# Patent

## Title
Antitumor agent using compounds having kinase inhibitory effect in combination

## Abstract

An antitumor agent for combined use of a compound or pharmaceutically acceptable salt thereof represented by Formula (I) and a compound or pharmaceutically acceptable salt thereof represented by Formula (II) exhibits an excellent antitumor effect compared to cases where these are individually used, and exhibits antitumor effects against various cancer types:




wherein R1 is azetidinyl and the like, R2 to R5 is a hydrogen atom or a halogen atom, R6 is C3-8 cycloalkyl and the like, R7 is a hydrogen atom and the like, and R8 is a halogen atom and the like.

## Background
TECHNICAL FIELD
The present invention relates to an antitumor agent for combined use of compounds having a kinase inhibitory effect. Particularly, the present invention relates to an antitumor agent for combined use of a compound having a HGFR inhibitory effect and a compound having a multi-tyrosine kinase inhibitory effect.
BACKGROUND ART




wherein R1 is azetidinyl and the like, R2 to R5 is a hydrogen atom or a halogen atom, R6 is C3-8 cycloalkyl and the like, R7 is a hydrogen atom and the like, and R8 is a halogen atom and the like.

The compound represented by Formula (I) has potent inhibitory effects against hepatocyte growth factor receptor (HGFR), and thus is useful as an antitumor agent, an angiogenesis inhibitor, and a tumor metastasis inhibitor (Patent Literature 1). HGFR is known to be overexpressed in a large number of tumor cells (Non Patent Literature 1) and involved in malignant alteration of tumors. Further, HGFR is also expressed in vascular endothelial cells, and is considered to cause the proliferation of tumors by promoting angiogenesis (Non Patent Literature 2).
On the other hand, the compound represented by Formula (II) has anti-angiogenic actions (Patent Literature 2), inhibitory effects (Patent Literatures 3 to 6) against tyrosine kinases which are reported to be involved in malignant alteration of tumors (Non-Patent Literatures 3 to 5), and the like; and is known as a therapeutic agent for various tumors such as thyroid cancer, lung cancer, melanoma, endometrial cancer, gastric cancer and bladder cancer.
In general, antitumor agents are often not effective for all of the patients when they were used individually. Thus, attempts have been made so far to increase the cure rate by combination of plural antitumor agents (Patent Literatures 7 to 9).
CITATION LIST
Patent Literature
Patent Literature 1: WO 2007/023768
Patent Literature 2: WO 2002/032872
Patent Literature 3: WO 2004/080462
Patent Literature 4: WO 2007/061130
Patent Literature 5: WO 2007/136103
Patent Literature 6: WO 2008/026748
Patent Literature 7: WO 2009/140549
Patent Literature 8: US Patent Application Publication No. 2004-259834
Patent Literature 9: U.S. Pat. No. 6,217,866
Non Patent Literature
Non Patent Literature 1: Oncology Reports, 5, 1013-1024, 1998.
Non Patent Literature 2: Advances in Cancer Research, 67, 257-279, 1995.
Non Patent Literature 3: Current Cancer Drug Targets, 6, 65-75, 2006.
Non Patent Literature 4: Nature Reviews, Cancer, 10, 116-129, 2010.
Non Patent Literature 5: Clinical Cancer Research, 15, 7119-7123, 2009.
SUMMARY OF INVENTION
Technical Problem
However, the therapeutic effects, which have been reported so far, obtained by combination of plural antitumor agents were insufficient, and hence development of a novel combination therapy using antitumor agents has been expected.
Solution to Problem
In view of such circumstances, the present inventors intensively studied to discover that administration of a combination of the compounds represented by Formula (I) and Formula (II) to a patient suffering from a tumor attains an unexpectedly excellent antitumor effect, thereby completing the present invention.
That is, the present invention provides [1] to [8] below.

    [1] An antitumor agent for combined use of:


a compound or pharmaceutically acceptable salt thereof represented by Formula (I):




wherein R1 is azetidinyl, piperidinyl, or a formula —NR11aR11b, each of which optionally have a substituent selected from Substituent group A, wherein R11a and R11b are the same or different and each is a hydrogen atom, C1-6 alkyl, or piperidinyl optionally having C1-6 alkyl, Substituent group A consists of hydroxyl, piperazinyl optionally having methyl, and azetidinyl optionally having dimethylamino, and

R2 to R5 are the same or different and each is a hydrogen atom or a fluorine atom; and

a compound or pharmaceutically acceptable salt thereof represented by Formula (II):




wherein R6 is C1-6 alkyl or C3-8 cycloalkyl,

R7 is a hydrogen atom, C1-6 alkyl, or C1-6 alkoxy, and

R8 is a hydrogen atom or a halogen atom.

