# Patent

## Title
Antitumor agent for undifferentiated gastric cancer

## Abstract

The present invention relates to a therapeutic agent represented by the General formula (I), or a pharmacologically acceptable salt thereof, or a solvate of the compound or the salt thereof:




The therapeutic agent comprises a substance having the activity of inhibiting kinase activity of fibroblast growth factor receptor 2 (“FGFR2”). The therapeutic agent can be used for treating undifferentiated gastric cancer, and can also be used to treat organisms comprising a cell overexpressing FGFR2 or a cell expressing mutant FGFR2, or both. The present invention further relates to a pharmaceutical composition comprising an FGFR2 inhibitory and methods of treatment therewith. The present invention also relates to a method for predicting the effect of an FGFR2 inhibitory substance on a patient.

## Background
This is a U.S. national phase application under 35 U.S.C. §371 of International Patent Application No. PCT/JP2007/07088 filed Aug. 27, 2007 and claims the benefit of Japanese Application No. JP2006-230816, filed Aug. 28, 2006. The International Application was published on Mar. 6, 2008 as International Publication No. WO/2008/026748 A1 under PCT Article 21(2). The content of both applications are incorporated herein in their entirety.
FIELD OF THE INVENTION
The present invention relates to a therapeutic agent and a therapeutic method for treating undifferentiated gastric cancer. The therapeutic agent comprises a substance having the activity of inhibiting kinase activity of fibroblast growth factor receptor 2 (“FGFR2”). The substance having such inhibitory activity is hereinafter also referred to as “FGFR2 inhibitory substance”. The present invention further relates to the use of the FGFR2 inhibitory substance for producing the therapeutic agent and to a FGFR2 inhibitory substance for the therapeutic agent.
In addition, the present invention relates to a pharmaceutical composition comprising a FGFR2 inhibitory substance to be administered to an organism comprising a cell overexpressing FGFR2, or a cell expressing mutant FGFR2, the pharmaceutical composition comprising a compound that inhibits FGFR2 kinase activity. The present invention also relates to a therapeutic method for a disease comprising administering an effective dosage of the FGFR2 inhibitory substance to the organism, to the use of the FGFR2 inhibitory substance for producing a pharmaceutical composition and to a FGFR2 inhibitory substance for the pharmaceutical composition.
Moreover, the present invention relates to a FGFR2 inhibitor.
The present invention also relates to a method for predicting the effect of a FGFR2 inhibitory substance on a patient by using either the expression level of FGFR2 in a cell, or the presence or absence of FGFR2 mutation in a cell as a biomarker.
BACKGROUND OF THE INVENTION
Gastric cancer is one of the major cancers that cause death. Gastric cancer is histopathologically classified into differentiated gastric cancer and undifferentiated gastric cancer, where the latter includes poorly differentiated adenocarcinoma, signet-ring cell carcinoma, mucinous carcinoma and the like.
In the case of undifferentiated gastric cancer, cancer cells are likely to diffuse and also likely to develop fibrosis that leads to scirrhous gastric cancer. Undifferentiated gastric cancer is commonly observed in young people, and is known to cause invasive proliferation and metastasis, indicating a poor prognosis (Clinical Cancer Research. 2(8), 1373-1381, 1996).
FGFR2 (also referred to as “K-sam”) is amplified in diffuse-type gastric cancer, namely, undifferentiated gastric cancer and is known to be involved in malignant alteration of cancer (Clinical Cancer Research. 2(8), 1373-1381, 1996, Journal of Cancer Research and Clinical Oncology. 127, 207-216, 2001). FGFR2 gene is reported to be amplified in 33% of patients with undifferentiated gastric cancer (Journal of Cancer Research and Clinical Oncology. 127, 207-216, 2001), and FGFR2 is reported to be positive in about 50% of patients with undifferentiated gastric cancer (Clinical Cancer Research. 2(8), 1373-1381, 1996).
FGFR2 gene induces transformation of NIH3T3 cell, and the transformed cell is reported to show tumorigenicity in nude mice (Cancer Research. 54, 3237-3241, 1994). Furthermore, FGFR2 truncated at C-terminal is reported to have strong transformation activity and predominantly expressed in undifferentiated gastric cancer cell line (Clinical Cancer Research. 2(8), 1373-1381, 1996, Cancer Research. 54, 3237-3241, 1994). For example, FGFR2 having residues downstream from tyrosine at 769 deleted is reported to have high transformation activity (Cancer Research. 54, 3237-3241, 1994).
It is also reported that FGFR2 gene is amplified in poorly differentiated gastric cancer, particularly scirrhous gastric cancer, while FGFR2 protein having C-terminal (including tyrosine residues 780, 784 and 813) deleted is specifically expressed in scirrhous gastric cancer (Cancer Research. 59(24), 6080-6086, 1999). Amplification of activated FGFR2 is reported to cause tumor growth in scirrhous gastric cancer (Cancer Research. 59(24), 6080-6086, 1999).
A FGFR2 inhibitory substance, diphenylamine derivative, is reported to dose-dependently suppress cell growth of a human scirrhous gastric cell line and show anti-tumor effect on a subcutaneous xenograft model of human scirrhous gastric cell line (Bioorganic and Medicinal Chemistry Letters. 14(4), 875-879, 2004).
A FGFR2 inhibitory substance, 4-[(4-fluoro-2-methylindol-5-yl)oxy]-6-methoxy-7-[3-(pyrrolidin-1-yl)propoxy]quinazoline, is suggested to be effective on gastric cancer overexpressing K-sam (Proceeding of the American Association for Cancer Research. 47, 890, 2006).
Accordingly, a FGFR2 inhibitory substance is suggested to show antiproliferative action and anti-tumor effect on undifferentiated gastric cancer, preferably poorly differentiated adenocarcinoma, signet-ring cell carcinoma, mucinous carcinoma and scirrhous gastric cancer.
Here, a compound represented by General Formula (I) is known as an antiangiogenic substance (International Publication No. 02/32872, International Publication No. 2004/080462 and International Publication No. 2005/063713). However, there is no report about the compound represented by General formula (I) having FGFR2 inhibitory activity.
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
The present invention was achieved regarding the circumstances described above and the problems to be solved by the invention are to provide a therapeutic agent and a therapeutic method for undifferentiated gastric cancer, and to provide a pharmaceutical composition and a therapeutic method which are more effective for an organism comprising at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2.
Another problem to be solved by the present invention is to provide a method for predicting the effect of a FGFR2 inhibitory substance.
Means for Solving the Problems
In order to solve the above problems, the present inventors have gone through keen examination, as a result of which the inventors found that the compound represented by General Formula (I), a pharmacologically acceptable salt thereof or a solvate thereof has FGFR2 inhibitory activity. The inventors also found that the compound represented by General Formula (I), a pharmacologically acceptable salt thereof or a solvate thereof exerts higher effect on undifferentiated gastric cancer. Furthermore, the inventors found that the compound represented by General Formula (I), a pharmacologically acceptable salt thereof or a solvate thereof exerts higher effect on an organism comprising at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2. The inventors also found that the effect of the compound represented by General formula (I), a pharmacologically acceptable salt thereof or a solvate thereof can be predicted by using at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in a cell as a biomarker.
Thus, the present invention relates to the following:
(1) A therapeutic agent for treating undifferentiated gastric cancer comprising a compound represented by General Formula (I), a pharmacologically acceptable salt thereof or a solvate thereof.
(2) A pharmaceutical composition to be administered to an organism comprising at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2, the pharmaceutical composition comprising a compound represented by General Formula (I), a pharmacologically acceptable salt thereof or a solvate thereof.
(3) A method for treating undifferentiated gastric cancer, characterized by administering an effective dosage of a compound represented by General Formula (I), a pharmacologically acceptable salt thereof or a solvate thereof to a patient.
(4) A method for inhibiting FGFR2 activation, characterized by administering an effective dosage of a compound represented by General Formula (I), a pharmacologically acceptable salt thereof or a solvate thereof to a patient.
(5) A method for treating a disease comprising administering an effective dosage of a compound represented by General Formula (I), a pharmacologically acceptable salt thereof or a solvate thereof to an organism comprising at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2.
(6) Use of a compound represented by General Formula (I), a pharmacologically acceptable salt thereof or a solvate thereof for producing a therapeutic agent for treating undifferentiated gastric cancer.
(7) Use of a compound represented by General Formula (I), a pharmacologically acceptable salt thereof or a solvate thereof for producing a pharmaceutical composition to be administered to an organism comprising at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2.
(8) A compound represented by General Formula (I), a pharmacologically acceptable salt thereof or a solvate thereof for a therapeutic agent for treating undifferentiated gastric cancer.
(9) A compound represented by General Formula (I), a pharmacologically acceptable salt thereof or a solvate thereof for a pharmaceutical composition to be administered to an organism comprising at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2.
(10) A FGFR2 inhibitor comprising a compound represented by General Formula (I), a pharmacologically acceptable salt thereof or a solvate thereof.
(11) A method for predicting whether a patient is highly sensitive to a compound represented by General Formula (I), a pharmacologically acceptable salt thereof or a solvate thereof, the method comprising using at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in a cell as a biomarker.
(12) A method for analyzing sensitivity of a cell to a compound represented by General Formula (I), a pharmacologically acceptable salt thereof or a solvate thereof, the method comprising determining at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in the cell.
(13) A method for selecting a cell having higher sensitivity to a compound represented by General Formula (I), a pharmacologically acceptable salt thereof or a solvate thereof, the method comprising determining at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in the cell.
(14) A method for selecting a patient having higher sensitivity to a compound represented by General Formula (I), a pharmacologically acceptable salt thereof or a solvate thereof, the method comprising determining at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in the cell.
(15) A method for classifying a patient, comprising analyzing sensitivity of the patient to a compound represented by General Formula (I), a pharmacologically acceptable salt thereof or a solvate thereof by determining at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in the cell, and classifying the patient based on the obtained results.
(16) A method for selecting a patient as a target of administering a compound represented by General Formula (I), a pharmacologically acceptable salt thereof or a solvate thereof, the method comprising determining at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in the cell, and selecting a patient having at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2 based on the obtained results.
(17) A method for predicting therapeutic effect of a compound represented by General Formula (I), a pharmacologically acceptable salt thereof or a solvate thereof on a patient, comprising determining at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in the cell.
(18) A method for determining at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in a cell from a patient in order to predict the degree of sensitivity of the patient to a compound represented by General Formula (I), a pharmacologically acceptable salt thereof or a solvate thereof.
The compound represented by General Formula (I), a pharmacologically acceptable salt thereof or a solvate thereof is as follows




[wherein, R1 represents a group represented by Formula —V1—V2—V3 (wherein, V1 represents an optionally substituted C1-6 alkylene group; V2 represents a single bond, an oxygen atom, a sulfur atom, a carbonyl group, a sulfinyl group, a sulfonyl group, a group represented by Formula —CONR6—, a group represented by Formula —SO2NR6—, a group represented by Formula —NR6SO2—, a group represented by Formula —NR6CO— or a group represented by Formula —NR6— (wherein, R6 represents a hydrogen atom, an optionally substituted C1-6 alkyl group or an optionally substituted C3-8 cycloalkyl group); V3 represents a hydrogen atom, an optionally substituted C1-6 alkyl group, an optionally substituted C2-6 alkenyl group, an optionally substituted C2-6 alkynyl group, an optionally substituted C3-8 cycloalkyl group, an optionally substituted C6-10 aryl group, an optionally substituted 5-10-membered heteroaryl group or an optionally substituted 3-10-membered nonaromatic heterocyclic group);

R2 represents a cyano group, an optionally substituted C1-6 alkoxy group, a carboxyl group, an optionally substituted C2-7 alkoxycarbonyl group or a group represented by Formula —CONVa11Va12 (wherein, Va11 represents a hydrogen atom, an optionally substituted C1-6 alkyl group, an optionally substituted C2-6 alkenyl group, an optionally substituted C2-6 alkynyl group, an optionally substituted C3-8 cycloalkyl group, an optionally substituted C6-10 aryl group, an optionally substituted 5-10-membered heteroaryl group or an optionally substituted 3-10-membered nonaromatic heterocyclic group; Va12 represents a hydrogen atom, an optionally substituted C1-6 alkyl group, an optionally substituted C2-6 alkenyl group, an optionally substituted C2-6 alkynyl group, an optionally substituted C3-8 cycloalkyl group, an optionally substituted C6-10 aryl group, an optionally substituted 5-10-membered heteroaryl group, an optionally substituted 3-10-membered nonaromatic heterocyclic group, a hydroxyl group, an optionally substituted C1-6 alkoxy group or an optionally substituted C3-8 cycloalkoxy group);

Y1 represents a group represented by either one of the following formulae





(wherein, R7 and R8 each independently represent a hydrogen atom, a halogen atom, a cyano group, a nitro group, an amino group, an optionally substituted C1-6 alkyl group, an optionally substituted C3-8 cycloalkyl group, an optionally substituted C1-6 alkoxy group, an optionally substituted C1-6 alkylthio group, a formyl group, an optionally substituted C2-7 acyl group, an optionally substituted C2-7 alkoxycarbonyl group or a group represented by Formula —CONVd1Vd2 (wherein, Vd1 and Vd2 each independently represent a hydrogen atom or an optionally substituted C1-6 alkyl group);

W1 and W2 each independently represent an optionally substituted carbon atom or nitrogen atom);

R3 and R4 each independently represent a hydrogen atom, an optionally substituted C1-6 alkyl group, an optionally substituted C2-6 alkenyl group, an optionally substituted C2-6 alkynyl group, an optionally substituted C3-8 cycloalkyl group, an optionally substituted C2-7 acyl group or an optionally substituted C2-7 alkoxycarbonyl group; and

