Nitrogen-containing aromatic heterocyclic compound

The present invention relates to compounds useful as active ingredients in pharmaceutical compositions, particularly in pharmaceutical compositions for the treatment of mutant FGFR3-positive bladder cancer.

The signaling pathway induced by fibroblast growth factors (FGFs) and their receptors, fibroblast growth factor receptors (FGFRs), is one of signaling pathways having the most important functions in the course of development from early embryogenesis to the formation of various organs. There are 18 genes of FGF ligands and four FGFR genes (FGFR1 to FGFR4), which are expressed in various cells and involved in cell growth, differentiation, and survival. In recent years, the importance of FGF signaling in the pathogenesis of diverse tumor types has been reported, and clinical reagents that specifically target the FGFs or FGF receptors are being developed (Nature Reviews Cancer 2010; 10, 116-129, J. Med. Chem. 2011; 54, 7066-7083, AACR 2011, No. 1643 AstraZeneca).

As for FGFR1, it is reported that FGFR1 gene is amplified in lung cancer (in particular, squamous cell cancer) and hormone therapy-resistant breast cancer, and it is also reported that these cell lines exhibit FGFR1-dependent cell growth (Sci. Transl. Med. 2010; 2(62): 62ra93, Breast Cancer Res. 2007; 9(2): R23, Cancer Res. 2010, 70 (5), 2085-2094).

As for FGFR2, the gene amplification in stomach cancer and triple negative breast cancer and the activating mutation in endometrial cancer are reported (Laboratory Investigation 1998, 78(9); 1143-1153, Virchows Arch. 1997, 431; 383-389, J. Cancer Res. Clin. Oncol., 1993, 119, 265-272, AACR 2011, No. 1643 AstraZeneca, Oncogene 2010; 29, 2013-2023). These cancer cells have been also confirmed to exhibit FGFR2-dependent growth.

Further, FGFR3 exhibits activating gene mutation in about 50% of cases of bladder cancer. Bladder cancer is largely divided into three types: non-invasive, invasive, and metastatic types. There have been issues on them that although non-invasive bladder cancer has a high 5-year survival rate of 70% or above, it frequently recurs or partly progresses to invasive cancer, and that invasive or metastatic bladder cancer has a low 5-year survival rate of 50% or below. Current therapies for non-invasive bladder cancer with FGFR3 mutation are transurethral resection of bladder tumor (TUR-BT) and postoperative BCG therapy or intravesical instillation of chemotherapeutic agents. However, their recurrence-preventing effect remains unsatisfactory, and their adverse effects such as hematuria and irritable bladder have been at issue. Meanwhile, total cystectomy and the systemic administration of chemotherapeutic agents have been used for the treatment of invasive or metastatic bladder cancer. However, there are issues on their effectiveness, and adverse effects. Bladder cancer is known to be characterized in that part of the cancer cells sloughs off from bladder tissues into urine, and, based on this characteristic, urine cytology is used for the diagnostic of bladder cancer. It was recently reported that FGFR3 mutation can be detected using the sediments in urine (Biochem. Biophys. Res. Commun. Nov. 3, 2007; 362(4): 865-71). Based on the presence or absence of this FGFR3 mutation, patients with FGFR3 mutation-positive bladder cancer can be selected, and the creation of an FGFR3 selective inhibitor has been demanded.

It is also reported that fusion genes combining FGFR genes and TACC (Transforming Acidic Coiled-coil) genes (FGFR3-TACC3 and FGFR1-TACC1) are expressed in the tumor of some glioblastoma patients (Science, Sep. 7, 2012; 337(6099): 1231-5). According to this report, the forced expression of FGFR3-TACC3 and FGFR1-TACC1 in astrocytes led to transformation and this result showed the oncogenicity of these fusion genes. It was also shown that FGFR3-TACC3 is localized in mitotic spindle poles and induces kinase activity-dependent chromosomal aneuploidy. Further, treatment of FGFR3-TACC3-expressing cells with an FGFR inhibitor suppressed chromosomal aneuploidy, thereby suppressing the growth of the cells. Thus, it is suggested that FGFR inhibitors might be effective for the treatment of glioblastoma patients with FGFR-TACC fusion genes.

It is also reported that human bladder cancer cell lines RT112, RT4, and LUCC2 express FGFR3-TACC3 fusion gene and that human bladder cancer cell line SW780 also expresses FGFR3-BAIAP2L1 fusion gene (Hum Mol Genet., 2013 Feb. 15, 22(4), 795-803). According to this report, the anchorage-independent growth of these fusion genes has been confirmed as a result of their introduction into NIH3T3 cells. Given that the growth of the foregoing bladder cancer cell lines expressing these FGFR3 fusion genes is inhibited by FGFR inhibitors, the detection of the presence of the fusion genes can be useful to select patients who can be treated effectively with FGFR inhibitors.

It is reported that the compounds of formula (A) shown below exhibit inhibition of various kinases and are useful as therapeutic agents for cancer and vascular disorders including myocardial infarction (Patent Document 1). Table 2 of the document discloses the test results of inhibition of kinases Yes, VEGFR, EphB4, PDGFRβ, and FGFR1 by some of the compounds, which discloses that IC 50 values for the FGFR1 inhibitory activity were higher than 1000 nM, showing that the activity was also lower than in the case of inhibition of the activity of the other kinases. Further, in the document, there is no specific disclosure about the compounds of formula (I) of the present invention described below.

(In this formula, each of A is CH, N, or the like; each of B is CH or the like; A 1 is O, CR 2 , or the like; R 0 is H or the like; A 2 is NR, O, or the like; L 1 is a bond, O, or the like; L 2 is a bond, C 1 -C 6 alkyl, or the like; R 1 is a 3- to 6-membered heterocyclic ring or the like; and each of R e and R f is H, C 1 -C 6 alkyl, hydroxyalkyl, or the like. For the other symbols, refer to the publication.)

It is reported that the compounds of formula (B) shown below exhibit Abl inhibitory action and are useful against various cancers (Patent Document 2). However, in the document, there is no specific description about FGFR inhibitory action. Further, the compounds of formula (I) of the present invention described below have group (R 1 ) p which differentiate the compounds in structure from the compounds of formula (B).

(In this formula, G is CH or the like; A is 3-hydroxyphenyl or the like; and Y is vinyl or ethylene. For the other symbols, refer to the publication.)

It is reported that the compounds of formula (C) shown below have inhibitory action on various kinases including Src, VEGFR2, Yes, Fyn, Lck, Abl, PDGFR, EGFR, and RET and are usable for the treatment of cancer, vascular disorders, and the like (Patent Document 3). However, there is no disclosure about FGFR inhibitory action in the document. In the document, there is also no specific disclosure about the compounds of formula (I) of the present invention described below.

(In this formula, G 1 is aryl optionally having a substituent, heteroaryl optionally having a substituent, or the like; L 1 is O, SO, SO 2 , optionally substituted alkyl, or the like; L 2 is optionally substituted alkyl, heterocyclic ring, or the like; A 1 is a bond, O, C(R a ) 2 , or the like; and A 2 is NR a , O, or the like. For the other symbols, refer to the publication.)

It is reported that the compounds of formula (D) shown below have TIE-2 and/or VEGFR-2 kinase inhibitory action and are useful in treatment of angiogenesis-related diseases including cancer (Patent Document 4). However, there is no specific description about FGFR inhibition in the document. Further, the compounds of formula (I) of the present invention described below differ in structure from the compounds of formula (D) in that the compounds of formula (I) have a group L 1 having no amino group and that the compounds also have two bonds positioned para to each other on a ring comprising X and Y.

(In this formula, W is N or CR; R is H or the like. For the other symbols, refer to the publication.)

It is reported that the compounds of formula (E) shown below exhibit inhibitory action on the activity of many receptor protein tyrosine kinases, particularly, FGFRs, and can be used for the treatment of various diseases related to aberrant or excessive activity of these enzymes (Patent Document 5). However, the compounds of formula (I) of the present invention described below differ in structure from the compounds of formula (E) in that the compounds of formula (I) have a group L 1 which does not represent a N atom and that the compounds also have two bonds positioned para to each other on a ring comprising X and Y.

(In this formula, two of X, Y, and Z are N and the third is CH or N. For the other symbols, refer to the publication.)

It is reported that the compounds of formula (F) shown below exhibit inhibitory action on various kinases and are useful against inflammation and autoimmune diseases (Patent Document 6). On the other hand, the compounds of formula (I) of the present invention described below differ in structure from the compounds of formula (F) in that the compounds of formula (I) have a group L 1 which is not amide and that the compounds also have two bonds positioned para to each other on a ring comprising X and Y.

(In this formula, A 1 , A 2 , A 3 , and A 4 are CR 4 , CR 5 , CR 6 , and CR 7 , respectively, or are N; L is —C(O)NR 7 —, —NR 7 C(O)—, or the like. For the other symbols, refer to the publication.)

It is reported that the compounds of formula (G) and those of formula (H) shown below exhibit FGFR inhibitory action and can be used for the treatment of various cancers (Patent Documents 7 and 8).

(In formula (G), ring B represents a 5- or 6-membered aromatic group that may comprise at least one heteroatom selected from O, S, and N. For the other symbols, refer to the publication.)

It is reported that the compounds of formula (J) shown below exhibit glucokinase activating effects and can be used for the treatment of diseases related to diabetes mellitus (Patent Document 9), and the structural feature is substitution with amino at the 2 position of the pyridine.

(For the symbols in this formula, refer to the publication.)

Also, the known compounds having the structures shown below are registered on the database as 1371065-79-0 and 1317903-92-6 in CAS registry number, respectively.

Patent Documents

Patent Document 1: International Publication No. WO 2006/101977

Patent Document 2: International Publication No. WO 2007/056075

Patent Document 3: International Publication No. WO 2008/008234

Patent Document 4: International Publication No. WO 2003/066601

Patent Document 5: International Publication No. WO 2007/071752

Patent Document 6: International Publication No. WO 2007/022380

Patent Document 7: International Publication No. WO 2008/075068

Patent Document 8: International Publication No. WO 2009/153592

Patent Document 9: International Publication No. WO 2009/046784

Technical Problem

The present invention provides compounds useful as active ingredients in pharmaceutical compositions, particularly in pharmaceutical compositions for the treatment of mutant FGFR3-positive bladder cancer.

Solution to Problem

As a result of intensive and extensive studies on compounds having FGFR inhibitory action, the present inventors have found that the nitrogen-containing aromatic heterocyclic compound of the present invention has inhibitory action on FGFR1, FGFR2, and FGFR3, particularly, good inhibitory action on mutant FGFR3. The present invention has been thus accomplished.

More specifically, the present invention relates to a compound of formula (I) or a salt thereof as well as to a pharmaceutical composition comprising a compound of formula (I) or a salt thereof and a pharmaceutically acceptable excipient.

(wherein

X and Y, the same or different from each other, are CH or N, with the proviso that X and Y are not N simultaneously;

L 1 is -lower alkylene-, -lower alkylene-O—, —O-lower alkylene-, or -lower alkynylene-;

Z is N or CH;

R 1 , the same or different from one another, are lower alkyl optionally substituted with halogen, —O-(lower alkyl optionally substituted with halogen), halogen, cyano, or —N(lower alkyl) 2 ;

p is an integer of 2 to 4;

ring W is an optionally substituted aromatic carbocyclic ring, an optionally substituted aromatic heterocyclic ring, or an optionally substituted non-aromatic heterocyclic ring;

Q is -L 2 -R 2 or R 3 ;

L 2 is an optionally substituted aromatic heterocyclic ring or an optionally substituted non-aromatic heterocyclic ring;

R 2 is a non-aromatic heterocyclic group optionally substituted with lower alkyl, optionally substituted cycloalkyl, lower alkyl optionally substituted with one or more groups selected from the group consisting of —OH and —O-lower alkyl, —C(O)—R 0 , —C(O)-optionally substituted cycloalkyl, —NH—R 0 , —N(lower alkyl)-R 0 , -L 3 -optionally substituted non-aromatic heterocyclic group, or H;

R 0 is lower alkyl optionally substituted with —OH;

R 3 is

(1) lower alkyl optionally substituted with one or more groups selected from the group consisting of —C(O)OH, —OH, —O—R 0 , amino optionally substituted with one or two R 0 , carbamoyl optionally substituted with one or two R 0 , an optionally substituted aromatic heterocyclic group, an optionally substituted non-aromatic heterocyclic group, and a —C(O)-optionally substituted non-aromatic heterocyclic group;

(2) —O-(lower alkyl optionally substituted with one or more groups selected from the group consisting of —C(O)OH, —OH, —O—R 0 , carbamoyl optionally substituted with one or two R 0 , an optionally substituted non-aromatic heterocyclic group, and a —C(O)-optionally substituted non-aromatic heterocyclic group);

(3) —NH-(lower alkyl optionally substituted with one or more groups selected from the group consisting of —OH, a non-aromatic heterocyclic group optionally substituted with lower alkyl, and carbamoyl optionally substituted with one or two R 0 );

(4) —N(lower alkyl)-(lower alkyl optionally substituted with one or more groups selected from the group consisting of —OH, a non-aromatic heterocyclic group optionally substituted with lower alkyl, and carbamoyl optionally substituted with one or two R 0 );

(5) —C(O)OH;

(6) —C(O)-optionally substituted non-aromatic heterocyclic group;

(7) —O-(a non-aromatic heterocyclic group optionally substituted with lower alkyl); or

(8) carbamoyl optionally substituted with one or two R 0 ; and

L 3 is a bond, —NH—, —N(lower alkyl)-, or lower alkylene.)

Unless otherwise specified, when symbols used in one chemical formula herein are also used in another chemical formula, the same symbols have identical meanings.

The present invention also relates to a pharmaceutical composition that comprises a compound of formula (I) or a salt thereof and a pharmaceutically acceptable excipient and which is available for the treatment of various cancers related to FGFR1, FGFR2, and/or FGFR3, such as FGFR1-related lung cancer and hormone therapy-resistant breast cancer, FGFR2-related stomach cancer, triple negative breast cancer, and endometrial cancer, and FGFR3-related bladder cancer and glioblastoma. It is to be noted that the pharmaceutical composition includes therapeutic agents for various cancers related to FGFR1, FGFR2, and/or FGFR3. One embodiment is a pharmaceutical composition for the treatment of FGFR3-related bladder cancer, which comprises a compound of formula (I) or a salt thereof and a pharmaceutically acceptable excipient. Another embodiment is a pharmaceutical composition for the treatment of mutant FGFR3-positive bladder cancer, which comprises a compound of formula (I) or a salt thereof and a pharmaceutically acceptable excipient. In the present specification, “mutant” includes point mutation, fusion mutation, deletion mutation and insertion mutation, and in an embodiment, “mutant” means general idea including point mutation and fusion mutation. In another embodiment, “mutant” means point mutation, and in yet another embodiment, “mutant” means fusion mutation.

Further, the present invention relates to: use of a compound of formula (I) or a salt thereof, for the manufacture of a pharmaceutical composition for the treatment of various cancers related to FGFR1, FGFR2, and/or FGFR3; use of a compound of formula (I) or a salt thereof, for the treatment of various cancers related to FGFR1, FGFR2, and/or FGFR3; a compound of formula (I) or a salt thereof, for the treatment of various cancers related to FGFR1, FGFR2, and/or FGFR3; and a method for treating various cancers related to FGFR1, FGFR2, and/or FGFR3, which comprises administering an effective amount of a compound of formula (I) or a salt thereof to a subject. The present invention also relates to: use of a compound of formula (I) or a salt thereof, for the manufacture of a pharmaceutical composition for the treatment of mutant FGFR3-positive bladder cancer; use of a compound of formula (I) or a salt thereof, for the treatment of mutant FGFR3-positive bladder cancer; a compound of formula (I) or a salt thereof, for the treatment of mutant FGFR3-positive bladder cancer; and a method for treating mutant FGFR3-positive bladder cancer, which comprises administering an effective amount of a compound of formula (I) or a salt thereof to a subject. It is to be noted that the “subject” referred to above is a human or another animal in need of the treatment, and is a human in need of the treatment in one embodiment.

Advantageous Effects of Invention

A compound of formula (I) or a salt thereof has inhibitory action on FGFR1, FGFR2, and/or FGFR3, particularly, mutant FGFR3, and can be used as a therapeutic agent for various cancers related to FGFR1, FGFR2, and/or FGFR3, such as lung cancer and hormone therapy-resistant breast cancer, stomach cancer, triple negative breast cancer, endometrial cancer, bladder cancer, and globlastoma, particularly as a therapeutic agent for mutant FGFR3-positive bladder cancer.

The present invention is described in detail below.

As used herein, the term “lower alkyl” refers to linear or branched alkyl having 1 to 8 carbon atoms (hereinafter abbreviated as C 1-8 ) including methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Another embodiment is C 1-4 alkyl, and yet another embodiment is methyl. Yet another embodiment is ethyl.

The term “lower alkylene” refers to linear or branched C 1-8 alkylene including methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, propylene, methylmethylene, ethylethylene, 1,2-dimethylethylene, 1,1,2,2-tetramethylethylene, and the like. Another embodiment is C 1-4 alkylene, and yet another embodiment is methylene. Yet another embodiment is ethylene.

The term “lower alkynylene” refers to linear or branched C 2-6 alkynylene including ethynylene, propynylene, butynylene, pentinylene, hexynylene, 1,3-butadiynylene, 1,3-pentadiynylene, and the like. Another embodiment is C 2-4 alkynylene, and yet another embodiment is ethynylene.

The term “cycloalkyl” refers to a C 3-10 saturated hydrocarbon ring group and it may be bridged. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, and the like. Another embodiment is C 3-8 cycloalkyl, and yet another embodiment is C 3-6 cycloalkyl. Yet another embodiment is cyclopropyl.

The term “aromatic carbocyclic ring” refers to a C 6-14 monocyclic to tricyclic aromatic hydrocarbon ring. Examples include benzene, naphthalene, and anthracene, and another embodiment is benzene.

The term “aromatic heterocyclic ring” refers to a 5- to 10-membered aromatic heterocyclic ring which has 1 to 4 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Examples include pyridine, pyrrole, pyrazine, pyrimidine, pyridazine, imidazole, pyrazole, thiazole, oxazole, isoxazole, thiophene, isothiazole, furan, oxadiazole, thiadiazole, indole, isoindole, indazole, benzofuran, benzothiophene, benzimidazole, benzoxazole, benzothiazole, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, thienopyridine, thienopyrimidine, thienopyrazine, and the like. Another embodiment is pyridine, pyrrole, pyrazine, pyrimidine, pyridazine, imidazole, pyrazole, thiazole, oxazole, thiophene, furan, oxadiazole, and indazole. Yet another embodiment is pyridine, pyrimidine, imidazole, pyrazole, thiazole, and indazole. Yet another embodiment is pyridine, imidazole, and pyrazole. Yet another embodiment is pyridine. Yet another embodiment is pyrazole. Yet another embodiment is imidazole.

The term “aromatic heterocyclic group” refers to a monovalent group of the “aromatic heterocyclic ring” described above. Examples include pyridyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, thienyl, furyl, 1,2,4-oxadiazolyl, and the like. Another embodiment is a 5- or 6-membered aromatic heterocyclic group which has 1 or 2 nitrogen atoms, and yet another embodiment is pyridyl.