    [2] An antitumor agent for simultaneous or separate administration of a compound or pharmaceutically acceptable salt thereof represented by the above Formula (I), and a compound or pharmaceutically acceptable salt thereof represented by the above Formula (II).
    [3] An antitumor agent comprising a compound or pharmaceutically acceptable salt thereof represented by the above Formula (I), and a compound or pharmaceutically acceptable salt thereof represented by the Formula (II).
    [4] A compound or pharmaceutically acceptable salt thereof represented by the Formula (II) for therapy of a tumor by combined use with a compound or pharmaceutically acceptable salt thereof represented by the above Formula (I).
    [5] A compound or pharmaceutically acceptable salt thereof represented by the Formula (I) for therapy of a tumor by combined use with a compound or pharmaceutically acceptable salt thereof represented by the above Formula (II).
    [6] A method of treating a tumor, wherein a compound or pharmaceutically acceptable salt thereof represented by the above Formula (I), and a compound or pharmaceutically acceptable salt thereof represented by the Formula (II) are used in combination.
    [7] A pharmaceutical composition comprising a compound or pharmaceutically acceptable salt thereof represented by the above Formula (I), a compound or pharmaceutically acceptable salt thereof represented by the Formula (II), and a vehicle.
    [8] A kit comprising:


a pharmaceutical composition comprising a compound or pharmaceutically acceptable salt thereof represented by the above Formula (I) and a vehicle; and
a pharmaceutical composition comprising a compound or pharmaceutically acceptable salt thereof represented by the Formula (II), and a vehicle.
The compound represented by the above Formula (I) is preferably one or more compounds selected from the group consisting of

    N-(2-fluoro-4-{[2-({[4-(4-methylpiperazin-1-yl)piperidin-1-yl]carbonyl}amino)pyridin-4-yl]oxy}phenyl)-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide:






    N-[4-({2-[({4-[3-(dimethylamino)azetidin-1-yl]piperidin-1-yl]carbonyl}amino)pyridin-4-yl}oxy)-2-fluorophenyl]-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide:






    N-{2,5-difluoro-4-[(2-{[(3-hydroxyazetidin-1-yl)carbonyl]amino}pyridin-4-yl)oxy]phenyl}-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide:






    N-{2,5-difluoro-4-[(2-{[4-(4-methylpiperazin-1-yl)piperidin-1-yl]carbonyl}amino)pyridin-4-yl]oxy}phenyl)-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide:






and

    N-(2,5-difluoro-4-{[2-({[methyl(1-methylpiperidin-4-yl)amino]carbonyl}amino)pyridin-4-yl]oxy}phenyl)-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide:






and more preferably

    N-(2-fluoro-4-{[2-({[4-(4-methylpiperazin-1-yl)piperidin-1-yl]carbonyl}amino)pyridin-4-yl]oxy}phenyl)-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide:






The compound represented by the above Formula (II) is preferably one or more compounds selected from the group consisting of

    4-[3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy]-7-methoxy-6-quinolinecarboxamide:






    4-[3-chloro-4-(methylaminocarbonyl)aminophenoxy]-7-methoxy-6-quinolinecarboxamide:






    4-[3-chloro-4-(ethylaminocarbonyl)aminophenoxy]-7-methoxy-6-quinolinecarboxamide:






    N6-methoxy-4-(3-chloro-4-{[(cyclopropylamino)carbonyl)amino]phenoxy}-7-methoxy-6-quinolinecarboxamide:






and

    N6-methoxy-4-(3-chloro-4-{[(ethylamino)carbonyl]amino}phenoxy)-7-methoxy-6-quinolinecarboxamide:






and more preferably

    4-[3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy]-7-methoxy-6-quinolinecarboxamide:






Advantageous Effects of Invention
The present invention provides an antitumor agent for combined use of a compound having a HGFR inhibitory effect and a compound having a multi-tyrosine kinase inhibitory effect. Such an antitumor agent exhibits an excellent antitumor effect compared to cases where these are individually used, and exhibits antitumor effects against various cancer types.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a graph showing a combined effect of Compound A and Compound B in a model animal to which human malignant melanoma cell line (SEKI) was transplanted.
FIG. 2 is a graph showing a combined effect of Compound A and Compound B in a model animal to which human pancreatic cancer cell line (KP-4) was transplanted.
FIG. 3 is a graph showing a combined effect of Compound A and Compound B in a model animal to which human gastric cancer cell line (IM95m) was transplanted.
FIG. 4 is a graph showing a combined effect of Compound A and Compound B in a model animal to which human ovarian cancer cell line (A2780) was transplanted.
FIG. 5 is a graph showing a combined effect of Compound A and Compound B in a model animal to which human glioblastoma cell line (U87MG) was transplanted.