R5 represents a hydrogen atom, an optionally substituted C1-6 alkyl group, an optionally substituted C2-6 alkenyl group, an optionally substituted C2-6 alkynyl group, an optionally substituted C3-8 cycloalkyl group, an optionally substituted C6-10 aryl group, an optionally substituted 5-10-membered heteroaryl group or an optionally substituted 3-10-membered nonaromatic heterocyclic group],

a pharmacologically acceptable salt thereof or a solvate thereof.
Preferably, the present invention relates to the following:
(19) A therapeutic agent for treating undifferentiated gastric cancer comprising 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide, a pharmacologically acceptable salt thereof or a solvate thereof.
(20) A pharmaceutical composition to be administered to an organism comprising at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2, the pharmaceutical composition comprising 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide, a pharmacologically acceptable salt thereof or a solvate thereof.
(21) A method for treating undifferentiated gastric cancer, characterized by administering an effective dosage of 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide, a pharmacologically acceptable salt thereof or a solvate thereof to a patient.
(22) A method for inhibiting FGFR2 activation, characterized by administering an effective dosage of 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide, a pharmacologically acceptable salt thereof or a solvate thereof to a patient.
(23) A method for treating a disease comprising administering an effective dosage of 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide, a pharmacologically acceptable salt thereof or a solvate thereof to an organism comprising at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2.
(24) Use of 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide, a pharmacologically acceptable salt thereof or a solvate thereof for producing a therapeutic agent for treating undifferentiated gastric cancer.
(25) Use of 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide, a pharmacologically acceptable salt thereof or a solvate thereof for producing a pharmaceutical composition to be administered to an organism comprising at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2.
(26) 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide, a pharmacologically acceptable salt thereof or a solvate thereof for a therapeutic agent for treating undifferentiated gastric cancer.
(27) 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide, a pharmacologically acceptable salt thereof or a solvate thereof for a pharmaceutical composition to be administered to an organism comprising at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2.
(28) A FGFR2 inhibitor comprising 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide, a pharmacologically acceptable salt thereof or a solvate thereof.
(29) A method for predicting whether or not a patient is highly sensitive to 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide, a pharmacologically acceptable salt thereof or a solvate thereof, the method comprising using at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in a cell as a biomarker.
(30) A method for analyzing sensitivity of a cell to 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide, a pharmacologically acceptable salt thereof or a solvate thereof, the method comprising determining at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in the cell.
(31) A method for selecting a cell having higher sensitivity to 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide, a pharmacologically acceptable salt thereof or a solvate thereof, the method comprising determining at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in the cell.
(32) A method for selecting a patient having higher sensitivity to 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide, a pharmacologically acceptable salt thereof or a solvate thereof, the method comprising determining at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in the cell.
(33) A method for classifying a patient, comprising analyzing sensitivity to 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide, a pharmacologically acceptable salt thereof or a solvate thereof by determining at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in the cell, and classifying the patient based on the obtained results.
(34) A method for selecting a patient as a target of administering 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide, a pharmacologically acceptable salt thereof or a solvate thereof, the method comprising determining at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in the cell, and selecting a patient having at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2 based on the obtained results.
(35) A method for predicting therapeutic effect of 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide, a pharmacologically acceptable salt thereof or a solvate thereof on a patient, comprising determining at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in the cell.
(36) A method for determining at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in a cell derived from a patient in order to predict the degree of sensitivity of the patient to 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide, a pharmacologically acceptable salt thereof or a solvate thereof.
The present invention further relates to the following:
(37) A therapeutic agent for treating undifferentiated gastric cancer comprising a FGFR2 inhibitory substance.
(38) A pharmaceutical composition comprising a FGFR2 inhibitory substance to be administered to an organism comprising at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2.
(39) A method for treating undifferentiated gastric cancer, characterized by administering an effective dosage of a FGFR2 inhibitory substance to a patient.
(40) A method for inhibiting FGFR2 activation, characterized by administering an effective dosage of a FGFR2 inhibitory substance to a patient.
(41) A method for treating a disease comprising administering an effective dosage of a FGFR2 inhibitory substance to an organism comprising at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2.
(42) Use of a FGFR2 inhibitory substance for producing a therapeutic agent for treating undifferentiated gastric cancer.
(43) Use of a FGFR2 inhibitory substance for producing a pharmaceutical composition to be administered to an organism comprising at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2.
(44) A FGFR2 inhibitory substance for a therapeutic agent for treating undifferentiated gastric cancer.
(45) A FGFR2 inhibitory substance for a pharmaceutical composition to be administered to an organism comprising at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2.
(46) A FGFR2 inhibitor comprising a FGFR2 inhibitory substance.
(47) A method for predicting whether a patient is highly sensitive to a FGFR2 inhibitory substance, the method comprising using at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in a cell as a biomarker.
(48) A method for analyzing sensitivity of a cell to a FGFR2 inhibitory substance, the method comprising determining at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in the cell.
(49) A method for selecting a cell having higher sensitivity to a FGFR2 inhibitory substance, the method comprising determining at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in the cell.
(50) A method for selecting a patient having higher sensitivity to a FGFR2 inhibitory substance, the method comprising determining at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in the cell.
(51) A method for classifying a patient, comprising analyzing sensitivity to a FGFR2 inhibitory substance by determining at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in the cell, and classifying the patient based on the obtained results.
(52) A method for selecting a patient as a target of administering a FGFR2 inhibitory substance, the method comprising determining at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in the cell, and selecting a patient having at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2 based on the obtained results.
(53) A method for predicting therapeutic effect of a FGFR2 inhibitory substance on a patient, comprising determining at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in the cell.
(54) A method for determining at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in a cell from a patient in order to predict the degree of sensitivity of the patient to a FGFR2 inhibitory substance.
The FGFR2 inhibitory substance may comprise the compound represented by General formula (I), a pharmacologically acceptable salt thereof or a solvate thereof.
The FGFR2 inhibitory substance may comprise at least one compound selected from the group consisting of:

    N-(3-trifluoromethyl-4-chlorophenyl)-N′-(4-(2-methylcarbamoylpyridine-4-yl)oxyphenyl)urea;
    6-[2-(methylcarbamoyl)phenylsulfanyl]-3-E-[2-(pyridine-2-yl)ethenyl]indazole;
    5-(5-fluoro-2-oxo-1,2-dihydroindole-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-diethylaminoethyl)amide; and
    N-{2-chloro-4-[(6,7-dimethoxy-4-quinolyl)oxy]phenyl}-N′-(5-methyl-3-isoxazolyl)urea,

a pharmacologically acceptable salt thereof or a solvate thereof.



Effect of the Invention
The present invention provides a therapeutic agent and a therapeutic method for undifferentiated gastric cancer comprising a FGFR2 inhibitory substance, use of a FGFR2 inhibitory substance for producing the therapeutic agent and a FGFR2 inhibitory substance for the therapeutic agent.
The present invention also provides a pharmaceutical composition comprising a FGFR2 inhibitory substance to be administered to an organism comprising at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2; a therapeutic method for a disease comprising administering an effective dosage of the FGFR2 inhibitory substance to an organism comprising at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2; use of the FGFR2 inhibitory substance for producing the pharmaceutical composition; and the FGFR2 inhibitory substance for the pharmaceutical composition.
In addition, the present invention provides a FGFR2 inhibitor.
The present invention also provides a method for predicting the effect of FGFR2 inhibitory substance.
More specifically, the effect of the FGFR2 inhibitory substance can be predicted by using at least one selected from the group consisting of an expression level of FGFR2 and (the presence or the absence of) FGFR2 mutation in a cell as a biomarker.
Since the predicting method of the invention induces prediction of the effect without administrating a compound to a patient, a patient who is expected to benefit higher therapeutic effect from the compound can be selected, thereby contributing to the improvement of QOL of the patients.



BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 shows the effect of 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide on FGFR2 activation (using phosphorylation as a biomarker) during cultivation of human undifferentiated gastric cancer cell lines. (A) KATO-III; (B) SNU-16; and (C)HSC-39.
FIG. 2 shows the effect of 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide on apoptosis (using fragmentation of PARP or caspase-3 as a biomarker) during cultivation of a human undifferentiated gastric cancer cell line HSC-39.
FIG. 3 shows the anti-tumor effect of 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide on subcutaneous transplanted (xenograft) models of human undifferentiated gastric cancer cell lines. (A) KATO-III; (B) SNU-16; and (C)HSC-39.
FIG. 4 shows the effect of 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide on FGFR2 activation (using phosphorylation as an indicator) in tumor tissues of subcutaneous transplanted (xenograft) models of human undifferentiated gastric cancer cell lines. (A) SNU-16; and (B) HSC-39.