The term “non-aromatic heterocyclic ring” refers to a 3- to 10-membered non-aromatic heterocyclic ring (or a 4- to 8-membered non-aromatic heterocyclic ring in one embodiment) having 1 to 4 heteroatoms which are selected from the group consisting of nitrogen, oxygen, and sulfur and which are the same or different. The non-aromatic heterocyclic ring may be fused to a benzene ring or a thiophene ring, be bridged by lower alkylene, be combined with another non-aromatic heterocyclic ring to form a spiro ring, or have an unsaturated bond on part of the own ring. The sulfur atom or nitrogen atom which is a ring-forming atom may be oxidized. Examples include aziridine, oxetane, azetidine, pyrrolidine, piperidine, azepane, diazepane, azocane, piperazine, 4-oxidopiperazine, homopiperazine, morpholine, oxazepane, thiomorpholine, 1,1-dioxidothiomorpholine, 1,1-dioxidotetrahydrothiopyran, 1,1-dioxidothiazolidine, thiazepane, 1-azabicyclo[2,2,2]octane, 7-oxabicyclo[2.2.1]heptane, 2,5-diazabicyclo[2.2.1]heptane, 3-azabicyclo[3.2.1]octane, 8-azabicyclo[3.2.1]octane, 9-azabicyclo[3.3.1]nonane, 3,9-diazabicyclo[3.3.1]nonane, 3,9-diazaspiro[5.5]undecane, 2,6-diazaspiro[3.3]heptane, 2-oxa-6-azaspiro[3.3]heptane, 2-oxa-7-azaspiro[3.5]nonane, tetrahydropyran, tetrahydrofuran, dioxane, dioxolan, tetrahydrothiophene, tetrahydrothiopyran, tetrahydrothienopyridine, tetrahydrobenzoazepine, tetrahydrobenzodiazepine, dihydrobenzofuran, dihydrobenzothiophene, dihydrobenzopyran, dihydrobenzodioxane, benzodioxane, dihydropyran, dihydropyrrole, dihydropyridine, tetrahydropyridine, tetrahydropyrazine, and the like. Another embodiment is a 5- to 7-membered non-aromatic heterocyclic ring having 1 or 2 heteroatoms which are selected from the group consisting of nitrogen, oxygen, and sulfur and which are the same or different. Yet another embodiment is a 5- to 7-membered nitrogen-containing non-aromatic heterocyclic ring which may have at least one nitrogen atom and have one additional heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur. Yet another embodiment is a 6-membered nitrogen-containing non-aromatic heterocyclic ring. Examples include piperazine, piperidine, morpholine, thiomorpholine, 1,1-dioxidothiomorpholine, and the like. Yet another embodiment is oxetane, piperidine, piperazine, morpholine, thiomorpholine, 4-oxidopiperazine, 1,1-dioxidothiomorpholine, tetrahydropyran, tetrahydrofuran, tetrahydrothiophene, tetrahydropyridine, 1-azabicyclo[2.2.2]octane, 8-azabicyclo[3.2.1]octane, 3,9-diazaspiro[5.5]undecane, 2,6-diazaspiro[3.3]heptane, 2-oxa-6-azaspiro[3.3]heptane, or 2-oxa-7-azaspiro[3.5]nonane. Yet another embodiment is morpholine, piperidine, piperazine, 4-oxidopiperazine, 3,9-diazaspiro[5.5]undecane, or 2,6-diazaspiro[3.3]heptane. Yet another embodiment is piperidine. Yet another embodiment is piperazine.

The term “non-aromatic heterocyclic group” refers to a monovalent group of a non-aromatic heterocyclic ring. The non-aromatic heterocyclic group is a 3- to 10-membered non-aromatic heterocyclic group having 1 to 4 heteroatoms which are selected from the group consisting of nitrogen, oxygen, and sulfur and which are the same or different. The non-aromatic heterocyclic group may be bridged by lower alkylene, have an unsaturated bond on part of the ring, or be combined with another non-aromatic heterocyclic ring to form a spiro ring. The sulfur atom or nitrogen atom which is a ring-forming atom may be oxidized. Examples include aziridinyl, azetidinyl, oxetanyl, pyrrolidinyl, piperidinyl, azepanyl, diazepanyl, azocanyl, piperazinyl, homopiperazinyl, morpholinyl, oxazepanyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl, thiazepanyl, tetrahydropyranyl, tetrahydrofuryl, dioxanyl, dioxolanyl, tetrahydrothienyl, tetrahydrothiopyranyl, 7-oxabicyclo[2.2.1]heptyl, 2,5-diazabicyclo[2.2.1]heptyl, 3-azabicyclo[3.2.1]octyl, 8-azabicyclo[3.2.1]octyl, 9-azabicyclo[3.3.1]nonyl, 3,9-diazabicyclo[3.3.1]nonyl, dihydropyranyl, dihydropyrrolyl, dihydropyridyl, tetrahydropyridyl, tetrahydropyrazyl, 9-diazaspiro[5.5]undec-3-yl, 1,9-diazaspiro[5.5]undec-9-yl, 2,8-diazaspiro[4.5]dec-8-yl, 1,4-dioxa-8-azaspiro[4.5]dec-8-yl, and the like. Another embodiment is a 5- to 7-membered non-aromatic heterocyclic group having 1 or 2 heteroatoms which are selected from the group consisting of nitrogen, oxygen, and sulfur and which are the same or different. Yet another embodiment is a 5- to 7-membered non-aromatic heterocyclic group having at least one nitrogen atom. Yet another embodiment is a 6-membered nitrogen-containing non-aromatic heterocyclic group. Examples include piperazinyl, piperidinyl, morpholinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl, and the like. Yet another embodiment is oxetanyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, 4-oxidopiperazinyl, 1,1-dioxidothiomorpholinyl, tetrahydropyranyl, tetrahydrofuryl, tetrahydrothienyl, tetrahydropyridyl, 1-azabicyclo[2.2.2]octyl, 8-azabicyclo[3.2.1]octyl, 3,9-diazaspiro[5.5]undec-3-yl, 2,6-diazaspiro[3.3]hept-2-yl, or 2-oxa-6-azaspiro[3.3]hept-6-yl. Yet another embodiment is piperidinyl or piperazinyl. Yet another embodiment is piperidinyl. Yet another embodiment is piperazinyl.

The term “halogen” refers to —F, —Cl, —Br, or —I. Another embodiment is —F, and yet another embodiment is —Cl.

A compound of formula (I) or a salt thereof, wherein L 1 in formula (I) is -lower alkylene-O—, means a compound of formula (II) or a salt thereof.

(In this formula, L 4 represents lower alkylene. The same applies hereinafter.)

Further, two to four R 1 in (R 1 ) p may be the same or different from one another.

The phrase “optionally substituted” as used herein means “unsubstituted” or “having 1 to 5 substituents”. When a plurality of substituents are contained, these substituents may be the same or different from one another. Further, for example, two R 0 on the nitrogen in the “carbamoyl optionally substituted with one or two R 0 ” may be the same lower alkyl or different lower alkyl from each other. Each R 0 may be substituted with —OH, or alternatively, either one may be substituted or neither one may be substituted.

As referred to herein, a substituent in “an optionally substituted aromatic carbocyclic ring”, “an optionally substituted aromatic heterocyclic ring”, or “an optionally substituted non-aromatic heterocyclic ring” as ring W in formula (I) is, for example, a group shown in group D1 described below.

Group D1 is a group consisting of:

(1) an aromatic heterocyclic group optionally substituted with one or more substituents selected from —OH and lower alkyl; (2) a non-aromatic heterocyclic group optionally substituted with one or more substituents selected from —OH and lower alkyl; (3) halogens; (4) —O-lower alkyl, —S-lower alkyl, —OH, and —SH; (5) —CN and —NO 2 ; (6) —CO 2 H and —CO 2 -lower alkyl; and (7) lower alkyl or —O-lower alkyl, each of which is optionally substituted with one or more groups selected from the group consisting of the groups shown in (1) to (6) above.

Another embodiment of group D1 is a group consisting of:

(1) an aromatic heterocyclic group optionally substituted with —OH; (2) halogens; (3) —OH; (4) —CN; (5) —CO 2 H; and (6) lower alkyl or —O-lower alkyl, each of which is optionally substituted with one or more substituents selected from the group consisting of the substituents shown in (1) to (5) above.

Yet another embodiment of group D1 is a group consisting of:

(1) lower alkyl optionally substituted with halogen; (2) —O-lower alkyl optionally substituted with an aromatic heterocyclic group optionally substituted with —OH; (3) halogens; and (4) —CN

Yet another embodiment of group D1 is a group consisting of lower alkyl optionally substituted with halogen; —O-(lower alkyl optionally substituted with one or more substituents selected from the group consisting of a non-aromatic heterocyclic group optionally substituted with oxo, an aromatic heterocyclic group optionally substituted with —OH, and halogens), halogens, cyano, and oxo.

A substituent acceptable in “an optionally substituted aromatic heterocyclic ring” or “an optionally substituted non-aromatic heterocyclic ring” referred to as L 2 in formula (I), “optionally substituted cycloalkyl” or “an optionally substituted non-aromatic heterocyclic group” referred to as R 2 in formula (I), and “an optionally substituted aromatic heterocyclic group” or “an optionally substituted non-aromatic heterocyclic group” referred to in R 3 in formula (I) is, for example, a substituent selected from group D2.

Group D2 is a group consisting of:

(1) halogens;

(2) —OH and —SH;

(3) —CN; and

(4) lower alkyl optionally substituted with one or more substituents selected from the group consisting of the substituents shown in (1) to (3) above.

Another embodiment of group D2 is a group consisting of:

(1) lower alkyl optionally substituted with —OH; and

(2) —OH

Some embodiments of the compounds of formula (I) or salts thereof are given below.

(1) A compound or a salt thereof, wherein X is N and Y is CH. Another embodiment is a compound or a salt thereof, wherein X is CH and Y is N. Yet another embodiment is a compound or a salt thereof, wherein X is CH and Y is CH.

(2) A compound or a salt thereof, wherein L 1 is lower alkylene or -lower alkylene-O—. Another embodiment is a compound or a salt thereof, wherein L 1 is -lower alkylene-. Yet another embodiment is a compound or a salt thereof, wherein L 1 is -lower alkylene-O—. Yet another embodiment is a compound or a salt thereof, wherein L 1 is ethylene or -methylene-O—. Yet another embodiment is a compound or a salt thereof, wherein L 1 is ethylene. Yet another embodiment is a compound or a salt thereof, wherein L 1 is -methylene-O—. Yet another embodiment is a compound or a salt thereof, wherein L 1 is ethynylene.

(3) A compound or a salt thereof, wherein Z is CH. Another embodiment is a compound or a salt thereof, wherein Z is N.

(4-1) A compound or a salt thereof, wherein p is 2 or 4. Another embodiment is a compound or a salt thereof, wherein p is 2. Yet another embodiment is a compound or a salt thereof, wherein p is 4.

(4-2) A compound or a salt thereof, wherein R 1 , the same or different from one another, are —O-lower alkyl or halogen. Another embodiment is a compound or a salt thereof, wherein R 1 , the same or different from one another, are —O-lower alkyl. Yet another embodiment is a compound or a salt thereof, wherein R 1 , the same or different from one another, are halogen. Yet another embodiment is a compound or a salt thereof, wherein R 1 , the same or different from one another, are —O-methyl or F. Yet another embodiment is a compound or a salt thereof, wherein R′, the same or different from one another, are —O-methyl or Cl. Yet another embodiment is a compound or a salt thereof, wherein all of R 1 are F.

(5) A compound or a salt thereof, wherein the 6-membered aromatic ring in formula (I) which is substituted with (R 1 ) p and which has Z as a ring-forming atom is 2,6-dichloro-3,5-dimethoxyphenyl or 2,6-difluoro-3,5-dimethoxyphenyl. Another embodiment is a compound or a salt thereof, wherein the 6-membered aromatic ring in formula (I) which is substituted with (R 1 ) p and which has Z as a ring-forming atom is 2,6-dichloro-3,5-dimethoxyphenyl. Another embodiment is a compound or a salt thereof, wherein the 6-membered aromatic ring in formula (I) which is substituted with (R 1 ) p and which has Z as a ring-forming atom is 2,6-difluoro-3,5-dimethoxyphenyl.

(6) A compound or a salt thereof, wherein ring W is an aromatic carbocyclic ring optionally substituted with one or more substituents selected from group D1 or is an aromatic heterocyclic ring optionally substituted with one or more substituents selected from group D1. Another embodiment is a compound or a salt thereof, wherein ring W is a benzene ring substituted with one or more substituents selected from group D1 or is pyrazole, pyridine, pyrimidine, thiazole, indazole, or imidazole which in each case is optionally substituted with one or more substituents selected from group D1. Yet another embodiment is a compound or a salt thereof, wherein ring W is a benzene ring optionally substituted with one or more substituents selected from group D1 or is pyrazole optionally substituted with one or more substituents selected from group D1. Yet another embodiment is a compound or a salt thereof, wherein ring W is a benzene ring optionally substituted with one or more substituents selected from group D1. Yet another embodiment is a compound or a salt thereof, wherein ring W is a benzene ring optionally substituted with one or more substituents selected from the group consisting of lower alkyl, —O-lower alkyl, and halogens. Yet another embodiment is a compound or a salt thereof, wherein ring W is a benzene ring optionally substituted with one or more substituents selected from the group consisting of methyl, —O-methyl, and halogens. Yet another embodiment is a compound or a salt thereof, wherein ring W is a benzene ring optionally substituted with —O-methyl. Yet another embodiment is a compound or a salt thereof, wherein ring W is pyrazole optionally substituted with one or more substituents selected from group D1. Yet another embodiment is a compound or a salt thereof, wherein ring W is pyrazole optionally substituted with lower alkyl. Yet another embodiment is a compound or a salt thereof, wherein ring W is pyrazole optionally substituted with methyl. Yet another embodiment is a compound or a salt thereof, wherein ring W is pyrazole substituted with methyl. Yet another embodiment is a compound or a salt thereof, wherein ring W is pyrazole.

(7) A compound or a salt thereof, wherein Q is -L 2 -R 2 . Another embodiment is a compound or a salt thereof, wherein Q is R 3 .

(8) A compound or a salt thereof, wherein L 2 is a non-aromatic heterocyclic ring optionally substituted with one or more substituents selected from group D2. Another embodiment is a compound or a salt thereof, wherein L 2 is a nitrogen-containing non-aromatic heterocyclic ring optionally substituted with one or more substituents selected from group D2. Yet another embodiment is a compound or a salt thereof, wherein L 2 is piperazine, 4-oxidopiperazine, piperidine, morpholine, azetidine, 3,9-diazaspiro[5.5]undecane, 2,6-diazaspiro[3.3]heptane, 2-oxa-6-azaspiro[3.3]heptane, 2-oxa-7-azaspiro[3.5]nonane, 8-azabicyclo[3.2.1]octane, or 1-azabicyclo[2.2.2]octane which in each case is optionally substituted with one or more substituents selected from group D2. Yet another embodiment is a compound or a salt thereof, wherein L 2 is piperazine optionally substituted with one or more methyl, piperidine optionally substituted with one or more methyl, or 3,9-diazaspiro[5.5]undecane. Yet another embodiment is a compound or a salt thereof, wherein L 2 is piperidine or 4-methylpyperazine.

(9) A compound or a salt thereof, wherein R 2 is lower alkyl optionally substituted with one or more groups selected from the group consisting of —OH and —O-lower alkyl, —NH-(lower alkyl optionally substituted with —OH), a non-aromatic heterocyclic group optionally substituted with one or more substituents selected from group D2, -lower alkylene-(a non-aromatic heterocyclic group optionally substituted with one or more substituents selected from the group D2), or H. Another embodiment is a compound or a salt thereof, wherein R 2 is lower alkyl optionally substituted with one or more groups selected from the group consisting of —OH and —O-lower alkyl, —NH-(lower alkyl optionally substituted with —OH), a non-aromatic heterocyclic group optionally substituted with lower alkyl (the lower alkyl is optionally substituted with —OH), or H. Yet another embodiment is a compound or a salt thereof, wherein R 2 is piperazine optionally substituted with methyl, piperidine optionally substituted with methyl, 2-hydroxyethylamino, or H. Yet another embodiment is a compound or a salt thereof, wherein R 2 is 4-methylpiperazine, 2-hydroxyethylamino, or H. Yet another embodiment is a compound or a salt thereof, wherein R 2 is 4-methylpiperazine. Yet another embodiment is a compound or a salt thereof, wherein R 2 is 2-hydroxyethylamino. Yet another embodiment is a compound or a salt thereof, wherein R 2 is H.

(10) A compound or a salt thereof, wherein R 3 is lower alkyl optionally substituted with one or more groups selected from the group consisting of —C(O)OH, carbamoyl optionally substituted with one or two R 0 , —OH, a non-aromatic heterocyclic group optionally substituted with one or more substituents selected from group D2, and —C(O)-(a non-aromatic heterocyclic group optionally substituted with one or more substituents selected from group D2) or wherein R 3 is —O-(lower alkyl optionally substituted with one or more groups selected from the group consisting of —C(O)OH, carbamoyl optionally substituted with one or two R 0 , —OH, a non-aromatic heterocyclic group optionally substituted with one or more substituents selected from group D2, and —C(O)-(a non-aromatic heterocyclic group optionally substituted with one or more substituents selected from group D2)). Another embodiment is a compound or a salt thereof, wherein R 3 is lower alkyl substituted with one or more groups selected from the group consisting of —C(O)OH, carbamoyl optionally substituted with one or two R 0 , —OH, a non-aromatic heterocyclic group optionally substituted with one or more substituents selected from group D2, and —C(O)-(a non-aromatic heterocylic group optionally substituted with one or more substituents selected from group D2). Yet another embodiment is a compound or a salt thereof, wherein R 3 is lower alkyl substituted with one or more substituents selected from the group consisting of —OH, a non-aromatic heterocyclic group optionally substituted with one or more substituents selected from —OH and lower alkyl, and —C(O)-(a non-aromatic heterocyclic group optionally substituted with one or more substituents selected from the group consisting of —OH and lower alkyl). Yet another embodiment is a compound or a salt thereof, wherein R 3 is lower alkyl substituted with one or more substituents selected from the group consisting of —OH, a non-aromatic heterocyclic group optionally substituted with lower alkyl, and —C(O)-(a non-aromatic heterocyclic group optionally substituted with —OH). Yet another embodiment is a compound or a salt thereof, wherein R 3 is lower alkyl substituted with one or more groups selected from the group consisting of —OH, piperazinyl optionally substituted with methyl, and —C(O)-(azetidinyl optionally substituted with —OH). Yet another embodiment is a compound or a salt thereof, wherein R 3 is 2-hydroxyethyl, 2,3-dihydroxypropyl, or 4-methylpiperazin-1-ylmethyl. Yet another embodiment is a compound or a salt thereof, wherein R 3 is 4-methylpiperazin-1-ylmethyl. Yet another embodiment is a compound or a salt thereof, wherein R 3 is lower alkyl optionally substituted with one or more —OH. Yet another embodiment is a compound or a salt thereof, wherein R 3 is 2-hydroxyethyl or 2,3-dihydroxypropyl.

(11) A compound or a salt thereof, which is a consistent combination of any two or more of the embodiments described in (1) to (10) above.

The present invention encompasses a compound or a salt thereof, which is a combination of any two or more of the embodiments described in (1) to (10) above, as described in (11) above. Specific examples include the embodiments described below.

(12) A compound or a salt thereof, wherein X is N; Y is CH; and L 1 is lower alkylene or -lower alkylene-O—.

(13) The compound according to (12) or a salt thereof, wherein Z is CH; R 1 , the same or different from one another, are —O-lower alkyl or halogen; p is 2 or 4; ring W is an optionally substituted aromatic carbocyclic ring or an optionally substituted aromatic heterocyclic ring.

(14) The compound according to (13) or a salt thereof, wherein L 1 is ethylene or -methylene-O—; p is 4; ring W is an optionally substituted benzene ring or optionally substituted pyrazole.

(15) The compound according to any one of (12) to (14) or a salt thereof, wherein Q is -L 2 -R 2 ; L 2 is an optionally substituted non-aromatic heterocyclic ring; R 2 is lower alkyl optionally substituted with one or more groups selected from the group consisting of —OH and —O-lower alkyl, —NH-(lower alkyl optionally substituted with —OH), an optionally substituted non-aromatic heterocyclic group, -lower alkylene-(an optionally substituted non-aromatic heterocyclic group), or H.