DESCRIPTION OF EMBODIMENTS
The compound or pharmaceutically acceptable salt thereof represented by Formula (I) according to the present invention can be produced by the method described in Patent Literature 1. Further, the compound or pharmaceutically acceptable salt thereof represented by Formula (II) according to the present invention can be produced by the method described in Patent Literature 2.
Examples of the pharmaceutically acceptable salt include salts with inorganic acids, salts with organic acids, salts with inorganic bases, salts with organic bases, and salts with acidic or basic amino acids.
Preferred examples of the salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like. Preferred examples of the salts with organic acids include salts with acetic acid, succinic acid, fumaric acid, maleic acid, tartaric acid, citric acid, lactic acid, stearic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid and the like.
Preferred examples of the salts with inorganic bases include alkaline metal salts such as a sodium salt and a potassium salt; alkaline earth metal salts such as a calcium salt and a magnesium salt; an aluminum salt; and an ammonium salt. Preferred examples of the salts with organic bases include salts with diethylamine, diethanolamine, meglumine, N,N-dibenzylethylenediamine and the like.
Preferred examples of the salts with acidic amino acids include salts with aspartic acid, glutamic acid and the like. Preferred examples of the salts with basic amino acids include salts with arginine, lysine, ornithine and the like.
Especially preferred pharmaceutically acceptable salts are salts with organic acids.
The antitumor agent of the present invention may be orally administered in the form of a solid formulation such as a tablet, granule, fine granule, powder or capsule, or in the form of a liquid, jelly, syrup or the like.
Further, the antitumor agent of the present invention may be parenterally administered in the form of an injection, suppository, ointment, cataplasm or the like.
The dose of the compound or pharmaceutically acceptable salt thereof represented by Formula (I) may be appropriately selected depending on the degrees of symptoms, age, sex and body weight of the patient, difference in sensitivity, route, time and interval of administration, type of pharmaceutical formulation, and/or the like. Usually, in cases where oral administration is carried out for an adult (60 kg body weight), the dose is 10 to 6000 mg, preferably 50 to 4000 mg per day. This may be administered at one time, or dividedly at 2 or 3 times per day.
The dose of the compound or pharmaceutically acceptable salt thereof represented by Formula (II) may be appropriately selected as in the case described above. Usually, in cases where oral administration is carried out for an adult (60 kg body weight), the dose is 1 to 600 mg, preferably 4 to 400 mg, more preferably 4 to 200 mg per day. This may be administered at one time, or dividedly at 2 or 3 times per day.
In cases where an oral solid formulation is prepared, a vehicle, and, as required, a binder, disintegrator, lubricant, coloring agent, flavoring agent and/or the like may be added to the principal component, that is, a compound or pharmaceutically acceptable salt thereof represented by Formula (I), and a compound or pharmaceutically acceptable salt thereof represented by Formula (II), to prepare, thereafter, a tablet, granule, fine granule, powder, capsule or the like according to a conventional method.
Examples of the vehicle include lactose, corn starch, white soft sugar, glucose, sorbitol, crystalline cellulose and silicon dioxide. Examples of the binder include polyvinyl alcohol, ethylcellulose, methylcellulose, gum Arabic, hydroxypropylcellulose and hydroxypropylmethylcellulose. Examples of the lubricant include magnesium stearate, talc and silica. Examples of the coloring agent include titanium oxide, iron sesquioxide, yellow iron sesquioxide, cochineal, carmine and riboflavin. Examples of the flavoring agent include cocoa powder, ascorbic acid, tartaric acid, peppermint oil, borneol and cinnamon powder. These tablets and granules may be coated as required.
In cases where an injection is prepared, a pH adjustor, buffering agent, suspending agent, solubilizer, stabilizer, isotonic agent, preservative and/or the like may be added as required to the principal component, to prepare an intravenous, subcutaneous or intramuscular injection, or an intravenous drip infusion. As required, these may be prepared into lyophilized products by conventional methods.
Examples of the suspending agent include methylcellulose, polysorbate 80, hydroxyethylcellulose, gum Arabic, powdered tragacanth, sodium carboxymethylcellulose and polyoxyethylene sorbitan monolaurate.
Examples of the solubilizer include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, macrogol and glycerin fatty acid ester.
Examples of the stabilizer include sodium sulfite and sodium metabisulfite. Examples of the preservative include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, sorbic acid, phenol, cresol and chlorocresol.
The antitumor agent of the present invention may be prepared by formulating a compound or pharmaceutically acceptable salt thereof represented by Formula (I), and a compound or pharmaceutically acceptable salt thereof represented by Formula (II) separately, and the both may be administered either at the same time or separately. Further, the two formulations may be placed in a single package, to provide the so called kit formulation. Further, the both compounds may be contained in a single formulation.
The type of the tumor to be treated with the antitumor agent of the present invention is not restricted, and examples thereof include fibroma, adipoma, myxoma, chondroma, osteoma, angioma, lymphoma, myeloma, melanoma, myoma, neuroma, glioma, osteosarcoma, myosarcoma, fibrosarcoma, papilloma, adenoma, brain tumor, and cancers such as cervical cancer, esophagus cancer, tongue cancer, lung cancer, breast cancer, pancreatic cancer, gastric cancer, small intestinal cancer in duodenum, jejunum, ileum and the like, large bowel cancer in colon, caecum, rectum and the like, bladder cancer, renal cancer, liver cancer, gallbladder cancer, prostate cancer, uterine cancer, ovarian cancer, thyroid cancer and pharyngeal cancer; and mixed tumors thereof.
EXAMPLES
The present invention is described in more detail by way of Examples below.
[List of Abbreviations]