BEST MODES FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described. The following embodiments are examples provided for illustrating the present invention, and the present invention is not intended to be limited thereto. The present invention may be carried out in various embodiments without departing from the spirit of the invention.
All of the publications cited herein, for example, prior art documents, laid-open patent publications, patent publications and other patent documents are entirely incorporated herein by reference. The present specification incorporates the content of the specification of Japanese Patent Application Publication No. 2006-230816, on which the present application claims priority.
1. Pharmaceutical Composition, Therapeutic Agent and Method of the Invention
(1) FGFR2
According to the present invention, FGFR2 comprises a polypeptide including an amino acid sequence identical or substantially identical to the amino acids 22-822 of the amino acid sequence represented by SEQ ID NO:2 (GenBank Accession No:NP—075259). The polypeptide including the amino acid sequence represented by SEQ ID NO:2 is coded, for example, by a polynucleotide including the base sequence represented by SEQ ID NO:1 (bases 648-3116 of GenBank Accession No:NM—022970). Usually, processing of the polypeptide comprising the amino acid sequence represented by SEQ ID NO:2 yields mature form (amino acids 22-822 of the amino acid sequence represented by SEQ ID NO:2).
Examples of a polypeptide having an amino acid sequence substantially identical to the amino acid sequence (amino acids 22-822) represented by SEQ ID NO:2 include those selected from the group consisting of (a) to (d) below:
(a) a polypeptide comprising the amino acid sequence (amino acids 22-822) represented by SEQ ID NO:2;
(b) a polypeptide comprising an amino acid sequence having one or more (e.g., one or a few) amino acids deleted, substituted, added or mutated by a combination thereof in the amino acid sequence (amino acids 22-822) represented by SEQ ID NO:2, and having substantially the same activity as FGFR2;
(c) a polypeptide encoded by a polynucleotide that hybridizes with a polynucleotide having a base sequence complementary to the base sequence represented by SEQ ID NO:1 under stringent conditions, and having substantially the same activity as FGFR2; and
(d) a polypeptide having an amino acid sequence having 90% or higher, preferably about 95% or higher and more preferably about 98% or higher identity (also referred to as “homology”) to the amino acid sequence (amino acids 22-822) represented by SEQ ID NO:2, and having substantially the same activity as FGFR2.
Herein, the phrase “having substantially the same activity as FGFR2” means that at least one of the intracellular signals caused by ligand (e.g., FGF) binding is identical to one of the signals of a protein having the amino acid sequence (amino acids 22-822) represented by SEQ ID NO:2, and that the activation level of the intracellular signal is comparable to that of the protein having the amino acid sequence (amino acids 22-822) represented by SEQ ID NO:2. Furthermore, the term “comparable” means, for example, that the activation level of the intracellular signal caused by ligand (e.g., FGF) binding is at least 10%, preferably at least 30% of the activation level of the intracellular signal of a protein having the amino acid sequence (amino acids 22-822) represented by SEQ ID NO:2, which can be referred to as substantially the same activity. Examples of intracellular signals caused by ligand binding include: FGFR2 phosphorylation; Raf, MEK, ERK1 and ERK2 phosphorylation owing to FGFR2 phosphorylation; phosphorylation of phosphatidylinositol 3 kinase; Akt phosphorylation; phosphorylation of phospholipase-C-γ; increase in inositol 1,4,5-trisphosphate (IP3); and increase in diacylglycerol (DAG).
The activity of an intracellular signal caused by ligand binding can be measured by a conventional method such as immunoprecipitation method and western blotting.
Examples of amino acid sequences having one or more (e.g., one or a few) amino acids deleted, substituted, added or mutated by a combination thereof in the amino acid sequence (amino acids 22-822) represented by SEQ ID NO:2 include:
(i) an amino acid sequence having 1-9 (e.g., 1-5, preferably 1-3, more preferably 1-2 and still more preferably one) amino acids deleted from the amino acid sequence (amino acids 22-822) represented by SEQ ID NO:2;
(ii) an amino acid sequence having 1-9 (e.g., 1-5, preferably 1-3, more preferably 1-2 and still more preferably one) amino acids added to the amino acid sequence (amino acids 22-822) represented by SEQ ID NO:2;
(iii) an amino acid sequence having 1-9 (e.g., 1-5, preferably 1-3, more preferably 1-2 and still more preferably one) amino acids substituted with other amino acids in the amino acid sequence (amino acids 22-822) represented by SEQ ID NO:2; and
(iv) an amino acid sequence mutated by a combination of (i) to (iii) above.
As used herein, “deletion” of an amino acid refers to a mutation where one or more amino acid residues in the sequence are deleted, including deletion of amino acid residues from the end of the amino acid sequence and deletion in the middle of the amino acid sequence.
As used herein, “addition” of an amino acid refers to a mutation where one or more amino acid residues are added to the sequence, including addition of amino acid residues to the end of the amino acid sequence and addition in the middle of the amino acid sequence. Addition in the middle of the sequence may also be referred to as “insertion”.
As used herein, “substitution” of an amino acid refers to a mutation where one or more amino acid residues in the sequence are substituted with different types of amino acid residues. When the amino acid sequence of FGFR2 is modified by such substitution, conservative substitution is preferable in order to retain the function of a protein. Conservative substitution means to alter a sequence to code an amino acid having similar property to the original amino acid. The property of amino acids can be classified, for example, into nonpolar amino acids (Ala, Ile, Leu, Met, Phe, Pro, Trp, Val), uncharged amino acids (Asn, Cys, Gln, Gly, Ser, Thr, Tyr), acidic amino acids (Asp, Glu), basic amino acids (Arg, His, Lys), neutral amino acids (Ala, Asn, Cys, Gln, Gly, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val), aliphatic amino acids (Ala, Gly), branched amino acids (Ile, Leu, Val), hydroxyamino acids (Ser, Thr), amide amino acids (Gln, Asn), sulfur-containing amino acids (Cys, Met), aromatic amino acids (His, Phe, Trp, Tyr), heterocyclic amino acids (His, Trp), imino acids (Pro, 4Hyp) and the like.
Thus, substitution, for example, between nonpolar amino acids or between uncharged amino acids, is preferable, among which, substitutions between Ala, Val, Leu and Ile; Ser and Thr; Asp and Glu; Asn and Gln; Lys and Arg; and Phe and Tyr are favorable as substitution that retains the property of the protein. There is no limitation to the number and the site of the amino acids to be mutated.
An example of a polypeptide having substantially the same amino acid sequence as the amino acid sequence (amino acids 22-822) represented by SEQ ID NO:2 includes a polypeptide encoded by a polynucleotide that hybridizes with a polynucleotide having a base sequence complementary to the base sequence represented by SEQ ID NO:1 under stringent conditions, and having substantially the same activity as FGFR2 as described above.
Herein, specific examples of polynucleotides that hybridize under stringent conditions include polynucleotides having base sequences with at least 90% or higher identity, preferably 95% or higher, more preferably 97% or higher, more preferably 98% or higher, or still more preferably 99% or higher identity with the base sequence represented by SEQ ID NO:1 when calculated using homology search software such as FASTA, BLAST, Smith-Waterman [Meth. Enzym., 164, 765 (1988)] with default (initial setting) parameters. Examples of such stringent conditions include “2×SSC, 0.1% SDS, 50° C.”, “2×SSC, 0.1% SDS, 42° C.,” and “1×SSC, 0.1% SDS, 37° C.”, and “2×SSC, 0.1% SDS, 65° C.,” “0.5×SSC, 0.1% SDS, 42° C.,” and “0.2×SSC, 0.1% SDS, 65° C.” for higher stringent conditions.
Hybridization can be carried out according to a known method. When a commercially available library is used, hybridization may be carried out according to the method described in the attached instruction.
Examples of polynucleotides that hybridize with a polynucleotide having a base sequence complementary to the base sequence represented by SEQ ID NO:1 under stringent conditions include polynucleotides comprising a base sequence having 90% or higher, preferably 95% or higher and more preferably 98% or higher identity with the base sequence represented by SEQ ID NO:1.
Examples of polynucleotides that hybridize with a polynucleotide having a base sequence complementary to the base sequence represented by SEQ ID NO:1 under stringent conditions include base sequences having one or more (e.g., one or several) nucleic acids mutated, for example, deleted, substituted or added in the base sequence represented by SEQ ID NO:1.
Examples of polynucleotides that hybridize with a polynucleotide having a base sequence complementary to the base sequence represented by SEQ ID NO:1 under stringent conditions include:
(i) a base sequence having 1-9 (e.g., 1-5, preferably 1-3, more preferably 1-2 and still more preferably one) nucleic acids deleted from the base sequence represented by SEQ ID NO:1;
(ii) a base sequence having 1-9 (e.g., 1-5, preferably 1-3, more preferably 1-2 and still more preferably one) nucleic acids added to the base sequence represented by SEQ ID NO:1;
(iii) a base sequence having 1-9 (e.g., 1-5, preferably 1-3, more preferably 1-2 and still more preferably one) nucleic acids substituted with other nucleic acids in the base sequence represented by SEQ ID NO:1; and
(iv) a base sequence mutated by a combination of (i) to (iii) above.
According to the present invention, FGFR2 also comprises a polypeptide comprising an amino acid sequence identical or substantially identical to the amino acids 22-821 of the amino acid sequence represented by SEQ ID NO:4 (GenBank Accession No: NP—00132). A polypeptide comprising the amino acid sequence represented by SEQ ID NO:4 is encoded, for example, by a polynucleotide comprising the base sequence represented by SEQ ID NO:3 (bases 648-3113 of GenBank Accession No: NM—000141). Usually, processing of the polypeptide comprising the amino acid sequence represented by SEQ ID NO:4 yields mature form (amino acids 22-821 of the amino acid sequence represented by SEQ ID NO:4).
An example of a polypeptide having an amino acid sequence substantially identical to the amino acid sequence (amino acids 22-821) represented by SEQ ID NO:4 includes one selected from the group consisting of (a) to (d) below:
(a) a polypeptide comprising the amino acid sequence (amino acids 22-821) represented by SEQ ID NO:4;
(b) a polypeptide comprising an amino acid sequence having one or more (e.g., one or a few) amino acids deleted, substituted, added or mutated by a combination thereof in the amino acid sequence (amino acids 22-821) represented by SEQ ID NO:4, and having substantially the same activity as FGFR2;
(c) a polypeptide encoded by a polynucleotide that hybridizes with a polynucleotide having a base sequence complementary to the base sequence represented by SEQ ID NO:3 under stringent conditions, and having substantially the same activity as FGFR2; and
(d) a polypeptide having an amino acid sequence having at least 90% or higher, preferably about 95% or higher and more preferably about 98% or higher identity (also referred to as “homology”) to the amino acid sequence (amino acids 22-821) represented by SEQ ID NO:4, and having substantially the same activity as FGFR2.
Herein, the phrase “having substantially the same activity as FGFR2” means that at least one of the intracellular signals caused by ligand (e.g., FGF) binding is identical to one of the signals of a protein having the amino acid sequence (amino acids 22-821) represented by SEQ ID NO:4, and that the activation level of the intracellular signal is comparable to that of the protein comprising the amino acid sequence (amino acids 22-821) represented by SEQ ID NO:4. Furthermore, the term “comparable” means, for example, that the activation level of the intracellular signal caused by ligand (e.g., FGF) binding is at least 10%, preferably at least 30% of the activation level of the intracellular signal of a protein having the amino acid sequence (amino acids 22-821) represented by SEQ ID NO:4, which can be referred to as substantially the same activity. Examples of intracellular signals caused by ligand binding include: FGFR2 phosphorylation; Raf, MEK, ERK1 and ERK2 phosphorylation owing to FGFR2 phosphorylation; phosphorylation of phosphatidylinositol 3 kinase; Akt phosphorylation; phosphorylation of phospholipase-C-γ; increase in inositol 1,4,5-trisphosphate (IP3); and increase in diacylglycerol (DAG).
The activity of an intracellular signal caused by ligand binding can be measured by a conventional method such as immunoprecipitation method and western blotting.
Examples of amino acid sequences having one or more (e.g., one or a few) amino acids deleted, substituted, added or mutated by a combination thereof in the amino acid sequence (amino acids 22-821) represented by SEQ ID NO:4 include:
(i) an amino acid sequence having 1-9 (e.g., 1-5, preferably 1-3, more preferably 1-2 and still more preferably one) amino acids deleted from the amino acid sequence (amino acids 22-821) represented by SEQ ID NO:4;
(ii) an amino acid sequence having 1-9 (e.g., 1-5, preferably 1-3, more preferably 1-2 and still more preferably one) amino acids added to the amino acid sequence (amino acids 22-821) represented by SEQ ID NO:4;
(iii) an amino acid sequence having 1-9 (e.g., 1-5, preferably 1-3, more preferably 1-2 and still more preferably one) amino acids substituted with other amino acids in the amino acid sequence (amino acids 22-821) represented by SEQ ID NO:4; and
(iv) an amino acid sequence mutated by a combination of (i) to (iii) above.
As used herein, “deletion” of an amino acid refers to a mutation where one or more amino acid residues in the sequence are deleted, including deletion of amino acid residues from the end of the amino acid sequence and deletion in the middle of the amino acid sequence.
As used herein, “addition” of an amino acid refers to a mutation where one or more amino acid residues are added to the sequence, including addition of amino acid residues to the end of the amino acid sequence and addition in the middle of the amino acid sequence. Addition in the middle of the sequence may also be referred to as “insertion”.
As used herein, “substitution” of an amino acid refers to a mutation where one or more amino acid residues in the sequence are substituted with different types of amino acid residues. When the amino acid sequence of FGFR2 is modified by such substitution, conservative substitution is preferable in order to retain the function of a protein. Conservative substitution means to alter a sequence to code an amino acid having similar property to the original amino acid. The property of amino acids can be classified, for example, into nonpolar amino acids (Ala, Ile, Leu, Met, Phe, Pro, Trp, Val), uncharged amino acids (Asn, Cys, Gln, Gly, Ser, Thr, Tyr), acidic amino acids (Asp, Glu), basic amino acids (Arg, His, Lys), neutral amino acids (Ala, Asn, Cys, Gln, Gly, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val), aliphatic amino acids (Ala, Gly), branched amino acids (Ile, Leu, Val), hydroxyamino acids (Ser, Thr), amide amino acids (Gln, Asn), sulfur-containing amino acids (Cys, Met), aromatic amino acids (His, Phe, Trp, Tyr), heterocyclic amino acids (His, Trp), imino acids (Pro, 4Hyp) and the like.
Thus, substitution, for example, between nonpolar amino acids or between uncharged amino acids, is preferable, among which, substitutions between Ala, Val, Leu and Ile; Ser and Thr; Asp and Glu; Asn and Gln; Lys and Arg; and Phe and Tyr are favorable as substitution that retains the property of the protein. There is no limitation to the number and the site of the amino acids to be mutated.
An example of a polypeptide having substantially the same amino acid sequence as the amino acid sequence (amino acids 22-821) represented by SEQ ID NO:4 includes, as described above, a polypeptide encoded by a polynucleotide that hybridizes with a polynucleotide having a base sequence complementary to the base sequence represented by SEQ ID NO:3 under stringent conditions, and having substantially the same activity as FGFR2.
Herein, specific examples of polynucleotides that hybridize under stringent conditions include polynucleotides having base sequences with at least 90% or higher identity, preferably 95% or higher, more preferably 97% or higher, more preferably 98% or higher, or still more preferably 99% or higher identity with the base sequence represented by SEQ ID NO:1 when calculated using homology search software such as FASTA, BLAST, Smith-Waterman [Meth. Enzym., 164, 765 (1988)] with default (initial setting) parameters. Examples of such stringent conditions include “2×SSC, 0.1% SDS, 50° C.”, “2×SSC, 0.1% SDS, 42° C.,” and “1×SSC, 0.1% SDS, 37° C.”, and “2×SSC, 0.1% SDS, 65° C.,” “0.5×SSC, 0.1% SDS, 42° C.,” and “0.2×SSC, 0.1% SDS, 65° C.” for higher stringent conditions.
Hybridization can be carried out according to a known method. When a commercially available library is used, hybridization may be carried out according to the method described in the attached instruction.
Examples of polynucleotides that hybridize with a polynucleotide having a base sequence complementary to the base sequence represented by SEQ ID NO:3 under stringent conditions include polynucleotides comprising a base sequence having 90% or higher, preferably 95% or higher and more preferably 98% or higher identity with the base sequence represented by SEQ ID NO:3.
Examples of polynucleotides that hybridize with a polynucleotide having a base sequence complementary to the base sequence represented by SEQ ID NO:3 under stringent conditions include a base sequence having one or more (e.g., one or several) nucleic acids mutated, for example, deleted, substituted or added in the base sequence represented by SEQ ID NO:3.
Examples of polynucleotides that hybridize with a polynucleotide having a base sequence complementary to the base sequence represented by SEQ ID NO:3 under stringent conditions include:
(i) a base sequence having 1-9 (e.g., 1-5, preferably 1-3, more preferably 1-2 and still more preferably one) nucleic acids deleted from the base sequence represented by SEQ ID NO:3;
(ii) a base sequence having 1-9 (e.g., 1-5, preferably 1-3, more preferably 1-2 and still more preferably one) nucleic acids added to the base sequence represented by SEQ ID NO:3;
(iii) a base sequence having 1-9 (e.g., 1-5, preferably 1-3, more preferably 1-2 and still more preferably one) nucleic acids substituted with other nucleic acids in the base sequence represented by SEQ ID NO:3; and
(iv) a base sequence mutated by a combination of (i) to (iii) above.
Herein, the term “identity” (also referred to as “homology”) of an amino acid sequence is used to indicate the degree of consistency of amino acid residues between the sequences to be compared. In order to calculate identity of a given amino acid sequence to an amino acid sequence to be compared, the presence of gaps and the property of the amino acids are considered (Wilbur, Natl. Acad. Sci. U.S.A. 80:726-730 (1983)). For the calculation of identity, a commercially available software BLAST (Altschul: J. Mol. Biol. 215:403-410 (1990)), FASTA (Peasron: Methods in Enzymology 183:63-69 (1990)) or the like can be used.
The “identity” value may be any value as long as it is obtained by using a homology search program known to those skilled in the art. For example, for such calculation, the default (initial setting) parameters can be used in homology algorithm BLAST (Basic local alignment search tool) http://www.ncbi.nlm.nih.gov/BLAST/ of the National Center for Biotechnology Information (NCBI).
According to the present invention, FGFR2 comprises mutant FGFR2 described below.
(2) Cell Overexpressing FGFR2
According to the present invention, a cell overexpressing FGFR2 comprises, for example, a cell expressing FGFR2 for a significant amount as compared to a normal cell. In addition, according to the present invention, a cell overexpressing FGFR2 comprises, for example, a cell expressing FGFR2 for at least 1.5 times higher, preferably at least 2 times higher, more preferably at least 3 times higher, still more preferably at least 4 times higher than a normal cell. Herein, according to the invention, a “normal cell” includes, for example, cells other than cancer (e.g., undifferentiated gastric cancer) cells.
According to the present invention, a cell overexpressing FGFR2 is preferably undifferentiated gastric cancer cell, more preferably at least one cell selected from the group consisting of poorly differentiated adenocarcinoma, signet-ring cell carcinoma, mucinous carcinoma and scirrhous gastric cancer.
An expression level of FGFR2 may be analyzed, for example, by measuring the protein and/or the mRNA of FGFR2 expressed in the cell.
Protein can be measured, for example, by an immunochemical method (e.g., immunohistochemistry method, immunoprecipitation, western blotting, flow cytometry, ELISA, RIA, etc.), mass spectrometry or the like, preferably an immunochemical method, particularly preferably flow cytometry. These methods may be carried out according to conventional techniques.
On the other hand, mRNA can be measured, for example, by a method such as in situ hybridization, northern blot analysis, DNA microarray, RT-PCR, quantitative RT-PCR or the like, preferably RT-PCR or quantitative RT-PCR. These methods may be carried out according to conventional techniques.
(3) Cell Overexpressing Mutant FGFR2
According to the present invention, mutant FGFR2 comprises polypeptides comprising an amino acid sequence having one or a few amino acids deleted, substituted, added or mutated by a combination thereof in a wild-type FGFR2 amino acid sequence such as the amino acid sequence (amino acids 22-822) represented by SEQ ID NO:2 or the amino acid sequence (amino acids 22-821) represented by SEQ ID NO:4. Preferably, the mutant FGFR2 comprises polypeptides comprising an amino acid sequence having one or a few amino acids deleted, substituted, added or mutated by a combination thereof in a wild-type FGFR2 amino acid sequence such as the amino acid sequence (amino acids 22-822) represented by SEQ ID NO:2 or the amino acid sequence (amino acids 22-821) represented by SEQ ID NO:4, and having substantially the same activity as FGFR2. According to the present invention, a cell expressing mutant FGFR2 comprises cells expressing the above-mentioned polypeptides.
Examples of mutant FGFR2 include polypeptides comprising an amino acid sequence where serine at position 267 of the amino acid sequence (amino acids 22-822) represented by SEQ ID NO:2 or the amino acid sequence (amino acids 22-821) represented by SEQ ID NO:4 is substituted by other amino acid, preferably proline (Cancer Research. 61, 3541-3543, 2001).
In addition, examples of mutant FGFR2 include polypeptides comprising a mutation site where a few amino acids at C-terminal are deleted in an amino acid sequence of wild-type FGFR2 such as amino acids 22-822 of the amino acid sequence represented by SEQ ID NO:2 or amino acids 22-821 of the amino acid sequence represented by SEQ ID NO:4. Examples of mutant FGFR2 include polypeptides comprising an amino acid sequence where amino acids downstream from at least Tyr at position 813 (Tyr at position 792 in the sequence of amino acids 22-822), preferably at least Tyr at position 784 (Tyr at position 763 in the sequence of amino acids 22-822), more preferably at least Tyr at position 780 (Tyr at position 759 in the sequence of amino acids 22-822), and still more preferably at least Tyr at position 770 (Tyr at position 749 in the sequence of amino acids 22-822) are deleted in the amino acid sequence (amino acids 22-822) represented by SEQ ID NO:2. In addition, examples of mutant FGFR2 include polypeptides comprising an amino acid sequence where amino acids downstream from at least Tyr at position 812 (Tyr at position 791 in the sequence of amino acids 22-821), preferably at least Tyr at position 783 (Tyr at position 762 in the sequence of amino acids 22-821), more preferably at least Tyr at position 779 (Tyr at position 758 in the sequence of amino acids 22-821), and still more preferably at least Tyr at position 769 (Tyr at position 748 in the sequence of amino acids 22-821) are deleted in the amino acid sequence (amino acids 22-821) represented by SEQ ID NO:4.
Preferably, mutant FGFR2 is activating mutant FGFR2. Activating mutant FGFR2 refers to mutant FGFR2 that causes autophosphorylation in a ligand-independent manner and that activates intracellular signals.
The presence or the absence of FGFR2 mutation can be determined by analyzing the gene sequence of FGFR2 or the sequence of the transcript, i.e., mRNA, of FGFR2. An example of the method for analyzing a sequence includes dideoxynucleotide chain termination technique (Sanger et al., (1977) Proc. Natl. Acad. Sci. USA 74:5463). A sequence can also be analyzed by employing an appropriate DNA sequencer.
Furthermore, the presence or the absence of FGFR2 mutation can be analyzed, for example, by in situ hybridization, northern blot analysis, DNA microarray, RT-PCR, SSCP-PCR (Single-Strand Conformation Polymorphism-PCR) or the like. These methods may be carried out according to conventional techniques.
The presence or the absence of FGFR2 mutation can be analyzed, for example, by an immunochemical method (e.g., an immunohistochemistry method, immunoprecipitation, western blotting, flow cytometry, ELISA, RIA, etc.). These methods may be carried out according to conventional techniques.
According to the present invention, a cell expressing mutant FGFR2 is preferably an undifferentiated gastric cancer cell, more preferably at least one cell selected from the group consisting of poorly differentiated adenocarcinoma, signet-ring cell carcinoma, mucinous carcinoma and a scirrhous gastric cancer.
(4) FGFR2 Inhibitor of the Invention
Herein, a “halogen atom” refers to a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.
Preferable examples of a “halogen atom” include a fluorine atom and a chlorine atom.
Herein, a “C1-6 alkyl group” refers to a linear or branched alkyl group with a carbon number of 1-6, specific examples including a methyl group, an ethyl group, a 1-propyl group (n-propyl group), a 2-propyl group (i-propyl group), a 2-methyl-1-propyl group (i-butyl group), a 2-methyl-2-propyl group (t-butyl group), a 1-butyl group (n-butyl group), a 2-butyl group (s-butyl group), a 1-pentyl group, a 2-pentyl group, a 3-pentyl group, a 2-methyl-1-butyl group, a 3-methyl-1-butyl group, a 2-methyl-2-butyl group, a 3-methyl-2-butyl group, a 2,2-dimethyl-1-propyl group, a 1-hexyl group, a 2-hexyl group, a 3-hexyl group, a 2-methyl-1-pentyl group, a 3-methyl-1-pentyl group, a 4-methyl-1-pentyl group, a 2-methyl-2-pentyl group, a 3-methyl-2-pentyl group, a 4-methyl-2-pentyl group, a 2-methyl-3-pentyl group, a 3-methyl-3-pentyl group, a 2,3-dimethyl-1-butyl group, a 3,3-dimethyl-1-butyl group, a 2,2-dimethyl-1-butyl group, a 2-ethyl-1-butyl group, a 3,3-dimethyl-2-butyl group and a 2,3-dimethyl-2-butyl group.
Preferable examples of a “C1-6 alkyl group” include a methyl group, an ethyl group, a 1-propyl group, a 2-propyl group, a 2-methyl-1-propyl group, a 2-methyl-2-propyl group, a 1-butyl group and a 2-butyl group.
Herein, a “C1-6 alkylene group” refers to a divalent group derived from a “C1-6 alkyl group” defined above by removing any one hydrogen atom therefrom, specific examples including a methylene group, a 1,2-ethylene group, a 1,1-ethylene group, a 1,3-propylene group, a tetramethylene group, a pentamethylene group and a hexamethylene group.
Herein, a “C2-6 alkenyl group” refers to a linear or branched alkenyl group having one double bond and a carbon number of 2-6, specific examples including an ethenyl group (vinyl group), a 1-propenyl group, a 2-propenyl group (allyl group), a 1-butenyl group, a 2-butenyl group, a 3-butenyl group, a pentenyl group and a hexenyl group.
Herein, a “C2-6 alkynyl group” refers to a linear or branched alkynyl group having one triple bond and a carbon number of 2-6, specific examples including an ethinyl group, a 1-propynyl group, a 2-propynyl group, a 1-butynyl group, a 2-butynyl group, a 3-butynyl group, a pentynyl group and a hexynyl group.
Herein, a “C3-8 cycloalkyl group” refers to a monocyclic or bicyclic saturated aliphatic hydrocarbon group with a carbon number of 3-8, specific examples including a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, a cyclooctyl group, a bicyclo[2.1.0]pentyl group, a bicyclo[3.1. 0]hexyl group, a bicyclo[2.1.1]hexyl group, a bicyclo[4.1.0]heptyl group, a bicyclo[2. 2.1]heptyl group (norbornyl group), a bicyclo[3.3.0]octyl group, a bicyclo[3.2.1]octyl group and a bicyclo[2.2.2]octyl group.
Preferable examples of a “C3-8 cycloalkyl group” include a cyclopropyl group, a cyclobutyl group and a cyclopentyl group.
Herein, a “C6-10 aryl group” refers to an aromatic hydrocarbon cyclic group with a carbon number of 6-10, specific examples including a phenyl group, a 1-naphthyl group, a 2-naphthyl group, an indenyl group and an azulenyl group.
A preferable example of a “C6-10 aryl group” includes a phenyl group.
Herein, a “heteroatom” refers to a nitrogen atom, an oxygen atom or a sulfur atom.
Herein, a “5-10-membered heteroaryl group” refers to an aromatic cyclic group having 5-10 atoms forming the ring and 1-5 heteroatoms included in the atoms forming the ring, specific examples including a furyl group, a thienyl group, a pyrrolyl group, an imidazolyl group, a triazolyl group, a tetrazolyl group, a thiazolyl group, a pyrazolyl group, an oxazolyl group, an isoxazolyl group, an isothiazolyl group, a furazanyl group, a thiadiazolyl group, an oxadiazolyl group, a pyridyl group, a pyrazinyl group, a pyridazinyl group, a pyrimidinyl group, a triazinyl group, a purinyl group, a pteridinyl group, a quinolyl group, an isoquinolyl group, a naphthiridinyl group, a quinoxalinyl group, a cinnolinyl group, a quinazolinyl group, a phthalazinyl group, an imidazopyridyl group, an imidazothiazolyl group, an imidazoxazolyl group, a benzothiazolyl group, a benzoxazolyl group, a benzimidazolyl group, an indolyl group, an isoindolyl group, an indazolyl group, a pyrrolopyridyl group, a thienopyridyl group, a furopyridyl group, a benzothiadiazolyl group, a benzoxadiazolyl group, a pyridopyrimidinyl group, a benzofuryl group, a benzothienyl group and a thienofuryl group.
Preferable examples of a “5-10-membered heteroaryl group” include a furyl group, a thienyl group, a pyrrolyl group, an imidazolyl group, a thiazolyl group, a pyrazolyl group, an oxazolyl group, an isoxazolyl group, an isothiazolyl group, a pyridyl group and a pyrimidinyl group.
Herein, a “3-10-membered nonaromatic heterocyclic group”:
(a) has 3-10 atoms forming the ring;
(b) has 1-2 heteroatoms included in the atoms forming the ring;
(c) may include 1-2 double bonds in the ring;
(d) may include 1-3 carbonyl groups, sulfinyl groups or sulfonyl groups in the ring; and
(e) is a nonaromatic monocyclic or bicyclic group where when a nitrogen atom is included in the atoms forming the ring, the nitrogen atom may have a bond.
Specific examples include an aziridinyl group, an azetidinyl group, a pyrrolidinyl group, a piperidinyl group, an azepanyl group, an azocanyl group, a piperazinyl group, a diazepanyl group, a diazocanyl group, a diazabicyclo[2.2.1]heptyl group, a morpholinyl group, a thiomorpholinyl group, a 1,1-dioxothiomorpholinyl group, an oxiranyl group, an oxetanyl group, a tetrahydrofuryl group, a dioxoranyl group, a tetrahydropyranyl group, a dioxanyl group, a tetrahydrothienyl group, a tetrahydrothiopyranyl group, an oxazolidinyl group and a thiazolidinyl group.
Preferable examples of a “3-10-membered nonaromatic heterocyclic group” include an aziridinyl group, an azetidinyl group, a pyrrolidinyl group, a piperidinyl group, an azepanyl group, a piperazinyl group, a diazepanyl group, a morpholinyl group, a thiomorpholinyl group, a 1,1-dioxothiomorpholinyl group, a tetrahydrofuryl group and a tetrahydropyranyl group.
Herein, a “C1-6 alkoxy group” refers to a group in which an oxygen atom is bound to the terminal of a “C1-6 alkyl group” defined above, specific examples including a methoxy group, an ethoxy group, a 1-propoxy group (n-propoxy group), a 2-propoxy group (i-propoxy group), a 2-methyl-1-propoxy group (i-butoxy group), a 2-methyl-2-propoxy group (t-butoxy group), a 1-butoxy group (n-butoxy group), a 2-butoxy group (s-butoxy group), a 1-pentyloxy group, a 2-pentyloxy group, a 3-pentyloxy group, a 2-methyl-1-butoxy group, a 3-methyl-1-butoxy group, a 2-methyl-2-butoxy group, a 3-methyl-2-butoxy group, a 2,2-dimethyl-1-propoxy group, a 1-hexyloxy group, a 2-hexyloxy group, a 3-hexyloxy group, a 2-methyl-1-pentyloxy group, a 3-methyl-1-pentyloxy group, a 4-methyl-1-pentyloxy group, a 2-methyl-2-pentyloxy group, a 3-methyl-2-pentyloxy group, a 4-methyl-2-pentyloxy group, a 2-methyl-3-pentyloxy group, a 3-methyl-3-pentyloxy group, a 2,3-dimethyl-1-butoxy group, a 3,3-dimethyl-1-butoxy group, a 2,2-dimethyl-1-butoxy group, a 2-ethyl-1-butoxy group, a 3,3-dimethyl-2-butoxy group and a 2,3-dimethyl-2-butoxy group.
Preferable examples of a “C1-6 alkoxy group” include a methoxy group, an ethoxy group, a 1-propoxy group, a 2-propoxy group, a 2-methyl-1-propoxy group, a 2-methyl-2-propoxy group, a 1-butoxy group and a 2-butoxy group.
Herein, a “C1-6 alkylthio group” refers to a group in which a sulfur atom is bound to the terminal of a “C1-6 alkyl group” defined above, specific examples including a methylthio group, an ethylthio group, a 1-propylthio group (n-propylthio group), a 2-propylthio group (i-propylthio group), a 2-methyl-1-propylthio group (i-butylthio group), a 2-methyl-2-propylthio group (t-butylthio group), a 1-butylthio group (n-butylthio group), a 2-butylthio group (s-butylthio group), a 1-pentylthio group, a 2-pentylthio group, a 3-pentylthio group, a 2-methyl-1-butylthio group, a 3-methyl-1-butylthio group, a 2-methyl-2-butylthio group, a 3-methyl-2-butylthio group, a 2,2-dimethyl-1-propylthio group, a 1-hexylthio group, a 2-hexylthio group, a 3-hexylthio group, a 2-methyl-1-pentylthio group, a 3-methyl-1-pentylthio group, a 4-methyl-1-pentylthio group, a 2-methyl-2-pentylthio group, a 3-methyl-2-pentylthio group, a 4-methyl-2-pentylthio group, a 2-methyl-3-pentylthio group, a 3-methyl-3-pentylthio group, a 2,3-dimethyl-1-butylthio group, a 3,3-dimethyl-1-butylthio group, a 2,2-dimethyl-1-butylthio group, a 2-ethyl-1-butylthio group, a 3,3-dimethyl-2-butylthio group and a 2,3-dimethyl-2-butylthio group.
Preferable examples of a “C1-6 alkylthio group” include a methylthio group, an ethylthio group, a 1-propylthio group (n-propylthio group), a 2-propylthio group (i-propylthio group), a 2-methyl-1-propylthio group (i-butylthio group), a 2-methyl-2-propylthio group (t-butylthio group), a 1-butylthio group (n-butylthio group) and a 2-butylthio group (s-butylthio group).
Herein, a “C3-8 cycloalkoxy group” refers to a group in which an oxygen atom is bound to the terminal of a “C3-8 cycloalkyl group” defined above, specific examples including a cyclopropoxy group, a cyclobutoxy group, a cyclopentyloxy group, a cyclohexyloxy group, a cycloheptyloxy group, a cyclooctyloxy group, a bicyclo[2.1. 0]pentyloxy group, a bicyclo[3.1.0]hexyloxy group, a bicyclo[2.1.1]hexyloxy group, a bicyclo[4.1.0]heptyloxy group, a bicyclo[2.2.1]heptyloxy group (norbornyloxy group), a bicyclo[3.3.0]octyloxy group, a bicyclo[3.2.1]octyloxy group and a bicyclo[2.2. 2]octyloxy group.
Preferable examples of a “C3-8 cycloalkoxy group” include a cyclopropoxy group, a cyclobutoxy group and a cyclopentyloxy group.
Herein, a “mono-C1-6 alkylamino group” refers to a group in which a hydrogen atom in an amino group is substituted with a “C1-6 alkyl group” defined above, specific examples including a methylamino group, an ethylamino group, a 1-propylamino group (n-propylamino group), a 2-propylamino group (i-propylamino group), a 2-methyl-1-propylamino group (i-butylamino group), a 2-methyl-2-propylamino group (t-butylamino group), a 1-butylamino group (n-butylamino group), a 2-butylamino group (s-butylamino group), a 1-pentylamino group, a 2-pentylamino group, a 3-pentylamino group, a 2-methyl-1-butylamino group, a 3-methyl-1-butylamino group, a 2-methyl-2-butylamino group, a 3-methyl-2-butylamino group, a 2,2-dimethyl-1-propylamino group, a 1-hexylamino group, a 2-hexylamino group, a 3-hexylamino group, a 2-methyl-1-pentylamino group, a 3-methyl-1-pentylamino group, a 4-methyl-1-pentylamino group, a 2-methyl-2-pentylamino group, a 3-methyl-2-pentylamino group, a 4-methyl-2-pentylamino group, a 2-methyl-3-pentylamino group, a 3-methyl-3-pentylamino group, a 2,3-dimethyl-1-butylamino group, a 3,3-dimethyl-1-butylamino group, a 2,2-dimethyl-1-butylamino group, a 2-ethyl-1-butylamino group, a 3,3-dimethyl-2-butylamino group and a 2,3-dimethyl-2-butylamino group.
Herein, a “di-C1-6 alkylamino group” refers to a group in which two hydrogen atoms in an amino group are substituted with an identical or different “C1-6 alkyl group” defined above, specific examples including a N,N-dimethylamino group, a N,N-diethylamino group, a N,N-di-n-propylamino group, a N,N-di-i-propylamino group, a N,N-di-n-butylamino group, a N,N-di-i-butylamino group, a N,N-di-s-butylamino group, a N,N-di-t-butylamino group, a N-ethyl-N-methylamino group, a N-n-propyl-N-methylamino group, a N-i-propyl-N-methylamino group, a N-n-butyl-N-methylamino group, a N-i-butyl-N-methylamino group, a N-s-butyl-N-methylamino group and a N-t-butyl-N-methylamino group.
Herein, a “C2-7 acyl group” refers to a carbonyl group bound with a “C1-6 alkyl group” defined above, specific examples including an acetyl group, a propionyl group, an isopropionyl group, a butyryl group, an isobutyryl group, a valeryl group, an isovaleryl group and a pivaloyl group.
Herein, a “C2-7 alkoxycarbonyl group” refers to a carbonyl group bound with a “C1-6 alkoxy group” defined above, specific examples including a methoxycarbonyl group, an ethoxycarbonyl group, a 1-propyloxycarbonyl group, a 2-propyloxycarbonyl group and a 2-methyl-2-propoxycarbonyl group.
Herein, “that may have a substituent” means “that may have one or more substituents in any combination at substitutable positions”, specific examples of the substituent including a halogen atom, a hydroxyl group, a thiol group, a nitro group, a cyano group, a formyl group, a carboxyl group, an amino group, a silyl group, a methanesulfonyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C2-6 alkynyl group, a C3-8 cycloalkyl group, a C6-10 aryl group, a 5-10-membered heteroaryl group, a 3-10-membered nonaromatic heterocyclic group, a C1-6 alkoxy group, a C1-6 alkylthio group, a C3-8 cycloalkoxy group, a mono-C1-6 alkylamino group, a di-C1-6 alkylamino group, a C2-7 acyl group and a C2-7 alkoxycarbonyl group. In this case, the C1-6 alkyl group, the C2-6 alkenyl group, the C2-6 alkynyl group, the C3-8 cycloalkyl group, the C6-10 aryl group, the 5-10-membered heteroaryl group, the 3-10-membered nonaromatic heterocyclic group, the C1-6 alkoxy group, the C1-6 alkylthio group, the C3-8 cycloalkoxy group, the mono-C1-6 alkylamino group, the di-C1-6 alkylamino group, the C2-7 acyl group and the C2-7 alkoxycarbonyl group may each independently have 1-3 groups selected from the group consisting from the following substituent groups.
<Substituent Groups>
A halogen atom, a hydroxyl group, a thiol group, a nitro group, a cyano group, a C1-6 alkyl group, a C3-8 cycloalkyl group, a C2-6 alkenyl group, a C2-6 alkynyl group, a C6-10 aryl group, a 5-10-membered heteroaryl group, a 3-10-membered nonaromatic heterocyclic group, a C1-6 alkoxy group and a C1-6 alkylthio group.
According to the present invention, a FGFR2 inhibitor comprises a compound represented by General formula (I) below.