(16) The compound according to (15) or a salt thereof, wherein p is 4; L 2 is piperazine optionally substituted with one or more methyl, piperidine optionally substituted with one or more methyl, or 3,9-diazaspiro[5.5]undecane; R 2 is piperazine optionally substituted with methyl, piperidine optionally substituted with methyl, 2-hydroxyethylamino, or H.

(17) The compound according to (16) or a salt thereof, wherein R 1 , the same or different from one another, are —O-methyl or F; L 1 is -methylene-O—; ring W is a benzene ring optionally substituted with —O-methyl; L 2 is piperidine or 4-methylpiperazine; R 2 is 4-methylpiperazine, 2-hydroxyethylamino, or H.

(18) The compound according to any one of (12) to (14) or a salt thereof, wherein ring W is optionally substituted pyrazole; Q is R 3 ; R 3 is lower alkyl substituted with one or more groups selected from the group consisting of —C(O)OH, carbamoyl optionally substituted with one or two R 0 , —OH, an optionally substituted non-aromatic heterocyclic group, and —C(O)-(an optionally substituted non-aromatic heterocyclic group).

(19) The compound according to (18) or a salt thereof, wherein p is 4 and R 3 is lower alkyl substituted with one or more substituents selected from the group consisting of —OH, a non-aromatic heterocyclic group optionally substituted with lower alkyl, and —C(O)-(a non-aromatic heterocyclic group optionally substituted with —OH).

(20) The compound according to (19) or a salt thereof, wherein R 1 , the same or different from one another, are —O-methyl or F; L 1 is -methylene-O—; ring W is pyrazole optionally substituted with methyl; R 3 is 2-hydroxyethyl, 2,3-dihydroxypropyl, or 4-methylpiperazin-1-ylmethyl.

Another embodiment of the compound of formula (I) or salt thereof is, for example, a compound or a salt thereof, wherein

X and Y, the same or different from each other, are CH or N, with the proviso that X and Y are not N simultaneously;

L 1 is -lower alkylene-, -lower alkylene-O—, —O-lower alkylene-, or lower alkynylene; Z is N or CH;

R 1 , the same or different from one another, are lower alkyl optionally substituted with halogen, —O-(lower alkyl optionally substituted with halogen), halogen, cyano, or —N(lower alkyl) 2 ;

p is an integer of 2 to 4;

ring W is an optionally substituted aromatic carbocyclic ring, an optionally substituted aromatic heterocyclic ring, or an optionally substituted non-aromatic heterocyclic ring;

Q is -L 2 -R 2 or R 3 ;

L 2 is an optionally substituted aromatic heterocyclic ring or an optionally substituted non-aromatic heterocyclic ring;

R 2 is lower alkyl optionally substituted with one or more groups selected from the group consisting of —OH and —O-lower alkyl, —C(O)-optionally substituted cycloalkyl, —NH-(lower alkyl optionally substituted with —OH), an -L 3 -optionally substituted non-aromatic heterocyclic group, or H;

R 3 is lower alkyl optionally substituted with one or more groups selected from the group consisting of —C(O)OH, —OH, —NH-lower alkyl, —N(lower alkyl) 2 , —C(O)—NH-lower alkyl, —C(O)—N(lower alkyl) 2 , an optionally substituted aromatic heterocyclic group, an optionally substituted non-aromatic heterocyclic group, and a —C(O)-optionally substituted non-aromatic heterocyclic group, —O-(lower alkyl optionally substituted with one or more groups selected from the group consisting of —OH, —C(O)—NH-lower alkyl, and —C(O)—N(lower alkyl) 2 ), —NH-(lower alkyl optionally substituted with one or more groups selected from the group consisting of —OH, —C(O)—NH-lower alkyl, and —C(O)—N(lower alkyl) 2 ), —N(lower alkyl)-(lower alkyl optionally substituted with one or more groups selected from the group consisting of —OH, —C(O)—NH-lower alkyl, and —C(O)—N(lower alkyl) 2 ), —C(O)OH, or a —C(O)-optionally substituted non-aromatic heterocyclic group; and L 3 is a bond or lower alkylene.

Examples of specific compounds falling within the scope of the compound of formula (I) or a salt thereof include the following compounds:

5-[2-(2,6-difluoro-3,5-dimethoxyphenyl)ethyl]-N-[3-methoxy-4-(4-methylpiperazin-1-yl)phenyl]pyrimidin-2-amine, (2S)-3-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]propane-1,2-diol, 5-[2-(2,6-difluoro-3,5-dimethoxyphenyl)ethyl]-N-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine, 5-[2-(2,6-difluoro-3,5-dimethoxyphenyl)ethyl]-N-{3-fluoro-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine, 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine, 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-[3-methoxy-4-(4-methylpiperazin-1-yl)phenyl]pyrimidin-2-amine, 5-[(2,6-dichloro-3,5-dimethoxybenzyl)oxy]-N-[1-(1-methylpiperidin-4-yl)-1H-pyrazol-4-yl]pyrimidin-2-amine, 5-[(2,6-dichloro-3,5-dimethoxybenzyl)oxy]-N-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]pyrimidin-2-amine, 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-{3-methoxy-4-[4-(1-methylpiperidin-4-yl)piperazin-1-yl]phenyl}pyrimidin-2-amine, 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-{3-methyl-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine, 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-{4-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-3-methoxyphenyl}pyrimidin-2-amine, N-[4-(3,9-diazaspiro[5.5]undec-3-yl)-3-methoxyphenyl]-5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-amine, 2-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]ethanol, 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-{1-[2-(4-methylpiperazin-1-yl)ethyl]-1H-pyrazol-4-yl}pyrimidin-2-amine, 2-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]-1-(3-hydroxyazetidin-1-yl)ethanone, (2R)-3-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]propane-1,2-diol, 2-({1-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)phenyl]piperidin-4-yl}amino)ethanol, 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-{1-methyl-5-[(4-methylpiperazin-1-yl)methyl]-1H-pyrazol-3-yl}pyrimidin-2-amine, and 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-[4-(4-methylpiperazin-1-yl)phenyl]pyrimidin-2-amine, and salts thereof.

In another embodiment, examples of specific compounds falling within the scope of the compound of formula (I) or a salt thereof include the following compounds:

5-[2-(2,6-difluoro-3,5-dimethoxyphenyl)ethyl]-N-[3-methoxy-4-(4-methylpiperazin-1-yl)phenyl]pyrimidin-2-amine, (2S)-3-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]propane-1,2-diol, 5-[2-(2,6-difluoro-3,5-dimethoxyphenyl)ethyl]-N-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine, 5-[2-(2,6-difluoro-3,5-dimethoxyphenyl)ethyl]-N-{3-fluoro-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine, 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine, 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-[3-methoxy-4-(4-methylpiperazin-1-yl)phenyl]pyrimidin-2-amine, 5-[(2,6-dichloro-3,5-dimethoxybenzyl)oxy]-N-[1-(1-methylpiperidin-4-yl)-1H-pyrazol-4-yl]pyrimidin-2-amine, 5-[(2,6-dichloro-3,5-dimethoxybenzyl)oxy]-N-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]pyrimidin-2-amine, 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-{3-methoxy-4-[4-(1-methylpiperidin-4-yl)piperazin-1-yl]phenyl}pyrimidin-2-amine, 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-{3-methyl-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine, 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-{4-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-3-methoxyphenyl}pyrimidin-2-amine, N-[4-(3,9-diazaspiro[5.5]undec-3-yl)-3-methoxyphenyl]-5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-amine, 2-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]ethanol, 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-{1-[2-(4-meth ylpiperazin-1-yl)ethyl]-1H-pyrazol-4-yl}pyrimidin-2-amine, 2-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]-1-(3-hydroxyazetidin-1-yl)ethanone, and (2R)-3-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]propane-1,2-diol, and salts thereof.

In yet another embodiment, examples of specific compounds falling within the scope of the compound of formula (I) or a salt thereof include the following compounds:

5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine, 2-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]ethanol, (2R)-3-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]propane-1,2-diol, 2-({1-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)phenyl]piperidin-4-yl}amino)ethanol, 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-{1-methyl-5-[(4-methylpiperazin-1-yl)methyl]-1H-pyrazol-3-yl}pyrimidin-2-amine, and 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-[4-(4-methylpiperazin-1-yl)phenyl]pyrimidin-2-amine, and salts thereof.

In yet another embodiment, examples of specific compounds falling within the scope of the compound of formula (I) or a salt thereof include the following compounds:

5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine, 2-[4-({15-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]ethanol, and (2R)-3-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]propane-1,2-diol, and salts thereof.

In yet another embodiment, examples of specific compounds falling within the scope of the compound of formula (I) or a salt thereof include the following compounds:

2-({1-[4-({5-[2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)phenyl]piperidin-4-yl}amino)ethanol, 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-{1-methyl-5-[(4-methylpiperazin-1-yl)methyl]-1H-pyrazol-3-yl}pyrimidin-2-amine, and 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-[4-(4-methylpiperazin-1-yl)phenyl]pyrimidin-2-amine, and salts thereof.

The compounds of formula (I) may have tautomers and/or geometrical isomers, depending on the type of substituents. Even when the compound of formula (I) appear herein only in one isomer form, the present invention encompasses the other isomers, and also encompasses separated isomers or mixtures thereof.

Further, since some compounds of formula (I) have an asymmetric carbon atom or axial asymmetry, optical isomers based on this asymmetry may also exist. The present invention also encompasses separated optical isomers of the compounds of formula (I) or mixtures thereof.

Furthermore, the present invention encompasses pharmaceutically acceptable prodrugs of the compounds represented by formula (I). The term “pharmaceutically acceptable prodrug” refers to a compound having a group that can be converted into an amino group, a hydroxyl group, a carboxyl group, or the like by solvolysis or under physiological conditions. Examples of a prodrug-forming group include those described in Prog. Med., 5, 2157-2161 (1985) and those described in “Development of Pharmaceuticals” (Hirokawa Publishing, 1990) vol. 7, Molecular Design, 163-198.

Likewise, salts of the compounds of formula (I) are pharmaceutically acceptable salts of the compounds of formula (I). The compounds of formula (I) may form acid addition salts or salts with bases, depending on the type of substituents. Specific examples include acid addition salts with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid) or with organic acids (e.g., formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, mandelic acid, tartaric acid, dibenzoyltartaric acid, ditoluoyltartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, aspartic acid, glutamic acid), salts with inorganic bases (e.g., sodium, potassium, magnesium, calcium, aluminum) or with organic bases (e.g., methylamine, ethylamine, ethanolamine, lysine, ornithine), salts with various amino acids and amino acid derivatives (e.g., acetylleucine), ammonium salts, and the like.

Moreover, the present invention encompasses the compounds of formula (I) and salts thereof in the form of various hydrates, solvates, and crystalline polymorphic substances. The present invention also encompasses the compounds labeled with various radioactive or non-radioactive isotopes.

(Preparation Processes)

The compounds of formula (I) and salts thereof can be prepared by applying various known synthesis methods on the basis of characteristics derived from their basic structure or the type of their substituents. In some cases, depending on the type of functional group, it is technically effective to replace such a functional group with an appropriate protecting group (a group that can be easily converted into the original functional group) between the starting material stage and the intermediate stage. Examples of the protecting group include those described in Wuts (P. G. M. Wuts) and Greene (T. W. Greene), “Greene's Protective Groups in Organic Synthesis (fourth edition, 2006)”, and the like, which may be selected and used as appropriate, depending on reaction conditions. In such a method, after introduction of the protecting group and subsequent reaction, the protecting group may be removed, if needed, to obtain a desired compound.

Likewise, a prodrug of the compound of formula (I) can be prepared by introducing a specific group between the starting material stage and the intermediate stage, as in the case of the above protecting group, or by subjecting the obtained compound of formula (I) to further reaction. The reaction may be accomplished by applying esterification, amidation, dehydration, or the like, which is a method that is common and known to those skilled in the art.

Described below are typical processes for preparing the compounds of formula (I). Each process may also be accomplished by reference to the documents cited in this description. It should be noted that the preparation processes of the present invention are not limited to the examples illustrated below.

(Preparation Process 1)

(In this formula, L 5 represents halogen, methylsulfinyl, or methylsulfonyl. The same applies hereinafter.)

The compound (I) of the present invention can be obtained by coupling reaction of compound (1a) and compound (2a).

In this reaction, compounds (1a) and (2a) are used in equal amounts or one of them is used in an excessive amount. A mixture of these compounds is stirred in the presence of a predetermined catalyst, in a solvent inert to the reaction or in the absence of a solvent, generally for 0.1 hour to 5 days under conditions between room temperature and heating to reflux. This reaction is preferably performed under an inert gas atmosphere. Examples of the solvent used in this process include, but are not particularly limited to, aromatic hydrocarbons (e.g., benzene, toluene, xylene), ethers (e.g., diethyl ether, tetrahydrofuran, dioxane, dimethoxyethane), halogenated hydrocarbons (e.g., dichloromethane, 1,2-dichloroethane, chloroform), N-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, ethyl acetate, acetonitrile, tert-butanol, and mixtures thereof. Examples of the predetermined catalyst include palladium acetate, tris(dibenzylideneacetone)dipalladium, and the like. Further, when a palladium catalyst is used, a ligand used for the catalyst may be triphenylphosphine, 1,1′-binaphthalene-2,2′-diylbis(diphenylphosphine), 2-(dicyclohexylphosphino)-2′,4′,6′-triisopropyl-1,1′-biphenyl, or 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene. The reaction may be performed in the presence of an organic base (e.g., triethylamine, N,N-diisopropylethylamine, or N-methylmorpholine) or an inorganic base (e.g., sodium tert-butoxide, potassium carbonate, sodium carbonate, cesium carbonate, or potassium hydroxide), because it is advantageous for smooth reaction in some cases. Heating the reaction mixture by microwave irradiation is advantageous for smooth reaction in some cases.

S. R. Sandler and W. Karo, “Organic Functional Group Preparations”, second edition, vol. 1, Academic Press Inc., 1991

The Chemical Society of Japan (ed.), “The Fifth Series of Experimental Chemistry”, vol. 14, MARUZEN Co., Ltd., 2005

(Preparation Process 2)

(In this formula, L 6 represents lower alkynylene. The same applies hereinafter.)

(Step 1)

This process is intended to prepare compound (I-1) of the present invention by Sonogashira coupling reaction of compound (1b) and a terminal alkyne derivative.

In this process, compound (1b) and a terminal alkyne derivative are used in equal amounts or one of them is used in an excessive amount. A mixture of these is stirred in the presence of a base, a palladium catalyst, and copper iodide, in a solvent inert to the reaction, generally for 0.1 hour to 5 days under conditions between room temperature and heating to reflux. This reaction is preferably performed under an inert gas atmosphere. Examples of the solvent used in this process include, but are not particularly limited to, aromatic hydrocarbons (e.g., benzene, toluene, xylene), ethers (e.g., diethyl ether, tetrahydrofuran, dioxane, dimethoxyethane), halogenated hydrocarbons (e.g., dichloromethane, 1,2-dichloroethane, or chloroform), alcohols (e.g., methanol, ethanol, 2-propanol, butanol), N,N-dimethylformamide, dimethyl sulfoxide, and mixtures thereof. The base is preferably an organic base (e.g., triethylamine, N,N-diisopropylethylamine, or N-methylmorpholine) or an inorganic base (e.g., potassium carbonate, sodium carbonate, cesium carbonate, or potassium hydroxide). The palladium catalyst is preferably tetrakis(triphenylphosphine)palladium, dichlorobis(triphenylphosphine)palladium, palladium chloride-1,1′-bis(diphenylphosphino)ferrocene, or the like. Heating the reaction mixture by microwave irradiation is advantageous for smooth reaction in some cases.

A. d. Meijere and F. Diederich (ed.), “Metal-Catalyzed Cross-Coupling Reactions”, first edition, VCH Publishers Inc., 1997

The Chemical Society of Japan (ed.), “The Fifth Series of Experimental Chemistry”, vol. 13, MARUZEN Co., Ltd., 2005

(Step 2)

This process is intended to prepare compound (I-2) of the present invention by reducing the alkyne moiety of compound (I-1) of the present invention to alkylene by hydrogenation or diimide reduction.

In this process, compound (I-1) of the present invention and palladium carbon are used in equal amounts or one of them is used in an excessive amount. A mixture of these is stirred in a solvent inert to the reaction, under a hydrogen atmosphere, generally for 0.1 hour to 5 days under conditions between room temperature and heating to reflux. Examples of the solvent used in this process include, but are not particularly limited to, ethers (e.g., diethyl ether, tetrahydrofuran, dioxane, dimethoxyethane), alcohols (e.g., methanol, ethanol, 2-propanol, butanol), and mixtures thereof.

Other than the hydrogenation reaction, compound (I-1) of the present invention and predetermined diimide are used in equal amounts or one of them is used in an excessive amount. A mixture of these is stirred in a solvent inert to the reaction, generally for 0.1 hour to 5 days under conditions between room temperature and heating to reflux. Examples of the solvent used in this process are the same as shown above. The predetermined diimide is, for example, 4-methylbenzenesulfonyl hydrazide.

The substituent(s) on ring W in the compound of formula (I) can be easily converted into other functional groups by the reaction described below in the Examples, reaction obvious to those skilled in the art, or a modified process thereof, using a compound of formula (I) as a starting material. For example, the conversion can be achieved by combining any processes that can be applied generally by those skilled in the art, such as reduction, halogenation, deprotection, hydrolysis, amidation, amination, oxidation, reductive amination, acylation, O-alkylation, N-alkylation, reductive alkylation, and epoxidation.

(Preparation of Starting Compound)

The starting compound used in the preparation process described above can be prepared, for example, by a process described below, the process in the Preparation Examples described later, a known process, or a modified process thereof.

(Starting Material Synthesis 1)

(In this formula, R 4 represents —OH or -lower alkylene-OH; L 7 represents halogen, —OH, -lower alkylene-OH, -lower alkylene-OMs, -lower alkylene-OTs, -lower alkylene-OTf, or -lower alkylene-halogen; L 8 represents -lower alkylene-O— or —O-lower alkylene-. The same applies hereinafter.)

This preparation process is intended to prepare compound (3c) which is starting compound (1a) of the Preparation Process 1 wherein L 1 is —O-lower alkylene- or -lower alkylene-O—.

In the case of compound (3a) wherein L 7 is halogen, -lower alkylene-OMs, -lower alkylene-OTs, -lower alkylene-OTf, or -lower alkylene-halogen, compounds (3a) and (3b) are used in equal amounts or one of them is used in an excessive amount. A mixture of these is stirred in the presence of a base in a solvent inert to the reaction, generally for 0.1 hour to 5 days under conditions between room temperature and heating to reflux. Examples of the solvent used in this process include, but are not particularly limited to, N-methylpyrrolidone, N,N-dimethylformamide, dimethyl sulfoxide, and the like. The base is preferably an inorganic base such as potassium carbonate, sodium carbonate, cesium carbonate, or potassium hydroxide.

In the case of compound (3a) wherein L 7 is —OH or -lower alkylene-OH, compounds (3a) and (3b) are used in equal amounts or one of them is used in an excessive amount. A mixture of these is stirred in the presence of a predetermined phosphine reagent and a predetermined condensing agent in a solvent inert to the reaction, generally for 0.1 hour to 5 days under conditions between room temperature and heating to reflux. Examples of the solvent used in this process include, but are not particularly limited to, ethers such as diethyl ether, tetrahydrofuran, dioxane, and dimethoxyethane. Examples of the predetermined phosphine reagent include tributylphosphine, triphenylphosphine, and the like. Examples of the predetermined condensing agent include diethyl azodicarboxylate, 1,1′-(azodicarbonyl)dipiperidine, and the like. Use of (cyanomethylene)trimethylphosphorane, instead of the predetermined phosphine and the predetermined condensing agent, is advantageous for smooth reaction in some cases.