    FBS: Fetal bovine serum
    EDTA: Ethylene diamine tetra acetic acid
    TV: Tumor volume
    RTV: Relative tumor volume
    Compound A: 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide mesylate
    Compound B: N-(2-fluoro-4-{[2-({[4-(4-methylpiperazin-1-yl)piperidin-1-yl]carbonyl}amino)pyridin-4-yl]oxy}phenyl)-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide tartrate


Example 1
A Combined Effect of Compound A and Compound B in a Model Animal to which Human Malignant Melanoma Cell Line (SEKI) was Transplanted
The human malignant melanoma cell line SEKI (JCRB Cell bank) was cultured using a 10% FBS-containing RPM 1640 medium (SIGMA) in a 5% CO2 incubator under the condition of 37° C. When the cells reached a state of approximately 80% confluency, the cells were collected using trypsin-EDTA. To these cells, a Hanks' Balanced Salt Solution containing 50% Matrigel was added to prepare a suspension at 5.0×107 cells/mL. The cell suspension thus obtained was subcutaneously transplanted at the lateral side of the body of a nude mouse (CAnN.Cg-Foxn1nu/CrlCrlj, Charles River Laboratories Japan, Inc.) in an amount of 0.1 mL, where each group contained six mice. From 11 days after the transplantation, Compound A (10 mg/kg, once daily, for 17 days) and Compound B (100 mg/kg, once daily, for 17 days) were orally administered, either individually or both in a row.
Setting the initial day of administration at Day 0, the major axis and the minor axis of a tumor developed in each mouse were measured using Digimatic caliper (Mitsutoyo Corporation) thereafter on Day 3, 7, 10, 14, and 17.
The tumor volume and the relative tumor volume were calculated according to the equations below.

TV=major axis (mm)×minor axis2 (mm2)/2

RTV=TV on the day of measurement/TV on the initial day of administration

The results of RTV were summarized in Table 1 and FIG. 1. The numbers in the Table indicate an average value±standard deviation (the same will apply to the following Tables). Compared to cases where Compound A and Compound B were each administered individually, the combined use of Compound A and Compound B exhibited a remarkably excellent antitumor effect. Also, as a result of performing two-way ANOVA with respect to log-transformed RTV by setting Compound A and Compound B as the factors, RTV on Day 17 was found to be statistically significant (p<0.05), whereby the synergistic effect of Compound A and Compound B was confirmed.