R1 represents a group represented by Formula —V1—V2—V3 (wherein, V1 represents an optionally substituted C1-6 alkylene group; V2 represents a single bond, an oxygen atom, a sulfur atom, a carbonyl group, a sulfinyl group, a sulfonyl group, a group represented by Formula —CONR6—, a group represented by Formula —SO2NR6—, a group represented by Formula —NR6SO2—, a group represented by Formula —NR6CO— or a group represented by Formula —NR6— (wherein, R6 represents a hydrogen atom, an optionally substituted C1-6 alkyl group or an optionally substituted C3-8 cycloalkyl group); V3 represents a hydrogen atom, an optionally substituted C1-6 alkyl group, an optionally substituted C2-6 alkenyl group, an optionally substituted C2-6 alkynyl group, an optionally substituted C3-8 cycloalkyl group, an optionally substituted C6-10 aryl group, an optionally substituted 5-10-membered heteroaryl group or an optionally substituted 3-10-membered nonaromatic heterocyclic group).
A preferable example of R1 includes a C1-6 alkyl group provided that R1 may have a substituent selected from a 3-10-membered nonaromatic heterocyclic group, a hydroxyl group, a C1-6 alkoxy group, an amino group, a mono-C1-6 alkylamino group and a di-C1-6 alkylamino group which may have a C1-6 alkyl group.
More preferable examples of R1 include a methyl group and a group represented by any one of the following formulae