(Starting Material Synthesis 2)

(In this formula, L 9 represents halogen. The same applies hereinafter.)

This preparation process is intended to prepare compound (4d) which is starting compound (1a) of the Preparation Process 1 wherein L 1 is lower alkylene.

(Step 1)

This process is intended to prepare compound (4b) by Sonogashira coupling reaction of compound (4a) and a terminal alkyne derivative.

The reaction conditions are the same as in Step 1 of the Preparation Process 2.

(Step 2)

This process is intended to prepare compound (4c) by reducing the alkyne moiety of compound (4b) to lower alkylene by hydrogenation.

The reaction conditions are the same as in Step 2 of the Preparation Process 2.

(Step 3)

This process is intended to prepare compound (4d) by converting the amino group of compound (4c) into halogen.

In this process, compound (4c) and a combination of copper chloride (II) and n-pentyl nitrite are used in equal amounts or one of them is used in an excessive amount. A mixture of these is stirred in a solvent inert to the reaction, generally for 0.1 hour to 5 days under conditions between ice cooling and heating to reflux. Examples of the solvent used in this process include, but are not particularly limited to, halogenated hydrocarbons such as dichloromethane, 1,2-dichloroethane, and chloroform.

(Starting Material Synthesis 3)

This preparation process is intended to prepare compound (5c) which is starting compound (1b) of the Preparation Process 2 wherein X is N.

This reaction is intended to prepare compound (5c) by ipso-substitution reaction of compounds (5a) and (5b).

Compounds (5a) and (5b) are used in equal amounts or one of them is used in an excessive amount. A mixture of these is stirred in a solvent inert to the reaction under a hydrogen atmosphere, generally for 0.1 hour to 5 days under conditions between ice cooling and heating to reflux. Examples of the solvent used in this process include, but are not particularly limited to, alcohols (e.g., methanol, ethanol, 2-propanol, butanol), N-methylpyrrolidone, N,N-dimethylformamide, dimethyl sulfoxide, and mixtures thereof. Use of an acid such as methanesulfonic acid, acetic acid, trifluoroacetic acid, hydrogen chloride, or sulfuric acid is advantageous for smooth reaction in some cases.

The pharmacological activity of the compounds of formula (I) was confirmed in the tests described below.

Test Example 1

FGFR1, FGFR2, and FGFR3 Enzyme Assay

In the enzyme assay, human recombinant FGFR1, FGFR2, and FGFR3 (Cama Biosciences; Catalog Nos. 08-133, 08-134, and 08-135) were used, and reactions were performed at room temperature (FGFR1 and FGFR2) or 30° C. (FGFR3). The measurement method is outlined below.

The compound was diluted with a solution of dimethyl sulfoxide (DMSO) (10-fold common ratio, 4 portions) before dilution with a reaction buffer (100 mM HEPES (pH7.5), 0.003% Brij-35, 0.004% Tween 20, 0.5 mM DTT, and 10 mM MgCl 2 ) so that the final DMSO concentration was 2%. To 4 μL of the compound solution in a 384-well plate, 2 μL each of FGFR1 enzyme (2 or 3 ng/μL), FGFR2 enzyme (2 ng/μL), or FGFR3 enzyme (6 ng/μL) which were diluted with the reaction buffer was added. In 20 minutes, 4 μL of a substrate-ATP solution (100 mM HEPES (pH7.5), 0.003% Brij-35, 0.004% Tween 20, 0.5 mM DTT, 10 mM MgCl 2 , 3.75 μM substrate-FL-peptide 22+ 500 μM (FGFR1) ATP, 188 μM (FGFR2) ATP, or 250 μM (FGFR3) ATP) was added before subsequent 30-minute reaction. After the reaction was stopped, the reaction mixture was measured with a LabChip EZ Reader. The IC 50 values were calculated by non-linear regression based on the inhibition rates obtained. The results of some compounds are shown in Table 1. The term “Ex” in the table denotes compound No. in the Examples described later.

Test Example 2

Growth Assay of Cells with Forced Expression of Mutant FGFR3 (FGFR3_S249C/NIH3T3)

FGFR3_S249C/NIH3T3 cells were added to a 96-well spheroid plate (U bottom) at a concentration of 3000 cells/well/90 μL, and the compound solution (10 μL) was added thereto on the next day (final DMSO concentration: 0.1%). The compound solution was prepared by serially diluting the compound with DMSO at a 3-fold common ratio (9 portions and DMSO only) from the maximum concentration of 10 mM and then diluted 100-fold with a culture medium (D-MEM, 10% FBS). 5 days after the addition of the compound, the growth inhibition caused by the compound was evaluated by Promega (G7573) CellTiter-Glo™ Luminescent Cell Viability Assay. The IC 50 value was calculated by non-linear regression, using DMSO-added wells as control and assuming count 0 to be 100% inhibition. The results of some compounds are shown in Table 2.

Test Example 3

Antitumor Test on UM-UC-14 (FGFR3_S249C-Positive Cells, Bladder Cancer)

3×10 6 UM-UC-14 cells per 0.1 mL (PBS+matrigel, 1:1) were inoculated subcutaneously into the right flank of nude mice (CAnN, Cg-Foxn1nu/CrlCrlj (nu/nu), male, 4- to 5-week-old), and when their tumor size reached about 250 mm 3 , drug administration was started (Day 1). The drug was administered once a day and the tumor size was measured with a caliper and the body weight was also measured every two or three days. The antitumor effect was finally determined based on the tumor volume (mm 3 ; minor axis (mm)×minor axis (mm)×major axis (mm)/2) at Day 11 (n=3-5). To the control group, 0.5% MC (methyl cellulose) was administered. For “% inhibition” in the table, for example, 100% inhibition indicates that the tumor growth of the control was inhibited to the level of the tumor volume at Day 1. “% regression” indicates what percentage of regression could be achieved compared with the tumor volume at Day 1. Here, the tumor volume at Day 1 means tumor volume immediately before drug administration. The results of some compounds administered orally (1 mg/kg/day for other than Ex 95 and 3 mg/kg/day for Ex 95) are shown in Table 3.

The test described above confirmed that the plural compounds of the Examples included in formula (I) of the present invention had inhibitory action on FGFR1, FGFR 2, and/or FGFR3. It was also confirmed that the plural compounds of the Examples included in formula (I) inhibited the growth of the cells with forced expression of mutant FGFR3 and that the compounds also inhibited the growth of bladder cancer or made bladder cancer itself regress, in the animal model bearing mutant FGFR3-positive bladder cancer. In light of the foregoing, the compound of formula (I) or a salt thereof can be used as a therapeutic agent for various cancers related to FGFR1, FGFR 2, and/or FGFR3, particularly, mutant FGFR3-positive bladder cancer.

Test Example 4

Isolation of FGFR3-TACC3_v1

cDNA was synthesized by reverse transcription reaction in 200 clinical specimens of lung cancer (Asterand plc.; US) using reverse transciptase (SuperScriptIII, Life Technologies, Corp.) and random primers (random primers, Life Technologies Corp.) in accordance with the protocol of the kit.

Next, PCR (30 cycles of 98° C. for 10 seconds, 55° C. for 15 seconds, 68° C. for 1.5 minutes) was carried out using primers FGFR3_TACC3_RT_F represented by SEQ ID No: 1 and FGFR3_TACC3_RT_R represented by SEQ ID No: 2, the cDNA obtained above as a template, and DNA polymerase (TaKaRa Ex Taq; Takara Bio Inc.). Additional PCR (30 cycles of 98° C. for 15 seconds, 55° C. for 15 seconds, 68° C. for 1 minute) was carried out using the PCR product described above which was diluted 10-fold as a template, primers FGFR3_TACC3_nested_F represented by SEQ ID No: 3 and FGFR3_TACC3_nested_R represented by SEQ ID No: 4, and the same DNA polymerase as shown above. Electrophoresis performed after the PCR reaction showed that a PCR product of about 500 bases was obtained from only sample Lg344 specimen.

After that, the PCR product was sequenced by dideoxy sequencing (BigDye Terminator v3.1 Cycle Sequencing Kit; Life Technologies Corp.). As a result, the PCR product of about 500 bases was found to be a sequence obtained by fusion of the 3′ end of exon 18 in the coding sequence (hereinafter, CDS) of FGFR3 (NM_001163213.1) registered in the NCBI to the 5′ end of exon 11 in the CDS of TACC3 (NM_006342.1).

cDNA was synthesized by reverse transcription reaction in the Lg344 specimen RNA which is the lung cancer tissue-derived RNA of a squamous cell lung cancer patient (Asterand plc.; US) using reverse transciptase (SuperScriptIII, Life Technologies, Corp.) and oligo(dT) primers (oligo(dT)20 primers, Life Technologies, Corp.) in accordance with the protocol of the kit.

Next, PCR (25 cycles of 98° C. for 15 seconds, 60° C. for 15 seconds, 68° C. for 3.5 minutes) was carried out using primers FGFR3-TACC3_cloning_F represented by SEQ ID No: 5 and FGFR3-TACC3_cloning_R represented by SEQ ID No: 6, the cDNA obtained above as a template, and DNA polymerase (KOD-plus-Ver. 2; Toyobo Co., Ltd.). Additional PCR (25 cycles of 98° C. for 15 seconds, 55° C. for 15 seconds, 68° C. for 3.5 minutes) was carried out using the PCR product described above which was diluted 10-fold as a template, primers FGFR3_TACC3_cloning_BamHI_F represented by SEQ ID No: 7 and FGFR3_TACC3_cloning_EcoRI_R represented by SEQ ID No: 8, and the same DNA polymerase as shown above. Electrophoresis performed after the PCR reaction showed that a PCR product of about 2.9 k bases was obtained. The PCR product was cloned into a cloning vector (TOPO XL PCR Cloning Kit; Life Technologies, Corp.). The insert was sequenced by dideoxy sequencing (BigDye Terminator v3.1 Cycle Sequencing Kit; Life Technologies, Corp.). As a result, it was found in the PCR product of about 2.9 k bases that there was a transcript obtained by fusion of the region between the 5′-terminus of the CDS of FGFR3 (NM_001163213.1) registered in the NCBI and the 3′ end of exon 18 to the region between the 5′ end of exon 11 in the CDS of TACC3 (NM_006342.1) and the 3′-terminus of the CDS (FGFR3-TACC3_v1) (SEQ ID No: 9). The polypeptide coded by SEQ ID No: 9 (FGFR3-TACC3_v1 fusion polypeptide) is shown in SEQ ID No: 10.

Test Example 5

Isolation of FGFR3-TACC3_v2

cDNA was synthesized by reverse transcription reaction in 59 specimens of bladder cancer (Asterand plc.; US) using reverse transciptase (SuperScriptIII, Life Technologies, Corp.) and random primers (random primers, Life Technologies Corp.) in accordance with the protocol of the kit.

Next, PCR (30 cycles of 98° C. for 10 seconds, 55° C. for 15 seconds, 68° C. for 1.5 minutes) was carried out using primers FGFR3_TACC3_RT_F represented by SEQ ID No: 1 and FGFR3_TACC3_RT_R represented by SEQ ID No: 2, the cDNA obtained above as a template, and DNA polymerase (TaKaRa Ex Taq; Takara Bio Inc.). Additional PCR (30 cycles of 98° C. for 15 seconds, 55° C. for 15 seconds, 68° C. for 1 minute) was carried out using the PCR product described above which was diluted 10-fold as a template, primers FGFR3_TACC3_nested_F represented by SEQ ID No: 3 and FGFR3_TACC3_nested_R represented by SEQ ID No: 4, and the same DNA polymerase as shown above. Electrophoresis performed after the PCR reaction showed that a PCR product of about 600 bases was obtained from sample Bd106 specimen.

After that, the PCR product was sequenced by dideoxy sequencing (BigDye Terminator v3.1 Cycle Sequencing Kit; Life Technologies Corp.). As a result, the PCR product of about 600 bases was found to be a sequence obtained by fusion of the 3′ end of exon 18 in the CDS of FGFR3 (NM_001163213.1) registered in the NCBI to the 5′ end of exon 10 in the CDS of TACC3 (NM_006342.1). cDNA was synthesized by reverse transcription reaction in the Bd106 specimen RNA which is the bladder cancer tissue-derived RNA of a bladder cancer patient (Asterand plc.; US) using reverse transciptase (SuperScriptIII, Life Technologies, Corp.) and oligo(dT) primers (oligo(dT)20 primers, Life Technologies, Corp.) in accordance with the protocol of the kit.

Next, PCR (25 cycles of 98° C. for 15 seconds, 60° C. for 15 seconds, 68° C. for 3.5 minutes) was carried out using primers FGFR3-TACC3_cloning_F represented by SEQ ID No: 5 and FGFR3-TACC3_cloning_R represented by SEQ ID No: 6, the cDNA obtained above as a template, and DNA polymerase (KOD-plus-Ver. 2; Toyobo Co., Ltd.). Additional PCR (25 cycles of 98° C. for 15 seconds, 55° C. for 15 seconds, 68° C. for 3.5 minutes) was carried out using the PCR product described above which was diluted 10-fold as a template, primers FGFR3_TACC3_cloning_BamHI_F represented by SEQ ID No: 7 and FGFR3_TACC3_cloning_EcoRI_R represented by SEQ ID No: 8, and the same DNA polymerase as shown above. Electrophoresis performed after the PCR reaction showed that a PCR product of about 3.0 k bases was obtained. The PCR product was cloned into a cloning vector (TOPO XL PCR Cloning Kit; Life Technologies, Corp.). The insert was sequenced by dideoxy sequencing (BigDye Terminator v3.1 Cycle Sequencing Kit; Life Technologies, Corp.). As a result, it was found in the PCR product of about 3.0 k bases that there was a transcript obtained by fusion of the region between the 5′-terminus of the CDS of FGFR3 (NM_001163213.1) registered in the NCBI and the 3′ end of exon 18 to the region between the 5′ end of exon 10 in the CDS of TACC3 (NM_006342.1) and the 3′-terminus of the CDS (FGFR3-TACC3_v2) (SEQ ID No: 11). The polypeptide coded by SEQ ID No: 11 (FGFR3-TACC3_v2 fusion polypeptide) is shown in SEQ ID No: 12.

Test Example 6

Isolation of FGFR3-TACC3_v3

cDNA was synthesized by reverse transcription reaction in 59 specimens of bladder cancer (Asterand plc.; US) using reverse transciptase (SuperScriptIII, Life Technologies, Corp.) and random primers (random primers, Life Technologies Corp.) in accordance with the protocol of the kit.

Next, PCR (30 cycles of 98° C. for 10 seconds, 55° C. for 15 seconds, 68° C. for 1.5 minutes) was carried out using primers FGFR3_TACC3_RT_F represented by SEQ ID No: 1 and FGFR3_TACC3_RT_R represented by SEQ ID No: 2, the cDNA obtained above as a template, and DNA polymerase (TaKaRa Ex Taq; Takara Bio Inc.). Additional PCR (30 cycles of 98° C. for 15 seconds, 55° C. for 15 seconds, 68° C. for 1 minute) was carried out using the PCR product described above which was diluted 10-fold as a template, primers FGFR3_TACC3_nested_F represented by SEQ ID No: 3 and FGFR3_TACC3_nested_R represented by SEQ ID No: 4, and the same DNA polymerase as shown above. Electrophoresis performed after the PCR reaction showed that a PCR product of about 650 bases was obtained from sample Bd021 specimen.

After that, the PCR product was sequenced by dideoxy sequencing (BigDye Terminator v3.1 Cycle Sequencing Kit; Life Technologies Corp.). As a result, the PCR product of about 650 bases was found to be a sequence obtained by fusion of a certain sequence of exon 19 in the CDS of FGFR3 (NM_001163213.1) registered in the NCBI to a part of intron 10-11 of TACC3 (NM_006342.1) and to the 5′ end of exon 11 in the CDS of TACC3.

cDNA was synthesized by reverse transcription reaction in the Bd021 specimen RNA which is the bladder cancer tissue-derived RNA of a bladder cancer patient (Asterand plc.; US) using reverse transciptase (SuperScriptIII, Life Technologies, Corp.) and oligo(dT) primers (oligo(dT)20 primers, Life Technologies, Corp.) in accordance with the protocol of the kit.

Next, PCR (25 cycles of 98° C. for 15 seconds, 60° C. for 15 seconds, 68° C. for 3.5 minutes) was carried out using primers FGFR3-TACC3_cloning_F represented by SEQ ID No: 5 and FGFR3-TACC3_cloning_R represented by SEQ ID No: 6, the cDNA obtained above as a template, and DNA polymerase (KOD-plus-Ver. 2; Toyobo Co., Ltd.). Additional PCR (25 cycles of 98° C. for 15 seconds, 55° C. for 15 seconds, 68° C. for 3.5 minutes) was carried out using the PCR product described above which was diluted 10-fold as a template, primers FGFR3_TACC3_cloning_BamHI_F represented by SEQ ID No: 7 and FGFR3_TACC3_cloning_EcoRI_R represented by SEQ ID No: 8, and the same DNA polymerase as shown above. Electrophoresis performed after the PCR reaction showed that a PCR product of about 3.0 k bases was obtained. The PCR product was cloned into a cloning vector (TOPO XL PCR Cloning Kit; Life Technologies, Corp.). The insert was sequenced by dideoxy sequencing (BigDye Terminator v3.1 Cycle Sequencing Kit; Life Technologies, Corp.). As a result, it was found in the PCR product of about 3.0 k bases that there was a transcript obtained by fusion of the region between the 5′-terminus of the CDS of FGFR3 (NM_001163213.1) registered in the NCBI and a certain sequence of exon 19 to part of intron 10-11 of TACC3 (NM_006342.1) and further to the region between the 5′ end of exon 11 in the CDS of TACC3 and the 3′-terminus of the CDS (FGFR3-TACC3_v3) (SEQ ID No: 13). The polypeptide coded by SEQ ID No: 13 (FGFR3-TACC3_v3 fusion polypeptide) is shown in SEQ ID No: 14.

Test Example 7

Isolation of FGFR3-TACC3 v1 from Bladder Cancer Patient-Derived Cell Line RT-112

cDNA was synthesized by reverse transcription reaction in RNA purified from bladder cancer patient-derived cell line RT-112 (purchased from Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH) using reverse transciptase (SuperScriptIII, Life Technologies, Corp.) and oligo(dT) primers (oligo(dT)20 primers, Life Technologies, Corp.) in accordance with the protocol of the kit.

Next, PCR (25 cycles of 98° C. for 15 seconds, 60° C. for 15 seconds, 68° C. for 3.5 minutes) was carried out using primers FGFR3-TACC3_cloning_F represented by SEQ ID No: 5 and FGFR3-TACC3_cloning_R represented by SEQ ID No: 6, the cDNA obtained above as a template, and DNA polymerase (KOD-plus-Ver. 2; Toyobo Co., Ltd.). Additional PCR (25 cycles of 98° C. for 15 seconds, 55° C. for 15 seconds, 68° C. for 3.5 minutes) was carried out using the PCR product described above which was diluted 10-fold as a template, primers FGFR3_TACC3_cloning_BamHI_F represented by SEQ ID No: 7 and FGFR3_TACC3_cloning_EcoRI_R represented by SEQ ID No: 8, and the same DNA polymerase as shown above. Electrophoresis performed after the PCR reaction showed that a PCR product of about 2.9 k bases was obtained. The PCR product was cloned into a cloning vector (TOPO XL PCR Cloning Kit; Life Technologies, Corp.), and the insert was sequenced by dideoxy sequencing (BigDye Terminator v3.1 Cycle Sequencing Kit; Life Technologies, Corp.). As a result, it was found that a transcript obtained was the same as the transcript obtained by fusion of the region between the N-terminus of the CDS of FGFR3 (NM_001163213.1) registered in the NCBI and the 3′ end of exon 18 to the region between the 5′ end of exon 11 in the CDS of TACC3 (NM_006342.1) and the C-terminus of the CDS (FGFR3-TACC3_v1) (SEQ ID No: 9).