TABLE 1








Day 3
Day 7
Day 10





Control group
1.63 ± 0.10
3.35 ± 0.56
4.95 ± 1.00


Compound A group
1.71 ± 0.19
2.88 ± 0.35
3.74 ± 0.53


Compound B group
1.76 ± 0.22
2.93 ± 0.57
4.06 ± 0.85


Combination group of
1.43 ± 0.06
2.10 ± 0.38
2.66 ± 0.19


Compound A and





Compound B






Day 14
Day 17





Control group
7.18 ± 1.66
8.65 ± 1.89



Compound A group
5.06 ± 0.49
5.92 ± 0.50



Compound B group
5.23 ± 0.20
5.80 ± 1.35



Combination group of
2.80 ± 0.27
2.77 ± 0.38



Compound A and





Compound B









Example 2
A Combined Effect of Compound A and Compound B in a Model Animal to which Human Pancreatic Cancer Cell Line (KP-4) was Transplanted
The human pancreatic cancer cell line KP-4 (acquired from National Hospital Organization Kyushu Cancer Center) was cultured using a 10% FBS-containing RPMI 1640 medium (SIGMA) in a 5% CO2 incubator under the condition of 37° C. When the cells reached a state of approximately 80% confluency, the cells were collected using trypsin-EDTA. To these cells, a Hanks' Balanced Salt Solution containing 50% Matrigel was added to prepare a suspension at 5.0×107 cells/mL. The cell suspension thus obtained was subcutaneously transplanted at the lateral side of the body of a nude mouse (CAnN.Cg-Foxn1nu/CrlCrlj, Charles River Laboratories Japan, Inc.) in an amount of 0.1 mL, where each group contained six mice. From 11 days after the transplantation, Compound A (10 mg/kg, once daily, for 17 days) and Compound B (100 mg/kg, once daily, for 17 days) were orally administered, either individually or both in a row.
Setting the initial day of administration at Day 0, the major axis and the minor axis of a tumor developed in each mouse were measured using Digimatic caliper (Mitsutoyo Corporation) thereafter on Day 3, 7, 10, 14, and 17.
The tumor volume and the relative tumor volume were calculated according to the equations below.

TV=major axis (mm)×minor axis2 (mm2)/2

RTV=TV on the day of measurement/TV on the initial day of administration

The results of RTV were summarized in Table 2 and FIG. 2. Compared to cases where Compound A and Compound B were each administered individually, the combined use of Compound A and Compound B exhibited a remarkably excellent antitumor effect. Also, as a result of performing two-way ANOVA with respect to log-transformed RTV by setting Compound A and Compound B as the factors, RTV on Day 17 was found to be statistically significant (p<0.05), whereby the synergistic effect of Compound A and Compound B was confirmed.











TABLE 2









Day 3
Day 7
Day 10






Control group
2.27 ± 0.25
4.68 ± 0.70
7.12 ± 1.35



Compound A group
1.67 ± 0.16
2.89 ± 0.74
3.77 ± 1.26



Compound B group
1.71 ± 0.26
3.33 ± 1.06
4.72 ± 1.55



Combination group of
1.40 ± 0.14
1.54 ± 0.24
1.64 ± 0.23



Compound A and






Compound B







Day 14
Day 17






Control group
9.65 ± 2.61
9.92 ± 3.07




Compound A group
4.83 ± 1.75
5.81 ± 2.17




Compound B group
6.53 ± 2.19
9.05 ± 3.71




Combination group of
1.79 ± 0.32
2.13 ± 0.52




Compound A and






Compound B









Example 3
A Combined Effect of Compound A and Compound B in a Model Animal to Which Human Gastric Cancer Cell Line (IM95m) was Transplanted
The human gastric cancer cell line IM95m (Health Science Research Resources Bank) was cultured using a DMEM medium (Wako Pure Chemical Industries, Ltd) containing 4500 mg/mL glucose, 10% FBS, and 10 μg/mL insulin in a 5% CO2 incubator under the condition of 37° C. When the cells reached a state of approximately 80% confluency, the cells were collected using trypsin-EDTA. To these cells, a Hanks' Balanced Salt Solution containing 50% Matrigel was added to prepare a suspension at 1.0×108 cells/mL. The cell suspension thus obtained was subcutaneously transplanted at the lateral side of the body of a nude mouse (CAnN.Cg-Foxn1nu/CrlCrlj, Charles River Laboratories Japan, Inc.) in an amount of 0.1 mL, where each group contained six mice. From 13 days after the transplantation, Compound A (10 mg/kg, once daily, for 21 days) and Compound B (100 mg/kg, once daily, for 21 days) were orally administered continuously, either individually or both in a row.
Setting the initial day of administration at Day 0, the major axis and the minor axis of a tumor developed in each mouse were measured using Digimatic caliper (Mitsutoyo Corporation) thereafter on Day 4, 7, 11, 14, 18 and 21.
The tumor volume and the relative tumor volume were calculated according to the equations below.

TV=major axis (mm)×minor axis2 (mm2)/2

RTV=TV on the day of measurement/TV on the initial day of administration

The results of RTV were summarized in Table 3 and FIG. 3. Compared to cases where Compound A and Compound B were each administered individually, the combined use of Compound A and Compound B exhibited a remarkably excellent antitumor effect. Although no statistical significance was shown by two-way ANOVA, an effect of complete inhibition of tumor proliferation was confirmed by the combined use of Compound A and Compound B.