(wherein, Ra3 represents a methyl group; Ra1 represents a hydrogen atom or a hydroxyl group; Ra2 represents a methoxy group, an ethoxy group, a 1-pyrrolidinyl group, a 1-piperidinyl group, a 4-morpholinyl group, a dimethylamino group or a diethylamino group).
Still more preferable examples of R1 include a methyl group and a 2-methoxyethyl group.
(ii) R2 
R2 represents a cyano group, an optionally substituted C1-6 alkoxy group, a carboxyl group, an optionally substituted C2-7 alkoxycarbonyl group or a group represented by Formula —CONVa11Va12 (wherein, Va11 represents a hydrogen atom, an optionally substituted C1-6 alkyl group, an optionally substituted C2-6 alkenyl group, an optionally substituted C2-6 alkynyl group, an optionally substituted C3-8 cycloalkyl group, an optionally substituted C6-10 aryl group, an optionally substituted 5-10-membered heteroaryl group or an optionally substituted 3-10-membered nonaromatic heterocyclic group; Va12 represents a hydrogen atom, an optionally substituted C1-6 alkyl group, an optionally substituted C2-6 alkenyl group, an optionally substituted C2-6 alkynyl group, an optionally substituted C3-8 cycloalkyl group, an optionally substituted C6-10 aryl group, an optionally substituted 5-10-membered heteroaryl group, an optionally substituted 3-10-membered nonaromatic heterocyclic group, a hydroxyl group, an optionally substituted C1-6 alkoxy group or an optionally substituted C3-8 cycloalkoxy group).
Preferable examples of R2 include a cyano group or a group represented by Formula —CONVa11Va12 (wherein, Va11 and Va12 have the same meaning as defined above).
More preferable examples of R2 include a cyano group or a group represented by Formula —CONHVa16 (wherein, Va16 represents a hydrogen atom, a C1-6 alkyl group, a C3-8 cycloalkyl group, a C1-6 alkoxy group or a C3-8 cycloalkoxy group, provided that Va16 may have a substituent selected from a halogen atom, a cyano group, a hydroxyl group and a C1-6 alkoxy group).
A still more preferable example of R2 includes a group represented by Formula —CONHVa17 (wherein, Va17 represents a hydrogen atom, a C1-6 alkyl group or a C1-6 alkoxy group).
The most preferable example of R2 includes a group represented by Formula —CONHVa18 (wherein, Va18 represents a hydrogen atom, a methyl group or a methoxy group).
(iii) Y1 
Y1 represents a group represented by the following formula




(wherein, R7 and R8 each independently represent a hydrogen atom, a halogen atom, a cyano group, a nitro group, an amino group, an optionally substituted C1-6 alkyl group, an optionally substituted C3-8 cycloalkyl group, an optionally substituted C1-6 alkoxy group, an optionally substituted C1-6 alkylthio group, a formyl group, an optionally substituted C2-7 acyl group, an optionally substituted C2-7 alkoxycarbonyl group or a group represented by Formula —CONVd1Vd2 (wherein, Vd1 and Vd2 each independently represent a hydrogen atom or an optionally substituted C1-6 alkyl group);

W1 and W2 each independently represent an optionally substituted carbon atom or nitrogen atom).
A preferable example of Y1 includes a group represented by the following formula




(wherein, R71 represents a hydrogen atom or a halogen atom).

(iv) R3 and R4 
R3 and R4 each independently represent a hydrogen atom, an optionally substituted C1-6 alkyl group, an optionally substituted C2-6 alkenyl group, an optionally substituted C2-6 alkynyl group, an optionally substituted C3-8 cycloalkyl group, an optionally substituted C2-7 acyl group or an optionally substituted C2-7 alkoxycarbonyl group.
A preferable example of R3 and R4 includes a hydrogen atom.
(v) R5 
R5 represents a hydrogen atom, an optionally substituted C1-6 alkyl group, an optionally substituted C2-6 alkenyl group, an optionally substituted C2-6 alkynyl group, an optionally substituted C3-8 cycloalkyl group, an optionally substituted C6-10 aryl group, an optionally substituted 5-10-membered heteroaryl group or an optionally substituted 3-10-membered nonaromatic heterocyclic group.
Preferable examples of R5 include a hydrogen atom, an optionally substituted C1-6 alkyl group, an optionally substituted C2-6 alkenyl group, an optionally substituted C2-6 alkynyl group, an optionally substituted C3-8 cycloalkyl group, an optionally substituted C6-10 aryl group and an optionally substituted 3-10-membered nonaromatic heterocyclic group.
More preferable examples of R5 include a hydrogen atom, a C1-6 alkyl group, a C3-8 cycloalkyl group and a C6-10 aryl group (provided that R5 may have at least one substituent selected from the group consisting of a halogen atom and a methanesulfonyl group).
More preferable examples of R5 include a methyl group, an ethyl group and a cyclopropyl group.
Moreover, preferable examples of the compound represented by Formula (I) include:

    N-(4-(6-cyano-7-(2-methoxyethoxy)-4-quinolyl)oxy-2-fluorophenyl)-N′-(4-fluorophenyl)urea;
    N-(2-chloro-4-((6-cyano-7-((1-methyl-4-piperidyl)methoxy)-4-quinolyl)oxy)phenyl)-N′-cyclopropylurea;
    N-(4-((6-cyano-7-(((2R)-3-(diethylamino)-2-hydroxypropyl)oxy)-4-quinolyl)oxy)phenyl)-N′-(4-fluorophenyl)urea;
    N-(4-((6-cyano-7-(((2R)-2-hydroxy-3-(1-pyrrolidino)propyl)oxy)-4-quinolyl)oxy)phenyl)-N′-(4-fluorophenyl)urea;
    4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide;
    4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-(2-methoxyethoxy)-6-quinolinecarboxamide;
    N6-cyclopropyl-4-(3-chloro-4-(((cyclopropylamino)carbonyl)amino)phenoxy)-7-methoxy-6-quinolinecarboxamide;
    N6-(2-methoxyethyl)-4-(3-chloro-4-(((cyclopropylamino)carbonyl)amino)phenoxy)-7-methoxy-6-quinolinecarboxamide;
    N6-(2-fluoroethyl)-4-(3-chloro-4-(((cyclopropylamino)carbonyl)amino)phenoxy)-7-methoxy-6-quinolinecarboxamide;
    N6-methoxy-4-(3-chloro-4-(((cyclopropylamino)carbonyl)amino)phenoxy)-7-methoxy-6-quinolinecarboxamide;
    N6-methyl-4-(3-chloro-4-(((cyclopropylamino)carbonyl)amino)phenoxy)-7-methoxy-6-quinolinecarboxamide;
    N6-ethyl-4-(3-chloro-4-(((cyclopropylamino)carbonyl)amino)phenoxy)-7-methoxy-6-quinolinecarboxamide;
    4-(3-fluoro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-(2-methoxyethoxy)-6-quinolinecarboxamide;
    4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-(2-hydroxyethoxy)-6-quinolinecarboxamide;
    4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-((2S)-2,3-dihydroxypropyl)oxy-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-(((ethylamino)carbonyl)amino)phenoxy)-7-methoxy-6-quinolinecarboxamide;
    4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-(2-ethoxyethoxy)-6-quinolinecarboxamide;
    4-(4-((cyclopropylamino)carbonyl)aminophenoxy)-7-(2-methoxyethoxy)-6-quinolinecarboxamide;
    N-(2-fluoro-4-((6-carbamoyl-7-methoxy-4-quinolyl)oxy)phenyl)-N′-cyclopropylurea;
    N6-(2-hydroxyethyl)-4-(3-chloro-4-(((cyclopropylamino)carbonyl)amino)phenoxy)-7-methoxy-6-quinolinecarboxamide;
    4-(3-chloro-4-(1-propylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide;
    4-(3-chloro-4-(cis-2-fluoro-cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide;
    N6-methyl-4-(3-chloro-4-(((cyclopropylamino)carbonyl)amino)phenoxy)-7-(2-methoxyethoxy)-6-quinolinecarboxamide;
    N6-methyl-4-(3-chloro-4-(((ethylamino)carbonyl)amino)phenoxy)-7-methoxy-6-quinolinecarboxamide;
    4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-(2-(4-morpholino)ethoxy)-6-quinolinecarboxamide;
    4-(3-chloro-4-(2-fluoroethylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide;
    N6-((2R)tetrahydro-2-furanylmethyl)-4-(3-chloro-4-(((methylamino)carbonyl)amino)phenoxy)-7-methoxy-6-quinolinecarboxamide;
    4-(3-fluoro-4-(ethylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide;
    4-(3-chloro-4-(((cyclopropylamino)carbonyl)amino)phenoxy)-7-((2R)-2-hydroxy-3-(1-pyrrolidino)propoxy)-6-quinolinecarboxamide;
    N6-methyl-4-(3-chloro-4-(((methylamino)carbonyl)amino)phenoxy)-7-((2R)-3-diethylamino-2-hydroxypropoxy)-6-quinolinecarboxamide;
    N6-methyl-4-(3-chloro-4-(((ethylamino)carbonyl)amino)phenoxy)-7-((2R)-3-diethylamino-2-hydroxypropoxy)-6-quinolinecarboxamide;
    N6-methyl-4-(3-chloro-4-(((methylamino)carbonyl)amino)phenoxy)-7-((2R)-2-hydroxy-3-(1-pyrrolidino)propoxy)-6-quinolinecarboxamide;
    N6-methyl-4-(3-chloro-4-(((ethylamino)carbonyl)amino)phenoxy)-7-((2R)-2-hydroxy-3-(1-pyrrolidino)propoxy)-6-quinolinecarboxamide;
    N6-methyl-4-(3-chloro-4-(((methylamino)carbonyl)amino)phenoxy)-7-((1-methyl-4-piperidyl)methoxy)-6-quinolinecarboxamide;
    N6-methyl-4-(3-chloro-4-(((ethylamino)carbonyl)amino)phenoxy)-7-((1-methyl-4-piperidyl)methoxy)-6-quinolinecarboxamide;
    N-(4-(6-cyano-7-(2-methoxyethoxy)-4-quinolyl)oxy-2-fluorophenyl)-N′-cyclopropylurea;
    N-(4-(6-cyano-7-(3-(4-morpholino)propoxy)-4-quinolyl)oxyphenyl)-N′-(3-(methylsulfonyl)phenyl)urea;
    4-(4-((cyclopropylamino)carbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide;
    4-(3-fluoro-4-((2-fluoroethylamino)carbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide;
    N6-(2-ethoxyethyl)-4-(3-chloro-4-(((methylamino)carbonyl)amino)phenoxy)-7-methoxy-6-quinolinecarboxamide;
    4-(4-(3-ethylureido)-3-fluoro-phenoxy)-7-methoxyquinoline-6-carboxylic acid (2-cyanoethyl)amide; and
    N-(4-(6-(2-cyanoethyl)carbamoyl-7-methoxy-4-quinolyl)oxy-2-fluorophenyl)-N′-cyclopropylurea.