Test Example 8

Isolation of FGFR3-TACC3_v4 from Bladder Cancer Patient-Derived Cell Line RT4

cDNA was synthesized by reverse transcription reaction in RNA purified from bladder cancer patient-derived cell line RT4 (purchased from ECACC (European Collection of Cell Cultures)) using reverse transciptase (SuperScriptIII, Life Technologies, Corp.) and oligo(dT) primers (oligo(dT)20 primers, Life Technologies, Corp.) in accordance with the protocol of the kit. Next, PCR (30 cycles of 98° C. for 15 seconds, 60° C. for 15 seconds, 68° C. for 5.5 minutes) was carried out using primers FGFR3-TACC3_cloning_F represented by SEQ ID No: 5 and FGFR3-TACC3_cloning_R represented by SEQ ID No: 6, the cDNA obtained above as a template, and DNA polymerase (KOD-plus-Ver. 2; Toyobo Co., Ltd.). Additional PCR (30 cycles of 98° C. for 15 seconds, 55° C. for 15 seconds, 68° C. for 5 minutes) was carried out using the PCR product described above which was diluted 10-fold as a template, primers FGFR3_TACC3_cloning_BamHI_F represented by SEQ ID No: 7 and FGFR3_TACC3_cloning_EcoRI_R represented by SEQ ID No: 8, and the same DNA polymerase as shown above. Electrophoresis performed after the PCR reaction showed that a PCR product of about 4.5 k bases was obtained. The PCR product was cloned into a cloning vector (TOPO XL PCR Cloning Kit; Life Technologies, Corp.), and the insert was sequenced by dideoxy sequencing (BigDye Terminator v3.1 Cycle Sequencing Kit; Life Technologies, Corp.). As a result, it was found that there was a transcript obtained by fusion of part of intron 18-19 sequence of FGFR3 (NM_001163213.1) registered in the NCBI to the region between the 5′-terminus of the CDS of the FGFR3 and the 3′ end of exon 18 and further to the region between a certain sequence of exon 4 of TACC3 (NM_006342.1) and the 3′-terminus of the CDS of the TACC3 (FGFR3-TACC3_v4). In the confirmed sequence, T at base position 882 was replaced by C (SNPs registration No.; rs2234909), C at base position 2484 by T, and G at base position 2663 by A (SEQ ID No: 15). The polypeptide coded by SEQ ID No: 15 (FGFR3-TACC3_v4 fusion polypeptide) is shown in SEQ ID No: 16.

Test Example 9

Preparation of Retrovirus Solutions of FGFR3-TACC3_v1, FGFR3-TACC3_v2, FGFR3-TACC3_v3, and FGFR3-TACC3_v4

To express, as proteins, the ORF full lengths of FGFR3-TACC3_v1, FGFR3-TACC3_v2, FGFR3-TACC3_v3, and FGFR3-TACC3_v4, enzyme reaction was performed at 37° C. for 3 hours using the cloning vectors prepared in Test Examples 4, 5, 6, and 8 and restriction enzyme BamHI, and restriction enzyme digested DNA fragments were obtained and purified. Another enzyme reaction was performed at 37° C. for 3 hours using EcoRI and the DNA fragments, and restriction enzyme digested DNA fragments were obtained and purified. The ORF-containing DNA fragments were cloned into BamHI and EcoRI sites in the multicloning site of an expression vector (pMXs-puro; Cosmo Bio) to construct expression plasmids (FGFR3-TACC3_v1/pMXs-puro, FGFR3-TACC3_v2/pMXs-puro, FGFR3-TACC3_v3/pMXs-puro, and FGFR3-TACC3_v4/pMXs-puro).

9 μg each of FGFR3-TACC3_v1/pMXs-puro, FGFR3-TACC3_v2/pMXs-puro, FGFR3-TACC3_v3/pMXs-puro, and FGFR3-TACC3_v4/pMXs-puro was transfected into Platinum-E cells, using a transfection reagent (FUGENE® HD, Roche). At 24 hours after the transfection, D-MEM media (Dulbecco's Modified Eagle Medium; Invitrogen) containing 10% bovine serum (Nichirei Biosciences) were replaced, and the culture supernatants were collected after 24 hours to prepare retrovirus solutions.

Test Example 10

Investigation of Anchorage-Independent Growth-Promoting Action of FGFR3-TACC3_v1, FGFR3-TACC3_v2, FGFR3-TACC3_v3, and FGFR3-TACC3_v4

To the virus solutions prepared using FGFR3-TACC3_v1/pMXs-puro, FGFR3-TACC3_v2/pMXs-puro, FGFR3-TACC3_v3/pMXs-puro, and FGFR3-TACC3_v4/pMXs-puro in Test Example 9, 4 μg/mL of polybrene (Polybrene; Sigma) was added followed by addition of the resulting mixtures to NIH3T3 cells for infection. At 6 hours after the addition, the media used were replaced by D-MEM media containing 10% bovine serum (Nichirei Biosciences), and, on the day after the infection, the media were replaced by D-MEM media (Invitrogen) containing 10% bovine serum (Nichirei Biosciences) and 1 μg/mL of puromycin (Sigma). The culture was continued in the presence of 5% CO 2 at 37° C. for 4 weeks to obtain NIH3T3 cells stably expressing each of FGFR3-TACC3_v1, FGFR3-TACC3_v2, FGFR3-TACC3_v3, and FGFR3-TACC3_v4 (these cells were designated as FGFR3-TACC3_v1-expressing NIH3T3 cells, FGFR3-TACC3_v2-expressing NIH3T3 cells, FGFR3-TACC3_v3-expressing NIH3T3 cells, and FGFR3-TACC3_v4-expressing NIH3T3 cells, respectively.)

To investigate the anchorage-independent growth-promoting ability of FGFR3-TACC3_v1-expressing NIH3T3 cells, FGFR3-TACC3_v2-expressing NIH3T3 cells, FGFR3-TACC3_v3-expressing NIH3T3 cells, and FGFR3-TACC3_v4-expressing NIH3T3 cells, these cells and NIH3T3 cells infected with a blank vector pMXs-puro (Mock/NIH3T3 cells) were each seeded at 1×10 3 cells per well in D-MEM media (Invitrogen) containing 10% bovine serum (Nichirei Biosciences) in a 96-well spheroid plate (Sumilon Celltight Spheroid 96U; Sumitomo Bakelite). The cells were cultured in the presence of 5% CO 2 at 37° C. and were counted on the next day (Day 1) and 4 days later (Day 4), using a cell counting reagent (CELLTITER-Glo™ Luminescent Cell Viability Assay; Promega) in accordance with the method described in the manual. A luminometer was used for detection. It was confirmed that the count of Mock/NIH3T3 cells did not increase between Day 1 and Day 4, while the counts of FGFR3-TACC3_v1-expressing NIH3T3 cells, FGFR3-TACC3_v2-expressing NIH3T3 cells, FGFR3-TACC3_v3-expressing NIH3T3 cells, and FGFR3-TACC3_v4-expressing NIH3T3 cells increased about 3.1-fold, about 2.8-fold, about 2.3-fold, and about 2.5-fold, respectively, between Day 1 and Day 4.

In light of the foregoing, it was found that FGFR3-TACC3_v1-expressing NIH3T3 cells, FGFR3-TACC3_v2-expressing NIH3T3 cells, FGFR3-TACC3_v3-expressing NIH3T3 cells, and FGFR3-TACC3_v4-expressing NIH3T3 cells exhibit anchorage-independent cell growth.

Test Example 11

Anchorage-Independent Cell Growth-Inhibitory Activity of Compounds on FGFR3-TACC3_v1-Expressing NIH3T3 Cells, FGFR3-TACC3_v2-Expressing NIH3T3 Cells, FGFR3-TACC3_v3-Expressing NIH3T3 Cells, FGFR3-TACC3_v4-Expressing NIH3T3 Cells, and Bladder Cancer Patient-Derived Cell Lines RT-112 and RT4

Measurement for anchorage-independent cell growth (colony method, etc.) is known to be a system for investigating the anticancer action (pharmacological effect) of compounds (Clinical Oncology, second edition, Cancer and Chemotherapy Publishers Inc.). As a method for measuring the non-adhesive growth of cells, there is the following method using a spheroid plate as referred to above in place of the colony method.

In a 96-well spheroid plate (Sumilon Celltight Spheroid 96U; Sumitomo Bakelite), FGFR3-TACC3_v1-expressing NIH3T3 cells, FGFR3-TACC3_v2-expressing NIH3T3 cells, FGFR3-TACC3_v3-expressing NIH3T3 cells, and FGFR3-TACC3_v4-expressing NIH3T3 cells were each seeded at 1×10 3 cells per well in D-MEM media (Invitrogen) containing 10% fetal bovine serum. Likewise, bladder cancer patient-derived cell line RT-112 was seeded at 1×10 3 cells per well in RPMI1640 medium containing 10% fetal bovine serum and 2 mM L-glutamine, and bladder cancer patient-derived cell line RT4 was seeded at 1×10 3 cells per well in RPMI1640 medium containing 10% fetal bovine serum. A well supplemented with only medium was also prepared for a positive control. Culturing was performed overnight in the presence of 5% CO 2 at 37° C. followed by addition of test compounds (final concentrations: 100 nM, 10 nM, and 1 nM). As a negative control, DMSO used as a solvent for the compounds was added at the same concentration (0.1%) as in the case of addition of the compounds. Then, culturing was performed in the presence of 5% CO 2 at 37° C. for 4 days, and a cell counting reagent (CellTiter-Glo™ Luminescent Cell Viability Assay; Promega) was added and the resulting mixture was stirred for 20 minutes followed by measurement with a luminometer. Assuming that the values of the positive control and the negative control were 100% inhibition and 0% inhibition, respectively, the growth inhibition rate (%) was calculated for each compound. As shown in Table 4, it was found out that some compounds of the present invention inhibited the anchorage-independent growth of FGFR3-TACC3_v1-expressing NIH3T3 cells, FGFR3-TACC3_v2-expressing NIH3T3 cells, FGFR3-TACC3_v3-expressing NIH3T3 cells, FGFR3-TACC3_v4-expressing NIH3T3 cells, and bladder cancer patient-derived cell lines RT-112 and RT4.

The results described above showed that the growth of cancer cells and tumors that express FGFR3-TACC3_v1, FGFR3-TACC3_v2, FGFR3-TACC3_v3, and FGFR3-TACC3_v4 can be inhibited by the compounds of the present invention.

Test Example 12

Inhibitory Activity of Compounds on the In Vitro Kinase Activity of FGFR3-TACC3 Fusion Polypeptide

(1) Construction of FLAG-Tag Fusion Expression Plasmids (FGFR3-TACC3_v1 (N-FLAG)/pcDNA3.1/Zeo(+), FGFR3-TACC3_v2 (N-FLAG)/pcDNA3.1/Zeo(+), and FGFR3-TACC3_v3 (N-FLAG)/pcDNA3.1/Zeo(+))

To obtain 5′-terminally FLAG-tagged FGFR3-TACC3 fusion polynucleotide, PCR was carried out for 5′-terminal FLAG tagging using the vectors cloned in Test Examples 4, 5, and 6 as templates. PCR (12 cycles of 98° C. for 15 seconds, 55° C. for 15 seconds, 68° C. for 3.5 minutes) was carried out using primers FGFR3_N_FLAG_BamHI represented by SEQ ID No: 17 and FGFR3_TACC3_cloning_EcoRI_R represented by SEQ ID No: 8 and DNA polymerase (KOD-plus-Ver. 2; Toyobo Co., Ltd.). PCR products obtained were cloned into cloning vectors (TOPO XL PCR Cloning Kit; Life Technologies, Corp.). The inserts were sequenced by dideoxy sequencing (BigDye Terminator v3.1 Cycle Sequencing Kit; Life Technologies, Corp.). As a result, it was confirmed that the PCR products were nucleic acid sequences of SEQ ID Nos: 9, 11, and 13 in which the three bases coding for the first methionine (ATG) were deleted and start codon and a nucleic acid sequence coding for FLAG tag (SEQ ID No: 24) were added to the 5′-terminus. Polypeptides coded by the above are referred to FGFR3-TACC3_v1 (N-FLAG) fusion polypeptide, FGFR3-TACC3_v2 (N-FLAG) fusion polypeptide and FGFR3-TACC3_v3 (N-FLAG) fusion polypeptide, respectively, and these polypeptides are collectively referred to FGFR3-TACC3 (N-FLAG) fusion polypeptide. Further, to construct an expression vector expressing, as a protein, each of the ORF full lengths of FGFR3-TACC3_v1 (N-FLAG), FGFR3-TACC3_v2 (N-FLAG), and FGFR3-TACC3_v3 (N-FLAG) which contained these FLAG sequences added, enzyme reaction was performed at 37° C. for 3 hours using the cloning vectors described above and restriction enzyme BamHI, and restriction enzyme digested DNA fragments were obtained and purified. Further, enzyme reaction was performed at 37° C. for 3 hours using EcoRI and the DNA fragments, and restriction enzyme digested DNA fragments were obtained and purified. These ORF-containing DNA fragments were cloned into BamHI and EcoRI sites in the multicloning site of an expression vector (pcDNA3.1/Zeo(+); Life Technologies, Corp.) to construct expression plasmids (FGFR3-TACC3_v1 (N-FLAG)/pcDNA3.1/Zeo(+), FGFR3-TACC3_v2 (N-FLAG)/pcDNA3.1/Zeo(+), and FGFR3-TACC3_v3 (N-FLAG)/pcDNA3.1/Zeo(+)).

(2) Preparation of FGFR3-TACC3 (N-FLAG) Fusion Polypeptide

On the day before transfection, 0.5×10 7 HEK293 cells per collagen-coated 15-cm dish were cultured in D-MEM medium containing 10% fetal bovine serum to prepare 10 dishes. On the day of transfection, 27 μg each of FGFR3-TACC3_v1 (N-FLAG)/pcDNA3.1/Zeo(+), FGFR3-TACC3_v2 (N-FLAG)/pcDNA3.1/Zeo(+), and FGFR3-TACC3_v3 (N-FLAG)/pcDNA3.1/Zeo(+) (Test Example 12) per dish was transfected into HEK293 cells, using 81 μL of a transfection reagent (FUGENE® HD, Roche). At 24 hours after the transfection, the media were removed, and after washing three times with PBS, 1 mL of PBS was added. The cells were scraped with a cell scraper (Corning Inc.) and then recovered in polypropylene tubes. After centrifugation at 1200 rpm for 5 minutes, the supernatant was removed, 1504 of a cell lysate (50 mM Tris-HCl (pH8.0), 150 mM NaCl, 1% NP-40, 1 mM EDTA, and protease inhibitor cocktail complete) was added, and the cells were incubated on ice for 30 minutes and lysed. Each of the FGFR3-TACC3_v1 (N-FLAG) fusion polypeptide, FGFR3-TACC3_v2 (N-FLAG) fusion polypeptide, and FGFR3-TACC3_v3 (N-FLAG) fusion polypeptide which were present in the supernatant obtained after the centrifugation was purified using M2 antibody affinity gel (ANTI-FLAG M2 Affinity Gel; Sigma-Aldrich) in accordance with the method described in the product information document. A wash liquid (50 mM Tris-HCl (pH8.0), 150 mM NaCl, 1% NP-40, 1 mM EDTA, and protease inhibitor cocktail complete) and an eluate (20 mM Tris-HCl (pH7.4), 10 mM MgCl 2 , 10 mM MnCl 2 , and 0.5 mg/mL of FLAG peptide) were used for washing and elution, respectively, to give 100 μL of eluates. The eluates were subjected to immunoblotting using an anti-FGFR3 antibody (Cell Signaling Technology) and an anti-FLAG M2 antibody (Sigma-Aldrich) and silver staining, and then confirmed that FGFR3-TACC3_v1 (N-FLAG) fusion polypeptide, FGFR3-TACC3_v2 (N-FLAG) fusion polypeptide, and FGFR3-TACC3_v3 (N-FLAG) fusion polypeptide were obtained.

(3) Detection of the In Vitro Kinase Activity of FGFR3-TACC3 (N-FLAG) Fusion Polypeptide

FGFR3-TACC3_v1 (N-FLAG) fusion polypeptide, FGFR3-TACC3_v2 (N-FLAG) fusion polypeptide, and FGFR3-TACC3_v3 (N-FLAG) fusion polypeptide, which were purified as described above were used to investigate their phosphorylating activity against a peptide substrate by using a kinase activity detection kit (HTRF KinEASE-TK; Cisbio). The reaction buffer was prepared by adding 1 mM (final concentration) of DTT and 5 mM (final concentration) of Mg to 5× kinase buffer enclosed in the kit using 1 μL of 1-fold, 3-fold and 10-fold diluted solutions of the above prepared elutates as enzyme solutions, respectively, in 384-well, low-volume black plate (Corning). Using 2.0 μM (final concentration) of TK Substrate enclosed in the kit as a substrate, the reaction was performed in a final volume of 5.0 μL at room temperature for 1 hour in each case of adding no ATP and adding 100 μM ATP (final concentration). After the reaction, Sa-XL665 solution and TK Antibody-Eu(K) solution were prepared in accordance with kit-recommended method and added each of 2.5 μL of the solutions. After the reaction was performed at room temperature for 1 hour, the HTRF counts (i.e., phosphorylation of the peptide substrate) were detected. As the results, it was showed that compared with ATP-free ones, the HTRF counts in ATP-added ones had increased about 38-fold, about 40-fold, and about 38-fold, respectively, in the case of adding 1 μL of 1-fold diluted solutions of the eluates described above including FGFR3-TACC3_v1 (N-FLAG) fusion polypeptide, FGFR3-TACC3_v2 (N-FLAG) fusion polypeptide, and FGFR3-TACC3_v3 (N-FLAG) fusion polypeptide, had increased about 27-fold, 34-fold, and 31-fold, respectively, in the case of adding 1 μL of 3-fold diluted solutions of the eluates, and had increased 5-fold, 18-fold, and 11-fold, respectively, in the case of adding 1 μL of 10-fold diluted solutions of the eluates.

As described above, the in vitro kinase activity of the respective fusion polypeptides could be detected by use of a kinase activity detection kit.

