 2. Use according to claim 1, wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidine-1- yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof and an additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof are administered simultaneously, separately or sequentially. 3. Use according to claim 1, wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidine-1- yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof and an additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof are administered by the same route of administration or administered by different routes of administration. 4. Use according to claim 1, wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidine-1- yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof is used in an amount of 0.0001 to 1 mol per mol of 5-fluorouracil. 5. Use according to claim 1, wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidine-1- yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof is used in an amount of 0.01 to 10 moles per mole of tegafur. 6. Use according to claim 1, wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidine-1- yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof is used in an amount of 0.001 to 1 mol per mol of gemcitabine. 7. Use according to claim 1, wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidine-1- yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof is used in an amount of 0.01 to 10 moles per mole of paclitaxel. 8. Use according to claim 1, wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidine-1- yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof is used in an amount of 0.01 to 50 moles per mole of cisplatin. 9. Use according to claim 1, wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidine-1- yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof is used in an amount of from 1 to 1000 moles per mole of everolimus. 10. Use according to claim 1, wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidine-1- yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof is used in an amount of from 1 to 1000 moles per mole of MK2206. 11. Use according to claim 1, wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidine-1- yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof is used in an amount of from 1 to 1000 moles per mole of trans-3-amino-1-methyl-3-(4-(3- phenyl-5H-imidazo[1,2-c]pyrido[3,4-e][1,3]oxazin-2-yl)phenyl)cyclobutanol. 12. Use according to claim 1, wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidine-1- yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof is used in an amount of 1 to 1000 moles per mole of gefitinib. 13. Use according to claim 1, wherein the tumor is selected from the group consisting of lung cancer, esophageal cancer, gastric cancer, duodenal cancer, liver cancer, hepatic cell cancer, bile duct cancer, pancreatic cancer, colorectal cancer, breast cancer cancer of the uterine body, ovarian cancer, kidney cancer, bladder cancer, prostate cancer, testicular tumor, thyroid cancer, bone or soft tissue tumor, leukemia, malignant lymphoma, multiple myeloma, head and neck cancer, brain tumor and skin cancer. 14. A method for treating a tumor, comprising administering (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl )pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof and one or more additional compounds having an antitumor effect, or a pharmaceutically acceptable salt(s) thereof, selected from 5-fluorouracil, tegafur/gimeracil /oteracil potassium, gemcitabine, paclitaxel, cisplatin, everolimus, MK2206 and trans-3-amino-1-methyl-3-(4-(3-phenyl-5H-imidazo[1,2-c]pyrido[3,4- e][1,3]oxazin-2-yl)phenyl)cyclobutanol, gefitinib, to a patient who needs it. 15. The method of claim 14, wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidine-1 -yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof and an additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof are administered simultaneously, separately or sequentially. 16. The method of claim 14, wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidine-1 -yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof and an additional compound having an antitumor effect or a pharmaceutically acceptable salt thereof are administered by the same route of administration or administered by different routes of administration. 17. The method of claim 14 wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidine-1- yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof is administered in an amount of 0.0001 to 1 mole per mole of 5-fluorouracil. 18. The method of claim 14 wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidine-1- yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof is administered in an amount of 0.01 to 10 moles per mole of tegafur. 19. The method of claim 14 wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidine-1- yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof is administered in an amount of 0.001 to 1 mol per mol of gemcitabine. 20. The method of claim 14 wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidine-1- yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof is administered in an amount of 0.01 to 10 moles per mole of paclitaxel. 21. The method of claim 14, wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidine-1 -yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof is administered in an amount of 0.01 to 50 moles per mole of cisplatin. 22. The method of claim 14, wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidine-1 -yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof is administered in an amount of 1 to 1000 moles per mole of everolimus. 23. The method of claim 14, wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidine-1 -yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof is administered in an amount of 1 to 1000 moles per mole of MK2206. 24. The method of claim 14, wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidine-1 -yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof is administered in an amount of from 1 to 1000 moles per mole of trans-3-amino-1-methyl-3-(4-(3 -phenyl-5H-imidazo[1,2-c]pyrido[3,4-e][1,3]oxazin-2-yl)phenyl)cyclobutanol. 25. The method of claim 14, wherein (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidine-1 -yl)pyrrolidin-1-yl)prop-2-en-1-one or a pharmaceutically acceptable salt thereof is administered in an amount of 1 to 1000 moles per mole of gefitinib. 26. The method according to claim 14, characterized in that the tumor is selected from the group consisting of lung cancer, esophageal cancer, gastric cancer, duodenal cancer, liver cancer, hepatic cell cancer, bile duct cancer, pancreatic cancer, colorectal cancer , breast cancer, endometrial cancer, ovarian cancer, kidney cancer, bladder cancer, prostate cancer, testicular tumor, thyroid cancer, bone or soft tissue tumor, leukemia, malignant lymphoma, multiple myeloma, head and neck cancer, tumors brain and skin cancer.

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