More preferable examples of the compound represented by Formula (I) include:

    4-(3-chloro-4-(cyclopropylaminocarbonyl)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;
    4-(3-chloro-4-(methylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide; and
    N6-methoxy-4-(3-chloro-4-(((ethylamino)carbonyl)amino)phenoxy)-7-methoxy-6-quinolinecarboxamide.


A still more preferable example of the compound represented by Formula (I) includes 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide (see Formula (II)).
The most preferable example of a FGFR2 inhibitory substance includes methanesulfonate of 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide.




A compound represented by General formula (I) may be produced by a known method such as a method described in either one of International Publication No. 02/32872 (WO02/32872) and International Publication No. 2005/063713 (WO2005/063713).
Moreover, examples of FGFR2 inhibitors according to the present invention also include
5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl)amide (hereinafter, also referred to as “SU11248”; Journal of Medicinal Chemistry., 46:1116-9, 2003, WO01/060814) (see Formula (III)),




N-(3-trifluoromethyl-4-chlorophenyl)-N′-(4-(2-methylcarbamoylpyridin-4-yl)oxyphenyl)urea (hereinafter, also referred to as “BAY 43-9006” or “sorafenib”; WO00/42012) (see Formula (IV)),




6-[2-(methylcarbamoyl)phenylsulfanyl]-3-E-[2-(pyridin-2-yl)ethenyl]indazole (hereinafter, also referred to as “AG013736”; WO01/002369) (see Formula (V)), and




N-{2-chloro-4-[(6,7-dimethoxy-4-quinolyl)oxy]phenyl}-N′-(5-methyl-3-isoxazolyl)urea (hereinafter, also referred to as “KRN951”; WO02/088110) (see Formula (VI))