(4) Inhibitory Action of Compounds on the In Vitro Kinase Activity of FGFR3-TACC3 (N-FLAG) Fusion Polypeptide

The inhibitory activity of the test compounds on the in vitro kinase activity of FGFR3-TACC3_v1 (N-FLAG) fusion polypeptide, FGFR3-TACC3_v2 (N-FLAG) fusion polypeptide, and FGFR3-TACC3_v3 (N-FLAG) fusion polypeptide was investigated using the kinase activity detection kit described above and 384-well plate of the same sort. The compounds were added so that the final concentrations were 100 nM, 10 nM, and 1 nM, and DMSO was added as a control so that the concentration was 0.1%. For FGFR3-TACC3_v1 (N-FLAG) fusion polypeptide, 1 μL of a 2-fold diluted solution of the eluate described above was added; for FGFR3-TACC3_v2 (N-FLAG) fusion polypeptide, 1 μL of a 3-fold diluted solution of the eluate described above was added; and for FGFR3-TACC3_v3 (N-FLAG) fusion polypeptide, 1 μL of a 3-fold diluted solution of the eluate described above was added. TK Substrate enclosed in the kit as a substrate was added in a final concentration of 2.0 μM, the reaction was performed at room temperature for 15 minutes. Then the reaction was performed in a final volume of 5.0 μL at room temperature for 60 minutes in each case of adding no ATP and adding 100 μM ATP (final concentration). After the other processes were performed by addition of each of 2.5 μL of Sa-XL665 solution and TK Antibody-Eu(K) solution prepared by using similar method to that described in (3) above, and the reaction was performed at room temperature for 1 hour, the HTRF counts were detected. Assuming that the phosphorylation counts with adding no ATP and adding ATP in the absence of the compounds (DMSO was added in a concentration of 0.1%, the concentration equal to the compounds) were 100% inhibition and 0% inhibition, respectively, the inhibition rates (%) of the kinase activity of FGFR3-TACC3_v1 (N-FLAG) fusion polypeptide, FGFR3-TACC3_v2 (N-FLAG) fusion polypeptide, and FGFR3-TACC3_v3 (N-FLAG) fusion polypeptide were calculated for the compounds, using the following formula:

[rate (%) of inhibiting kinase activity by compound]=(1−[phosphorylation count with adding compound and adding ATP−phosphorylation count with adding no compound and adding no ATP]/[phosphorylation count with adding no compound and adding ATP−phosphorylation count with adding no compound and adding no ATP])×100

As a result, as shown in Table 5, it was found out that some compounds of the present invention inhibit the phosphorylating activity of purified FGFR3-TACC3_v1 (N-FLAG) fusion polypeptide, purified FGFR3-TACC3_v2 (N-FLAG) fusion polypeptide, and purified FGFR3-TACC3_v3 (N-FLAG) fusion polypeptide against the peptide substrate.

Test Example 13

Isolation of FGFR3-BAIAP2L1 from Bladder Cancer Patient-Derived Cell Line SW780

cDNA was synthesized by reverse transcription reaction in RNA purified from bladder cancer patient-derived cell line SW780 (purchased from ATCC) using reverse transciptase (SuperScriptIII, Life Technologies, Corp.) and oligo(dT) primers (oligo(dT)20 primers, Life Technologies, Corp.) in accordance with the protocol of the kit.

Next, PCR (30 cycles of 98° C. for 15 seconds, 60° C. for 15 seconds, 68° C. for 5 minutes) was carried out using primers FGFR3-BAIAP2L1_cloning_F represented by SEQ ID No: 18 and FGFR3-BAIAP2L1_cloning_R represented by SEQ ID No: 19, the cDNA obtained above as a template, and DNA polymerase (KOD-plus-Ver. 2; Toyobo Co., Ltd.). Additional PCR (30 cycles of 98° C. for 15 seconds, 55° C. for 15 seconds, 68° C. for 4 minutes) was carried out using the PCR product described above which was diluted 10-fold as a template, primers FGFR3_BAIAP2L1_cloning_BamHI_F represented by SEQ ID No: 20 and FGFR3_BAIAP2L1_cloning_NotI_R represented by SEQ ID No: 21, and the same DNA polymerase as shown above. Electrophoresis performed after the PCR reaction showed that a PCR product of about 3.8 k bases was obtained. The PCR product was cloned into a cloning vector (TOPO XL PCR Cloning Kit; Life Technologies, Corp.), and the insert was sequenced by dideoxy sequencing (BigDye Terminator v3.1 Cycle Sequencing Kit; Life Technologies, Corp.). As a result, the product was found to be a transcript obtained by fusion of the region between the 5′-terminus of the CDS of FGFR3 (NM_001163213.1) registered in the NCBI and the 3′ end of exon 18 to the region between the 5′ end of exon 2 in the CDS of BAIAP2L1 (NM_018842.4) and the 3′-terminus of the CDS (FGFR3-BAIAP2L1). In the confirmed sequence, G at base position 3558 was replaced by A (SNPs registration No.: rs1045916), C at base position 3723 by T, and G at base position 3747 by A (SEQ ID No: 22). The polypeptide coded by SEQ ID No: 22 is shown in SEQ ID No: 23.

Test Example 14

Preparation of Retrovirus Solution of FGFR3-BAIAP2L1

To construct an expression plasmid expressing, as a protein, the ORF full length of FGFR3-BAIAP2L1, enzyme reaction was performed at 37° C. for 3 hours using the cloning vector described above and restriction enzyme BamHI, and restriction enzyme digested DNA fragments were obtained and purified. Further, enzyme reaction was performed at 37° C. for 3 hours using NotI and the DNA fragments, and restriction enzyme digested DNA fragments were obtained and purified. This ORF-containing DNA fragment was cloned into BamHI and NotI sites in the multicloning site of an expression vector (pMXs-puro; Cosmo Bio) to construct an expression plasmid (FGFR3-BAIAP2L1/pMXs-puro). The prepared FGFR3-BAIAP2L1/pMXs-puro was used to prepare a retrovirus solution in accordance with the method used in Test Example 9.

Test Example 15

Investigation of Anchorage-Independent Growth of FGFR3-BAIAP2L1

The virus solution prepared using FGFR3-BAIAP2L1/pMXs-puro in Test Example 14 was used to obtain NIH3T3 cells expressing FGFR3-BAIAP2L1 stably in accordance with the method used in Test Example 10 (designated as FGFR3-BAIAP2L1-expressing NIH3T3 cells).

To investigate the anchorage-independent growth-promoting ability of FGFR3-BAIAP2L1-expressing NIH3T3 cells, the same method as in Test Example 10 was applied. It was confirmed that the count of Mock/NIH3T3 cells did not increase between Day 1 and Day 4, while the count of FGFR3-BAIAP2L1-expressing NIH3T3 cells increased about 2.5-fold between Day 1 and Day 4. In light of the foregoing, it was shown that FGFR3-BAIAP2L1-expressing NIH3T3 cells exhibit anchorage-independent cell growth.

Test Example 16

Inhibitory Activity on Anchorage-Independent Cell Growth of FGFR3-BAIAP2L1-Expressing NIH3T3 Cells

In a 96-well spheroid plate (Sumilon Celltight Spheroid 96U; Sumitomo Bakelite), FGFR3-BAIAP2L1-expressing NIH3T3 cells were seeded at 1×10 3 cells per well in D-MEM medium containing 10% fetal bovine serum. A well supplemented with only medium was also prepared for a positive control. Culturing was performed overnight in the presence of 5% CO 2 at 37° C. followed by addition of test compounds (final concentrations: 100 nM, 10 nM, and 1 nM). As a negative control, DMSO used as a solvent for the compounds was added at the same concentration (0.1%) as in the case of addition of the compounds. Then, culturing was performed in the presence of 5% CO 2 at 37° C. for 4 days, and a cell counting reagent (CellTiter-Glo™ Luminescent Cell Viability Assay; Promega) was added and the resulting mixture was stirred for 20 minutes followed by measurement with a luminometer. Assuming that the values of the positive control and the negative control were 100% inhibition and 0% inhibition, respectively, the growth inhibition rate (%) was calculated for each compound. As shown in Table 6, it was found out that some compounds of the present invention inhibit the anchorage-independent growth of FGFR3-BAIAP2L1-expressing NIH3T3 cells.

The results described above showed that the growth of cancer cells and tumors that express FGFR3-BAIAP2L1 can be inhibited by the compounds of the present invention.

A pharmaceutical composition which comprises one or more of the compounds of formula (I) or salts thereof, as active ingredient, can be prepared in a conventional manner by using an excipient commonly used in the art, more specifically, a pharmaceutical excipient, pharmaceutical carrier, or another additive.

Any mode of administration may be used: namely, either oral administration in the form of tablets, pills, capsules, granules, powders, solutions or the like, or parenteral administration in the form of injections (e.g., intraarticular, intravenous, or intramuscular injection), suppositories, eye drops, eye ointments, percutaneous solutions, ointments, percutaneous patches, transmucosal solutions, transmucosal patches, inhalants, intravesical instillation or the like.

Solid compositions used for oral administration include tablets, powders, granules, and the like. In these solid compositions, one or more active ingredients are mixed with at least one inert excipient. The compositions may also comprise inert additives such as lubricants, disintegrating agents, stabilizers, and/or solubilizers, as in usual cases. Tablets or pills may be coated with sugar or a gastrosoluble or enteric film, if needed.

Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs, and the like, and comprise a commonly-used inert diluent such as purified water or ethanol. These liquid compositions may comprise auxiliaries (e.g., solubilizers, wetting agents, suspending agents), sweeteners, flavors, aromatics, and/or antiseptics, in addition to such an inert diluent.

Injections for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of aqueous solvents include injectable distilled water and physiological saline. Examples of non-aqueous solvents include alcohols such as ethanol. These compositions may further comprise isotonizing agents, antiseptics, wetting agents, emulsifiers, dispersants, stabilizers or solubilizers. They are sterilized, for example, by filtration through a bacteria-retaining filter, by incorporation of disinfectants, or by irradiation. Alternatively, they may be formulated into a sterile solid composition and reconstituted for use by being dissolved or suspended in sterile water or a sterile injectable solvent before use.

Formulations for external use include ointments, plasters, creams, jellies, cataplasms, sprays, lotions, eye drops, eye ointments, and the like. They include commonly-used ointment bases, lotion bases, aqueous or non-aqueous solutions, suspensions, emulsions, or the like.

Transmucosal formulations such as inhalants or transnasal formulations are used in solid, liquid, or semi-solid form and can be prepared in a conventionally known manner. For example, such formulations may be supplemented as appropriate with known excipients, and further with pH adjusters, antiseptics, surfactants, lubricants, stabilizers, thickeners, or the like. For their administration, an appropriate device for inhalation or insufflation may be used. For example, using a known device (e.g., a metered-dose inhalation device) or a nebulizer, the compound(s) may be administered alone or as a powder of a formulated mixture or as a solution or suspension in combination with a pharmaceutically acceptable carrier. Dry powder inhalators and the like may be for single or multiple administration use, and dry powders or powder-containing capsules may be used in such devices. Alternatively, they may be in the form of pressurized aerosol sprays or the like which use an appropriate propellant, for example, a preferred gas such as chlorofluoroalkane or carbon dioxide.

In general, for oral administration, the daily dosage is desirably about 0.001 to 100 mg/kg body weight, preferably 0.1 to 30 mg/kg body weight, more preferably 0.1 to 10 mg/kg body weight, given as a single dose or in 2 to 4 divided doses. For intravenous administration, the daily dosage is desirably about 0.0001 to 10 mg/kg body weight, given in one or several doses per day. Likewise, for transmucosal formulations, the daily dosage is about 0.001 to 100 mg/kg body weight, given in one or several doses per day. The dosage may be determined as appropriate for each case in consideration of symptom, age, sex, and the like.

The pharmaceutical composition of the present invention comprises one or more of the compounds of formula (I) or salts thereof, as active ingredients in an amount of 0.01 to 100 wt. % (0.01 to 50 wt. % in one embodiment), which varies depending on administration route, dosage form, administration site, or the types of excipients and additives.

The compounds of formula (I) can be used in combination with various therapeutic or prophylactic agents for diseases against which the compounds of formula (I) would be effective. In such combination therapy, drugs may be administered simultaneously or separately in succession or at desired time intervals. Formulations for simultaneous administration may be in either mixed form or separate form.

The processes for preparing the compounds of formula (I) are described in more detail with reference to the examples shown below. It should be noted that the present invention is not limited to the compounds described in the examples shown below. In addition, the processes for preparing the starting compounds are shown in preparation examples. Processes for preparing the compounds of formula (I) are not limited only to those actually described in the examples shown below, and the compounds of formula (I) may also be prepared by any combination of these processes or by any processes obvious to those skilled in the art.

In the examples, preparation examples and tables shown below, the following abbreviations are used as needed.

PEx: Preparation Example No., Ex: Example No., PSyn: Preparation Example No. of compound prepared in the same manner, Syn: Example No. of compound prepared in the same manner, Str: chemical structural formula (Me: methyl, Et: ethyl, i Pr: isopropyl, t Bu: tert-butyl, Boc: tert-butoxycarbonyl, Bn: benzyl, THP: tetrahydropyranyl), DAT: physical and chemical data, ESI+: m/z value in mass analysis (ionization method ESI, (M+H) + unless otherwise specified), ESI−: m/z value (ionization method ESI, (M−H) − unless otherwise specified), EI: m/z value in mass analysis (ionization method EI, (M) + unless otherwise specified), APCI/ESI+: m/z value in mass analysis (simultaneous measurement by ionization methods APCI and ESI, (M+H) + unless otherwise specified), NMR1: δ (ppm) in 1 H-NMR in dimethyl sulfoxide-d 6 , NMR2: δ (ppm) in 1 H-NMR in CDCl 3 , NMR3: δ (ppm) in 1 H-NMR in CD 3 OD, “M” in Preparation Example and Example: which indicates mol/L. “HCl” in a structural formula indicates hydrochloride and the number in front of the term “HCl” indicates molar ratio. For example, 2HCl means a dihydrochloride salt. The symbol “*” in the tables in Preparation Examples and Examples indicates that the compounds given the symbol are optically active substances.

Preparation Example 1

Under an argon atmosphere, to a mixture of 3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]aniline (300 mg) and ethanol (6 mL), methanesulfonic acid (128 μL) was added followed by stirring at room temperature for 30 minutes. Subsequently, 5-bromo-2-chloropyrimidine (229 mg) was added thereto and the resulting mixture was stirred at 100° C. for 4 hours. Additional 5-bromo-2-chloropyrimidine (95 mg) was added thereto and the resulting mixture was stirred at 100° C. for 12 hours. To the reaction mixture, a saturated aqueous sodium hydrogen carbonate solution was added followed by extraction with chloroform. An organic layer obtained was dried over anhydrous sodium sulfate and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by basic silica gel column chromatography (ethyl acetate/methanol) to give 5-bromo-N-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine (352 mg).

Preparation Example 2

To a mixture of 3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]aniline (253 mg) and isopropanol (6 mL), methanesulfonic acid (162 μL) was added followed by stirring at room temperature for 30 minutes. After that, 2-chloro-5-iodopyrimidine (200 mg) was added thereto, and the resulting mixture was stirred at 90° C. for 12 hours and further stirred at 130° C. for 2 hours under microwave irradiation. To the reaction mixture, a saturated aqueous sodium hydrogen carbonate solution was added followed by extraction with chloroform. An organic layer obtained was dried over anhydrous sodium sulfate and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (chloroform/methanol) to give 5-iodo-N-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine (282 mg).

Preparation Example 3

Under an argon atmosphere, a mixture of 1-ethynyl-3,5-dimethoxybenzene (3 g) and acetonitrile (30 mL) was ice cooled, and then sulfuryl chloride (3.15 mL) was added thereto followed by stirring at room temperature for 4 hours. Additional sulfuryl chloride (449 μL) was added thereto followed by stirring at room temperature for 12 hours. After the reaction mixture was concentrated under reduced pressure, ethyl acetate and a saturated aqueous sodium hydrogen carbonate solution were added to the resulting residue followed by stirring at room temperature for 30 minutes. The resulting solid was collected by filtration, washed with ethyl acetate, and then dried under reduced pressure to give 2,4-dichloro-3-ethynyl-1,5-dimethoxybenzene (1.99 g).

Preparation Example 4

A mixture of 1-ethynyl-3,5-dimethoxybenzene (4 g) and acetonitrile (80 mL) was ice cooled, and N-fluoro-N′-(chloromethyl)triethylenediamine bis(tetrafluoroborate) (19.4 g) was added thereto. The resulting mixture was gradually warmed and stirred at room temperature for 12 hours. To the reaction mixture, a saturated aqueous sodium hydrogen carbonate solution was added followed by extraction with ethyl acetate. An organic layer obtained was washed with saturated brine, and then dried over anhydrous sodium sulfate and filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (ethyl acetate/hexane) and subsequently purified by silica gel column chromatography (chloroform/hexane) to give 3-ethynyl-2,4-difluoro-1,5-dimethoxybenzene (798 mg, Preparation Example No. PEx. 4-1, which is described later) and 1-ethynyl-2-fluoro-3,5-dimethoxybenzene (375 mg, Preparation Example No. PEx. 4-2, which is described later).

Preparation Example 5

Under an argon atmosphere, a mixture of 1-ethynyl-2-fluoro-3,5-dimethoxybenzene (800 mg) and acetonitrile (8 mL) was ice cooled, and sulfuryl chloride (378 μL) was added thereto followed by stirring at room temperature for 12 hours. To the reaction mixture, ethyl acetate and a saturated aqueous sodium hydrogen carbonate solution were added followed by extraction with ethyl acetate. An organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was solidified with ethyl acetate/diisopropyl ether to give 2-chloro-3-ethynyl-4-fluoro-1,5-dimethoxybenzene (787 mg).

Preparation Example 6

To a mixture of 2,6-difluoro-3-methoxybenzaldehyde (500 mg), potassium carbonate (803 mg), and methanol (10 mL), dimethyl (1-diazo-2-oxopropyl)phosphonate (523 μL) was added at room temperature under an argon atmosphere followed by stirring for 5 hours. To the reaction mixture, water was added followed by extraction with ethyl acetate. An organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (ethyl acetate/hexane) to give 2-ethynyl-1,3-difluoro-4-methoxybenzene (452 mg).

Preparation Example 7

To a mixture of 2-amino-5-iodopyrimidine (1 g), 3-ethynyl-2,4-difluoro-1,5-dimethoxybenzene (897 mg), tetrakistriphenylphosphine palladium (261 mg), copper iodide (43 mg), and N,N-dimethylformamide (20 mL), N,N-diisopropylethylamine (1.55 mL) was added under an argon atmosphere followed by stirring at 80° C. for 1 hour. The reaction mixture was concentrated under reduced pressure, and to the obtained residue were added chloroform and water, and insoluble materials were removed by filtration through celite. After the filtrate was extracted with chloroform, the organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (chloroform/methanol) to give 5-[(2,6-difluoro-3,5-dimethoxyphenyl)ethynyl]pyrimidin-2-amine (1.07 g).

Preparation Example 8

To a mixture of 5-[(2,6-difluoro-3,5-dimethoxyphenyl)ethynyl]pyrimidin-2-amine (400 mg), methanol (4 mL), and tetrahydrofuran (4 mL), 10% palladium-carbon (73 mg) was added under an argon atmosphere. After the resulting mixture was stirred at 60° C. for 8 hours under a hydrogen atmosphere, insoluble materials were removed by filtration through celite. The filtrate was concentrated under reduced pressure to give 5-[(2,6-difluoro-3,5-dimethoxyphenyl)ethyl]pyrimidin-2-amine (402 mg).

Preparation Example 9

To a mixture of 5-[(2,6-difluoro-3,5-dimethoxyphenyl)ethyl]pyrimidin-2-amine (100 mg) and acetonitrile (2 mL) under an argon atmosphere were added copper chloride (II) (68 mg) and n-pentyl nitrite (69 μL), followed by stirring at 60° C. for 4 hours. To the reaction mixture, ethyl acetate was added and insoluble materials were removed by filtration. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (ethyl acetate/hexane) to give 2-chloro-5-[2-(2,6-difluoro-3,5-dimethoxyphenyl)ethyl]pyrimidine (20 mg).

Preparation Example 10

A mixture of (2-chloropyrimidin-5-yl)methanol (120 mg), 3,5-dimethoxyphenol (186 mg), tributylphosphine (297 μL), and tetrahydrofuran (2.4 mL) was ice cooled, and 1,1′-(azodicarbonyl)dipiperidine (305 mg) was added thereto followed by stirring at room temperature for 12 hours. Insoluble materials were removed by filtration and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate/hexane) to give 2-chloro-5-[(3,5-dimethoxyphenoxy)methyl]pyrimidine (119 mg).