TABLE 3








Day 4
Day 7
Day 11





Control group
1.97 ± 0.16
2.87 ± 0.20
4.91 ± 0.64


Compound A group
1.53 ± 0.12
2.10 ± 0.18
2.65 ± 0.37


Compound B group
1.12 ± 0.08
1.24 ± 0.15
1.75 ± 0.17


Combination group of
0.92 ± 0.12
0.89 ± 0.22
0.76 ± 0.09


Compound A and





Compound B






Day 14
Day 18
Day 21





Control group
6.27 ± 0.83
8.38 ± 1.41
10.36 ± 1.74 


Compound A group
2.65 ± 0.49
2.80 ± 0.47
3.18 ± 0.57


Compound B group
1.85 ± 0.16
3.09 ± 0.48
4.02 ± 1.05


Combination group of
0.73 ± 0.15
0.91 ± 0.14
1.00 ± 0.25


Compound A and





Compound B









Example 4
A Combined Effect of Compound A and Compound B in a Model Animal to Which Human Ovarian Cancer Cell Line (A2780) was Transplanted
The human ovarian cancer cell line A2780 (ATCC) was cultured using a 10% FBS-containing RPMI 1640 medium (SIGMA) in a 5% CO2 incubator under the condition of 37° C. When the cells reached a state of approximately 80% confluency, the cells were collected using trypsin-EDTA. To these cells, a Hanks' Balanced Salt Solution containing 50% Matrigel was added to prepare a suspension at a concentration of 5.0×107 cells/mL. The cell suspension thus obtained was subcutaneously transplanted at the lateral side of the body of a nude mouse (CAnN.Cg-Foxn1nu/CrlCrlj, Charles River Laboratories Japan, Inc.) in an amount of 0.1 mL, where each group contained six mice. Compound A (10 mg/kg, once daily, for 10 days) and Compound B (100 mg/kg, once daily, for 10 days) were orally administered, either individually or both in a row.
Setting the initial day of administration at Day 0, the major axis and the minor axis of a tumor developed in each mouse were measured using Digimatic caliper (Mitsutoyo Corporation) thereafter on Day 3, 5, 8, and 10.
The tumor volume and the relative tumor volume were calculated according to the equations below.

TV=major axis (mm)×minor axis2 (mm2)/2

RTV=TV on the day of measurement/TV on the initial day of administration

The results of RTV were summarized in Table 4 and FIG. 4. Compared to cases where Compound A and Compound B were each administered individually, the combined use of Compound A and Compound B exhibited a remarkably excellent antitumor effect. Also, as a result of performing two-way ANOVA with respect to log-transformed RTV by setting Compound A and Compound B as the factors, RTV on Day 10 was found to be statistically significant (p<0.05), whereby the synergistic effect of Compound A and Compound B was confirmed.









TABLE 4








Day 3
Day 5





Control group
2.37 ± 0.60
7.52 ± 1.45


Compound A group
1.92 ± 0.17
4.77 ± 0.85


Compound B group
2.23 ± 0.42
7.01 ± 1.54


Combination group of
1.38 ± 0.12
1.95 ± 0.27


Compound A and




Compound B






Day 8
Day 10





Control group
17.47 ± 3.75
20.41 ± 6.02


Compound A group
 9.51 ± 2.44
12.37 ± 3.53


Compound B group
15.70 ± 2.27
21.29 ± 2.76


Combination group of
 2.50 ± 0.76
 3.34 ± 1.30


Compound A and




Compound B









Example 5
A Combined Effect of Compound A and Compound B in a Model Animal to Which Human Glioblastoma Cell Line (U87MG) was Transplanted
The human glioblastoma cell line (U87MG) (ATCC) was cultured using a 10% FBS-containing E-MEM medium (SIGMA) in a 5% CO2 incubator under the condition of 37° C. When the cells reached a state of approximately 80% confluency, the cells were collected using trypsin-EDTA. To these cells, a Hanks' Balanced Salt Solution containing 50% Matrigel was added to prepare a suspension at a concentration of 5.0×107 cells/mL. The cell suspension thus obtained was subcutaneously transplanted at the lateral side of the body of a nude mouse (CAnN.Cg-FOXn1nu/CrlCrlj, Charles River Laboratories Japan, Inc.) in an amount of 0.1 mL, where each group contained six mice. Compound A (10 mg/kg, once daily, for 21 days) and Compound B (100 mg/kg, once daily, for 21 days) were orally administered, either individually or both in a row.
Setting the initial day of administration at Day 0, the major axis and the minor axis of a tumor developed in each mouse were measured using Digimatic caliper (Mitsutoyo Corporation) thereafter on Day 2, 5, 7, 9, 12, 14, 16, 19, and 21.
The tumor volume and the relative tumor volume were calculated according to the equations below.