SU11248, BAY 43-9006, AG013736 and KRN951 may be produced by a known method such as methods described in each publication mentioned above.
According to the present invention, a FGFR2 inhibitory substance may form a pharmacologically acceptable salt with acid or base. The FGFR2 inhibitory substance of the invention also comprises such pharmacologically acceptable salts. Examples of salts formed with acids include inorganic acid salts such as hydrochloride salts, hydrobromate salts, sulfate salts and phosphate salts, and organic acid salts such as formic acid, acetic acid, lactic acid, succinic acid, fumaric acid, maleic acid, citric acid, tartaric acid, stearic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and trifluoroacetic acid. Examples of salts formed with bases include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, organic base salts such as trimethylamine, triethylamine, pyridine, picoline, dicyclohexylamine, N,N′-dibenzyl ethylenediamine, arginine and lysine and ammonium salts.
Furthermore, according to the present invention, the FGFR2 inhibitory substance may exist as a solvate or an optical isomer. According to the present invention, the FGFR2 inhibitory substance comprises such solvates and optical isomers. Examples of solvates include hydrates and nonhydrates, preferably hydrates. Examples of solvents include water, alcohols (for example, methanol, ethanol and n-propanol) and dimethylformamide.
Moreover, according to the present invention, the FGFR2 inhibitory substance may be crystalline or amorphous. If a crystalline polymorph is present, it may be one type of any crystalline forms or mixture of thereof.
According to the present invention, the FGFR2 inhibitory substance also comprises metabolites that underwent metabolism such as oxidation, reduction, hydrolysis and conjugation in vivo. According to the present invention, the FGFR2 inhibitory substance also comprises compounds that generate the FGFR2 inhibitory substance by undergoing metabolism such as oxidation, reduction and hydrolysis in vivo.
Preferably, the FGFR2 inhibitory substance of the invention is a substance having activity of inhibiting FGFR2 kinase activity (hereinafter, also referred to as “FGFR2 inhibitory activity”). Herein, “FGFR2 kinase activity” refers to activity of FGFR2 to phosphorylate a tyrosine residue of own protein or other protein.
Examples of methods for determining FGFR2 inhibitory activity of the FGFR2 inhibitory substance include cell free kinase assay, western blotting, cell growth assay and viability assay. Examples of the cell growth assay include tritium thymidine uptake method, MTT method, XTT method (cell counting kit-8 (Dojindo Laboratories)), AlamarBlue technique, Neutral Red technique, BrdU technique, Ki67 staining and PCNA staining. Examples of the viability assay include TUNNEL staining, Caspase-3 cleavage detection and PARP cleavage detection. These methods may be carried out according to conventional techniques (Blood. 2005, 105, 2941-2948., Molecular Cancer Therapeutics. 2005, 4, 787-798).
Hereinafter, an exemplary method for determining FGFR2 inhibitory activity will be described.
The FGFR2 inhibitory activity can be determined by cell free kinase assay.
FGFR2 can be prepared by a gene-engineering technique according to a conventional method. For example, according to the method of Baculovirus Expression System, human recombinant GST fusion protein, human recombinant histidine-tag fusion protein or the like may be expressed in an insect cell (Spondoptera frugiperda 9 (Sf9)). Furthermore, the expressed recombinant protein can be purified by affinity chromatography (e.g., GSH-agarose (Sigma) or Ni—NTH-agarose (Qiagen)). The purity and identification of the protein can be confirmed by SDS-PAGE, silver staining and western blotting using an antibody specific to FGFR2.
The cell free kinase assay may be carried out as follows.
First, to each well of a plate (e.g., 96-well, 384-well, etc.), 25 μl of a solution containing an ATP solution, a test substance, 5-10 mU of FGFR2 recombinant protein and 0.1 mg/ml Poly(Glu, Tyr)4:1 may be added. To this mixture, MgATP is added to initiate reaction.
25 μl of this mixture may contain 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 2.5 mM MnCl2, 10 mM Mg acetate and the like. ATP used in this case may be labeled with a radioisotope such as [γ-32P]-ATP and [γ-33P]-ATP.
The reaction may be terminated by adding 5 μL of 3% phosphoric acid following incubation for a given period of time.
Each well may be subjected to an appropriate washing procedure.
FGFR2 inhibitory activity can be assessed by determining the amount of ATP incorporation. When a radioactive isotope-labeled ATP mentioned above is used, the amount of ATP incorporation can be assessed by determination of the radioactivity captured on the plate with a scintillation counter. Alternatively, a certain amount of the reaction solution may be spotted onto a filter to measure the radioactivity thereof with a scintillation counter. The filter may be subjected to an appropriate washing procedure.
The FGFR2 inhibitory activity of compounds can be assessed by this method.
(5) Pharmaceutical Composition, Therapeutic Agent and Therapeutic Method
The therapeutic agent of the invention comprises a FGFR2 inhibitory substance and is an agent for treating undifferentiated gastric cancer. Preferably, the therapeutic agent of the invention is used for an organism comprising at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2.
Herein, undifferentiated gastric cancer comprises poorly differentiated adenocarcinoma, signet-ring cell carcinoma and mucinous carcinoma (Japanese Classification of Gastric Carcinoma “Igan-Toriatsukai Kiyaku” (13th ed.)). In the case of undifferentiated gastric cancer, cancer cells are likely to diffuse and likely to develop fibrosis that leads to scirrhous gastric cancer. Thus, the therapeutic agent of the invention is effective against at least one gastric cancer selected from the group consisting of poorly differentiated adenocarcinoma, signet-ring cell carcinoma, mucinous carcinoma and scirrhous gastric cancer.
The therapeutic agent of the invention may be administered to a mammal (e.g., human, rat, rabbit, sheep, pig, bovine, cat, dog, monkey, etc.).
The pharmaceutical composition of the invention comprises a FGFR2 inhibitory substance to be administered to an organism comprising at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2.
The pharmaceutical composition of the invention may be used as a therapeutic agent for treating a disease comprising comprising at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2. An example of such a disease includes undifferentiated gastric cancer.
The pharmaceutical composition of the invention may be administered to an organism, i.e., a mammal (e.g., human, rat, rabbit, sheep, pig, bovine, cat, dog, monkey, etc.). The organisms of the present invention may comprise either a cell overexpressing FGFR2 or a cell expressing mutant FGFR2, or both of them.
According to the present invention, the therapeutic agent comprises agents for improving prognosis, agents for preventing of recurrence and the like. When the therapeutic agent is an agent for cancer, it may comprise antitumoral agents and agents for suppressing cancer metastasis.
The effect of treatment may be confirmed by observation of an x-ray picture, CT or the like, or by histopathological diagnosis by biopsy, or measurement of values of disease markers
Where a therapeutic agent or a pharmaceutical composition of the invention is used, the given dose of the FGFR2 inhibitory substance differs depending on the degree of the symptom, age, sex, weight and sensitivity difference of the patient, administration mode, administration period, administration interval, nature, prescription and the type of the pharmaceutical formulation, and the type of the active element. Usually, but without limitation, the dose of the compound is 0.1-1000 mg/day, preferably 0.5-100 mg/day, more preferably 1-30 mg/day for an adult (weight 60 kg), which may be administered once to three times a day.
Although the pharmaceutical composition or the therapeutic agent comprising the FGFR2 inhibitory substance of the invention as an active ingredient may be used alone, it is usually mixed with appropriate additives and made into a formulation.
Examples of such additives include excipients, binders, lubricants, disintegrants, colorants, flavoring agents, emulsifiers, surfactants, solubilizing agents, suspending agents, tonicity agents, buffers, antiseptic agents, antioxidant agents, stabilizers, absorption promoters and the like that are generally used for medicine. If required, they may be used in combination. Examples of such additive are mentioned below.
Excipients: lactose, sucrose, glucose, cornstarch, mannitol, sorbitol, starch, alpha-starch, dextrin, crystalline cellulose, light anhydrous silicic acid, aluminum silicate, calcium silicate, magnesium aluminometasilicate and calcium hydrogen phosphate;
Binders: for example, polyvinyl alcohol, methyl cellulose, ethyl cellulose, gum arabic, tragacanth, gelatin, shellack, hydroxypropyl methylcellulose, hydroxypropylcellulose, carboxymethylcellulose sodium, polyvinylpyrrolidone and macrogol;
Lubricants: magnesium stearate, calcium stearate, sodium stearyl fumarate, talc, polyethyleneglycol and colloid silica;
Disintegrants: crystalline cellulose, agar, gelatin, calcium carbonate, sodium hydrogen carbonate, calcium citrate, dextrin, pectin, low substituted hydroxypropylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, carboxymethyl starch and carboxymethyl starch sodium.
Colorants: ferric oxide, yellow ferric oxide, carmine, caramel, beta-carotene, titanium oxide, talc, riboflavin sodium phosphate, yellow aluminum lake and the like that are approved as additives in drugs;
Flavoring agents: cocoa powder, menthol, aromatic acid, peppermint oil, borneol and cinnamon powder;
Emulsifiers and surfactants: stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionate, lecithin, glycerine monostearate, sucrose fatty acid ester and glycerine fatty acid ester;
Solubilizing agents: polyethyleneglycol, propylene glycol, benzyl benzoate, ethanol, cholesterol, triethanolamine, sodium carbonate, sodium citrate, Polysorbate 80 and nicotine acid amide;
Suspending agents: for example, in addition to the surfactants mentioned above, hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose and the like;
Tonicity agents: glucose, sodium chloride, mannitol and sorbitol;
Buffers: buffers such as phosphate, acetate, carbonate, citrate and the like;
Antiseptic agents: methylparaben, propylparaben, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid and sorbic acid;
Antioxidant agents: sulfate, ascorbic acid and alpha-tocopherol;
Stabilizers: those generally used for medicine; and
Absorption promoters: those generally used for medicine.
If required, components such as vitamins and amino acids may be blended.
Examples of the above-mentioned formulations include oral formulations such as tablets, powder, granule, fine granule, capsule, syrup, lozenge and inhaler; external formulations such as suppository, ointment, eye ointment, poultice strip, eye-drops, nasal drops, eardrops, gel patch and lotion; and injectable formulations.
The oral formulations mentioned above may be formulated by appropriately combining the additives mentioned above. If necessary, surface of these formulations may be coated.
The external formulations mentioned above may be formulated by appropriately combining the additives mentioned above, particularly excipients, binders, flavoring agents, emulsifiers, surfactants, solubilizing agents, suspending agent, tonicity agents, antiseptic agents, antioxidant agents, stabilizers and absorption promoters.
The injectable formulations mentioned above may be formulated by appropriately combining the additives mentioned above, particularly emulsifiers, surfactants, solubilizing agents, suspending agents, tonicity agents, buffers, antiseptic agents, antioxidant agents, stabilizers and absorption promoters. The injectable formulations may be used through means such as infusion, intramuscular injection, subcutaneous injection, intradermal injection and intravenous injection.
The present invention comprises a method for treating undifferentiated gastric cancer, characterized by administering an effective dosage of a FGFR2 inhibitory substance to a patient.
In addition, the present invention comprises a method for treating a disease, characterized by administering an effective dosage of a FGFR2 inhibitory substance to an organism including at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2. According to the present invention, the disease is preferably undifferentiated gastric cancer.
According to the therapeutic method of the invention, the route and the method for administering the FGFR2 inhibitory substance are not particularly limited and reference may be made to the description of the pharmaceutical composition or the therapeutic agent of the invention above.
The present invention comprises use of a FGFR2 inhibitory substance for producing a therapeutic agent for treating undifferentiated gastric cancer.
The present invention also comprises use of a FGFR2 inhibitory substance for producing a pharmaceutical composition to be administered to an organism including at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2. In the use according to the present invention, the pharmaceutical composition is useful as a therapeutic agent for treating undifferentiated gastric cancer.
The present invention comprises a FGFR2 inhibitory substance for a therapeutic agent for treating undifferentiated gastric cancer.
The present invention also comprises a FGFR2 inhibitory substance for a pharmaceutical composition to be administered to an organism including at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2. According to the present invention, the pharmaceutical composition is useful as a therapeutic agent for treating undifferentiated gastric cancer.
Furthermore, the present invention provides a FGFR2 inhibitor comprising a compound represented by General Formula (I), a pharmacologically acceptable salt thereof or a solvate thereof. The FGFR2 inhibitor has an effect of inhibiting kinase activity of FGFR2.
The compound represented by General Formula (I) is as described above, and it is preferably 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide. More preferably, the present invention provides a FGFR2 inhibitor comprising methanesulfonate of 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide.
The present invention also provides a FGFR2 inhibitor comprising at least one compound selected from the group consisting of N-(3-trifluoromethyl-4-chlorophenyl)-N′-(4-(2-methylcarbamoylpyridin-4-yl)oxyphenyl)urea (BAY 43-9006), 6-[2-(methylcarbamoyl)phenylsulfanyl]-3-E-[2-(pyridin-2-yl)ethenyl]indazole (AG013736), 5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl)amide (SU 11248) and N-{2-chloro-4-[(6,7-dimethoxy-4-quinolyl)oxy]phenyl}-N′-(5-methyl-3-isoxazolyl)urea (KRN951), a pharmacologically acceptable salt thereof or a solvate thereof.
The FGFR2 inhibitory activity of a FGFR2 inhibitor of the invention may be determined as described above.
As the FGFR2 inhibitor of the invention, the compound of the invention may be used alone, or it may be mixed and formulated with appropriate additives mentioned above.
As to the usage and the dosage of the FGFR2 inhibitor, reference may be made to the description of the pharmaceutical composition or the therapeutic agent above.
The present invention also comprises use of at least one compound selected from the group consisting of a compound represented by General Formula (I), SU11248, BAY43-9006, AG013736 and KRN951, a pharmacologically acceptable salt thereof or a solvate thereof for producing a FGFR2 inhibitor.
The present invention further comprises at least one compound selected from the group consisting of a compound represented by General Formula (I), SU 11248, BAY43-9006, AG013736 and KRN951, a pharmacologically acceptable salt thereof or a solvate thereof for a FGFR2 inhibitor.
The present invention yet further comprises a method for inhibiting FGFR2, preferably a method for inhibiting kinase activity of FGFR2 with at least one compound selected from the group consisting of a compound represented by General Formula (I), SU 11248, BAY43-9006, AG013736 and KRN951, a pharmacologically acceptable salt thereof or a solvate thereof. According to the method of the invention, the usage and the dosage of the compound are not particularly limited and reference may be made to the description of the pharmaceutical composition or the therapeutic agent above.
2. Method for Predicting Sensitivity
The present invention provides a method for predicting whether or not a patient is highly sensitive to a FGFR2 inhibitory substance of the invention by using at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in the cell as a biomarker. Patients highly sensitive to a FGFR2 inhibitory substance is expected to benefit higher therapeutic effect from the substance.
According to the method of the invention, the patient is preferably an undifferentiated gastric cancer patient, more preferably a patient suffering from at least one selected from the group consisting of poorly differentiated adenocarcinoma, signet-ring cell carcinoma, mucinous carcinoma and scirrhous gastric cancer.
(1) Step of determining at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in a cell
In this step, the cell is preferably a cell taken from a patient. The cell may be obtained by removal from the patient, for example, through surgical procedure (e.g., biopsy, bone marrow puncture, etc.).
The cell is preferably a tumor cell. In the case of genetically-caused undifferentiated gastric cancer, the cell used is preferably a blood cell.
Herein, the meaning of a cell overexpressing FGFR2 or a cell expressing mutant FGFR2 is described in “1. Pharmaceutical composition, therapeutic agent and method of the invention” above. An expression level of FGFR2 and the presence or the absence of FGFR2 mutation may be determined according to the method described in “1. Pharmaceutical composition, therapeutic agent and method of the invention” above.
In this step, either the expression level of FGFR2 or the presence or the absence of FGFR2 mutation, or both of them may be determined.
(2) Step of predicting whether or not a patient is highly sensitive to FGFR2 inhibitory substance
In this step, whether or not a patient is highly sensitive to a FGFR2 inhibitory substance may be predicted by using at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in a cell as a biomarker. Specifically, when the measured cell meets at least one of the case where the cell is overexpressing FGFR2 or the case where the cell is expressing mutant FGFR2, the patient is judged to be highly sensitive to the FGFR2 inhibitory substance.
Another aspect of the invention is a method for analyzing sensitivity of a cell to a FGFR2 inhibitory substance by using the measurement result from (1) as a biomarker. Based on the measurement results from (1), when the cell is at least one of a cell overexpressing FGFR2 or a cell expressing mutant FGFR2, this cell may be judged to have higher sensitivity to the FGFR2 inhibitory substance compared to cells that are neither of the above cells.
Yet another aspect of the invention is a method for selecting a cell or a patient having higher sensitivity to a FGFR2 inhibitory substance by using the measurement result from (1) as a biomarker. Based on the measurement results from (1), when the cell is at least one of a cell overexpressing FGFR2 or a cell expressing mutant FGFR2, this cell or the patient having this cell may be judged to have higher sensitivity to the FGFR2 inhibitory substance as described above. Thus, such a cell or a patient may be selected as a cell or a patient having higher sensitivity to the FGFR2 inhibitory substance.
Still yet another aspect of the present invention is a method for classifying a patient, comprising analyzing sensitivity of the patient to a FGFR2 inhibitory substance by using the measurement result from (1) as a biomarker, and classifying the patient based on the obtained result. Specifically, according to the method of the invention, sensitivity to a FGFR2 inhibitory substance is analyzed based on the measurement results from (1) as described above and the patient is classified accordingly. For example, a patient may be classified into a group comprising at least one of a cell overexpressing FGFR2 or a cell expressing mutant FGFR2, or a group comprising neither of the cells. Alternatively, a patient may be classified into a group having higher sensitivity to a FGFR2 inhibitory substance or a group other than those.
Yet another aspect of the invention is a method for selecting a patient as a target of administering a FGFR2 inhibitory substance, the method comprising selecting a patient having at least one of a cell overexpressing FGFR2 or a cell expressing mutant FGFR2 based on the measurement results from (1). A patient comprising at least one of a cell overexpressing FGFR2 or a cell expressing mutant FGFR2 may be a target of administering the FGFR2 inhibitory substance.
Yet another aspect of the invention is a method for predicting therapeutic effect of a FGFR2 inhibitory substance on a patient based on the measurement results from (1). According to the method of the invention, when a cell is at least one of a cell overexpressing FGFR2 or a cell expressing mutant FGFR2 based on the measurement results from (1), the cell may be judged to have higher sensitivity to the FGFR2 inhibitory substance and thus the substance may be predicted to have higher therapeutic effect on this cell or a patient having this cell.
The present invention also comprises a method for determining at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation in a cell from a patient in order to predict the degree of sensitivity of the patient to a FGFR2 inhibitory substance. This determination method is described in (1) above.
Determination of either or both of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation induces prediction of the degree of sensitivity of a patient to a FGFR2 inhibitory substance.
In this step, FGFR2 inhibitory substances are as described above, and it is preferably 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide, a pharmacologically acceptable salt thereof or a solvate thereof.
The method of the invention may be used for predicting the degree of efficacy of a FGFR2 inhibitory substance on a patient in advance of administering the FGFR2 inhibitory substance to the patient. Furthermore, treatment of a disease can be carried out by selecting a patient who is more expected to benefit the effect of the FGFR2 inhibitory substance. Thus, the present invention is clinically very useful.
The present invention provides a test kit used for the method of the invention for determining at least one selected from the group consisting of an expression level of FGFR2 and the presence or the absence of FGFR2 mutation. The test kit of the invention comprises the reagents mentioned above used for the determination. The test kit of the invention induces prediction of whether or not a patient is highly sensitive to a FGFR2 inhibitory substance.
The present invention also comprises use of the test kit for the prediction.
EXAMPLES
Hereinafter, the present invention will be illustrated by way of specific examples, although the invention should not be limited thereto.
Example 1
Determination of FGFR2 Kinase Inhibitory Activity of FGFR2 Inhibitory Substance
FGFR2 kinase inhibitory activity of the test substance was tested by Upstate (UK) on our request. Specifically, the FGFR2 kinase inhibitory activity was measured as follows.
MgATP was added to 25 μL of solution containing 5-10 mU of FGFR2 recombinant protein, 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 2.5 mM MnCl2, 0.1 mg/ml poly (Glu, Tyr)4:1, 10 mM Mg Acetate, 500 cpm/mol [γ-33P]-ATP and a test substance to initiate reaction.
The test substance used was 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide
(methanesulfonate), 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine (hereinafter, also referred to as “PTK787/Z 222584”), SU11248, BAY 43-9006, AG013736 or KRN951.
PTK787/Z 222584 is a compound known as a VEGF receptor kinase inhibitory substance.
4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide was produced according to the descriptions of International Publications Nos. 02/32872 (WO02/32872) and 2005/063713 (WO2005/063713).
PTK787/ZK222584 was produced according to the description of International Publication No. 98/035958. SU11248 was produced according to the description of International Publication No. 01/060814. BAY43-9006 was produced according to International Publication No. 00/42012. AG013736 was produced according to the description of International Publication No. 01/002369. KRN951 was produced according to the description of International Publication No. 02/088110.
Following 40 minutes of reaction at room temperature, 5 μL of 3% phosphoric acid was added to terminate the reaction. 10 μL of the reaction solution was spotted on Filtermat A, which was washed with 75 mM phosphoric acid for five times and once with ethanol and then dried. The radioactivity of the spot was determined (measured).
The concentration (IC50) of the test substance required to inhibit FGFR2 kinase activity by 50% was calculated by referring to radioactivity with respect to 33P at each concentration.
In this regard, a value obtained when substrate Poly(Glu, Tyr)4:1 was contained but not the FGFR2 recombinant protein was assumed 0% value while a value obtained when the FGFR2 recombinant protein and substrate Poly(Glu, Tyr)4:1 were contained but not the test substance was assumed 100% value.
The kinase activity in the presence of the test substance at each concentration was calculated as percentage of the value obtained by subtracting the 0% value from each radioactivity value to the value obtained by subtracting the 0% value from the 100% value. From this ratio (%), the concentration (IC50) of the test substance required to inhibit the FGFR kinase activity by 50% was calculated.
As a result, 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide was found to have a FGFR2 kinase inhibitory activity (IC50<10 nM). In addition, SU11248, BAY 43-9006, AG013736 and KRN951 were also found to have FGFR2 kinase inhibitory activity (IC50=83, 168, 17 and 124 nM, respectively). An IC50 value of PTK787/Z 222584 had 54200 nM.
Example 2
Effect of FGFR2 Inhibitory Substance on FGFR2 Phosphorylation in Human Undifferentiated Gastric Cancer Cell Lines (KATO-III, HSC-39 and SNU-16)
1. Preparation of Cell Extract
Human undifferentiated gastric cancer cell lines (KATO-III (purchased from ATCC)), SNU-16 (purchased from ATCC) and HSC-39 (purchased from Immuno-Biological Laboratories)) were suspended in RPMI1640 media (purchased from Sigma) containing 10% FBS (fetal bovine serum). KATO-III, SNU-16 and HSC-39 are cells showing FGFR2 gene amplification (Laboratory Investigation, 78, 1143-1153, 1998), and are also known as scirrhous gastric cancer cell lines. Each 10 mL of the cell suspensions (5×105 cells/mL) was placed into a 75 cm2 cell culture flask (purchased from FALCON) and cultured in a 5% CO2 incubator (37° C.) overnight. In the case of KATO-III, the medium was replaced by an RMPI1640 medium containing 1% FBS. To this, 10 mL of the test substance 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide (methanesulfonate) diluted in RPMI1640 containing 1% or 10% FBS was added and cultured in a 5% CO2 incubator (37° C.) for an hour. The culture solution was collected, each flask was washed with 5 mL PBS, and the mixture of the collected culture and the washing solution was centrifuged at 1,000 rpm, at 4° C. for 5 minutes. For immunoprecipitation, 100 μL of a lysis buffer (50 mM Hepes (pH 7.4), 150 mM NaCl, 10% (v/v) glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA (pH 8.0), 100 mM NaF, 1 mM PMSF, 10 μg/mL Aprotinin, 50 μg/mL Leupeptin, 1 μg/mL Pepstatin A, 1 mM Na3VO4) was added to solubilize the cells. This solution was collected and centrifugated at 15,000 rpm, at 4° C. for 15 minutes. 1000 μg/1000 μL (KATO-III), 800 μg/500 μL (SNU-16) and 1000 μg/500 μL (HSC-39) of the supernatants were prepared as cell extracts.
2. Immunoprecipitation
To each cell extract, 10 μL of anti-FGFR2 antibody (purchased from Sigma) and 100 μL of protein A agarose (purchased from Upstate) were added and incubated at 4° C. overnight. This solution was washed with 1 mL phosphate buffer for three times and an SDS buffer was added followed by 5 minutes of treatment at 94° C. to solubilize the protein, which was prepared as a cell sample solution.
3. Electrophoresis and Western Blotting
Fifteen μL of the cell sample solution was subjected to electrophoresis on 4-20% gradient polyacrylamide gel (purchased from Daiichi Pure Chemicals). Following electrophoresis, the sample was transferred to a PVDF membrane (purchased from Amersham Pharmacia Biotech) according to a conventional technique. The resulting membrane was subjected to immunoblotting using anti-FGFR2 antibody or anti-phosphotyrosine antibody (4G10, purchased from Upstate) as primary antibody and horse radish peroxidase-labeled anti-rabbit IgG antibody (anti-rabbit IgG, HRP-linked Antibody (purchased from Cell Signaling)) or horse radish peroxidase-labeled anti-mouse IgG antibody (anti-mouse IgG, HRP-linked Antibody (purchased from Cell Signaling)) as secondary antibody. The membrane was washed and then allowed to develop color with Super Signal (purchased from PIERCE).
As a result, 1 μM of 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide almost completely inhibited phosphorylation of FGFR2 for every cell (FIG. 1).
Example 3
Effect of FGFR2 Inhibitory Substance on Cell Proliferation of Human Undifferentiated Gastric Cancer Cell Lines (KATO-III, HSC-39 and SNU-16)
Human undifferentiated gastric cancer cell lines (KATO-III (purchased from ATCC)), SNU-16 (purchased from ATCC) and HSC-39 (purchased from Immuno-Biological Laboratories)) were suspended in RPMI1640 media (purchased from Sigma) containing 10% FBS except that KATO-III was suspended in an RMPI1640 medium containing 1% FBS. The cell suspensions (KATO-III: 6×104 cells/mL; SNU-16 and HSC-39: 2×104 cells/mL) were placed into a 96-well cell culture plate (purchased from NUNC) at 0.1 mL/well and cultured in a 5% CO2 incubator (37° C.) overnight. Following cultivation, 0.1 mL of the test substance 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide (methanesulfonate) diluted in an RPMI1640 medium containing 1% or 10% FBS was added to each well and further cultured in a 5% CO2 incubator (37° C.) for three days. Following cultivation, 20 μL of Cell Counting Kit-8 (purchased from DOJINDO) was added to each well and allowed to develop color in a 5% CO2 incubator (37° C.) to determine (measure) the absorbance of each well by using a plate reader MTP-500 (Corona Electric) at a measurement wavelength of 450 nm and a reference wavelength of 660 nm. The concentration (IC50) of the test substance required to inhibit cell proliferation by 50% was calculated by referring to the ratio (%) of absorbance of each well containing the test substance to absorbance of the well without the test substance.
As a result, 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide was found to have inhibitory activity against the proliferation of the human undifferentiated gastric cancer cell lines (KATO-III, SNU-16 and HSC-39), with IC50 values of 141, 251 or 157 nM respectively.
Example 4
Effect of FGFR2 Inhibitory Substance on Apoptosis of Human Undifferentiated Gastric Cancer Cell Line (HSC-39)
1. Preparation of Cell Extract
Human undifferentiated gastric cancer cell line HSC-39 (purchased from Immuno-Biological Laboratories) was suspended in an RPMI1640 medium (purchased from Sigma) containing 10% FBS. Five mL of the cell suspension (4×105 cells/mL) was placed into a 75 cm cell culture flask (purchased from FALCON). To this, 5 mL of the test substance 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide (methanesulfonate) diluted in an RPMI1640 medium containing 10% FBS was added and cultured in a 5% CO2 incubator (37° C.) for one or three days. The culture solution was collected and each flask was washed with 5 mL PBS. The mixture of the collected culture solution and the washing solution was centrifuged at 1,000 rpm, at 4° C. for 5 minutes. For precipitation, 100 μL of a lysis buffer (50 mM Hepes (pH 7.4), 150 mM NaCl, 10% (v/v) glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA (pH 8.0), 100 mM NaF, 1 mM PMSF, 10 μg/mL Aprotinin, 50 μg/mL Leupeptin, 1 μg/mL Pepstatin A, 1 mM Na3VO4) was added to solubilize the cells. This solution was collected and centrifugated at 15,000 rpm, at 4° C. for 15 minutes. An SDS buffer was added to the supernatant to prepare a 25 μg/15 μL solution and the resultant was treated at 94° C. for 5 minutes to solubilize the protein, which was prepared as a cell sample solution.
2. Electrophoresis and Western Blotting
Fifteen μL of the cell sample solution was subjected to electrophoresis on 4-20% gradient polyacrylamide gel (purchased from Daiichi Pure Chemicals). Following electrophoresis, the sample was transferred to a PVDF membrane (purchased from Amersham Pharmacia Biotech) according to a conventional technique. The resulting membrane was subjected to immunoblotting using anti-PARP antibody (purchased from Cell Signaling) or anti-cleaved-caspase-3 antibody (purchased from Cell Signaling) as primary antibody and horse radish peroxidase-labeled anti-rabbit IgG antibody (anti-rabbit IgG, HRP-linked Antibody (purchased from Cell Signaling)) as secondary antibody. PARP (Poly-ADP-Ribose-Polymerase) is a protein known as an apoptosis marker since it is fragmented by caspase upon induction of apoptosis. Caspase-3 is also known to be fragmented upon induction of apoptosis.
The membrane was washed and then allowed to develop color with Super Signal (purchased from PIERCE).
As a result, cleaved-PARP and cleaved-caspase-3 were clearly detected with 1 μM of 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide (FIG. 2). Hence, 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide was shown to have an effect of inducing apoptosis.
Example 5
Anti-Tumor Effect of FGFR2 Inhibitory Substance on Subcutaneous Transplanted (Xenograft) Models of Human Undifferentiated Gastric Cancer Cell Lines (KATO-III, HSC-39 and SNU-16)
Human undifferentiated gastric cancer cell lines (KATO-III (purchased from ATCC)), SNU-16 (purchased from ATCC) and HSC-39 (purchased from Immuno-Biological Laboratories) were cultured with RPMI1640 (containing 10% FBS) in a 5% CO2 incubator at 37° C. The cells were collected with trypsin-EDTA according to a conventional method. The cells were suspended in a phosphate buffer to prepare a 5×107 cells/mL suspension. Each 0.1 mL of the resulting cell suspensions was subcutaneously transplanted into the side of body of nude mice (purchased from Charles River).
Following transplantation, the test substance 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide (methanesulfonate) dissolved in distilled water for injection (purchased from Otsuka Pharmaceutical) was orally administered at 1, 3, 10, 30 or 100 mg/kg once a day on a four-weeks schedule (except for KATO-III, 30 or 100 mg/kg, once a day, two-weeks schedule) once the tumor volume became (reached) about 100-200 mm3. The major and minor axes of tumors were measured with Digimatic caliper (Mitsutoyo), and tumor volumes were calculated according to the following formulae.
Tumor Volume (TV)=Major axis of tumor (mm)×(Minor axis of tumor)2 (mm2)/2
As a result, 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide was found to have dose-dependent anti-tumor effect on subcutaneous transplanted (xenograft) models of human undifferentiated gastric cancer cell lines (KATO-III, SNU-16 and HSC-39) (FIG. 3).
Example 6
Effect of FGFR2 Inhibitory Substance Against FGFR2 Phosphorylation in Subcutaneous Transplanted (Xenograft) Models of Human Undifferentiated Gastric Cancer Cell Lines (HSC-39 and SNU-16)
1. Preparation of Tumor and Solubilization of Tumor
Human undifferentiated gastric cancer cell lines (SNU-16 (purchased from ATCC) and HSC-39 (purchased from Immuno-Biological Laboratories) were cultured with RPMI1640 (containing 10% FBS) in a 5% CO2 incubator (37° C.) and cells were collected with trypsin-EDTA according to a conventional method. The cells were suspended in a phosphate buffer to prepare a 5×107 cells/mL suspension. Each 0.1 mL of the resulting cell suspensions was subcutaneously transplanted into the side of body of nude mice (purchased from Charles River).
Following transplantation, the test substance 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide (methanesulfonate) dissolved in distilled water for injection (purchased from Otsuka Pharmaceutical) was orally administered at 10, 30 or 100 mg/kg once the tumor volume became (reached) about 400-800 mm3. Tumors were removed two hours after the administration, to which a lysis buffer (50 mM Hepes (pH 7.4), 150 mM NaCl, 10% (v/v) glycerol, 1% TritonX-100, 1.5 mM MgCl2, 1 mM EDTA (pH 8.0), 100 mM NaF, 1 mM PMSF, 10 μg/mL Aprotinin, 50 μg/mL Leupeptin, 1 μg/mL Pepstatin A, 1 mM Na3VO4) and 25 mM β-glycerophosphate and phosphatase inhibitor cocktail II (SIGMA)) was added for homogenization. The resultant was centrifugated at 15,000 rpm, at 4° C. for 15 minutes. 1000 μg/500 μL of The supernatant was prepared as a tumor extract.
2. Immunoprecipitation
To the tumor extract, 10 μL of anti-FGFR2 antibody (purchased from Sigma) and 100 μL of protein A agarose (purchased from Upstate) were added and incubated at 4° C. overnight. This solution was washed with a 1 mL phosphate buffer for three times and an SDS buffer was added followed by 5 minutes of treatment at 94° C. to solubilize the protein, which was prepared as a tumor sample solution.
3. Electrophoresis and Western Blotting
Fifteen μL of the tumor sample solution was subjected to electrophoresis on 4-20% gradient polyacrylamide gel (purchased from Daiichi Pure Chemicals). Following electrophoresis, the sample was transferred to a PVDF membrane (purchased from Amersham Pharmacia Biotech) according to a conventional technique. The resulting membrane was subjected to immunoblotting using anti-FGFR2 antibody or anti-phosphotyrosine antibody (4G10, purchased from Upstate) as primary antibody and horse radish peroxidase-labeled anti-rabbit IgG antibody (anti-rabbit IgG, HRP-linked Antibody (purchased from Cell Signaling)) or horse radish peroxidase-labeled anti-mouse IgG antibody (anti-mouse IgG, HRP-linked Antibody (purchased from Cell Signaling)) as secondary antibody. The membrane was washed and then allowed to develop color with Super Signal (purchased from PIERCE).
As a result, 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide was found to inhibit phosphorylation of FGFR2 at an administered dosage that showed anti-tumor effect in the subcutaneous transplanted (xenograft) models of human undifferentiated gastric cancer cell lines (SNU-16 and HSC-39) (FIG. 4).
From the above results, the FGFR2 inhibitory substance of the invention was expected to have higher effect against undifferentiated gastric cancers.
Moreover, the FGFR2 inhibitory substance of the invention was expected to have higher effect on an organism comprising at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2.
Furthermore, the effect of the compound of the invention can be predicted without administrating it to a patient by determining at least one selected from the group consisting of an expression level of FGFR2 and (the presence or the absence of) FGFR2 mutation in a cell and using either one or a combination of the determined expression level of FGFR2 and the presence or the absence of FGFR2 mutation in the cell as a biomarker. Accordingly, the method of the invention induces selection of a patient who is expected to benefit higher therapeutic effect from the compound without administrating the compound to the patient, thereby contributing to the improvement of QOL of the patients.
Reference Example
Hereinafter, a method for producing a formulation of one of the FGFR2 inhibitory substances, i.e., 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide, will be described as a reference example.
(Production of Pharmaceutical Composition)
(1) 1 mg tablet
24 g of crystal (C) of methanesulfonate of 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide (hereinafter, also referred to as “crystal (C)”, which was produced according to the method described in Example 7 of WO2005/063713) and 192 g of light anhydrous silicic acid (antigelling agent sold under the trade name of AEROSIL (Registered Trademark) 200, Nippon Aerosil) were mixed with 20 L Super Mixer, and then 1236 g of D-mannitol (excipient, Towa-Kasei), 720 g of crystalline cellulose (excipient sold under the trade name of Avicel PH101, Asahi Kasei) and 72 g of hydroxypropylcellulose (binder sold under the trade name of HPC-L, Nippon Soda) were further added and mixed together. Subsequently, a suitable amount of anhydrous ethanol was added to obtain a granulated body containing crystal (C). This granulated body was dried in a rack dryer (60° C.), and then size-regulated using PowerMILL to obtain granules. Together with the granules, 120 g of croscarmellose sodium (disintegrant sold under the trade name of Ac-Di-Sol, FMC International Inc.) and 36 g of sodium stearyl fumarate (lubricant, JRS Pharma LP) were placed and mixed together in a 20 L tumbler mixer, and molded with a tablet machine to obtain tablets with a total mass of 100 mg per tablet. Furthermore, the tablets were coated using aqueous 10% Opadry yellow (OPADRY 03F42069 YELLOW, Colorcon Japan) solution as a coating solution with a tablet coating machine, thereby obtaining coated tablets with a total mass of 105 mg per tablet.
(2) 10 mg Tablet
Sixty grams of crystal (C) and 192 g of light anhydrous silicic acid (antigelling agent sold under the trade name of AEROSIL (Registered Trademark) 200, Nippon Aerosil) were mixed with 20 L Super Mixer, and then 1200 g of D-mannitol (excipient, Towa-Kasei), 720 g of crystalline cellulose (excipient sold under the trade name of Avicel PH101, Asahi Kasei) and 72 g of hydroxypropylcellulose (binder sold under the trade name of HPC-L, Nippon Soda) were further added and mixed together. Subsequently, a suitable amount of anhydrous ethanol was added to obtain a granulated body containing crystal (C). This granulated body was dried in a rack dryer (60° C.), and then size-regulated using PowerMILL to obtain granules. Together with the granules, 120 g of croscarmellose sodium (disintegrant sold under the trade name of Ac-Di-Sol, FMC International Inc.) and 36 g of sodium stearyl fumarate (lubricant, JRS Pharma LP) were placed and mixed together in a 20 L tumbler mixer, and molded with a tablet machine to obtain tablets with a total mass of 400 mg per tablet. Furthermore, the tablets were coated using aqueous 10% Opadry yellow (OPADRY 03F42069 YELLOW, Colorcon Japan) solution as a coating solution with a tablet coating machine, thereby obtaining coated tablets with a total mass of 411 mg per tablet.
(3) 100 mg Tablet
31.4 g of crystal (C) and 4 g of light anhydrous silicic acid (antigelling agent sold under the trade name of AEROSIL (Registered Trademark) 200, Nippon Aerosil) were mixed with 1 L Super Mixer, and then 40.1 g of anhydrous calcium hydrogen phosphate (excipient, Kyowa Chemical Industry), 10 g of low substituted hydroxypropylcellulose (binder sold under the trade name of L-HPC (LH-21), Shin-Etsu Chemical) and 3 g of hydroxypropylcellulose (binder sold under the trade name of HPC-L, Nippon Soda) were further added and mixed together. Subsequently, a suitable amount of anhydrous ethanol was added to obtain a granulated body containing crystal (C). This granulated body was dried in a rack dryer (60° C.), and then granulated using PowerMILL to obtain granules. Together with the granules, 10 g of croscarmellose sodium (disintegrant sold under the trade name of Ac-Di-Sol, FMC International Inc.) and 1.5 g of sodium stearyl fumarate (lubricant, JRS Pharma LP) were mixed and molded with a tablet machine to obtain tablets with a total mass of 400 mg per tablet.
INDUSTRIAL APPLICABILITY
According to the present invention, there is provided a therapeutic agent and a therapeutic method for undifferentiated gastric cancer comprising a FGFR2 inhibitory substance, use of the FGFR2 inhibitory substance for producing the therapeutic agent, and a FGFR2 inhibitory substance for the therapeutic agent.
In addition, the present invention also provides a pharmaceutical composition comprising a FGFR2 inhibitory substance to be administered to an organism comprising at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2; a therapeutic method for a disease comprising administering an effective dosage of the FGFR2 inhibitory substance to an organism comprising at least one selected from the group consisting of a cell overexpressing FGFR2 and a cell expressing mutant FGFR2; use of a FGFR2 inhibitory substance for producing the pharmaceutical composition; and a FGFR2 inhibitory substance for the pharmaceutical composition.
Moreover, the present invention provides a FGFR2 inhibitor.
The present invention also provides a method for predicting the effect of a FGFR2 inhibitory substance.
More specifically, effect of a FGFR2 inhibitory substance can be predicted by using at least one selected from the group consisting of an expression level of FGFR2 and (the presence or the absence of) FGFR2 mutation in a cell as a biomarker.
Since the predicting method of the invention induces prediction of the effect without administrating the compound to a patient, a patient who is expected to benefit higher effect from the compound can be selected, thereby contributing to the improvement of QOL of the patients.

## Claims

1. A method for treating undifferentiated gastric cancer, characterized by administering, to a patient having undifferentiated gastric cancer, an effective dosage of:
4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide;
or a pharmacologically acceptable salt thereof.

2. A method according to claim 1, wherein methanesulfonate salt form of 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide is administered.

3. A method according to claim 1, wherein the undifferentiated gastric cancer overexpresses FGFR2 or expresses a mutant FGFR2.

4. A method according to claim 1, wherein the undifferentiated gastric cancer is at least one gastric cancer selected from the group consisting of poorly differentiated adenocarcinoma, signet-ring cell carcinoma, mucinous carcinoma and scirrhous gastric cancer.