Preparation Example 13

A mixture of 2-chloro-5-hydroxypyrimidine (278 mg), potassium carbonate (453 mg), and N,N-dimethylformamide (3 mL) was ice cooled, and 3,5-dimethoxybenzyl bromide (541 mg) was added thereto followed by stirring at room temperature for 7 hours. To the reaction mixture, water was added followed by extraction with ethyl acetate. An organic layer obtained was washed with saturated brine, dried over anhydrous magnesium sulfate, and then filtered. The resulting residue was purified by silica gel column chromatography (ethyl acetate/hexane) to give 2-chloro-5-[(3,5-dimethoxybenzyl)oxy]pyrimidine (360 mg).

Preparation Example 14

To a mixture of 2-chloro-5-[(3,5-dimethoxybenzyl)oxy]pyrimidine (4.17 g) and N,N-dimethylformamide (40 mL), N-chlorosuccinimide (4.05 g) was added followed by stirring at room temperature for 2 hours and stirring at 60° C. for 2 hours. To the reaction mixture, water was added, and the resulting solid was collected by filtration, washed with water, and then dried under reduced pressure. The obtained solid was suspended in ethyl acetate (40 mL) and heated to 80° C. The solid was collected by filtration, and then dried under reduced pressure to give 2-chloro-5-[(2,6-dichloro-3,5-dimethoxybenzyl)oxy]pyrimidine (3.99 g).

Preparation Example 15

A mixture of 2-chloro-5-hydroxypyrimidine (487 mg) and 1-(3,5-dimethoxyphenyl)ethanol (680 mg), tributylphosphine (1.37 mL), and tetrahydrofuran (14 mL) was ice cooled, and 1,1′-(azodicarbonyl)dipiperidine (1.4 g) was added thereto followed by stirring at room temperature for 12 hours and stirring at 50° C. for 3 hours. Insoluble materials were removed by filtration and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate/hexane) to give 2-chloro-5-[1-(3,5-dimethoxyphenyl)ethoxy]pyrimidine (415 mg).

Preparation Example 16

A mixture of methyl 3,5-dimethoxybenzoate (1 g) and acetonitrile (20 mL) was ice cooled, and N-fluoro-N′-(chloromethyl)triethylenediamine bis(tetrafluoroborate) (4.09 g) was added thereto followed by stirring at room temperature overnight. To the reaction mixture, a saturated aqueous sodium hydrogen carbonate solution was added followed by extraction with ethyl acetate. An organic layer obtained was washed with saturated brine, added anhydrous sodium sulfate and basic silica gel followed by stirring for 30 minutes, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (ethyl acetate/hexane) to give methyl 2,6-difluoro-3,5-dimethoxybenzoate (292 mg: Preparation Example 16-1) and methyl 2-fluoro-3,5-dimethoxybenzoate (232 mg: Preparation Example 16-2).

Preparation Example 17

A mixture of methyl 2,6-difluoro-3,5-dimethoxybenzoate (10 g) and tetrahydrofuran (50 mL) was ice cooled, and lithium borohydride (3.0M tetrahydrofuran solution, 43 mL) was added thereto followed by stirring at room temperature for 65 hours. The reaction mixture was ice cooled again, and additional lithium borohydride (3.0M tetrahydrofuran solution, 14 mL) was added thereto followed by stirring at room temperature for 22 hours. The reaction mixture was ice cooled and slowly added into ice water (300 mL). Further, concentrated hydrochloric acid (25 mL) was slowly added thereto, and the resulting mixture was stirred at room temperature for 1 hour and extracted with toluene/ethyl acetate (1:1). An organic layer obtained was washed with a saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over anhydrous sodium sulfate, and then filtered. The filtrate was concentrated under reduced pressure to give (2,6-difluoro-3,5-dimethoxyphenyl)methanol (8.67 g).

Preparation Example 18

A mixture of (2,6-difluoro-3,5-dimethoxyphenyl)methanol (1.71 g), triethylamine (2.57 mL), and tetrahydrofuran (34 mL) was ice cooled, and methanesulfonyl chloride (716 μL) was added thereto followed by stirring for 1 hour. To the reaction mixture, water was added followed by extraction with ethyl acetate. An organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. The filtrate was concentrated under reduced pressure to give 2,6-difluoro-3,5-dimethoxybenzyl methanesulfonate (2.32 g).

Preparation Example 19

To a mixture of 2-chloro-5-hydroxypyrimidine (4.38 g), potassium carbonate (9.27 g), and N,N-dimethylformamide (79 mL), 2,6-difluoro-3,5-dimethoxybenzyl methanesulfonate (7.89 g) was added followed by stirring at 60° C. for 1 hour. To the reaction mixture, water was added, and the resulting solid was collected by filtration, washed with water, and then dried under reduced pressure to give 2-chloro-5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidine (8.53 g).

Preparation Example 20

A mixture of 2,3,5,6-tetrafluoropyridine (1.5 g) and methanol (15 mL) was ice cooled, and sodium methoxide (4.03 g) was added thereto followed by stirring at room temperature for 2 hours and stirring at 50° C. overnight. To the reaction mixture, water was added followed by extraction with diethyl ether. An organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. The filtrate was concentrated under reduced pressure to give 3,5-difluoro-2,6-dimethoxypyridine (1.47 g).

Preparation Example 21

A mixture of diisopropylamine (745 μL) and tetrahydrofuran (5 mL) was cooled to −78° C., and n-butyl lithium (1.6M hexane solution, 3.02 mL) was added thereto followed by stirring at 0° C. for 30 minutes. The reaction mixture was cooled to −78° C., and a mixture of 3,5-difluoro-2,6-dimethoxypyridine (770 mg) and tetrahydrofuran (5 mL) was added thereto dropwise followed by stirring for 1 hour. After N,N-dimethylformamide (440 μL) was added thereto, the resulting mixture was warmed to room temperature and stirred for 1 hour. To the reaction mixture, water was added followed by extraction with ethyl acetate. An organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. The resulting residue was purified by silica gel column chromatography (ethyl acetate/hexane) to give 3,5-difluoro-2,6-dimethoxyisonicotinaldehyde (406 mg).

Preparation Example 22

A mixture of 3,5-difluoro-2,6-dimethoxyisonicotinaldehyde (400 mg) and methanol (4 mL) was ice cooled, and sodium borohydride (82 mg) was added thereto followed by stirring for 1 hour. To the reaction mixture, 1M hydrochloric acid was added followed by extraction with ethyl acetate. An organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. The filtrate was concentrated to give (3,5-difluoro-2,6-dimethoxypyridin-4-yl)methanol (403 mg).

Preparation Example 23

To a mixture of 2-chloro-5-[(2,6-dichloro-3,5-dimethoxybenzyl)oxy]pyrimidine (235 mg), tert-butyl 4-(4-amino-3-methoxyphenyl)piperidine-1-carboxylate (306 mg), 1,1′-binaphthalene-2,2′-diylbis(diphenylphosphine) (138 mg), cesium carbonate (660 mg), and dioxane (10 mL), palladium acetate (30 mg) was added at room temperature under an argon atmosphere. The resulting mixture was stirred at 100° C. for 3 hours. To the reaction mixture, water was added followed by extraction with ethyl acetate. An organic layer obtained was washed with saturated brine, dried over anhydrous magnesium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (ethyl acetate/hexane) to give tert-butyl 4-[4-({5-[(2,6-dichloro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-3-methoxyphenyl]piperidine-1-carboxylate (298 mg).

Preparation Example 24

To a mixture of 2-fluoro-5-nitrotoluene (500 mg), potassium carbonate (2.0 g), and N,N-dimethylformamide (15 mL), 4-piperidin-4-ylthiomorpholine 1,1-dioxide bistrifluoroacetate (2.16 g) was added followed by stirring at 80° C. for 20 hours. To the reaction mixture, water was added followed by extraction with ethyl acetate. An organic layer obtained was washed with saturated brine, dried over anhydrous magnesium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (chloroform/methanol) to give 4-[1-(2-methyl-4-nitrophenyl)piperidin-4-yl]thiomorpholine 1,1-dioxide (870 mg).

Preparation Example 25

To a mixture of 4-[1-(2-methyl-4-nitrophenyl)piperidin-4-yl]thiomorpholine 1,1-dioxide (1.5 g) and acetic acid (30 mL), 10% palladium-carbon (452 mg) was added under an argon atmosphere. After stirring for 13 hours under a hydrogen atmosphere, insoluble materials were removed by filtration through celite. The filtrate was concentrated under reduced pressure, and then a saturated aqueous sodium hydrogen carbonate solution was added to the resulting residue. The resulted solid was collected by filtration, washed with water, and then dried under reduced pressure to give 4-[4-(1,1-dioxidothiomorpholin-4-yl)piperidin-1-yl]-3-methylaniline (1.26 g).

Preparation Example 26

To a mixture of 1-chloro-2-(difluoromethoxy)-4-nitrobenzene (920 mg), potassium carbonate (1.7 g), and N,N-dimethylformamide (10 mL), 1-methyl-4-piperidin-4-ylpiperazine (1.13 g) was added followed by stirring at 100° C. overnight. The reaction mixture was concentrated under reduced pressure, and water was added to the resulting residue followed by extraction with ethyl acetate. An organic layer obtained was washed with saturated brine, dried over anhydrous magnesium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (chloroform/methanol/conc. aqueous ammonia solution) to give 1-{1-[2-(difluoromethoxy)-4-nitrophenyl]piperidin-4-yl}-4-methylpiperazine (1.38 g).

Preparation Example 27

To a mixture of 1-{1-[2-(difluoromethoxy)-4-nitrophenyl]piperidin-4-yl}-4-methylpiperazine (1.38 g) and ethanol (54 mL), 10% palladium-carbon (397 mg) was added under an argon atmosphere. After stirring for 1 hour under a hydrogen atmosphere, insoluble materials were removed by filtration through celite. The filtrate was concentrated under reduced pressure to give 3-(difluoromethoxy)-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]aniline (1.25 g).

Preparation Example 28

A mixture of benzyl piperazine-1-carboxylate (10 g), 2,2,6,6-tetramethylpiperidin-4-one (7.05 g), and dichloromethane (100 mL) was ice cooled, and sodium triacetoxy borohydride (11.5 g) was added thereto followed by stirring at room temperature overnight. To the reaction mixture, a saturated aqueous sodium hydrogen carbonate solution was added followed by extraction with chloroform. An organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by basic silica gel column chromatography (ethyl acetate/hexane) to give benzyl 4-(2,2,6,6-tetramethylpiperidin-4-yl)piperazine-1-carboxylate (7.18 g).

Preparation Example 29

To a mixture of benzyl 4-(2,2,6,6-tetramethylpiperidin-4-yl)piperazine-1-carboxylate (7.18 g) and ethanol (60 mL), 10% palladium-carbon (2.0 g) was added under an argon atmosphere. After stirring for 7 hours under a hydrogen atmosphere, insoluble materials were removed by filtration through celite. The filtrate was concentrated under reduced pressure to give 1-(2,2,6,6-tetramethylpiperidin-4-yl)piperazine (4.35 g).

Preparation Example 30

To a mixture of 2-chloro-5-[(2,3,5,6-tetrafluorobenzyl)oxy]pyrimidine (202 mg), tert-butyl 9-(4-amino-2-methoxyphenyl)-3,9-diazaspiro[5,5]undecane-3-carboxylate (311 mg), 2-(dicyclohexylphosphino)-2′,4′,6′-triisopropyl-1,1′-biphenyl (30 mg), potassium carbonate (134 mg), and tert-butanol (10 mL), tris(dibenzylideneacetone)dipalladium (19 mg) was added at room temperature under an argon atmosphere. The resulting mixture was stirred at 100° C. for 4 hours. Insoluble materials were removed by filtration and washed with ethyl acetate. The filtrate was concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography (ethyl acetate/hexane) to give tert-butyl 4-[4-({5-[(2,3,5,6-tetrafluorobenzyl)oxy]pyrimidin-2-yl}amino)-2-methoxyphenyl]-3,9-diazaspiro[5,5]undecane-3-carboxylate (259 mg).

Preparation Example 31

To a mixture of N-[3-(1,4-dioxa-8-azaspiro[4,5]dec-8-yl)phenyl]-5-[(2,3,5,6-tetrafluorobenzyl)oxy]pyrimidin-2-amine (596 mg), acetic acid (9 mL), and water (9 mL), concentrated hydrochloric acid (0.5 mL) was added followed by stirring at 80° C. for 7 hours. The reaction mixture was ice cooled, and a 1M aqueous sodium hydroxide solution (155 mL) and a saturated aqueous sodium hydrogen carbonate solution were added thereto, and then the resulting solid was collected by filtration. Chloroform was added thereto, and the resulting mixture was dried over anhydrous sodium sulfate and then filtered. The filtrate was concentrated under reduced pressure to give 1-[3-({5-[(2,3,5,6-tetrafluorobenzyl)oxy]pyrimidin-2-yl}amino)phenyl]piperidin-4-one (512 mg).

Preparation Example 32

A mixture of 2-[3-({5-[(2,3,5,6-tetrafluorobenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]ethanol (116 mg), triethylamine (84 μL), and tetrahydrofuran (4 mL) was ice cooled, and methanesulfonyl chloride (47 μL) was added thereto followed by stirring for 3 hours. To the reaction mixture, water was added followed by extraction with ethyl acetate. An organic layer obtained was washed with saturated brine, dried over anhydrous magnesium sulfate, and then filtered. The filtrate was concentrated under reduced pressure to give 2-[3-({5-[(2,3,5,6-tetrafluorobenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]ethyl methanesulfonate (129 mg).

Preparation Example 33

A mixture of 4-(4-nitro-1H-pyrazol-1-yl)piperidine (250 mg), 1-methylpiperidin-4-one (220 μL), and dichloromethane (5 mL) was ice cooled, and sodium triacetoxy borohydride (810 mg) was added thereto followed by stirring at room temperature for 4 hours. To the reaction mixture, a saturated aqueous sodium hydrogen carbonate solution was added followed by extraction with chloroform. An organic layer obtained was washed with saturated brine, dried over anhydrous magnesium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (chloroform/methanol/conc. aqueous ammonia solution) to give 1′-methyl-4-(4-nitro-1H-pyrazol-1-yl)-1,4′-bipiperidine (342 mg).

Preparation Example 34

To a mixture of 1-(2-chloro-4-nitrophenyl)-4-(1-methylpiperidin-4-yl)piperazine (3.7 g), ammonium chloride (352 mg), ethanol (94 mL), tetrahydrofuran (47 mL), and water (47 mL), iron powder (3.06 g) was added followed by stirring at 70° C. for 4 hours. After insoluble materials were removed by filtration, the filtrate was concentrated under reduced pressure. To the resulting residue, a saturated aqueous sodium hydrogen carbonate solution was added followed by extraction with ethyl acetate. An organic layer obtained was dried over anhydrous magnesium sulfate and then filtered. The filtrate was concentrated under reduced pressure to give 3-chloro-4-[4-(1-methylpiperidin-4-yl)piperazin-1-yl]aniline (1.03 g).

Preparation Example 35

To a mixture of (3R,5S)-1-(2-methoxy-4-nitrophenyl)-3,5-dimethylpiperazine (3.0 g), N,N-diisopropylethylamine (2.32 mL), di-tert-butyldicarbonate (2.71 g), and dioxane (20 mL), 4-dimethylaminopyridine (69 mg) was added followed by stirring at 80° C. overnight. To the reaction mixture, a saturated aqueous sodium hydrogen carbonate solution was added followed by extraction with ethyl acetate. An organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (ethyl acetate/hexane) to give tert-butyl (2R,6S)-4-(2-methoxy-4-nitrophenyl)-2,6-dimethylpiperazine-1-carboxylate (1.73 g).

Preparation Example 36

To a mixture of 2-(2-bromoethoxy)-1-chloro-4-nitrobenzene (3.0 g), cesium carbonate (5.23 g), N-methylpyrrolidone (30 mL), 1H-pyrazole (874 mg) was added followed by stirring at 60° C. for 6 hours. To the reaction mixture, water was added, and the resulting solid was collected by filtration. The solid was washed with water and dried under reduced pressure to give 1-[2-(2-chloro-5-nitrophenoxy)ethyl]-1H-pyrazole (2.57 g).

Preparation Example 37

To a mixture of 1-[2-(2-chloro-5-nitrophenoxy)ethyl]-1H-pyrazole (1.3 g), cesium carbonate (1.0 g), N-methylpyrrolidone (8 mL), cis-2,6-dimethylpiperazine (832 mg) was added followed by stirring at 130° C. overnight. To the reaction mixture, water was added, and the resulting solid was collected by filtration. The solid was washed with water and dried under reduced pressure to give (3R,5S)-3,5-dimethyl-1-{4-nitro-2-[2-(1H-pyrazol-1-yl)ethoxy]phenyl}piperazine (1.15 g).

Preparation Example 38

A mixture of 2-chloro-5-[(3,5-dimethoxybenzyl)oxy]pyridine (500 mg) and acetonitrile (10 mL) was ice cooled, and sulfuryl chloride (297 μL) was added thereto followed by stirring at room temperature for three days. After the reaction mixture was concentrated under reduced pressure, a saturated aqueous sodium hydrogen carbonate solution was added to the residue obtained. The resulting solid was collected by filtration, washed with water, and then dried under reduced pressure to give 2-chloro-5-[(2,6-dichloro-3,5-dimethoxybenzyl)oxy]pyridine (596 mg).

Preparation Example 39

To a mixture of 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-[1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl]pyrimidin-2-amine (3.6 g) and methanol (20 mL), a 4M hydrogen chloride/dioxane solution (40 mL) was added followed by stirring at room temperature for 6 hours. After the reaction mixture was concentrated under reduced pressure, a saturated aqueous sodium hydrogen carbonate solution was added to the residue obtained. The resulting solid was collected by filtration, washed with diethyl ether, and then dried under reduced pressure to give 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-(1H-pyrazol-4-yl)pyrimidin-2-amine (2.9 g).

Preparation Example 40

To a mixture of 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-(1H-pyrazol-4-yl)pyrimidin-2-amine (4.0 g), potassium carbonate (4.6 g), and N,N-dimethylformamide (80 mL), ethyl bromoacetate (2.4 mL) was added followed by stirring at 80° C. for 3 hours. To the reaction mixture, water was added followed by extraction with ethyl acetate. An organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (ethyl acetate/hexane) to give ethyl [4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]acetate (4.2 g).

Preparation Example 41

To a mixture of 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-(1H-pyrazol-4-yl)pyrimidin-2-amine (50 mg), potassium carbonate (57 mg), and N,N-dimethylformamide (1 mL), [(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]methyl 4-methylbenzenesulfonate (98 μL) was added followed by stirring at 60° C. for 1 hour and stirring at 110° C. for 4 days. To the reaction mixture, water was added followed by extraction with ethyl acetate. An organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (ethyl acetate/hexane) to give 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-(1-{[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]methyl}-1H-pyrazol-4-yl)pyrimidin-2-amine (45 mg).

Preparation Example 111

To a mixture of 4-nitro-1H-pyrazole (500 mg), tert-butyl (3-endo)-3-[(methylsulfonyl)oxy]-8-azabicyclo[3,2,1]octane-8-carboxylate (1.35 g) and N-methylpyrrolidone (6 mL), cesium carbonate (2.16 g) was added followed by stirring at 100° C. for 6 hours. To the reaction mixture, water was added, and the resulting solid was collected by filtration, washed with water, and then dried under reduced pressure to give tert-butyl (3-exo)-3-(4-nitro-1H-pyrazol-1-yl)-8-azabicyclo[3,2,1]octane-8-carboxylate (1.07 g).

Preparation Example 118

A mixture of 4-nitro-1H-pyrazol (3 g), quinuclidin-3-ol (4.05 g), triphenylphosphine (9.05 g), and tetrahydrofuran (60 mL) was ice cooled, and diisopropyl azodicarboxylate (6.84 mL) was added thereto followed by stirring at room temperature overnight. After the reaction mixture was concentrated under reduced pressure, 1M hydrochloric acid (50 mL) was added to the resulting residue. The aqueous layer obtained was washed with ethyl acetate, and then a 1M aqueous sodium hydroxide solution (60 mL) was added for basification. After extraction with chloroform, an organic layer obtained was dried over anhydrous sodium sulfate and then filtered. The filtrate was concentrated under reduced pressure and then the resulting residue was purified by basic silica gel column chromatography (ethyl acetate) to give 3-(4-nitro-1H-pyrazol-1-yl)quinuclidine (5.15 g).

Preparation Example 133

A mixture of tert-butyl (4-amino-2-methoxyphenyl)[2-(4-methylpiperazin-1-yl)ethyl]carbamate (1.21 g) and tetrahydrofuran (24 mL) was ice cooled, and lithium aluminum hydride (629 mg) was added thereto followed by stirring for 1 hour under heating to reflux. To the reaction mixture, water (0.63 mL), a 1M aqueous sodium hydroxide solution (0.63 mL), and water (1.89 mL) in that order were added. After insoluble materials were removed by filtration through celite, the filtrate was extracted with ethyl acetate. An organic layer obtained was washed with saturated brine, dried over anhydrous magnesium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by basic silica gel column chromatography (chloroform/methanol) to give 2-methoxy-N 1 -methyl-N 1 -[2-(4-methylpiperazin-1-yl)ethyl]benzene-1,4-diamine (922 mg).

Preparation Example 138

To a mixture of 2-chloro-5-nitropyrimidine (798 mg), potassium carbonate (1.04 g), and N,N-dimethylformamide (16 mL), 1-methyl-4-(piperidin-4-yl)piperazine (1.1 g) was added followed by stirring at room temperature for 3 hours. To the reaction mixture, water was added followed by extraction with ethyl acetate. An organic layer obtained was washed with saturated brine, dried over anhydrous magnesium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (chloroform/methanol) to give 2-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]-5-nitropyrimidine (542 mg).

Preparation Example 143

To a mixture of tert-butyl 4-(2-amino-1,3-thiazol-5-yl)piperidine-1-carboxylate (1.13 g) and ethyl acetate (8 mL), 4M hydrogen chloride/ethyl acetate solution (8 mL) was added followed by stirring at room temperature for 3 hours. The solvent was concentrated under reduced pressure to give 5-(piperidin-4-yl)-1,3-thiazol-2-amine hydrochloride (877 mg).

Preparation Example 144

To a mixture of 5-(piperidin-4-yl)-1,3-thiazol-2-amine hydrochloride (519 mg), dichloromethane (5 mL), and methanol (5 mL), 1H-benzotriazol-1-ylmethanol (423 mg), sodium acetate (388 mg), and sodium triacetoxy borohydride (1.0 g) in that order were added followed by stirring at room temperature for 2 hours. To the reaction mixture, a saturated aqueous sodium hydrogen carbonate solution and basic silica gel were added followed by concentration of the solvent under reduced pressure. The resulting residue was purified by basic silica gel column chromatography (chloroform/methanol) to give 5-(1-methylpiperidin-4-yl)-1,3-thiazol-2-amine (411 mg).

Preparation Example 145

A mixture of 5-nitropyridin-2(1H)-one (700 mg), (R)-2,2-dimethyl-1,3-dioxolane-4-methanol (661 mg), triphenylphosphine (1.97 g), and tetrahydrofuran (20 mL) was ice cooled, diisopropyl azodicarboxylate (1.49 mL) was added followed by stirring at room temperature for 5 hours. To the reaction mixture, water was added followed by extraction with ethyl acetate. An organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (hexane/ethyl acetate) to give (R)-2-[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]-5-nitropyridine (541 mg).

Preparation Example 152

To a mixture of (S)-2,2-dimethyl-1,3-dioxolane-4-methanol (661 mg) and N,N-dimethylformamide (23 mL), sodium hydride (218 mg) was added followed by stirring at room temperature for 10 minutes. To the reaction mixture, 2-chloro-5-nitropyridine (793 mg) was added followed by stirring at room temperature for 2 hours. After water was added to the reaction mixture, extraction with ethyl acetate was performed. An organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (hexane/ethyl acetate) to give (S)-2-[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]-5-nitropyridine (810 mg).

Preparation Example 162

To a mixture of 2-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]ethyl methanesulfonate (320 mg) and N-methylpyrrolidone (6 mL), tert-butyl piperazine-1-carboxylate (1.31 g) was added followed by stirring at 80° C. overnight and additional stirring at 120° C. overnight. To the reaction mixture, water and a saturated aqueous sodium hydrogen carbonate solution were added followed by extraction with ethyl acetate. An organic layer obtained was washed with saturated brine, dried over anhydrous magnesium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (chloroform/methanol) and then solidified with ethyl acetate/diisopropyl ether to give tert-butyl 4-{2-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]ethyl}piperazine-1-carboxylate (202 mg).

Preparation Example 175

A mixture of 5-methyl-1H-pyrazol-3-amine (522 mg) and N,N-dimethylformamide (10 mL) was ice cooled, and sodium hydride (473 mg) was added thereto followed by stirring for 30 minutes. To the reaction mixture, 2-(2-bromoethoxy)tetrahydro-2H-pyran (893 μL) was added followed by stirring at room temperature for 12 hours. After saturated aqueous ammonium chloride solution was added to the reaction mixture, extraction with ethyl acetate was performed. An organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (hexane/ethyl acetate) to give 5-methyl-1-[2-(tetrahydro-2H-pyran-2-yloxy)ethyl]-1H-pyrazol-3-amine (427 mg: Preparation Example 175-1) and 3-methyl-1-[2-(tetrahydro-2H-pyran-2-yloxy)ethyl]-1H-pyrazol-5-amine (199 mg: Preparation Example 175-2).

Preparation Example 176

To a mixture of 5-[2-(benzyloxy)ethyl]-3-(2,5-dimethyl-1H-pyrrol-1-yl)-1-methyl-1H-pyrazole (640 mg) and ethanol (9.7 mL), hydroxylamine (1.37 mL) and p-toluenesulfonic acid monohydrate (1.95 g) in that order were added followed by stirring at 95° C. overnight. The reaction mixture was concentrated under reduced pressure, and then water was added to the resulting residue followed by extraction with chloroform. An organic layer obtained was washed with saturated brine, dried over anhydrous magnesium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (chloroform/methanol) to give 5-[2-(benzyloxy)ethyl]-1-methyl-1H-pyrazol-3-amine (470 mg).

Preparation Example 183

To a mixture of (1-methyl-3-nitro-1H-pyrazol-5-yl)methanol (398 mg), 3,4-dihydro-2H-pyran (459 μL), and ethyl acetate (8 mL), p-toluenesulfonic acid monohydrate (96 mg) was added followed by stirring at room temperature for 1.5 hours. Additional 3,4-dihydro-2H-pyran (459 μL) and p-toluenesulfonic acid monohydrate (96 mg) were added thereto followed by stirring at room temperature for 1.5 hours. After water was added to the reaction mixture, extraction with ethyl acetate was performed. An organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (hexane/ethyl acetate) to give 1-methyl-3-nitro-5-[(tetrahydro-2H-pyran-2-yloxy)methyl]-1H-pyrazole (487 mg).

Preparation Example 186

A mixture of 4-nitro-1H-pyrazole (300 mg), 2-phenyl-1,3-dioxan-5-ol (717 mg), triphenylphosphine (1.11 g) and tetrahydrofuran (4.5 mL) was ice cooled, and then diisopropyl azodicarboxylate (842 μL) was added thereto followed by stirring at room temperature for 12 hours. After the reaction mixture was concentrated under reduced pressure, the resulting residue was purified by silica gel chromatography (ethyl acetate/hexane) to give 4-nitro-1-(2-phenyl-1,3-dioxan-5-yl)-1H-pyrazol (121 mg).

Preparation Example 189

To a mixture of 5-nitropyridine-2-carbaldehyde (761 mg), 2-(piperazin-1-yl)ethanol (1.23 mL), acetic acid (570 μl), and dichloromethane (20 mL), sodium triacetoxy borohydride (2.23 g) was added followed by stirring at room temperature for 16 hours. To the reaction mixture, a saturated aqueous sodium hydrogen carbonate solution was added followed by extraction with chloroform/2-propanol. An organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (chloroform/methanol) to give 2-{4-[(5-nitropyridin-2-yl)methyl]piperazin-1-yl}ethanol (726 mg).

Preparation Example 191

To a mixture of methyl 5-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-3-carboxylate (871 mg), ethanol (8.7 mL), and tetrahydrofuran (8.7 mL), a 1M aqueous sodium hydroxide solution (3.45 mL) was added followed by stirring at 60° C. for 2 hours. To the reaction mixture, 1M hydrochloric acid was added, and the resulting solid was collected by filtration, washed with water, and then dried under reduced pressure to give 5-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-3-carboxylic acid (846 mg).

Preparation Example 193

A mixture of 5-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-3-carboxylic acid (300 mg) and dioxane (8.5 mL) was ice cooled, and 1,1′-carbonyldiimidazole (99 mg) was added thereto followed by stirring at room temperature for 2 hours and stirring at 60° C. for 2 hours. Additional 1,1′-carbonyldiimidazole (99 mg) was added thereto followed by stirring at 60° C. for 2 hours. Further, 1,1′-carbonyldiimidazole (297 mg) was added thereto followed by stirring at room temperature for 1 hour. The reaction mixture was ice cooled and sodium borohydride (230 mg) was added thereto followed by stirring at room temperature for 12 hours. Water was added to the reaction mixture and extraction with ethyl acetate was performed. An organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (hexane/ethyl acetate) to give [5-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl]methanol (126 mg).

Preparation Example 201

To a mixture of 1-methyl-3-nitro-1H-pyrazole-5-carbaldehyde (850 mg) and tetrahydrofuran (50 mL), methyl (triphenylphosphoranylidene)acetate (3.66 g) was added followed by stirring at 60° C. for 3 hours. After the reaction mixture was concentrated under reduced pressure, water was added to the resulting residue followed by extraction with chloroform. An organic layer obtained was washed with saturated brine, dried over anhydrous magnesium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the residue obtained was washed with chloroform and the resulting solid was collected by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (chloroform/methanol) and then combined with the solid obtained earlier to give methyl (E)-3-(1-methyl-3-nitro-1H-pyrazol-5-yl)acrylate (1.15 g).

Preparation Example 202

Under an argon atmosphere, to a mixture of methyl (E)-3-(1-methyl-3-nitro-1H-pyrazol-5-yl)acrylate (1.15 g) and ethanol (50 mL) was added 10% palladium-carbon (580 mg). After stirring under a hydrogen atmosphere in 1 atm for 12 hours and in 2.7 atm for 4 hours, insoluble materials were removed by filtration through celite. The resulting filtrate was concentrated under reduced pressure to give methyl 3-(3-amino-1-methyl-1H-pyrazol-5-yl)propanoate (955 mg).

Preparation Example 204

To a mixture of 2-[(tert-butoxycarybonyl)amino]-1,3-thiazole-5-carboxylic acid (500 mg), N-[3-(diethylamino)propyl]-N′-ethylcarbodiimide hydrochloride (589 mg), 1H-benzotriazol-1-ol (415 mg), and N,N-dimethylformamide (10 mL), 1-methylpiperazine (451 μL) was added followed by stirring at room temperature for 3 days. To the reaction mixture, water was added followed by extraction with ethyl acetate. An organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (chloroform/methanol) to give tert-butyl {5-[(4-methylpiperazin-1-yl)carbonyl]-1,3-thiazol-2-yl}carbamate (560 mg).

Preparation Example 205

A mixture of 4-aminopyridin-2(1H)-one (400 mg) and N-methylpyrrolidone (15 mL) was ice cooled, and sodium hydride (218 mg) was added thereto followed by stirring at room temperature for 30 minutes. To the reaction mixture, (S)-2,2-dimethyl-1,3-dioxolan-4-ylmethyl p-toluenesulfonate (1.14 g) and sodium iodide (109 mg) in that order were added followed by stirring at room temperature for 4 hours. After sodium hydride (218 mg) was added to the reaction mixture followed by stirring at 80° C. overnight. To the reaction mixture, a saturated aqueous ammonium chloride solution was added, and then the resulting mixture was saturated with sodium chloride, and extraction with methanol/chloroform was performed. An organic layer obtained was dried over anhydrous sodium sulfate and then filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by basic silica gel column chromatography (ethyl acetate/methanol) to give (R)-4-amino-1-[(2,2-dimethyl-1,3-dioxolan-4-yl)methyl]pyridin-2(1H)-one (136 mg).

Preparation Example 209

A mixture of [5-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl]methanol (126 mg), triethylamine (147 μL), dichloromethane (6 mL), and tetrahydrofuran (6 mL) was ice cooled, and then methanesulfonyl chloride (82 μL) was added thereto followed by stirring at room temperature for 3 hours. To the reaction mixture, N,N-dimethylformamide (6 mL) was added followed by stirring at room temperature for 12 hours. Water was added to the reaction mixture and extraction with chloroform was performed. An organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. The filtrate was concentrated under reduced pressure to give N-[3-(chloromethyl)-1H-pyrazol-5-yl]-5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-amine (109 mg).

Preparation Example 210

To a mixture of tert-butyl {5-[(4-methylpiperazin-1-yl)carbonyl]-1,3-thiazol-2-yl}carbamate (560 mg) and ethyl acetate (8 mL) was added 4M hydrogen chloride/ethyl acetate solution (8 mL) followed by stirring at room temperature for 3 hours. After the reaction mixture was concentrated under reduced pressure, the resulted residue was purified by basic silica gel chromatography (methanol/chloroform) to give (2-amino-1,3-thiazol-5-yl)(4-methylpiperazin-1-yl)methanone (357 mg).

Preparation Example 211

To a mixture of (5-nitro-1H-pyrazol-3-yl)methanol (1.86 g), 3,4-dihydro-2H-pyran (4.7 mL), and acetonitrile (28 mL), trifluoroacetic acid (40 μL) was added followed by stirring at 70° C. for 3 hours. To the reaction mixture, a saturated aqueous sodium hydrogen carbonate solution was added followed by extraction with ethyl acetate. An organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (hexane/ethyl acetate) to give 5-nitro-1-(tetrahydro-2H-pyran-2-yl)-3-[(tetrahydro-2H-pyran-2-yloxy)methyl]-1H-pyrazole (3.98 g).

Preparation Example 214

A mixture of [5-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-3-yl]methanol (200 mg) and 1,2-dichloroethane (12 mL) was ice cooled, and manganese dioxide (442 mg) was added thereto followed by stirring at room temperature for 30 minutes and then stirring at 90° C. for 2 hours. After insoluble materials were removed by filtration, the filtrate was concentrated under reduced pressure to give 5-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazole-3-carbaldehyde (142 mg).

Preparation Example 229

A mixture of methyl 2-chloro-6-fluoro-3,5-dimethoxybenzoate (682 mg) and tetrahydrofuran (25 mL) was ice cooled, and lithium aluminum hydride (104 mg) was added thereto followed by stirring at room temperature for 3 hours. To the reaction mixture, diethylether was added for dilution under ice cooling, and then a saturated aqueous sodium sulfate solution was added thereto. Insoluble materials were separated by filtration and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane/ethyl acetate) to give (2-chloro-6-fluoro-3,5-dimethoxyphenyl)methanol (363 mg).

Preparation Example 232

To a mixture of 2-bromo-5-nitroanisole (3.15 g), tert-butyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-8-azabicyclo[3.2.1]oct-2-ene-8-carboxylate (5.00 g), and dioxane (40 mL), [1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride dichloromethane complex (554 mg) and potassium carbonate (2.81 g) in that order were added under an argon atmosphere followed by stirring at 80° C. for 21 hours. To the reaction mixture, water was added followed by extraction with ethyl acetate. An organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (hexane/ethyl acetate) to give tert-butyl 3-(2-methoxy-4-nitrophenyl)-8-azabicyclo[3.2.1]oct-2-ene-8-carboxylate (2.78 g).

Preparation Example 252

A mixture of tert-butyl 4,4-bis(acetoxymethyl)-1,4′-bipiperidin-1′-carboxylate (712 mg) and dichloromethane (6 mL) was ice cooled, and then trifluoroacetic acid (3 mL) was added thereto followed by stirring at room temperature for 3 hours. To the reaction mixture, a saturated aqueous sodium hydrogen carbonate solution was added followed by extraction with dichloromethane. An organic layer obtained was washed with brine, dried over anhydrous sodium sulfate, and then filtered. The filtrate was concentrated under reduced pressure to give 1,4′-bipiperidin-4,4-diylbis(methylene) diacetate (529 mg).

Preparation Example 255

A mixture of tert-butyl 4,4-bis(hydroxymethyl)piperidine-1-carboxylate (1.01 g), triethylamine (861 μL), and dichloromethane (10 mL) was ice cooled, and acetic anhydride (950 μL) was added thereto followed by stirring for 2 hours. To the reaction mixture, water was added followed by extraction with dichloromethane. An organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (hexane/ethyl acetate) to give tert-butyl 4,4-bis(acetoxymethyl)piperidine-1-carboxylate (1.38 g).

Preparation Example 295

After a mixture of 2-[3-(2,5-dimethyl-1H-pyrrol-1-yl)-1-methyl-1H-pyrazol-5-yl]ethanol (630 mg), benzyl bromide (376 μL), and tetrahydrofuran (8 mL) was ice cooled, sodium hydride (173 mg) was added thereto followed by stirring at room temperature for 6 hours. To the reaction mixture, water was added followed by extraction with ethyl acetate. An organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (ethyl acetate/hexane) to give 5-[2-(benzyloxy)ethyl]-3-(2,5-dimethyl-1H-pyrrol-1-yl)-1-methyl-1H-pyrazole (640 mg).

Preparation Example 296

After a mixture of 3-(2,5-dimethyl-1H-pyrrol-1-yl)-1-methyl-1H-pyrazole (2 g) and tetrahydrofuran (60 mL) was cooled to −78° C., n-butyl lithium (1.6M hexane solution, 8.56 mL) was added thereto followed by stirring for 2 hours. To the reaction mixture, oxirane (1.1M tetrahydrofuran solution, 15.6 mL) and borontrifluoride tetrahydrofuran complex (1.51 mL) were added followed by stirring for 30 minutes. After that, the mixture obtained was warmed to room temperature and stirred for 6 hours. To the reaction mixture, a saturated aqueous ammonium chloride solution was added followed by extraction with ethyl acetate. An organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (ethyl acetate/hexane) to give 2-[3-(2,5-dimethyl-1H-pyrrol-1-yl)-1-methyl-1H-pyrazol-5-yl]ethanol (630 mg).

The compounds shown in Tables 7 to 62 below were prepared in the same manner as in the preparation examples described above. Tables 7 to 62 also show the processes for preparing the compounds of the preparation examples and the structures and physical and chemical data of the compounds.

The compound of formula (I) or a salt thereof according to the present invention has inhibitory action on FGFR1, FGFR2, and/or FGFR3, particularly, mutant FGFR3, and can be used as a therapeutic agent for various cancers related to FGFR1, FGFR2, and/or FGFR3, such as lung cancer and hormone therapy-resistant breast cancer, stomach cancer, triple negative breast cancer, endometrial cancer, and bladder cancer, particularly as a therapeutic agent for mutant FGFR3-positive bladder cancer.

The numerical heading <223> in the Sequence Listing shown below contains an explanation of “Artificial Sequence”. More specifically, the base sequences represented by SEQ ID NOs: 7, 8, 17, 20, and 21 in the Sequence Listing are artificially synthesized primer sequences. The base sequence represented by SEQ ID NO: 24 in the Sequence Listing is an artificially synthesized FLAG tag sequence.