TV=major axis (mm)×minor axis2 (mm2)/2

RTV=TV on the day of measurement/TV on the initial day of administration

The results of RTV were summarized in Table 5 and FIG. 5. Compared to cases where Compound A and Compound B were each administered individually, the combined use of Compound A and Compound B exhibited a remarkably excellent antitumor effect. Also, although no statistical significance was shown by two-way ANOVA performed with respect to log-transformed RTV by setting Compound A and Compound B as the factors, an effect of complete inhibition of tumor proliferation was confirmed by the combined use of Compound A and Compound B.










TABLE 5








Day 2
Day 5
Day 7





Control group
1.30 ± 0.19
1.86 ± 0.45
2.45 ± 0.71


Compound A group
0.95 ± 0.08
1.27 ± 0.07
1.59 ± 0.16


Compound B group
0.69 ± 0.05
0.61 ± 0.05
0.56 ± 0.10


Combination group of
0.59 ± 0.05
0.49 ± 0.10
0.44 ± 0.09


Compound A and





Compound B






Day 9
Day 12
Day 14





Control group
3.19 ± 0.89
5.71 ± 1.58
8.88 ± 2.26


Compound A group
1.85 ± 0.13
3.29 ± 0.32
4.76 ± 0.49


Compound B group
0.57 ± 0.07
0.65 ± 0.08
0.73 ± 0.12


Combination group of
0.36 ± 0.11
0.48 ± 0.16
0.46 ± 0.17


Compound A and





Compound B






Day 16
Day 19
Day 21





Control group
12.13 ± 3.46 
18.47 ± 6.88 
23.08 ± 8.72


Compound A group
6.19 ± 0.95
9.60 ± 1.99
11.53 ± 2.57


Compound B group
0.93 ± 0.13
1.65 ± 0.37
 2.23 ± 0.51


Combination group of
0.59 ± 0.20
0.78 ± 0.26
 0.95 ± 0.38


Compound A and





Compound B

## Claims

1. A method of treating a tumor, comprising administering to a patient in need thereof a combination of:
a compound represented by Formula (I) or a pharmaceutically acceptable salt thereof:





wherein R1 is azetidinyl, piperidinyl, or a formula —NR11aR11b, each of which optionally have a substituent selected from Substituent group A, wherein R11a and R11b are the same or different and each is a hydrogen atom, C1-6 alkyl, or piperidinyl optionally having C1-6 alkyl, Substituent group A consists of hydroxyl, piperazinyl optionally having methyl, and azetidinyl optionally having dimethylamino, and
R2 to R5 are the same or different and each is a hydrogen atom or a fluorine atom; and
a compound represented by Formula (II) or a pharmaceutically acceptable salt thereof:





wherein R6 is C1-6 alkyl or C3-8 cycloalkyl,
R7 is a hydrogen atom, C1-6 alkyl, or C1-6 alkoxy, and
R8 is a hydrogen atom or a halogen atom.

2. The method of claim 1, wherein the compound represented by Formula (I) is selected from the group consisting of:
N-(2-fluoro-4-{[2-({[4-(4-methylpiperazin-1-yl)piperidin-1-yl]carbonyl}amino)pyridin-4-yl]oxy}phenyl)-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide:




N-[4-({2-[({4-[3-(dimethylamino)azetidin-1-yl]piperidin-1-yl}carbonyl)amino]pyridin-4-yl}oxy)-2-fluorophenyl]-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide:




N-{2,5-difluoro-4-[(2-{[(3-hydroxyazetidin-1-yl)carbonyl]amino}pyridin-4-yl)oxy]phenyl}-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide:




N-{2,5-difluoro-4-[(2-{[4-(4-methylpiperazin-1-yl)piperidin-1-yl]carbonyl}amino)pyridin-4-yl]oxy}phenyl)-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide:




and
N-(2,5-difluoro-4-{[2-({[methyl(1-methylpiperidin-4-yl)amino]carbonyl}amino)pyridin-4-yl]oxy}phenyl)-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide:




and the compound represented by Formula (II) is selected from the group consisting of:
4-[3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy]-7-methoxy-6-quinolinecarboxamide:




4-[3-chloro-4-(methylaminocarbonyl)aminophenoxy]-7-methoxy-6-quinolinecarboxamide:




4-[3-chloro-4-(ethylaminocarbonyl)aminophenoxy]-7-methoxy-6-quinolinecarboxamide:




N6-methoxy-4-(3-chloro-4-{[(cyclopropylamino)carbonyl)amino]phenoxy}-7-methoxy-6-quinolinecarboxamide:




and
N6-methoxy-4-(3-chloro-4-{[(ethylamino)carbonyl]amino}phenoxy)-7-methoxy-6-quinolinecarboxamide:

3. The method of claim 1, wherein the compound represented by Formula (I) is N-(2-fluoro-4-{[2-({[4-(4-methylpiperazin-1-yl)piperidin-1-yl]carbonyl}amino)pyridin-4-yl]oxy}phenyl)-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide:



and the compound represented by Formula (II) is 4-[3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy]-7-methoxy-6-quinolinecarboxamide:

4. The method of claim 1, comprising simultaneous administration of the compound represented by Formula (I), or a pharmaceutically acceptable salt thereof, and the compound represented by Formula (II), or a pharmaceutically acceptable salt thereof.

5. The method of claim 1, comprising separate administration of the compound represented by Formula (I), or a pharmaceutically acceptable salt thereof, and the compound represented by Formula (II), or a pharmaceutically acceptable salt thereof.

6. The method of claim 1, wherein the tumor is melanoma, pancreatic cancer, gastric cancer, ovarian cancer, or glioblastoma.

7. A pharmaceutical composition comprising:
a compound represented by Formula (I) or a pharmaceutically acceptable salt thereof:





wherein R1 is azetidinyl, piperidinyl, or a formula —NR11aR11b, each of which optionally have a substituent selected from Substituent group A, wherein R11a and R11b are the same or different and each is a hydrogen atom, C1-6 alkyl, or piperidinyl optionally having C1-6 alkyl,
Substituent group A consists of hydroxyl, piperazinyl optionally having methyl, and azetidinyl optionally having dimethylamino, and
R2 to R5 are the same or different and each is a hydrogen atom or a fluorine atom; and
a compound represented by Formula (II) or a pharmaceutically acceptable salt thereof:





wherein R6 is C1-6 alkyl or C3-8 cycloalkyl,
R7 is a hydrogen atom, C1-6 alkyl, or C1-6 alkoxy, and
R8 is a hydrogen atom or a halogen atom.

8. The pharmaceutical composition of claim 7, wherein the compound represented by Formula (I) is selected from the group consisting of:
N-(2-fluoro-4-{[2-({[4-(4-methylpiperazin-1-yl)piperidin-1-yl]carbonyl}amino)pyridin-4-yl]oxy}phenyl)-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide:




N-[4-({2-[({4-[3-(dimethylamino)azetidin-1-yl]piperidin-1-yl}carbonyl)amino]pyridin-4-yl}oxy)-2-fluorophenyl]-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide:




N-{2,5-difluoro-4-[(2-{[(3-hydroxyazetidin-1-yl)carbonyl]amino}pyridin-4-yl)oxy]phenyl}-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide:




N-{2,5-difluoro-4-[(2-{[4-(4-methylpiperazin-1-yl)piperidin-1-yl]carbonyl}amino)pyridin-4-yl]oxy}phenyl)-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide:




and
N-(2,5-difluoro-4-{[2-({[methyl(1-methylpiperidin-4-yl)amino]carbonyl}amino)pyridin-4-yl]oxy}phenyl)-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide:




and the compound represented by Formula (II) is selected from the group consisting of:
4-[3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy]-7-methoxy-6-quinolinecarboxamide:




4-[3-chloro-4-(methylaminocarbonyl)aminophenoxy]-7-methoxy-6-quinolinecarboxamide:




4-[3-chloro-4-(ethylaminocarbonyl)aminophenoxy]-7-methoxy-6-quinolinecarboxamide:




N6-methoxy-4-(3-chloro-4-{[(cyclopropylamino)carbonyl)amino]phenoxy}-7-methoxy-6-quinolinecarboxamide:




and
N6-methoxy-4-(3-chloro-4-{[(ethylamino)carbonyl]amino}phenoxy)-7-methoxy-6-quinolinecarboxamide:

9. The pharmaceutical composition of claim 7, wherein the compound represented by Formula (I) is N-(2-fluoro-4-{[2-({[4-(4-methylpiperazin-1-yl)piperidin-1-yl]carbonyl}amino)pyridin-4-yl]oxy}phenyl)-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide:



and the compound represented by Formula (II) is 4-[3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy]-7-methoxy-6-quinolinecarboxamide: