# Patent

## Title
Nitrogen-containing aromatic heterocyclic compound

## Abstract

Provided is a compound useful as a prophylactic and/or therapeutic agent for bladder cancer.
As a result of studies on compounds having FGFR inhibitory action, the present inventors have found that the nitrogen-containing aromatic heterocyclic compounds of the present invention have inhibitory action on FGFR1, FGFR2, and/or FGFR3, particularly, mutant FGFR3, and thus, the present invention has been accomplished. The nitrogen-containing aromatic heterocyclic compound of the present invention 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 glioblastoma, particularly as a prophylactic and/or therapeutic agent for mutant FGFR3-positive bladder cancer.

## Background
TECHNICAL FIELD
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.
BACKGROUND ART
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 IC50 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; A1 is O, CR2, or the like; R0 is H or the like; A2 is NR, O, or the like; L1 is a bond, O, or the like; L2 is a bond, C1-C6 alkyl, or the like; R1 is a 3- to 6-membered heterocyclic ring or the like; and each of Re and Rf is H, C1-C6 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 (R1)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, G1 is aryl optionally having a substituent, heteroaryl optionally having a substituent, or the like; L1 is O, SO, SO2, optionally substituted alkyl, or the like; L2 is optionally substituted alkyl, heterocyclic ring, or the like; A1 is a bond, O, C(Ra)2, or the like; and A2 is NRa, 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 L1 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 L1 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 L1 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, A1, A2, A3, and A4 are CR4, CR5, CR6, and CR7, respectively, or are N; L is —C(O)NR7—, —NR7C(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.




CITATION LIST
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


SUMMARY OF INVENTION
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;

L1 is -lower alkylene-, -lower alkylene-O—, —O-lower alkylene-, or -lower alkynylene-;

Z is N or CH;

R1, 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 -L2-R2 or R3;

L2 is an optionally substituted aromatic heterocyclic ring or an optionally substituted non-aromatic heterocyclic ring;

R2 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)—R0, —C(O)-optionally substituted cycloalkyl, —NH—R0, —N(lower alkyl)-R0, -L3-optionally substituted non-aromatic heterocyclic group, or H;

R0 is lower alkyl optionally substituted with —OH;

R3 is

(1) lower alkyl optionally substituted with one or more groups selected from the group consisting of —C(O)OH, —OH, —O—R0, amino optionally substituted with one or two R0, carbamoyl optionally substituted with one or two R0, 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—R0, carbamoyl optionally substituted with one or two R0, 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 R0);

(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 R0);

(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 R0; and

L3 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.


DESCRIPTION OF EMBODIMENTS
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 C1-8) 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 C1-4 alkyl, and yet another embodiment is methyl. Yet another embodiment is ethyl.
The term “lower alkylene” refers to linear or branched C1-8 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 C1-4 alkylene, and yet another embodiment is methylene. Yet another embodiment is ethylene.
The term “lower alkynylene” refers to linear or branched C2-6 alkynylene including ethynylene, propynylene, butynylene, pentinylene, hexynylene, 1,3-butadiynylene, 1,3-pentadiynylene, and the like. Another embodiment is C2-4 alkynylene, and yet another embodiment is ethynylene.
The term “cycloalkyl” refers to a C3-10 saturated hydrocarbon ring group and it may be bridged. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, and the like. Another embodiment is C3-8 cycloalkyl, and yet another embodiment is C3-6 cycloalkyl. Yet another embodiment is cyclopropyl.
The term “aromatic carbocyclic ring” refers to a C6-14 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 L1 in formula (I) is -lower alkylene-O—, means a compound of formula (II) or a salt thereof.




(In this formula, L4 represents lower alkylene. The same applies hereinafter.)

Further, two to four R1 in (R1)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 R0 on the nitrogen in the “carbamoyl optionally substituted with one or two R0” may be the same lower alkyl or different lower alkyl from each other. Each R0 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 —NO2;
    (6) —CO2H and —CO2-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) —CO2H; 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 L2 in formula (I), “optionally substituted cycloalkyl” or “an optionally substituted non-aromatic heterocyclic group” referred to as R2 in formula (I), and “an optionally substituted aromatic heterocyclic group” or “an optionally substituted non-aromatic heterocyclic group” referred to in R3 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 L1 is lower alkylene or -lower alkylene-O—. Another embodiment is a compound or a salt thereof, wherein L1 is -lower alkylene-. Yet another embodiment is a compound or a salt thereof, wherein L1 is -lower alkylene-O—. Yet another embodiment is a compound or a salt thereof, wherein L1 is ethylene or -methylene-O—. Yet another embodiment is a compound or a salt thereof, wherein L1 is ethylene. Yet another embodiment is a compound or a salt thereof, wherein L1 is -methylene-O—. Yet another embodiment is a compound or a salt thereof, wherein L1 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 R1, the same or different from one another, are —O-lower alkyl or halogen. Another embodiment is a compound or a salt thereof, wherein R1, the same or different from one another, are —O-lower alkyl. Yet another embodiment is a compound or a salt thereof, wherein R1, the same or different from one another, are halogen. Yet another embodiment is a compound or a salt thereof, wherein R1, 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 R1 are F.
(5) A compound or a salt thereof, wherein the 6-membered aromatic ring in formula (I) which is substituted with (R1)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 (R1)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 (R1)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 -L2-R2. Another embodiment is a compound or a salt thereof, wherein Q is R3.
(8) A compound or a salt thereof, wherein L2 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 L2 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 L2 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 L2 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 L2 is piperidine or 4-methylpyperazine.
(9) A compound or a salt thereof, wherein R2 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 R2 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 R2 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 R2 is 4-methylpiperazine, 2-hydroxyethylamino, or H. Yet another embodiment is a compound or a salt thereof, wherein R2 is 4-methylpiperazine. Yet another embodiment is a compound or a salt thereof, wherein R2 is 2-hydroxyethylamino. Yet another embodiment is a compound or a salt thereof, wherein R2 is H.
(10) A compound or a salt thereof, wherein R3 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 R0, —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 R3 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 R0, —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 R3 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 R0, —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 R3 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 R3 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 R3 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 R3 is 2-hydroxyethyl, 2,3-dihydroxypropyl, or 4-methylpiperazin-1-ylmethyl. Yet another embodiment is a compound or a salt thereof, wherein R3 is 4-methylpiperazin-1-ylmethyl. Yet another embodiment is a compound or a salt thereof, wherein R3 is lower alkyl optionally substituted with one or more —OH. Yet another embodiment is a compound or a salt thereof, wherein R3 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 L1 is lower alkylene or -lower alkylene-O—.
(13) The compound according to (12) or a salt thereof, wherein Z is CH; R1, 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 L1 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 -L2-R2; L2 is an optionally substituted non-aromatic heterocyclic ring; R2 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; L2 is piperazine optionally substituted with one or more methyl, piperidine optionally substituted with one or more methyl, or 3,9-diazaspiro[5.5]undecane; R2 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 R1, the same or different from one another, are —O-methyl or F; L1 is -methylene-O—; ring W is a benzene ring optionally substituted with —O-methyl; L2 is piperidine or 4-methylpiperazine; R2 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 R3; R3 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 R0, —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 R3 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 R1, the same or different from one another, are —O-methyl or F; L1 is -methylene-O—; ring W is pyrazole optionally substituted with methyl; R3 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;
L1 is -lower alkylene-, -lower alkylene-O—, —O-lower alkylene-, or lower alkynylene; Z is N or CH;
R1, 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 -L2-R2 or R3;
L2 is an optionally substituted aromatic heterocyclic ring or an optionally substituted non-aromatic heterocyclic ring;
R2 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 -L3-optionally substituted non-aromatic heterocyclic group, or H;

R3 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 L3 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, L5 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.
DOCUMENTS


    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, L6 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.
DOCUMENTS


    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, R4 represents —OH or -lower alkylene-OH; L7 represents halogen, —OH, -lower alkylene-OH, -lower alkylene-OMs, -lower alkylene-OTs, -lower alkylene-OTf, or -lower alkylene-halogen; L8 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 L1 is —O-lower alkylene- or -lower alkylene-O—.
In the case of compound (3a) wherein L7 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 L7 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, L9 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 L1 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 MgCl2) 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 MgCl2, 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 IC50 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.












TABLE 1








FGFR1
FGFR2
FGFR3



Ex
IC50 (nM)
IC50 (nM)
IC50 (nM)





















7
2
3
1



11
1
—
2



27
2
1
<1



33
2
3
2



56
2
2
1



57
1
2
2



71
2
3
2



72
—
2
2



84
2
2
1



87
—
3
2



92
—
2
1



95
—
3
2



113
1
—
2



114
2
—
1



115
2
—
2



116
1
—
2



122
—
2
2



248
4
—
5



299
<1  
—
2










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-GloTM Luminescent Cell Viability Assay. The IC50 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.










TABLE 2







Ex
IC50 (nM)



















7
18



11
13



27
13



33
26



56
13



57
5



71
10



72
24



84
16



87
20



92
7



95
57



113
11



114
7



115
36



116
8



122
7



248
50



299
10










Test Example 3
Antitumor Test on UM-UC-14 (FGFR3_S249C-Positive Cells, Bladder Cancer)
3×106 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 mm3, 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 (mm3; 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.










TABLE 3







Ex
Antitumor Activity




















7
33%
inhibition



11
53%
regression



27
62%
inhibition



33
70%
inhibition



56
4%
regression



57
77%
inhibition



71
50%
inhibition



72
30%
inhibition



84
34%
regression



87
72%
inhibition



92
49%
inhibition



95
97%
inhibition



113
4%
regression



114
33%
regression



115
70%
inhibition



116
54%
regression



122
15%
regression



248
95%
inhibition



299
15%
regression










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% CO2 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 1×103 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% CO2 at 37° C. and were counted on the next day (Day 1) and 4 days later (Day 4), using a cell counting reagent (CELLTITER-GloTM 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 1×103 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 1×103 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 1×103 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% CO2 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% CO2 at 37° C. for 4 days, and a cell counting reagent (CellTiter-GloTM 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.















TABLE 4





Ex


v1
v2
v3
v4
RT-112
RT4























56
100
nM
92
91
91
90
90
64



10
nM
84
79
78
82
83
42



1
nM
22
21
20
22
29
5


113
100
nM
91
91
87
88
89
64



10
nM
53
42
32
73
77
39



1
nM
4
2
3
13
23
8


116
100
nM
91
90
86
89
89
63



10
nM
44
31
24
70
72
39



1
nM
5
0
3
11
21
8


122
100
nM
90
88
89
89
89
63



10
nM
84
79
79
82
80
43



1
nM
26
23
25
19
23
6


248
100
nM
84
79
81
83
81
43



10
nM
28
29
20
26
33
10



1
nM
7
11
6
−5
5
3


299
100
nM
92
91
89
90
89
63



10
nM
77
63
51
82
84
45



1
nM
9
4
5
16
31
8









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, 0.5×107 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 MgCl2, 10 mM MnCl2, 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.














TABLE 5







Ex


v1
v2
v3























56
100
nM
92
94
93




10
nM
77
86
85




1
nM
49
33
47



113
100
nM
92
94
96




10
nM
79
74
81




1
nM
28
24
35



116
100
nM
95
95
96




10
nM
79
73
86




1
nM
31
22
41



122
100
nM
94
95
97




10
nM
80
80
85




1
nM
34
27
45



248
100
nM
86
78
91




10
nM
40
25
55




1
nM
7
6
30



299
100
nM
94
95
96




10
nM
84
77
88




1
nM
35
20
47










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 1×103 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% CO2 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% CO2 at 37° C. for 4 days, and a cell counting reagent (CellTiter-GloTM 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.












TABLE 6










FGFR3-BAIAP2L1-



Ex


expressing NIH3T3 Cells





















56
100
nM
90




10
nM
80




1
nM
24



113
100
nM
89




10
nM
70




1
nM
14



116
100
nM
87




10
nM
74




1
nM
15



122
100
nM
90




10
nM
83




1
nM
17



248
100
nM
82




10
nM
19




1
nM
−3



299
100
nM
91




10
nM
81




1
nM
15










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.
EXAMPLES
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, iPr: isopropyl, tBu: 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 1H-NMR in dimethyl sulfoxide-d6, NMR2: δ (ppm) in 1H-NMR in CDCl3, NMR3: δ (ppm) in 1H-NMR in CD3OD, “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-N1-methyl-N1-[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.
Example 1
To a mixture of 5-bromo-N-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine (104 mg), 1-ethynyl-3,5-dimethoxybenzene (37 mg), tetrakistriphenylphosphine palladium (13 mg), copper iodide (4 mg), and N,N-dimethylformamide (2 mL), triethylamine (157 μL) was added under an argon atmosphere followed by stirring at 120° C. for 30 minutes. Further, a mixture of 1-ethynyl-3,5-dimethoxybenzene (146 mg) and N,N-dimethylformamide (1 mL) was added thereto followed by stirring at 120° C. for 2 hours. The reaction mixture was diluted with ethyl acetate, and insoluble materials were removed by filtration through celite. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (chloroform/methanol) and subsequently purified by basic silica gel column chromatography (ethyl acetate/methanol), and then solidified with ethyl acetate to give 5-[(3,5-dimethoxyphenyl)ethynyl]-N-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine (23 mg).
Example 2
To a mixture of 5-[(3,5-dimethoxyphenyl)ethynyl]-N-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine (72 mg), methanol (2 mL), and tetrahydrofuran (2 mL), 10% palladium-carbon (25 mg) was added under an argon atmosphere. After stirring for 4 hours under a hydrogen atmosphere (3 atm), insoluble materials were removed by filtration through celite. After the filtrate was concentrated under reduced pressure, the resulting residue was purified by silica gel column chromatography (chloroform/methanol) and then solidified with diethyl ether to give 5-[2-(3,5-dimethoxyphenyl)ethyl]-N-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine (17 mg).
Example 3
To a mixture of 5-iodo-N-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine (100 mg), 2,4-dichloro-3-ethynyl-1,5-dimethoxybenzene (55 mg), tetrakistriphenylphosphine palladium (23 mg), copper iodide (2 mg), and N,N-dimethylformamide (2 mL), N,N-diisopropylethylamine (67 μL) was added under an argon atmosphere followed by stirring at 100° C. for 4 hours. The reaction mixture was diluted with ethyl acetate, and insoluble materials were removed by filtration through celite. To the filtrate, a saturated aqueous sodium hydrogen carbonate solution was added followed by extraction with ethyl acetate. An organic layer obtained was washed with 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) and subsequently purified by basic silica gel column chromatography (ethyl acetate/methanol), and then solidified with ethyl acetate to give 5-[(2,6-dichloro-3,5-dimethoxyphenyl)ethynyl]-N-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine (56 mg).
Example 4
To a mixture of 5-[(2,6-dichloro-3,5-dimethoxyphenyl)ethynyl]-N-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine (92 mg) and ethyl acetate (6 mL), a 4M hydrogen chloride/ethyl acetate solution (1 mL) was added followed by stirring at room temperature for 4 hours. The resulting solid was collected by filtration and dried under reduced pressure to give 5-[(2,6-dichloro-3,5-dimethoxyphenyl)ethynyl]-N-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine trihydrochloride (101 mg).
Example 5
A mixture of 5-[2-(3,5-dimethoxyphenyl)ethyl]-N-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine (131 mg) and acetonitrile (1.3 mL) was ice cooled, and then sulfuryl chloride (41 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 column chromatography (chloroform/methanol) and then washed with diisopropyl ether to give N-{2-chloro-5-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}-5-[2-(2,6-dichloro-3,5-dimethoxyphenyl)ethyl]pyrimidin-2-amine (29 mg).
Example 6
Under an argon atmosphere, a mixture of 5-[(2,6-difluoro-3,5-dimethoxyphenyl)ethynyl]-N-{3-methoxy-4-[4-(1-methylpiperidin-4-yl)piperazin-1-yl]phenyl}pyrimidin-2-amine (164 mg), 4-methylbenzenesulfonyl hydrazide (2.63 g), and 1,2-dimethoxyethane (3 mL) was stirred at 110° C., and a mixture of sodium acetate (1.16 g) and water (1 mL) was added thereto. After 2 hours, 4-methylbenzenesulfonyl hydrazide (1.32 g) was added thereto, and then an additional mixture of sodium acetate (581 mg) and water (1 mL) was added thereto followed by stirring at 110° C. for 2 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 magnesium 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/conc. aqueous ammonia solution) and then solidified with ethyl acetate/diisopropyl ether to give 5-[2-(2,6-difluoro-3,5-dimethoxyphenyl)ethyl]-N-{3-methoxy-4-[4-(1-methylpiperidin-4-yl)piperazin-1-yl]phenyl}pyrimidin-2-amine (114 mg).
Example 7
A mixture of 5-[(2,6-difluoro-3,5-dimethoxyphenyl)ethynyl]-N-[3-methoxy-4-(4-methylpiperazin-1-yl)phenyl]pyrimidin-2-amine (100 mg), tetrahydrofuran (5 mL), and methanol (5 mL) was reacted using H-Cube (trademark) (10% palladium-carbon, 0.5 mL/min, 50° C., 1 atm). The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (chloroform/methanol) and then solidified with ethyl acetate/diisopropyl ether to give 5-[2-(2,6-difluoro-3,5-dimethoxyphenyl)ethyl]-N-[3-methoxy-4-(4-methylpiperazin-1-yl)phenyl]pyrimidin-2-amine (29 mg).
Example 8
To a mixture of ethyl [4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]acetate (292 mg), tetrahydrofuran (6 mL), and ethanol (6 mL), a 1M aqueous sodium hydroxide solution (1.3 mL) was added at room temperature followed by stirring for 5 hours. The reaction mixture was neutralized with 1M hydrochloric acid and the resulting solid was collected by filtration. The solid was washed with water and dried under reduced pressure to give [4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]acetic acid (267 mg).
Example 9
To a mixture of 2-chloro-5-[2-(2,6-difluoro-3,5-dimethoxyphenyl)ethyl]pyrimidine (56 mg), 1-(1-methylpiperidin-4-yl)-1H-pyrazol-4-amine (48 mg), 1,1′-binaphthalene-2,2′-diylbis(diphenylphosphine) (33 mg), cesium carbonate (174 mg), and dioxane (2.2 mL), palladium acetate (8 mg) was added at room temperature under an argon atmosphere followed by stirring at 100° C. for 4 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 (chloroform/methanol) and then solidified with ethyl acetate/diisopropyl ether to give 5-[2-(2,6-difluoro-3,5-dimethoxyphenyl)ethyl]-N-[1-(1-methylpiperidin-4-yl)-1H-pyrazol-4-yl]pyrimidin-2-amine (43 mg).
Example 10
To a mixture of 1-(bromomethyl)-2,6-difluorobenzene (14 mg), 2-chloro-5-hydroxypyrimidine (9.1 mg), and N,N-dimethylformamide (1 mL), potassium carbonate (16 mg) was added followed by stirring at room temperature overnight. To the reaction mixture, water was added followed by extraction with chloroform. An organic layer obtained was concentrated under reduced pressure. To the resulting residue, a mixture of 3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]aniline (30 mg), cesium carbonate (65 mg), palladium acetate-X-Phos (Pd:P=1:2) ChemDose (trademark) tablet, and tert-butyl alcohol (0.5 mL) was added followed by stirring at 120° C. overnight under a nitrogen atmosphere. To the reaction mixture, water was added followed by extraction with chloroform. An organic layer obtained was concentrated under reduced pressure. The resulting residue was purified by HPLC (0.1% aqueous formic acid solution/methanol) to give 5-[(2,6-difluorobenzyl)oxy]-N-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine (17 mg).
Example 11
To a mixture of 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) and tetrahydrofuran (2 mL), 1M hydrochloric acid (1 mL) was added followed by stirring at 50° C. for 3 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 sodium sulfate, and then filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (chloroform/methanol) and then solidified with ethyl acetate to give (2S)-3-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]propane-1,2-diol (27 mg).
Example 12
A mixture of tert-butyl 4-[4-({5-[(2,6-dichloro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-3-methoxyphenyl]piperidin-1-carboxylate (298 mg) and chloroform (6 mL) was ice cooled, and trifluoroacetic acid (1 mL) was added thereto followed by stirring at room temperature for 4 hours. After the reaction mixture was ice cooled, a 1M aqueous sodium hydroxide solution (10 mL) and a saturated aqueous sodium hydrogen carbonate solution were added thereto for basification followed by extraction with chloroform. 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 a crude product (273 mg). Further, the crude product (60 mg) was purified by silica gel chromatography (chloroform/methanol/conc. aqueous ammonia solution), and then solidified with ethyl acetate/diisopropyl ether to give 5-[(2,6-dichloro-3,5-dimethoxybenzyl)oxy]-N-[2-methoxy-4-(piperidin-4-yl)phenyl]pyrimidin-2-amine (23 mg).
Example 13
To a mixture of 5-[(2,6-dichloro-3,5-dimethoxybenzyl)oxy]-N-[2-methoxy-4-(piperidin-4-yl)phenyl]pyrimidin-2-amine (63 mg), dichloromethane (2 mL), and methanol (1 mL), 1H-benzotriazol-1-ylmethanol (20 mg) was added followed by stirring at room temperature for 1 hour. Subsequently, sodium triacetoxy borohydride (51 mg) was added thereto followed by stirring at room temperature for 2 hours. To the reaction mixture, a saturated aqueous sodium hydrogen carbonate solution was added and extraction with chloroform was performed. An organic layer obtained was washed with saturated brine, dried over anhydrous magnesium sulfate, and then filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (chloroform/methanol/conc. aqueous ammonia solution) and then solidified with ethyl acetate/diisopropyl ether to give 5-[(2,6-dichloro-3,5-dimethoxybenzyl)oxy]-N-[2-methoxy-4-(1-methylpiperidin-4-yl)phenyl]pyrimidin-2-amine (28 mg).
Example 14
To a mixture of 5-[(2,6-dichloro-3,5-dimethoxybenzyl)oxy]-N-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]pyrimidin-2-amine (200 mg), ethanol (3 mL), and N,N-dimethylformamide (3 mL), 2,2-dimethyloxyrane (112 μL) was added 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, 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) and then solidified with ethyl acetate to give 1-{4-[4-({5-[(2,6-dichloro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]piperidin-1-yl}-2-methylpropan-2-ol (93 mg).
Example 15
A mixture of 5-[(2,6-dichloro-3,5-dimethoxybenzyl)oxy]-N-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]pyrimidin-2-amine (150 mg), triethylamine (131 μL), and dichloromethane (4 mL) was ice cooled, and cyclopropanecarbonyl chloride (29 μL) 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. The filtrate was concentrated under reduced pressure, and the resulting residue was solidified with ethyl acetate to give cyclopropyl {4-[4-({5-[(2,6-dichloro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]piperidin-1-yl}methanone (159 mg).
Example 16
To a mixture of 5-[(2,6-dichloro-3,5-dimethoxybenzyl)oxy]-N-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]pyrimidin-2-amine (150 mg), potassium carbonate (130 mg), and N,N-dimethylformamide (4 mL), 2-bromoethyl methyl ether (32 μL) was added followed by stirring at room temperature overnight and stirring at 60° C. for 3 hours. Additional 2-bromoethyl methyl ether (12 μL) was added thereto followed by stirring at 60° C. for 4 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. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by basic silica gel column chromatography (ethyl acetate/hexane) and then solidified with ethyl acetate to give 5-[(2,6-dichloro-3,5-dimethoxybenzyl)oxy]-N-{1-[1-(2-methoxyethyl)piperidin-4-yl]-1H-pyrazol-4-yl}pyrimidin-2-amine (41 mg).
Example 17
To a mixture of ethyl 1-methyl-5-({5-[(2,3,5,6-tetrafluorobenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-3-carboxylate (663 mg), ethanol (6.6 mL), and tetrahydrofuran (6.6 mL), a 1M aqueous sodium hydroxide solution (3.2 mL) was added followed by stirring at room temperature for 4 hours. To the reaction mixture, 1M hydrochloric acid (3.2 mL) was added, and the resulting solid was collected by filtration, washed with water, and dried under reduced pressure to give 1-methyl-5-({5-[(2,3,5,6-tetrafluorobenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-3-carboxylic acid (464 mg).
Example 18
To a mixture of 1-methyl-5-({5-[(2,3,5,6-tetrafluorobenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-3-carboxylic acid (100 mg), 1-methylpiperazine (83 μL), 1H-benzotriazol-1-ol (68 mg), and N,N-dimethylformamide (2 mL), N-[3-(diethylamino)propyl]-N′-ethylcarbodiimide hydrochloride (97 mg) was added 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, dried over anhydrous magnesium sulfate, and then filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography (chloroform/methanol/conc. aqueous ammonia solution) to give (4-methylpiperazin-1-yl)[1-methyl-5-({5-[(2,3,5,6-tetrafluorobenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-3-yl]methanone (79 mg).
Example 19
A mixture of 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 (232 mg) and dichloromethane (3 mL) was ice cooled, and trifluoroacetic acid (0.5 mL) was added thereto followed by stirring at room temperature for 1 hour. 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. The resulting solid was collected by filtration, washed with ethyl acetate, and then dried under reduced pressure to give N-[4-(3,9-diazaspiro[5,5]undec-3-yl)-3-methoxyphenyl]-5-[(2,3,5,6-tetrafluorobenzyl)oxy]pyrimidin-2-amine (167 mg).
Example 20
To a mixture of 2-[3-({5-[(2,3,5,6-tetrafluorobenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]ethyl methanesulfonate (160 mg) and N-methylpyrrolidone (6 mL), 1-methylpiperazine (382 μL) was added followed by stirring at 80° C. for 2 hours. 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 sodium sulfate, and then filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (chloroform/methanol/conc. aqueous ammonia solution) and then solidified with ethyl acetate/diisopropyl ether to give N-{1-[2-(4-methylpiperazin-1-yl)ethyl]-1H-pyrazol-3-yl}-5-[(2,3,5,6-tetrafluorobenzyl)oxy]pyrimidin-2-amine (64 mg).
Example 21
A mixture of N-[3-methoxy-4-(4-methylpiperazin-1-yl)phenyl]-5-[(2,3,5,6-tetrafluorobenzyl)oxy]pyrimidin-2-amine (100 mg) and chloroform (4 mL) was ice cooled, and m-chloroperbenzoic acid (43 mg) was added thereto followed by stirring at 4 to 10° C. for 3 hours and stirring at room temperature for 2 hours. To the reaction mixture, an aqueous sodium thiosulfate solution was added, and the resulting mixture was stirred at room temperature for 1 hour followed by extraction with chloroform. An organic layer obtained was washed with saturated brine, dried over anhydrous magnesium sulfate, and then filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by basic silica gel column chromatography (chloroform/methanol) and then solidified with ethyl acetate/diisopropyl ether to give N-[3-methoxy-4-(4-methyl-4-oxidopiperazin-1-yl)phenyl]-5-[(2,3,5,6-tetrafluorobenzyl)oxy]pyrimidin-2-amine (16 mg).
Example 22
To a mixture of 2-chloro-5-[(2,6-dichloro-3,5-dimethoxybenzyl)oxy]pyridine (100 mg), 3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]aniline (87 mg), 2-(dicyclohexylphosphino)-2′,4′,6′-triisopropyl-1,1′-biphenyl (27 mg), sodium tert-butoxide (41 mg), and N-methylpyrrolidone (3 mL), palladium acetate (6.4 mg) was added under an argon atmosphere followed by stirring at 160° C. for 2 hours under microwave irradiation. 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, and the resulting residue was purified by basic silica gel column chromatography (chloroform/methanol) to give 5-[(2,6-dichloro-3,5-dimethoxybenzyl)oxy]-N-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyridin-2-amine (27 mg).
Example 23
A mixture of 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-(1H-pyrazol-4-yl)pyrimidin-2-amine (200 mg), cesium carbonate (215 mg), (2S)-2-methyloxylane (128 mg), and N-methylpyrrolidone (4 mL) was stirred at 130° C. for 30 minutes under microwave irradiation. 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 magnesium sulfate, and then filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (ethyl acetate/hexane) and then washed with ethyl acetate/diisopropyl ether to give (2S)-1-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]propan-2-ol (171 mg).
Example 24
To a mixture of [4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]acetic acid (50 mg), ammonium chloride (25 mg), triethylamine (66 μL), 1H-benzotriazol-1-ol (32 mg) and N,N-dimethylformamide (1 mL) was added N-[3-(diethylamino)propyl]-N′-ethylcarbodiimide hydrochloride (45 mg) followed by stirring at room temperature for 12 hours. To the reaction mixture, water was added followed by extraction with ethyl acetate. An organic layer obtained was concentrated under reduced pressure, and the resulted residue was solidified with diisopropyl ether to give 2-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]acetamide (48 mg).
Example 64
To a mixture of 5-[(2,6-dichloro-3,5-dimethoxybenzyl)oxy]-N-[2-methoxy-4-(piperidin-4-yl)phenyl]pyrimidin-2-amine (62 mg), acetone (118 μL), and dichloromethane (3 mL), sodium triacetoxy borohydride (51 mg) was added 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 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) and then solidified with ethyl acetate/diisopropyl ether to give 5-[(2,6-dichloro-3,5-dimethoxybenzyl)oxy]-N-[4-(1-isopropylpiperidin-4-yl)-2-methoxyphenyl]pyrimidin-2-amine (14 mg).
Example 106
To a mixture of 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-{4-(piperazin-1-yl)-3-[2-(1H-pyrazol-1-yl)ethoxy]phenyl}pyrimidin-2-amine (229 mg), formaldehyde (37% aqueous solution, 164 μL), acetic acid (231 μL), and dichloromethane (6 mL), sodium triacetoxy borohydride (257 mg) was added 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/methanol) and then solidified with ethyl acetate/diisopropyl ether to give 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-{4-(4-methylpiperazin-1-yl)-3-[2-(1H-pyrazol-1-yl)ethoxy]phenyl}pyrimidin-2-amine (72 mg).
Example 120
To a mixture of 1-[3-({5-[(2,3,5,6-tetrafluorobenzyl)oxy]pyrimidin-2-yl}amino)phenyl]piperidin-4-one (209 mg), 1-methylpiperazine (103 μL), and dichloromethane (4 mL), sodium triacetoxy borohydride (298 mg) was added 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 silica gel column chromatography (chloroform/methanol/conc. aqueous ammonia solution) and then solidified with ethyl acetate/diisopropyl ether to give N-{3-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}-5-[(2,3,5,6-tetrafluorobenzyl)oxy]pyrimidin-2-amine (98 mg).
Example 161
To a mixture of 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-[4-(piperidin-4-yl)phenyl]pyrimidin-2-amine (52 mg), glycolic acid (26 mg), 1H-benzotriazol-1-ol (31 mg), and N,N-dimethylformamide (1 mL), N-[3-(diethylamino)propyl]-N′-ethylcarbodiimide hydrochloride (44 mg) was added followed by stirring at room temperature for 2 days. 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 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 1-{4-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)phenyl]piperidin-1-yl}-2-hydroxyethanone (10 mg).
Example 162
To a mixture of 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-{3-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine (123 mg) and ethanol (3 ml), fumaric acid (26 mg) was added followed by heating to reflux. To the reaction mixture, water was added followed by stirring at room temperature overnight, and the resulting solid was collected by filtration. The solid was washed with ethanol and then dried under reduced pressure to give 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-{3-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine hemifumarate (82 mg).
Example 166
To a mixture of tert-butyl 4-[4-({5-[2-(2,6-difluoro-3,5-dimethoxyphenyl)ethyl]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]piperidine-1-carboxylate (276 mg) and ethyl acetate (2 mL), a 4M hydrogen chloride/ethyl acetate solution (2 mL) was added followed by stirring at room temperature for 3 hours. After the reaction mixture was concentrated under reduced pressure, a saturated aqueous sodium hydrogen carbonate solution was added to the resulting residue 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 basic silica gel column chromatography (chloroform/methanol) and then solidified with ethyl acetate/diisopropyl ether to give 5-[2-(2,6-difluoro-3,5-dimethoxyphenyl)ethyl]-N-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]pyrimidin-2-amine (119 mg).
Example 190
To a mixture of tert-butyl {2-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]ethyl}carbamate (101 mg) and ethyl acetate (2 mL), a 4M hydrogen chloride/ethyl acetate solution (2 mL) was added followed by stirring at room temperature for 3 hours. The resulting solid was collected by filtration and then dried under reduced pressure to give N-[1-(2-aminoethyl)-1H-pyrazol-4-yl]-5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-amine trihydrochloride (100 mg).
Example 212
To a mixture of 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-{1-[3-(tetrahydro-2H-pyran-2-yloxy)propyl]-1H-pyrazol-4-yl}pyrimidin-2-amine (1.6 g), tetrahydrofuran (6.9 mL), and water (3.4 mL), acetic acid (13.8 mL) was added followed by stirring at 70° C. for 2 days. After the reaction mixture was concentrated under reduced pressure, a saturated aqueous sodium hydrogen carbonate solution was added to the resulting residue followed by extraction with chloroform. An organic layer obtained was dried over anhydrous magnesium sulfate and then filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was dissolved in methanol (30 mL). Potassium carbonate (656 mg) was added thereto followed by stirring at 60° C. for 5 hours. To the reaction mixture, water was added and extraction with chloroform was performed. An organic layer obtained was dried over anhydrous magnesium sulfate and then filtered. The filtrate was concentrated under reduced pressure to give 3-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]propan-1-ol (510 mg).
Example 213
To a mixture of 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-{6-[(2-phenyl-1,3-dioxan-5-yl)oxy]pyridin-3-yl}pyrimidin-2-amine (335 mg) and acetic acid (10 mL), water (2 mL) was added followed by stirring at 60° C. for 16 hours. After the solvent was concentrated under reduced pressure, a saturated aqueous sodium hydrogen carbonate solution was added to the resulting residue 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 silica gel column chromatography (chloroform/methanol) and then solidified with ethyl acetate to give 2-{[5-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)pyridin-2-yl]oxy}propane-1,3-diol (92 mg).
Example 214
To a mixture of 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-{6-[2-(tetrahydro-2H-pyran-2-yloxy)ethoxy]pyridin-3-yl}pyrimidin-2-amine (1.49 g) and methanol (5 mL), a 4M hydrogen chloride/dioxane solution (5 mL) was added followed by stirring at room temperature for 2 hours. After the reaction mixture was concentrated under reduced pressure, a saturated aqueous sodium hydrogen carbonate solution was added to the resulting residue followed by extraction with chloroform. An organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. The filtrate was concentrated under reduced pressure, and the resulted residue was solidified with ethyl acetate. The solid was collected by filtration to give 2-{[5-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)pyridin-2-yl]oxy}ethanol (452 mg). Further the filtrate was purified by silica gel column chromatography (chloroform/methanol) to give the product (701 mg).
Example 217
To a mixture of 1-[5-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)pyridin-2-yl]piperidin-4-one (256 mg), 2-aminoethanol (131 μL), acetic acid (200 μL), and dichloromethane (9.3 mL), sodium triacetoxy borohydride (243 mg) was added 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/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 basic silica gel column chromatography (chloroform/methanol) and then solidified with ethyl acetate to give 2-({1-[5-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)pyridin-2-yl]piperidin-4-yl}amino)ethanol (137 mg).
Example 239
A mixture of N-{5-[2-(benzyloxy)ethyl]-1-methyl-1H-pyrazol-3-yl}-5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-amine (283 mg) and dichloromethane (47 mL) was cooled to −78° C., and boron tribromide (1.0M dichloromethane solution, 830 μL) was added thereto followed by stirring at −78° C. for 1 hour and stirring at 0° C. for 1 hour. 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) to give 2-[3-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1-methyl-1H-pyrazol-5-yl]ethanol (38 mg).
Example 246
A mixture of 5-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1-methyl-1H-pyrazol-3-carboxylic acid (320 mg) and dioxane (6 mL) was ice cooled, and 1,1′-carbonyldiimidazole (616 mg) was added thereto followed by stirring at room temperature for 2 hours. To the reaction mixture, sodium borohydride (287 mg) was added followed by stirring at room temperature for 12 hours. To the reaction mixture, water and chloroform were added, and insoluble materials were removed by filtration through celite, and then the filtrate was extracted 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 silica gel column chromatography (hexane/ethyl acetate) to give [5-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1-methyl-1H-pyrazol-3-yl]methanol (125 mg).
Example 253
To a mixture of 5-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1-(2-hydroxyethyl)pyridin-2(1H)-one (90 mg), triethylamine (50 μL), and dichloromethane (3 mL), methanesulfonyl chloride (20 μL) was added followed by stirring at room temperature for 1 hour. To the reaction mixture, 1-methylpiperazine (50 μL) and N,N-dimethylformamide (3 mL) were added followed by stirring at 50° C. for 20 hours. To the reaction mixture, water was added 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 basic silica gel column chromatography (ethyl acetate/chloroform) and then solidified with diethyl ether to give 5-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1-[2-(4-methylpiperazin-1-yl)ethyl]pyridin-2(1H)-one (28 mg).
Example 254
To a mixture of 2-chloro-5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidine (270 mg), 1-(1-methylpiperidin-4-yl)-1H-imidazol-4-amine (231 mg), 1,1′-binaphthalene-2,2′-diylbis(diphenylphosphine) (80 mg), cesium carbonate (556 mg), and dioxane (5.4 mL), palladium acetate (19 mg) was added under an argon atmosphere followed by stirring at 150° C. for 30 minutes under microwave irradiation. To the reaction mixture, water 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 silica gel column chromatography (chloroform/methanol) and then solidified with ethanol/diethyl ether to give 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-[1-(1-methylpiperidin-4-yl)-1H-imidazol-4-yl]pyrimidin-2-amine (241 mg).
Example 278
To a mixture of 2-chloro-5-[(2-fluoro-3,5-dimethoxybenzyl)oxy]pyrimidine (200 mg), 2-(4-amino-1H-pyrazol-1-yl)ethanol (170 mg), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (39 mg), cesium carbonate (655 mg), and dioxane (4 mL), tris(dibenzylideneacetone)dipalladium (31 mg) was added under an argon atmosphere followed by stirring at 80° C. overnight. 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 basic silica gel column chromatography (ethyl acetate/methanol) and then solidified with ethanol to give 2-[4-({5-[(2-fluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]ethanol (58 mg).
Example 282
To a mixture of 5-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-3-carbaldehyde (100 mg), morpholine (67 μL), and N,N-dimethylformamide (2 mL), sodium triacetoxy borohydride (243 mg) was added followed by stirring at room temperature 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 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 (chloroform/methanol) and purified by silica gel column chromatography (chloroform/methanol) and then solidified with ethanol/diisopropyl ether to give 5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-[3-(morpholin-4-ylmethyl)-1H-pyrazol-5-yl]pyrimidin-2-amine (42 mg).
Example 286
To a mixture of 2-chloro-5-[(2,6-dichloro-3,5-dimethoxybenzyl)oxy]pyrimidine (159 mg), 3-methoxy-4-(1-methylpiperidin-4-yl)aniline (100 mg), and tert-butanol (5 mL), tris(dibenzylideneacetone)dipalladium (13 mg), 2-(dicyclohexylphosphino)-2′,4′,6′-triisopropyl-1,1′-biphenyl (20 mg), and potassium carbonate (88 mg) were added under an argon atmosphere followed by stirring at 100° C. for 8 hours. Insoluble materials were removed by filtration, washed with ethyl acetate, and then the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform/methanol) to give 5-[(2,6-dichloro-3,5-dimethoxybenzyl)oxy]-N-[3-methoxy-4-(1-methylpiperidin-4-yl)phenyl]pyrimidin-2-amine (35 mg).
Example 302
To a mixture of 2-{4-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)phenyl]piperazin-1-yl}ethyl methanesulfonate (100 mg) and methanol (3 mL), sodium methoxide (25% methanol solution, 3 mL) was added followed by stirring at 90° C. for 15 minutes under microwave irradiation. After the reaction mixture was concentrated under reduced pressure, 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 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-dimethoxybenzyl)oxy]-N-{4-[4-(2-methoxyethyl)piperazin-1-yl]phenyl}pyrimidin-2-amine (46 mg).
Example 315
To a mixture of {1′-[4-({15-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-2-methoxyphenyl]-1,4′-bipiperidine-4,4-diyl}bis(methylene)diacetate (46 mg) and methanol (3 mL), sodium methoxide (25% methanol solution, 0.2 mL) was added followed by stirring at room temperature for 14 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (chloroform/methanol) to give {1′-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-2-methoxyphenyl]-1,4′-bipiperidine-4,4-diyl}dimethanol (34 mg).
Example 336
A mixture of ethyl 4-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-2-fluorophenyl]butanoate (150 mg) and tetrahydrofuran (3 mL) was ice cooled, and lithium aluminum hydride (11 mg) was added thereto followed by stirring at room temperature for 3 hours. The reaction mixture was diluted with diethyl ether 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 (chloroform/methanol) to give 4-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-2-fluorophenyl]butan-1-ol (70 mg).
Example 349
A mixture of ethyl 4-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-2-fluorophenyl]butanoate (150 mg) and tetrahydrofuran (3 mL) was ice cooled, and then methyl magnesium bromide (1.0M tetrahydrofuran solution, 1.2 mL) was added thereto followed by stirring for 3 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 (chloroform/methanol) to give 5-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-2-fluorophenoxy]-2-methylpentan-2-ol (104 mg).
Example 356
To a mixture of 2-(4-aminophenoxy)-2-methylpropionic acid (14.6 mg), cesium carbonate (49 mg), 2-chloro-5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidine (15.8 mg) and tert-butanol (0.5 mL) was added palladium(II) acetate-2-(dicyclohexylphosphino)-2′,4′,6′-triisopropyl-1,1′-biphenyl (Pd:P 1:2) ChemDose (trademark) tablet under a nitrogen atmosphere followed by stirring at 120° C. overnight. To the reaction mixture, water was added followed by extraction with chloroform (2 mL), and then the solvent was concentrated under reduced pressure. The resulting residue was purified by HPLC (0.1% aqueous formic acid solution/methanol) to give 2-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)phenoxy]-2-methylpropionic acid (0.7 mg).
Example 375
To a mixture of 4-amino-1-(1-tert-butoxycarbonyl-azetidin-3-yl)-1H-pyrazole (17.9 mg), cesium carbonate (49 mg), 2-chloro-5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidine (15.8 mg), tert-butanol (0.34 mL) and N,N-dimethylformamide (0.16 mL) was added palladium(II) acetate-2-(dicyclohexylphosphino)-2′,4′,6′-triisopropyl-1,1′-biphenyl (Pd:P 1:2) ChemDose (trademark) tablet under a nitrogen atmosphere followed by stirring at 120° C. overnight. To the reaction mixture, water was added followed by extraction with chloroform (2 mL), and then the solvent was concentrated under reduced pressure. To the resulting residue, ethanol (1 mL) and a 4M hydrogen chloride/ethyl acetate solution (0.5 mL) were added followed by stirring at room temperature overnight. After that, the solvent was concentrated under reduced pressure. The resulting residue was purified by HPLC (0.1% aqueous formic acid solution/methanol) to give N-[1-(azetidin-3-yl)-1H-pyrazol-4-yl]-5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-amine (1.7 mg).
In the same manner as in the examples shown above, the compounds shown in Tables 63 to 137 below were prepared. Tables 63 to 137 show the structures of the compounds of the examples and Tables 138 to 156 show the preparation processes and physical and chemical data of the compounds of the examples.










TABLE 7





PEx
PSyn
Str
DAT







1
1




APCI/ESI+: 461,463


 


2
2




APCI/ESI+: 509


 


3
3




NMR2: 3.68 (1H,  s), 3.92 (6H, s), 6.58 (1H, s)


 


4-1
4




APCI/ESI+: 199


 


4-2
4




APCI/ESI+: 181


 


5
5




NMR2: 3.60 (1H,  S), 3.89 (3H, S), 3.91 (3H, S),  6.61 (1H, d, J =  7.5 Hz)



















TABLE 8





PEx
PSyn
Str
DAT







6
6




EI: 168


 


7
7




APCI/ESI+: 292


 


8
8




APCI/ESI+: 296


 


9
9




APCI/ESI+: 315


 


10
10




APCI/ESI+: 281



















TABLE 9





PEx
PSyn
Str
DAT







13
13




APCI/ESI+: 281


 


14
14




APCI/ESI+: 349


 


15
15




ESI+: 295


 


16-1
16




ESI+: 233


 


16-2
16




ESI+: 215



















TABLE 10





PEx
PSyn
Str
DAT







17
17




ESI+: 205


 


18
18




NMR2: 3.04 (3H,  s), 3.88 (6H, s),  5.34 (2H, s),  6.72 (1H, t, J =  8.2 Hz)


 


19
19




APCI/ESI+: 317


 


20
20




APCI/ESI+: 176


 


21
21




NMR2: 4.03  (6H, s), 10.3  (1H, s)


 


22
22




APCI/ESI+: 206


 


23
23




APCI/ESI+: 619



















TABLE 11





PEx
PSyn
Str
DAT







24
24




ESI+: 354


 


25
25




ESI+: 324


 


26
26




ESI+: 371


 


27
27




APCI/ESI+: 341


 


28
28




NMR2: 1.10-1.19 (2H, m), 1.12 (6H, s), 1.19 (6H, s), 1.69-1.76 (2H, m), 2.27  (1H, brs), 2.55-2.59 (4H,  m), 2.76-2.87 (1H, m),  3.48-3.55 (4H, m), 5.13 (2H, s), 7.23-7.40 (5H, m)


 


29
29




APCI/ESI+: 226



















TABLE 12





PEx
PSyn
Str
DAT







30
30




ESI+: 632


 


31
31




ESI+: 447


 


32
32




APCI/ESI+:  462


 


33
33




APCI/ESI+:  294


 


34
34




ESI+: 309


 


35
35




APCI/ESI+:  366



















TABLE 13





PEx
PSyn
Str
DAT







36
36




APCI/ESI+:  268


 


37
37




APCI/ESI+:  346


 


38
38




APCI/ESI+:  348


 


39
39




APCI/ESI+:  364


 


40
40




APCI/ESI+:  450


 


41
41




APCI/ESI+:  478



















TABLE 14





PEx
PSyn
Str
DAT







42
2




APCI/ESI+:  497


 


43
2




APCI/ESI+:  509


 


44
6




EI: 138


 


45
6




EI: 174


 


46
14




APCI/ESI+:  363


 


47
18




APCI/ESI+:  284



















TABLE 15





PEx
PSyn
Str
DAT







48
19




APCI/ESI+:  318


 


49
13




APCI/ESI+:  293


 


50
24




NMR2: 1.06-1.26  (14H, m), 1.77-1.84  (2H, m), 2.74-2.91  (6H, m), 3.22-3.31  (4H, m), 3.95 (3H,  s), 6.88 (1H, d, J =  8.8 Hz), 7.70 (1H,  s), 7.86 (1H, d, J =  8.8 Hz)


 


51
27




APCI/ESI+:  347


 


52
23




APCI/ESI+:  579


 


53
23




APCI/ESI+:  426



















TABLE 16





PEx
PSyn
Str
DAT







54
24




NMR2: 1.47-1.71 (17H, m), 3.17- 3.20 (4H, m),  3.40-3.43 (4H,  m), 3.95 (3H, s), 6.89 (1H, d, J =  8.8 Hz), 7.69  (1H, d, J = 2.7  Hz), 7.85 (1H, dd, J = 8.8, 2.4  Hz)


 


55
27




NMR2: 1.46-1.87 (13H, m), 2.88- 2.90 (4H, m),  3.38-3.41 (4H,  m), 3.73-3.81  (6H, m), 6.23- 6.26 (2H, m),  6.78 (1H, d,  J = 8.1 Hz)


 


56
24




ESI+: 320


 


57
27




ESI+: 290


 


58
23




ESI+: 491



















TABLE 17





PEx
PSyn
Str
DAT







59
23




ESI+: 549


 


60
24




NMR2: 1.45 (9H, s), 3.84 (3H, s), 4.10 (4H, s), 4.26 (4H, s), 6.22 (1H, d, J = 9.0  Hz), 7.60 (1H, d,  J = 2.4 Hz), 7.82  (1H, dd, J = 8.8, 2.4 Hz)


 


61
27




NMR2: 1.44 (9H, s),  3.40 (2H, brs), 3.74 (3H, s), 3.87 (4H, s), 4.05 (4H, s), 6.21-6.31 (3H, m)


 


62
30




ESI+: 576


 


63
27




APCI/ESI+:  264


 


64
33




APCI/ESI+:  281


 


65
27




ESI+:  251



















TABLE 18





PEx
PSyn
Str
DAT







66
24




ESI+: 330


 


67
27




ESI+: 300


 


68
27




ESI+: 311


 


69
24




ESI+: 339


 


70
24




APCI/ ESI+: 266


 


71
27




APCI/ ESI+: 336



















TABLE 19





PEx
PSyn
Str
DAT







72
23




APCI/ ESI+: 616


 


73
28




APCI/ ESI+: 332


 


74
35




APCI/ ESI+: 432


 


75
29




APCI/ ESI+: 298


 


76
24




APCI/ ESI+: 449


 


77
27




APCI/ ESI+: 419



















TABLE 20





PEx
PSyn
Str
DAT







78
23




APCI/ ESI+: 699


 


79
24




NMR2: 1.23 (6H,  d, J = 6.3 Hz),  2.44-2.50 (2H, m),  3.50-3.52 (2H, m), 3.85-3.90 (2H, m),  3.95 (3H, s), 6.85  (1H, d, J = 9.0 Hz),  7.71 (1H, d, J =  2.4 Hz), 7.85 (1H,  dd, J = 8.8, 2.7 Hz)


 


80
27




NMR2: 1.20 (6H,  d, J = 6.3 Hz),  2.27-2.32 (2H, m),  3.15-3.18 (2H, m), 3.49 (2H, brs),  3.82-3.93 (5H, m),  6.23-6.27 (2H, m),  6.74 (1H, d, J = 8.1 Hz)


 


81
30




ESI+: 656


 


82
30




ESI+: 600



















TABLE 21





PEx
PSyn
Str
DAT







83
24




ESI+: 349


 


84
27




ESI+: 319


 


85
28




ESI+: 338


 


86
27




ESI+: 308


 


87
35




APCI/ ESI+: 446


 


88
27




APCI/ ESI+: 416



















TABLE 22





PEx
PSyn
Str
DAT







89
23




APCI/ESI+: 696


 


90
37




APCI/ESI+: 418


 


91
27




APCI/ESI+: 388


 


92
23




APCI/ESI+: 668


 


93
36




APCI/ESI+: 295



















TABLE 23





PEx
PSyn
Str
DAT







94
37




APCI/ESI+: 373


 


95
35




APCI/ESI+: 473


 


96
27




APCI/ESI+: 443


 


97
23




ESI+: 723



















TABLE 24





PEx
PSyn
Str
DAT


















98
30




ESI+: 688


 


99
30




ESI+: 632


 


100
13




APCI/ESI+: 280


 


101
2




APCI/ESI+: 509


 


102
2




APCI/ESI+: 426


 


103
2




APCI/ESI+: 456



















TABLE 25





PEx
PSyn
Str
DAT







104
32




APCI/ESI+: 486


 


105
23




APCI/ESI+: 448


 


106
41




APCI/ESI+: 478


 


107
19




ESI+: 317


 


108
32




APCI/ESI+: 462



















TABLE 26





PEx
PSyn
Str
DAT


















109
23




APCI/ESI+: 545


 


110
31




APCI/ESI+: 501


 


111
111




NMR2: 1.50 (9H, s), 1.72-1.81  (2H, m), l. 95-2.24 (6H, m),  4.32-4.49 (2H, m), 4.61-4.72  (1H, m), 8.06 (1H, s), 8.12 (1H, s)


 


112
27




NMR2: 1.49 (9H, s), 1.71-1.81 (2H,  m), 1.90-2.12 (6H, m), 2.87 (2H, brs),  4.20-4.45 (2H, m), 4.50-4.61 (1H, m),  6.98 (1H, s), 7.12 (1H, s)



















TABLE 27





PEx
PSyn
Str
DAT







113
23




APCI/ESI+: 605


 


114
23




APCI/ESI+: 523


 


115
23




APCI/ESI+: 547


 


116
23




ESI+: 557


 


117
23




APCI/ESI+: 515



















TABLE 28





PEx
PSyn
Str
DAT


















118
118




APCI/ESI+: 223


 


119
27




APCI/ESI+: 193


 


120
23




APCI/ESI+: 558


 


121
23




APCI/ESI+: 576


 


122
23




APCI/ESI+: 614


 


123
23




APCI/ESI+: 543



















TABLE 29





PEx
PSyn
Str
DAT







124
31




APCI/ESI+: 499


 


125
24




APCI/ESI+: 295


 


126
17




NMR2: 1.74-1.80 (1H,  m), 3.79 (3H, s), 3.86 (3H, s), 4.74 (2H, dd, J = 6.2, 1.2 Hz), 6.47 (1H, dd, J = 7.0, 3.0  Hz), 6.51 (1H, dd, J =  4.8, 3.0 Hz)


 


127
23




APCI/ESI+: 586


 


128
35




APCI/ESI+: 395


 


129
27




APCI/ESI+: 365



















TABLE 30





PEx
PSyn
Str
DAT


















130
27




APCI/ESI+: 265


 


131
18




NMR2: 3.01 (3H,  s), 3.79 (3H, s), 3.87 (3H, s), 5.26 (2H, d, J = 1.6 Hz), 6.47 (1H, dd, J =  4.7, 3.0 Hz), 6.57 (1H, d, J = 7.0, 3.0 Hz)


 


132
19




APCI/ESI+: 299


 


133
133




APCI/ESI+: 279


 


134
31




APCI/ESI+: 471



















TABLE 31





PEx
PSyn
Str
DAT


















135
23




APCI/ESI+: 450


 


136
23




APCI/ESI+: 573


 


137
23




APCI/ESI+: 545


 


138
138




APCI/ESI+: 307


 


139
27




APCI/ESI+: 277


 


140
27




APCI/ESI+: 208



















TABLE 32





PEx
PSyn
Str
DAT


















141
24




APCI/ESI+: 320


 


142
27




APCI/ESI+: 290


 


143
143




ESI+: 184


 


144
144




ESI+: 198


 


145
145




ESI+: 255


 


146
189




ESI+: 671


 


147
40




APCI/ESI+: 507



















TABLE 33





PEx
PSyn
Str
DAT


















148
27




APCI/ESI+: 225


 


149
143




APCI/ESI+: 197


 


150
23




APCI/ESI+: 505


 


151
40




ESI+: 575


 


152
152




ESI+: 255


 


153
27




ESI+: 225


 


154
152




ESI+: 303



















TABLE 34





PEx
PSyn
Str
DAT







155
27




ESI+: 273


 


156
24




APCI/ESI+: 336


 


157
27




APCI/ESI+: 306


 


158
40




APCI/ESI+: 464


 


159
40




APCI/ESI+: 464


 


160
40




APCI/ESI+: 478



















TABLE 35





PEx
PSyn
Str
DAT


















161
40




APCI/ESI+: 464


 


162
162




ESI+: 576


 


163
23




APCI/ESI+: 505


 


164
111




ESI+: 256


 


165
152




ESI+: 269


 


166
27




ESI+: 226



















TABLE 36





PEx
PSyn
Str
DAT







167
27




APCI/ESI+: 155(−THP)


 


168
23




APCI/ESI+: 519


 


169
23




ESI+: 506


 


170
23




APCI/ESI+: 553


 


171
23




APCI/ESI+: 516



















TABLE 37





PEx
PSyn
Str
DAT


















172
32




ESI+: 500


 


173
31




APCI/ESI+: 472


 


174
40




ESI+: 561


 


175-1
175




APCI/ESI+: 226


 


175-2
175




APCI/ESI+: 226


 


176
176




ESI+: 232



















TABLE 38





PEx
PSyn
Str
DAT







177
23




ESI+: 512


 


178
23




APCI/ESI+: 506


 


179
23




APCI/ESI+: 506


 


180
23




APCI/ESI+: 506



















TABLE 39





PEx
PSyn
Str
DAT


















181
23




APCI/ESI+: 450


 


182
27




ESI+: 226


 


183
183




APCI/ESI+: 242


 


184
27




APCI/ESI+: 212


 


185
23




APCI/ESI+: 492


 


186
186




APCI/ESI+: 276


 


187
27




APCI/ESI+: 246



















TABLE 40





PEx
PSyn
Str
DAT


















188
23




APCI/ESI+: 526


 


189
189




APCI/ESI+: 267


 


190
27




APCI/ESI+: 237


 


191
191




APCI/ESI−: 490


 


192
27 + 23




APCI/ESI+: 435(−THP)



















TABLE 41





PEx
PSyn
Str
DAT


















193
193




APCI/ESI+: 478


 


194
175




APCI/ESI+: 239


 


195
32




NMR2: 2.96(3 H, s), 3.85(3H, s), 3.89(6H, s), 5.16 (2H, s), 5.25(2 H, s), 6.68(1H, t, J = 8.0 Hz), 6.91 (1H, s), 7.63(1H, brs), 8.24(2H, s)


 


196
193




APCI/ESI+: 158


 


197
214




NMR1: 4.22(3 H, s), 7.77(1H, s), 9.91(1H, s)


 


198
144




APCI/ESI+: 211



















TABLE 42





PEx
PSyn
Str
DAT


















199
27




APCI/ESI+: 181


 


200
23




ESI+: 519


 


201
201




ESI+: 212


 


202
202




ESI+: 184


 


203
23




ESI+: 464


 


204
204




APCI/ESI+: 327


 


205
205




ESI+: 225



















TABLE 43





PEx
PSyn
Str
DAT


















206
205




ESI+: 225


 


207
23




ESI+: 505


 


208
23




ESI+: 505


 


209
209




APCI/ESI+: 412


 


210
210




APCI/ESI+: 227



















TABLE 44





PEx
PSyn
Str
DAT


















211
211




NMR2: 1.36- 1.94(9H, m), 1.99- 2.07(1H, m), 2.09- 2.20(1H, m), 2.35- 2.54(1H, m), 3.44- 3.75(2H, m), 3.78- 4.04(2H, m), 4.51- 4.72(2H, m), 4.78- 4.90(1H, m), 5.52- 5.65(1H, m), 6.87- 6.94(1H, m)


 


212
27




NMR2: 1.31- 1.94(10H, m), 2.00- 2.11(1H, m), 2.29- 2.44(1H, m), 3.27- 3.77(4H, m), 3.81- 3.97(1H, m), 4.00- 4.11(1H, m), 4.47- 4.59(1H, m), 4.61- 4.73(2H, m), 5.20- 5.33(1H, m), 5.66(1 H, s)


 


213
23




APCI/ESI+: 562


 


214
214




APCI/ESI+: 392



















TABLE 45





PEx
PSyn
Str
DAT


















215
214




APCI/ESI+: 406


 


216
204




APCI/ESI+: 324


 


217
27




APCI/ESI+: 294


 


218
23




APCI/ESI+: 574



















TABLE 46





PEx
PSyn
Str
DAT







219
32




ESI+: 500


 


220
27




ESI+: 221


 


221
24




ESI+: 251


 


222
27




ESI+: 294


 


223
28




ESI+: 324


 


224
24




NMR2: 2.64(4 H, t, J = 6.0 Hz), 3.54(4H, t, J = 6.0 Hz), 3.99(3H, s), 6.93(1H, d, J = 8.8 Hz), 7.75(1H, d, J = 2.4 Hz), 7.88 (1H, dd, J = 8.8, 2.4 Hz)



















TABLE 47





PEx
PSyn
Str
DAT


















225
27




ESI+: 322


 


226
28




ESI+: 352


 


227
13




EI: 332


 


228
18




EI: 298


 


229
229




EI: 220


 


230
30




ESI+: 613



















TABLE 48





PEx
PSyn
Str
DAT


















231
27




ESI+: 333


 


232
232




ESI+: 361


 


233
27




ESI+: 294


 


234
28




ESI+: 324


 


235
30




ESI+: 616


 


236
27




ESI+: 294


 


237
28




ESI+: 324



















TABLE 49





PEx
PSyn
Str
DAT







238
32




ESI+: 580


 


239
27




ESI+: 336


 


240
28




ESI+: 366


 


241
27




EI: 248


 


242
24




ESI+: 279


 


243
30




ESI+: 588



















TABLE 50





PEx
PSyn
Str
DAT







244
19




NMR2: 3.94(6H, s), 5.35(2H, s), 6.62(1H, s), 7.24 (1H, d, J = 8.8 Hz), 7.31(1H, dd, J = 8.8, 3.2 Hz), 8.16(1H, d, J = 3.2 Hz)


 


245
30




ESI+: 680


 


246
27




ESI+: 400


 


247
30




ESI+: 430


 


248
19




NMR2: 3.94(6H, s), 5.63(2H, s), 6.63(1H, s), 8.02 (1H, s), 8.15(1H, s)



















TABLE 51





PEx
PSyn
Str
DAT


















249
30




ESI+: 714


 


250
27




NMR2: 1.93- 1.96(6H, m), 2.06- 2.09(2H, m), 2.08 (6H, s), 2.54-2.60 (3H, m), 2.95-2.97 (4H, m), 3.42-3.45 (2H, m), 3.81(3H, s), 4.06(4H, s), 6.22- 6.26(2H, m), 6.74 (1H, d, J = 8.0 Hz)


 


251
24




ESI+: 464


 


252
252




ESI+: 313



















TABLE 52





PEx
PSyn
Str
DAT







253
 28




ESI+: 413


 


254
252




ESI+: 230


 


255
255




NMR2: 1.46-1.52(13H, m), 2.07 (6H, s), 3.40-3.43 (4H, m), 4.03(4H, s)


 


256
 27




NMR3: 3.97(2H, t, J = 5.2 Hz), 4.42(2H, t, J = 5.2 Hz), 6.85(1H, dd, J = 2.0, 0.8 Hz), 6.92(1H, dd, J = 8.8, 2.0 Hz), 7.41(1H, dd, J = 8.8, 0.8 Hz), 7.89(1H, d, J = 0.8 Hz)



















TABLE 53





PEx
PSyn
Str
DAT







257
40




NMR2: 2.91(1H, t, J = 6.0 Hz), 4.15- 4.19(2H, m), 4.61 (2H, t, J = 4.8 Hz), 7.74 (1H, d, J = 9.2 Hz), 8.11(1H, dd, J = 9.2, 2.0 Hz), 8.29 (1H, s), 8.73 (1H, d, J = 2.4 Hz)


 


258
27




NMR3: 3.95(2H, t, J = 5.6 Hz), 4.37 (2H, t, J = 5.6 Hz), 6.65(1H, dd, J = 8.8, 2.0 Hz), 6.72 (1H, m), 7.44(1H, dd, J = 8.8, 0.8 Hz), 7.99(1H, s)


 


259
40




NMR2: 2.87(1H, t, J = 6.0 Hz), 4.17- 4.20(2H, m), 4.62 (2H, t, J = 4.8 Hz), 7.77(1H, dd, J = 9.2, 0.8 Hz), 7.91 (1H, dd, J = 9.2, 2.0 Hz), 8.13(1H, s), 8.67-8.68(1H, m)



















TABLE 54





PEx
PSyn
Str
DAT







260
30




ESI+: 631


 


261
27




ESI+: 351


 


262
24




ESI+: 381


 


263
30




ESI+: 482



















TABLE 55





PEx
PSyn
Str
DAT







264
30




ESI+: 522


 


265
27




ESI+: 264 [M + Na]+


 


266
19




EI: 271


 


267
30




ESI+: 522


 


268
27




ESI+: 242


 


269
30




ESI+: 490



















TABLE 56





PEx
PSyn
Str
DAT







270
27




NMR2: 2.09(3H, s), 2.29 (3H, s), 2.48-2.65(8H, m), 2.83(2H, t, J = 5.6 Hz), 3.54(2H, brs), 4.06 (2H, t, J = 5.6 Hz), 6.20- 6.22(2H, m), 6.88(1H, d, J = 8.4 Hz)


 


271
36




NMR2: 2.30-2.66(14H, m), 2.89(2H, t, J = 5.4 Hz), 4.19(2H, t, J = 5.6 Hz), 7.24(1H, d, J = 8.0 Hz), 7.66(1H, d, J = 2.0 Hz), 7.75(1H, dd, J = 8.0, 2.0 Hz)


 


272
30




ESI+: 490



















TABLE 57





PEx
PSyn
Str
DAT







273
27




NMR3: 1.75-1.83(2H, m), 1.95-2.01(6H, m), 2.53-2.59 (2H, m), 2.86-2.99(7H, m), 3.81(3H, s), 4.45(4H, s), 6.27(1H, dd, J = 8.4, 2.4 Hz), 6.42(1H, d, J = 2.4 Hz), 6.78(1H, d, J = 8.0 Hz)


 


274
24




NMR2: 1.69-1.79(2H, m), 1.85-1.90(6H, m), 2.43-2.49 (5H, m), 2.67-2.73(2H, m), 3.73-3.76(2H, m), 3.94(3H, s), 4.41(4H, s), 6.87(1H, d, J = 8.8 Hz), 7.69(1H, d, J = 2.4 Hz), 7.84 (1H, dd, J = 8.8, 2.4 Hz).


 


275
252 




ESI+: 211


 


276
28




ESI+: 311



















TABLE 58





PEx
PSyn
Str
DAT







277
30




EI: 497


 


278
27




EI: 217


 


279
36




ESI+: 648


 


280
27




ESI+: 250


 


281
28




ESI+: 280


 


282
30




ESI+: 518



















TABLE 59





PEx
PSyn
Str
DAT







283
27




ESI+: 238


 


284
19




EI: 267


 


285
27




ESI+: 197


 


286
30




ESI+: 506


 


287
27




ESI+: 264


 


288
28




ESI+: 294


 


289
30




ESI+: 631



















TABLE 60





PEx
PSyn
Str
DAT







290
27




NMR2: 1.14-1.16(2H, m), 1.45(9H, s), 1.63-1.87(5H, m), 2.70-2.71 (2H, m), 3.48-3.49(2H, m), 3.81 (3H, s), 3.95-4.07(4H, m), 6.21 (1H, dd, J = 8.0, 2.4 Hz), 6.30(1H, d, J = 2.4 Hz), 6.71(1H, d, J = 8.4 Hz)


 


291
13




NMR2: 1.18-1.22(2H, m), 1.48(9H, s), 1.59(2H, br-s), 1.69-1.75(3H, m), 1.82-1.87(2H, m), 2.71-2.73 (2H, m), 3.95(3H, s), 4.16(2H, t, J = 6.4 Hz), 6.89(1H, d, J = 8.8 Hz), 7.74(1H, d, J = 2.4 Hz), 7.89 (1H, dd, J = 8.8, 2.4 Hz)


 


292
19




NMR2: 2.22(3H, s), 3.89(3H, s), 3.93(3H, s), 5.33(2H, s), 6.57 (1H, s), 8.37(2H, s)


 


293
18




EI: 294



















TABLE 61





PEx
PSyn
Str
DAT







294
229




NMR2: 1.85(1H, t, J = 6.4 Hz), 2.26(3H, s), 3.84(3H, s), 3.90(3H, s), 4.86(2H, d, J = 6.4 Hz), 6.49(1H, s)


 


295
295




ESI+: 310


 


296
296




ESI+: 220


 


297
 27




ESI+: 336


 


298
 35




ESI+: 366



















TABLE 62





PEx
PSyn
Str
DAT







299
28




ESI+: 266


 


300
23




APCI/ESI+: 558


 


301
175 




APCI/ESI−: 268


 


302
23




ESI+: 476


 


303
24




ESI+: 267


 


304
27




ESI+: 237

















TABLE 63





Ex
Str







1






 


2






 


3






 


4






 


5





















TABLE 63





Ex
Str
















6






 


7






 


8






 


9






 


10






 


11





















TABLE 65





Ex
Str







12






 


13






 


14






 


15






 


16





















TABLE 66





Ex
Str







17






 


18






 


19






 


20






 


21





















TABLE 67





Ex
Str







22






 


23






 


24






 


25






 


26





















TABLE 68





Ex
Str







27






 


28






 


29






 


30






 


31





















TABLE 69





Ex
Str







32






 


33






 


34






 


35






 


36





















TABLE 70





Ex
Str







37






 


38






 


39






 


40






 


41





















TABLE 71





Ex
Str







42






 


43






 


44






 


45






 


46





















TABLE 72





Ex
Str







47






 


48






 


49






 


50






 


51





















TABLE 73





Ex
Str







52






 


53






 


54






 


55






 


56





















TABLE 74





Ex
Str







57






 


58






 


59






 


60






 


61





















TABLE 75





Ex
Str







62






 


63






 


64






 


65






 


66





















TABLE 76





Ex
Str







67






 


68






 


69






 


70






 


71





















TABLE 77





Ex
Str







72






 


73






 


74






 


75






 


76





















TABLE 78





Ex
Str







77






 


78






 


79






 


80






 


81





















TABLE 79





Ex
Str







82






 


83






 


84






 


85





















TABLE 80





Ex
Str







86






 


87






 


88






 


89






 


90





















TABLE 81





Ex
Str







91






 


92






 


93






 


94






 


95





















TABLE 82





Ex
Str







96






 


97






 


98






 


99






 


100





















TABLE 83





Ex
Str







101






 


102






 


103






 


104






 


105





















TABLE 84





Ex
Str







106






 


107






 


108






 


109






 


110





















TABLE 85





Ex
Str







111






 


112






 


113






 


114






 


115





















TABLE 86





Ex
Str







116






 


117






 


118






 


119






 


120





















TABLE 87





Ex
Str







121






 


122






 


123






 


124






 


125





















TABLE 88





Ex
Str







126






 


127






 


128






 


129






 


130





















TABLE 89





Ex
Str







131






 


132






 


133






 


134






 


135





















TABLE 90





Ex
Str







136






 


137






 


138






 


139






 


140





















TABLE 91





Ex
Str







141






 


142






 


143






 


144






 


145





















TABLE 92





Ex
Str







146






 


147






 


148






 


149






 


150






 


151





















TABLE 93





Ex
Str







152






 


153






 


154






 


155






 


157





















TABLE 94





Ex
Str







158






 


159






 


160






 


161






 


162





















TABLE 95





Ex
Str







165






 


166






 


167






 


168






 


169





















TABLE 96





Ex
Str







170






 


171






 


172






 


173






 


174





















TABLE 97





Ex
Str







175






 


176






 


177






 


178






 


179





















TABLE 98





Ex
Str







180






 


181






 


182






 


183






 


184





















TABLE 99





Ex
Str







185






 


186






 


187






 


188






 


189





















TABLE 100





Ex
Str







190






 


191






 


192






 


193






 


194





















TABLE 101





Ex
Str







195






 


196






 


197






 


198






 


199





















TABLE 102





Ex
Str







200






 


201






 


202






 


203






 


204





















TABLE 103





Ex
Str







205






 


206






 


207






 


208






 


209





















TABLE 104





Ex
Str







210






 


211






 


212






 


213






 


214





















TABLE 105





Ex
Str







215






 


216






 


217






 


218






 


219





















TABLE 106





Ex
Str







220






 


221






 


222






 


223






 


224





















TABLE 107





Ex
Str







225






 


226






 


227






 


228






 


229





















TABLE 108





Ex
Str







230






 


231






 


232






 


233






 


234





















TABLE 109





Ex
Str







235






 


236






 


237






 


238






 


239





















TABLE 110





Ex
Str







240






 


241






 


242






 


243






 


244





















TABLE 111





Ex
Str







245






 


246






 


247






 


248






 


249





















TABLE 112





Ex
Str







250






 


251






 


252






 


253






 


254





















TABLE 113





Ex
Str







255






 


256






 


257






 


258






 


259





















TABLE 114





Ex
Str







260






 


261






 


262






 


263






 


264





















TABLE 115





Ex
Str







265






 


266






 


267






 


268






 


269





















TABLE 116





Ex
Str







270






 


271






 


272






 


273






 


274





















TABLE 117





Ex
Str







275






 


276






 


277






 


278






 


279





















TABLE 118





Ex
Str







280






 


281






 


282






 


283






 


284





















TABLE 119





Ex
Str







285






 


286






 


287






 


288






 


289





















TABLE 120





Ex
Str







290






 


291






 


292






 


293






 


294





















TABLE 121





Ex
Str







295






 


296






 


297






 


298






 


299






 


300





















TABLE 122





Ex
Str







301






 


302






 


303






 


304






 


305





















TABLE 123





Ex
Str







306






 


307






 


308






 


309






 


310





















TABLE 124





Ex
Str







311






 


312






 


313






 


314






 


315





















TABLE 125





Ex
Str







316






 


317






 


318






 


319






 


320





















TABLE 126





Ex
Str







321






 


322






 


323






 


324






 


325





















TABLE 127





Ex
Str







326






 


327






 


328






 


329






 


330





















TABLE 128





Ex
Str







331






 


332






 


333






 


334






 


335






 


336





















TABLE 129





Ex
Str







337






 


338






 


339






 


340






 


341






 


342





















TABLE 130





Ex
Str







343






 


344






 


345






 


346






 


347






 


348





















TABLE 131





Ex
Str







349






 


350






 


351






 


352






 


353





















TABLE 132





Ex
Str







354






 


355






 


356






 


357






 


358






 


359





















TABLE 133





Ex
Str







360






 


361






 


362






 


363






 


364






 


365





















TABLE 134





Ex
Str







366






 


367






 


368






 


369






 


370






 


371





















TABLE 135





Ex
Str







372






 


373






 


374 






 


375






 


376






 


377





















TABLE 136





Ex
Str







378






 


379






 


380






 


381






 


382






 


383





















TABLE 137





Ex
Str







384






 


385






 


386






 


387






 


388






















TABLE 138





Ex
Syn
DAT

















1
1
ESI+: 543




NMR1: 1.49-1.61(2H, m), 1.77-1.85(2H, m), 2.19(3H, s),




2.22-2.71(11H, m), 3.28-3.43(2H, m), 3.76(3H, s),




3.77(6H, s), 6.55(1H, t, J = 2.3 Hz), 6.69(2H, d,




J = 2.3 Hz), 6.83(1H, d, J = 8.6 Hz), 7.27(1H, dd,




J = 8.6, 2.3 Hz), 7.31(1H, d, J = 2.3 Hz),




8.61(2H, s), 9.79(1H, s)


2
2
ESI+: 547




NMR1: 1.46-1.59(2H, m), 1.74-1.83(2H, m), 2.14(3H, s),




2.19-2.54(11H, m), 2.71-2.82(4H, m), 3.26-3.36(2H, m),




3.70(6H, s), 3.74(3H, s), 6.30(1H, t, J = 2.3 Hz),




6.38(2H, d, J = 2.3 Hz), 6.79(1H, d, J = 8.6 Hz),




7.24(1H, dd, J = 8.6, 2.3 Hz), 7.35(1H, d, J =




2.3 Hz), 8.26(2H, s), 9.22(1H, s)


3
3
ESI+: 611


4
4
ESI+: 611


5
5
ESI+: 649, 651


6
6
ESI+: 583


7
7
ESI+: 500




NMR1: 2.21(3H, s), 2.35-2.59(4H, m), 2.66-2.76(2H, m),




2.81-3.00(6H, m), 3.74(3H, s), 3.81(6H, s), 6.78(1H,




d, J = 8.4 Hz), 6.85(1H, t, J = 8.4 Hz), 7.24(1H, dd,




J = 8.4, 2.4 Hz), 7.36(1H, d, J = 2.4 Hz), 8.18(2H,




s), 9.26(1H, s)


8
8
APCI/ESI+: 422


9
9
APCI/ESI+: 459


10
10
ESI+: 525


11
11
ESI+: 438




NMR1: 3.28-3.37(2H, m), 3.72-3.80(1H, m),




3.83-3.91(7H, m), 4.15(1H, dd, J = 13.8, 4.1 Hz),




4.67(1H, t, J = 5.6 Hz), 4.91(1H, d, J = 5.3 Hz),




5.14(2H, s), 7.06(1H, t, J = 8.4 Hz), 7.45(1H, d,




J = 0.6 Hz), 7.87(1Hd, J = 0.6 Hz), 8.26(2H, s),




9.21(1H, s)


12
12
ESI+: 519


















TABLE 139





Ex
Syn
DAT

















13
13
ESI+: 533


14
14
ESI+: 551


15
15
ESI+: 547


16
16
ESI+: 537


17
17
APCI/ESI+: 398


18
18
ESI+: 480


19
19
ESI+: 532




NMR1: 1.59-1.61(8H, m), 2.84-2.86(4H, m),




3.04-3.05(4H, m), 3.74(3H, s), 5.26(2H, s), 6.82(1H,




d, J = 8.4 Hz), 7.24(1H, d, J = 8.4 Hz), 7.32(1H, s),




7.97-8.03(1H, m), 8.34(2H, s), 8.53(1H, brs),




9.27(1H, s)


20
20
ESI+: 466


21
21
ESI+: 494


22
22
ESI+: 616


23
23
ESI+: 422




NMR1: 0.97-1.05(3H, m), 3.87(6H, s), 3.92-3.96(3H, m),




4.82-4.86(1H, m), 5.14(2H, s), 7.06(1H, t, J = 8.4 Hz),




7.44(1H, d, J = 0.6 Hz), 7.86(1H, d, J = 0.6 Hz),




8.26(2H, s), 9.21(1H, s)


24
24
ESI+: 421




NMR1: 3.87(6H, s), 4.69(2H, s), 5.15(2H, s), 7.06(1H,




t, J = 8.4 Hz), 7.19(1H, brs), 7.34(1H, brs), 7.46(1H,




s), 7.89(1H, s), 8.26(2H, s), 9.26(1H, s)


25
3
ESI+: 579


26
4
ESI+: 579


















TABLE 140





Ex
Syn
DAT

















27
2
ESI+: 583




NMR1: 1.46-1.59(2H, m), 1.74-1.82(2H, m), 2.14(3H, s),




2.18-2.58(11H, m), 2.65-2.75(2H, m), 2.84-2.93(2H, m),




3.24-3.38(2H, m), 3.74(3H, s), 3.81(6H, s), 6.79(1H,




d, J = 8.8 Hz), 6.85(1H, t, J = 8.4 Hz), 7.23(1H, dd,




J = 8.8, 2.4 Hz), 7.34(1H, d, J = 2.4 Hz), 8.17(2H,




s), 9.25(1H, s)


28
7 + 4
ESI+: 530


29
3
ESI+: 579


30
3
ESI+: 496


31
3
ESI+: 526


32
3
ESI+: 567


33
6
APCI/ESI+: 571




NMR1: 1.48-1.61(2H, m), 1.77-1.87(2H,




m), 2.13(3H, s), 2.20-2.64(11H, m), 2.69-2.76(2H, m),




2.85-2.93(2H, m), 3.22-3.34(2H, m), 3.81(6H, s),




6.85(1H, t, J = 8.4 Hz), 6.95(1H, dd, J = 9.9,




9.0 Hz), 7.32(1H, dd, J = 8.8, 1.8 Hz), 7.65(1H, dd,




J = 15.2, 2.4 Hz), 8.21(2H, s), 9.50(1H, s)


34
3
ESI+: 579


35
4
ESI+: 579


36
7
ESI+: 583


37
3
ESI+: 561


38
6
ESI+: 565


















TABLE 141





Ex
Syn
DAT

















39
3
ESI+: 595


40
4
ESI+: 595


41
6
ESI+: 599


42
6
ESI+: 615


43
3
ESI+: 549


44
6
ESI+: 553


45
3
ESI+: 519


46
6
ESI+: 523


47
3
ESI+: 555


48
6
ESI+: 559


49
9
ESI+: 549


50
9
ESI+: 549




NMR1: 1.43-1.62(2H, m), 1.68-1.87(2H, m), 2.14(3H, s),




2.17-2.70(11H, m), 3.23-3.35(2H, m), 3.74(6H, s),




3.74(3H, s), 5.07(2H, s), 6.46(1H, t, J = 2.4 Hz),




6.60(2H, d, J = 2.4 Hz), 6.78(1H, d, J = 8.6 Hz),




7.23(1H, dd, J = 8.6, 2.2 Hz), 7.32(1H, d, J =




2.2 Hz), 8.29(2H, s), 9.15(1H, s)


51
9
ESI+: 466


52
9
ESI+: 617




NMR1: 1.40-1.60(2H, m), 1.73-1.84(2H, m), 2.14(3H, s),




2.17-2.70(11H, m), 3.24-3.36(2H, m), 3.75(3H, s),




3.94(6H, s), 5.29(2H, s), 6.79(1H, d, J = 8.6 Hz),




7.00(1H, s), 7.24(1H, dd, J = 8.6, 2.3 Hz), 7.33(1H,




d, J = 2.3 Hz), 8.32(2H, s), 9.21(1H, s)


53
9
ESI+: 534


















TABLE 142





Ex
Syn
DAT

















54
9
ESI+: 631


55
9
ESI+: 548


56
9
ESI+: 585




NMR1: 1.45-1.60(2H, m), 1.73-1.84(2H, m), 2.14(3H, s),




2.17-2.58(11H, m), 3.24-3.36(2H, m), 3.75(3H, s),




3.87(6H, s), 5.16(2H, s), 6.79(1H, d, J = 8.8 Hz),




7.07(1H, t, J = 8.4 Hz), 7.24(1H, dd, J = 8.8,




2.4 Hz), 7.32(1H, d, J = 2.4 Hz), 8.29(2H, s),




9.21(1H, s)


57
9
ESI+: 502




NMR1: 2.21(3H, s), 2.37-2.53(4H, m), 2.83-2.94(4H, m),




3.75(3H, s), 3.87(6H, s), 5.16(2H, s), 6.79(1H, d,




J = 8.4 Hz), 7.07(1H, t, J = 8.4 Hz), 7.25(1H, dd,




J = 8.4, 2.4 Hz), 7.34(1H, d, J = 2.4 Hz), 8.30(2H,




s), 9.23(1H, s)


58
9
ESI+: 561


59
9
ESI+: 478


60
9
ESI+: 586


61
9
ESI+: 522


62
9
ESI+: 601


63
9
ESI+: 601


64
64
ESI+: 561


65
64
ESI+: 616


66
9
ESI+: 572


















TABLE 143





Ex
Syn
DAT

















67
9
ESI+: 636


68
9
ESI+: 653


69
9
ESI+: 605


70
9
APCI/ESI+: 659


71
9
ESI+: 493




NMR1: 1.80-2.12(6H, m), 2.19(3H, s), 2.77-2.89(2H, m),




3.94(6H, s), 3.98-4.10(1H, m), 5.27(2H, s), 7.00(1H,




s), 7.46(1H, s), 7.88(1H, s), 8.28(2H, s), 9.19(1H, s)


72
12
ESI+: 479




NMR1: 1.66-1.78(2H, m), 1.85-1.95(2H, m),




2.50-2.61(2H, m), 2.98-3.06(2H, m), 3.94(6H, s),




4.06-4.16(1H, m), 5.27(2H, s), 7.00(1H, s), 7.45(1H,




s), 7.87(1H, s), 8.29(2H, s), 9.19(1H, s)


73
64
ESI+: 535


74
9
ESI+: 437


75
9
ESI+: 549


76
64
ESI+: 546


77
64
ESI+: 574




NMR2: 1.07(6H, d, J = 6.8 Hz), 1.51-1.72(8H, m),




2.51(4H, t, J = 5.2 Hz), 2.69-2.73(1H, m), 2.95(4H,




t, J = 5.2 Hz), 3.87(3H, s), 5.14(2H, d, J = 1.2 Hz),




6.91-7.03(3H, m), 7.10-7.15(1H, m), 7.20(1H, d,




J = 2.4 Hz), 8.20(2H, s)


78
286
ESI+: 546




NMR2: 1.67-1.71(4H, m), 1.76-1.78(4H, m),




2.31(3H, s), 2.54(2H, s), 2.71-2.72(4H, m),




2.85-3.00(4H, m), 5.14(2H, s), 6.84(1H, s),




7.05-7.15(2H, m), 7.30(1H, brs), 7.34-7.60




(1H, m), 8.19(2H, s)


79
64
ESI+: 490




NMR2: 2.43(3H, s), 3.56(4H, brs), 3.81(3H, s),




3.96(4H, s), 5.14(2H, s), 6.41(1H, d, J = 8.3 Hz),




6.81(1H, brs), 6.93(1H, dd, J = 8.3, 2.4 Hz),




7.10-7.12(2H, m), 8.17(2H, s)


80
64
ESI+: 518




NMR2: 0.95(6H, d, J = 5.4 Hz), 2.30(1H, brs),




3.37(4H, brs), 3.80-3.94(7H, m), 5.14(2H, s),




6.41(1H, d, J = 8.1 Hz), 6.81(1H, brs), 6.93(1H,




d, J = 8.5 Hz), 7.11-7.14(2H, m), 8.17(2H, s).


81
9
ESI+: 461




NMR1: 1.83-2.07(6H, m), 2.19(3H, s), 2.78-2.88(2H, m),




3.87(6H, s), 3.98-4.09(1H, m), 5.14(2H, s), 7.07(1H,




t, J = 8.4 Hz), 7.45(1H, s), 7.88(1H, s), 8.25(2H,




s), 9.19(1H, s)


















TABLE 144





Ex
Syn
DAT

















82
9
ESI+: 544


83
9
ESI+: 531


84
9
ESI+: 585




NMR1: 1.34-1.50(2H, m), 1.68-1.89(4H, m),




2.07-2.19(4H, m), 2.47-2.64(4H, m), 2.73-2.95(6H, m),




3.74(3H, s), 3.87(6H, s), 5.16(2H, s), 6.78(1H, d,




J = 8.6 Hz), 7.07(1H, t, J = 8.4 Hz), 7.25(1H, dd,




J = 8.6, 2.2 Hz), 7.33(1H, d, J = 2.2 Hz), 8.29(2H,




s), 9.22(1H, s)


85
19
ESI+: 476


86
9
ESI+: 384


87
9
ESI+: 569




NMR1: 1.45-1.63(2H, m), 1.75-1.88(2H, m), 2.14(3H, s),




2.16-2.70(14H, m), 2.92-3.08(2H, m), 3.87(6H, s),




5.16(2H, s), 6.92(1H, d, J = 8.4 Hz), 7.07(1H, t,




J = 8.4 Hz), 7.40-7.50(2H, m), 8.28(2H, s),




9.19(1H, s)


88
9
ESI+: 501


89
9
ESI+: 580


90
9
ESI+: 591


91
9
ESI+: 589


92
12
ESI+: 516




NMR1: 0.96(6H, d, J = 6.4 Hz), 1.98-2.10(2H, m),




2.82-2.95(2H, m), 3.06-3.15(2H, m), 3.74(3H, s),




3.87(6H, s), 5.16(2H, s), 6.76(1H, d, J = 8.8 Hz),




7.07(1H, t, J = 8.4 Hz), 7.24(1H, dd, J = 8.8,




2.4 Hz), 7.32(1H, d, J = 2.4 Hz), 8.29(2H, s),




9.21(1H, s)


















TABLE 145





Ex
Syn
DAT

















93
12
ESI+: 599


94
286
ESI+: 517


95
12
ESI+: 556




NMR2: 1.76-1.81(8H, m), 2.97(4H, brs), 3.15(4H, brs),




3.88(9H, s), 5.14(2H, s), 6.67(1H, t, J = 8.2 Hz),




6.88-6.90(2H, m), 7.01(1H, dd, J = 8.4, 2.4 Hz),




8.20(2H, s), 9.26(1H, brs)


96
64
ESI+: 570




NMR2: 1.62-1.68(8H, m), 2.32(3H, s), 2.43-2.45(4H,




m), 2.94- 2.97(4H, m), 3.88(9H, s), 5.14(2H, s),




6.67(1H, t, J = 8.2 Hz), 6.88-7.00(3H, m),




7.21-7.22(1H, m), 8.19(2H, t, J = 2.2 Hz)


97
12
ESI+: 500




NMR2: 3.81-4.01(17H, m), 5.13(2H, s), 6.40(1H, d,




J = 8.3 Hz), 6.64-6.68(1H, m), 6.77(1H, d, J =




7.1 Hz), 6.92(1H, d, J-8.5 Hz), 7.12(1H, brs),




8.17(2H, s)


98
64
ESI+: 514




NMR2: 2.38(3H, s), 3.47-3.58(4H, m), 3.81-3.94(13H,




m), 5.13(2H, s), 6.40(1H, d, J = 8.5 Hz),




6.64-6.68(1H, m), 6.78(1H, brs), 6.92(1H, dd, J =




8.5, 2.0 Hz), 7.12(1H, d, J = 2.7 Hz), 8.17(2H, s)


99
64
ESI+: 542




NMR2: 0.90-0.94(6H, m), 2.10(1H, brs),




3.40-3.96(17H, m), 5.13(2H, s), 6.41(1H, d, J =




8.3 Hz), 6.66(1H, d, J = 8.0 Hz), 6.76(1H, brs),




6.91-6.93(1H, m), 7.13(1H, s), 8.17(2H, s)


100
286
ESI+: 599




NMR2: 1.66-1.69(4H, m), 2.29(3H, s), 2.30(2H, s),




2.40(3H, s), 2.45(4H, brs), 2.70(4H, brs),




2.84-2.87(2H, m), 2.96-3.01(2H, m), 3.88(6H, s),




5.13(2H, s), 6.67(1H, t, J = 8.2 Hz), 6.87(1H, s),




7.04(1H, d, J = 8.8 Hz), 7.29(1H, d, J = 2.8 Hz),




7.34(1H, d, J = 8.4 Hz), 8.18(2H, s)


101
286
ESI+: 588




NMR3: 1.08(3H, d, J = 6.4 Hz), 1.12(3H, s),




1.20(3H, s), 1.49-1.52(1H, m), 1.64-1.66(1H, m),




1.94-1.96(1H, m), 2.03-2.04(1H, m), 2.59-2.68(4H,




m), 3.29-3.34(2H, m), 3.87(9H, s), 5.15(2H, d,




J = 2.0 Hz), 6.89-6.93(2H, m), 7.10-7.13(1H, m),




7.37(1H, d, J = 2.0 Hz), 8.19(2H, d, J = 2.0 Hz)


















TABLE 146





Ex
Syn
DAT

















102
286
ESI+: 470


103
9
ESI+: 502


104
19
ESI+: 596


105
12
ESI+: 568


106
106
ESI+: 582


107
12
ESI+: 623


108
19
ESI+: 588




NMR2: 1.76-1.81(8H, m), 2.96-2.97(4H, m),




3.15-3.16(4H, m), 3.88(3H, s), 3.94(6H, s),




5.33(2H, s), 6.61(1H, s), 6.89-6.91(2H, m),




7.01(1H, dd, J = 8.0, 2.4 Hz), 7.22-7.26(1H, m),




8.23(2H, s)


109
19
ESI+: 532


















TABLE 147





Ex
Syn
DAT

















110
64
ESI+: 546




NMR2: 2.36(3H, brs), 3.45-3.50(4H, m), 3.81(3H, s),




3.93(10H, s), 5.32(2H, s), 6.40(1H, d, J = 8.5 Hz),




6.60(1H, s), 6.76(1H, brs), 6.92-6.95(1H, m),




7.14(1H, s), 8.20(2H, s)


111
4
ESI+: 649


112
10
ESI+: 543


113
9
ESI+: 408




NMR1: 3.69(2H, dd, J = 11.0, 5.6 Hz), 3.87(6H, s),




4.07(2H, t, J = 5.6 Hz), 4.83(1H, t, J = 5.4 Hz),




5.14(2H, s), 7.07(1H, t, J = 8.4 Hz), 7.45(1H, d,




J = 0.6 Hz), 7.88(1H, d, J = 0.6 Hz), 8.26(2H s),




9.20(1H, s)


114
20
ESI+: 490




NMR1: 2.13(3H, s), 2.17-2.54(8H, m), 2.66(2H, t,




J = 6.8 Hz), 3.87(6H, s), 4.14(2H, t, J = 6.8 Hz),




5.14(2H, s), 7.06(1H, t, J = 8.4 Hz), 7.43(1H, d,




J = 0.6 Hz), 7.89(1H, d, J = 0.6 Hz), 8.25(2H, s),




9.20(1H, s)


115
18
ESI+: 477




NMR1: 3.58-3.65(1H, m), 3.75-3.84(1H, m), 3.87(6H,




s), 4.04-4.10 (1H, m), 4.17-4.24(1H, m),




4.41-4.49(1H, m), 4.78(2H, s), 5.15(2H, s),




5.72(1H, s), 7.07(1H, t, J = 8.4 Hz), 7.46(1H, d,




J = 0.4 Hz), 7.88(1H, d, J = 0.4 Hz), 8.26(2H, s),




9.27(1H, s)


116
11
ESI+: 438




NMR1: 3.23-3.38(2H, m), 3.72-3.80(1H, m),




3.84-3.96(7H, m), 4.15(1H, dd, J = 13.8, 4.1 Hz),




4.67(1H, t, J = 5.6 Hz), 4.91(1H, d, J = 5.3 Hz),




5.14(2H, s), 7.06(1H, t, J = 8.4 Hz), 7.45(1H, d,




J = 0.6 Hz), 7.87(1H, d, J = 0.6 Hz), 8.26(2H, s),




9.21(1H, s)


117
286
ESI+: 502


118
9
ESI+: 463


119
12
APCI/ESI+: 449


120
120
ESI+: 531


121
13
ESI+: 463


122
9
ESI+: 472




NMR1: 2.21(3H, s), 2.41-2.48(4H, m), 2.98-3.08(4H,




m), 3.87(6H, s), 5.15(2H, s), 6.81-6.90(2H, m),




7.07(1H, t, J = 8.4 Hz), 7.47-7.55(2H, m), 8.26(2H,




s), 9.15(1H, s)




















TABLE 148







Ex
Syn
DAT




















123
9
ESI+: 555



124
9
ESI+: 531



125
20
ESI+: 466



126
20
ESI+: 453



127
9
APCI/ESI+: 384



128
20
ESI+: 453



129
9
ESI+: 599



130
9
ESI+: 603



131
120
ESI+: 599



132
120
ESI+: 613



133
120
ESI+: 572



134
12
ESI+: 505



135
9
ESI+: 499



136
12
ESI+: 423



137
12
ESI+: 447



138
9
ESI+: 573



139
9
ESI+: 470



140
9
ESI+: 505



141
120
ESI+: 597



142
12
ESI+: 458



143
12
ESI+: 476



144
166 + 4 
ESI+: 514



145
12
ESI+: 486



146
12
ESI+: 457



147
13 + 4 
ESI+: 611



148
9 + 4
ESI+: 583



149
9 + 4
ESI+: 543



150
9 + 4
ESI+: 557



151
13
ESI+: 471



152
64
ESI+: 554



153
9
ESI+: 567





















TABLE 149







Ex
Syn
DAT




















154
9
ESI+: 474



155
9
ESI+: 487



157
15
ESI+: 499



158
16
ESI+: 501



159
64
ESI+: 541



160
17
ESI+: 422



161
161
ESI+: 515



162
120 + 162 
ESI+: 555



165
9
ESI+: 487



166
166
ESI+: 445



167
18
ESI+: 504



168
12 + 162
ESI+: 473



169
18
ESI+: 465



170
13 + 162
ESI+: 487



171
9
ESI+: 557



172
18
ESI+: 491



173
20
ESI+: 477



174
20
ESI+: 504



175
23
ESI+: 436



176
9
ESI+: 461



177
9
ESI+: 556



178
9
ESI+: 488





















TABLE 150







Ex
Syn
DAT




















179
9
APCI/ESI+: 487



180
18
ESI+: 465



181
18
ESI+: 509



182
9
ESI+: 483



183
9
ESI+: 570



184
23
ESI+: 422



185
9
ESI+: 556



186
17
ESI+: 448



187
166
ESI+: 458



188
16
ESI+: 422



189
16
ESI+: 378



190
190
APCI/ESI+: 407



191
64
ESI+: 555



192
106
ESI+: 502



193
19
ESI+: 571



194
12
ESI+: 475



195
9
ESI+: 586



196
18
ESI+: 491



197
18
ESI+: 491



198
18
ESI+: 505





















TABLE 151







Ex
Syn
DAT




















199
18
ESI+: 504



200
18
ESI+: 518



201
11
ESI+: 465



202
13
ESI+: 489



203
20
ESI+: 504



204
20
ESI+: 435



205
11
ESI+: 465



206
20 + 4
ESI+: 463



207
12 + 4
ESI+: 476



208
9
ESI+: 461



209
20
ESI+: 504



210
 20 + 162
ESI+: 518



211
9
ESI+: 448



212
212
ESI+: 422



213
213
ESI+: 465



214
214
ESI+: 435



215
12
ESI+: 461



216
120
ESI+: 531



217
217
ESI+: 517



218
17
ESI+: 436



219
17
ESI+: 450



220
17
ESI+: 436



221
17
ESI+: 450



222
18
ESI+: 491



223
18
ESI+: 505



224
18
ESI+: 491



225
18
ESI+: 505



226
13
ESI+: 475



















TABLE 152





Ex
Syn
DAT

















227
18
ESI+: 495


228
18
ESI+: 479


229
9
ESI+: 503


230
214
ESI+: 422


231
20 + 162
ESI+: 504


232
217
ESI+: 531


233
214
ESI+: 422


234
217
ESI+: 547


235
213
ESI+: 438


236
9 + 4 
ESI+: 517


237
9
ESI+: 517


238
17
APCI/ESI+: 422


239
239
ESI+: 422


240
214
ESI+: 408


241
20 + 162
ESI+: 504


242
18
ESI+: 504


243
18
ESI+: 505


244
24
ESI+: 421


245
214
ESI+: 408


246
246
APCI/ESI+: 408


247
214
ESI+: 435


248
20
ESI+: 490




NMR1: 2.14(3H, s), 2.18-2.53(8H, m),




3.45(2H, s), 3.67(3H, s), 3.87(6H, s), 5.15(2H,




s), 6.46(1H, s), 7.06(1H, t, J = 8.4 Hz),




8.26(2H, s), 9.42(1H, s)


249
20 + 4 
ESI+: 491


250
64
ESI+: 514


251
214
ESI+: 435


252
9
ESI+: 478


253
253
ESI+: 517


254
254
ESI+: 461


255
253
ESI+: 517


256
17
ESI+: 450




















TABLE 153







Ex
Syn
DAT




















257
246
ESI+: 436



258
24
ESI+: 449



259
11
ESI+: 465



260
11
ESI+: 465



261
18
ESI+: 505



262
18
ESI+: 532



263
20
ESI+: 476



264
254
ESI+: 507



265
254
ESI+: 507



266
18
ESI+: 491



267
18
ESI+: 463



268
18
ESI+: 504



269
24
ESI+: 407



270
214
APCI/ESI+: 394



271
20
ESI+: 504



272
20
ESI+: 477



273
20
ESI+: 461



274
20
ESI+: 504



275
20
ESI+: 504



276
20
ESI+: 520



277
20
ESI+: 477



278
278
ESI+: 390



279
20
ESI+: 490



280
282
ESI+: 447



281
282 + 4
ESI+: 490



282
282
ESI+: 463



283
282
ESI+: 490



284
282
ESI+: 506



285
214
ESI+: 490



286
286
ESI+: 533



















TABLE 154





Ex
Syn
DAT

















287
286
ESI+: 501


288
286
ESI+: 530


289
286
ESI+: 574


290
286
ESI+: 602


291
286
ESI+: 601


292
12
ESI+: 513


293
64
ESI+: 527


294
64
ESI+: 555


295
286
ESI+: 574




NMR3: 1.23(6H, s), 1.61-1.64(2H, m), 1.97-1.99(2H,




m), 2.57-2.62(5H, m), 3.31-3.34(2H, m), 3.87(9H, s),




5.16(2H, t, J = 1.6 Hz), 6.89-6.94(2H, m), 7.11(1H,




dd, J = 8.8, 2.4 Hz), 7.36(1H, d, J = 2.4 Hz),




8.20(2H, s)


296
16
ESI+: 544


297
286
ESI+: 532


298
286
ESI+: 502




NMR2: 2.61(2H, t, J = 5.6 Hz), 2.69(4H, t, J =




4.8 Hz), 3.16(4H, t, J = 4.8 Hz), 3.66(2H, t,




J = 5.6 Hz), 3.88(6H, s), 5.13(2H, s), 6.66(1H, t,




J = 8.0 Hz), 6.80(1H, br-s), 6.92(2H, d, J =




9.2 Hz), 7.43(2H, d, J = 9.2 Hz), 8.18(2H, s)


299
12
ESI+: 516




NMR2: 1.67-1.69(2H, m), 2.06-2.09(2H, m),




2.70-2.81(3H, m), 2.98(2H, t, J = 5.2 Hz), 3.60(2H,




d, J = 12.4 Hz), 3.76-3.77(2H, m), 3.88(6H, s),




5.13(2H, s), 6.66(1H, t, J = 8.0 Hz), 6.82(1H, s),




6.92(2H, d, J = 8.8 Hz), 7.41(2H, d, J = 8.8 Hz),




8.18(2H, s)


300
286
ESI+: 574


301
16
ESI+: 571


302
302
ESI+: 516


303
64
ESI+: 540


304
286
ESI+: 616


305
4
ESI+: 529


306
286
ESI+: 529


307
16
ESI+: 546


308
12
ESI+: 488


309
286
ESI+: 533


310
336
ESI+: 560


311
12
ESI+: 580


312
64
ESI+: 594


313
64
ESI+: 622


314
286
ESI+: 534


















TABLE 155





Ex
Syn
DAT

















315
315
ESI+: 630


316
286
ESI+: 585


317
286
ESI+: 458


318
286
ESI+: 458


319
286
ESI+: 458


320
286
ESI+: 458


321
315
ESI+: 547


322
16
ESI+: 533


323
16
ESI+: 546


324
11
ESI+: 482


325
17
ESI+: 494


326
17
ESI−: 460


327
286
ESI+: 448


328
18
ESI+: 531


329
286
ESI+: 530


330
286
ESI+: 517


331
17
ESI−: 460


332
18
ESI+: 544


333
286
ESI+: 516


334
286
ESI+: 612


335
16
ESI+: 549


336
336
ESI+: 480


337
12
ESI+: 548


338
286
ESI+: 530


339
11
ESI+: 478


340
286
ESI+: 408


341
286
ESI+: 490


342
286
ESI+: 477


343
17
ESI−: 476


344
286
ESI+: 438




NMR1: 3.26-3.40(2H, m), 3.76-3.78(1H, m), 3.87(6H,




s), 3.89-3.94(1H, m), 4.15(1H, dd, J = 14.0,




4.0 Hz), 4.71(1H, t, J = 5.6 Hz), 4.95(1H, d,




J = 5.2 Hz), 5.27(2H, s), 7.05(1H, t, J = 8.4 Hz),




7.41(1H, s), 7.74(1H, d, J = 1.6 Hz), 7.84(1H, d,




J = 1.6 Hz), 7.88(1H, s), 8.95(1H, brs)


345
64
ESI+: 562


346
286
ESI+: 544


347
18
ESI+: 563


348
18
ESI+: 521


349
349
ESI+: 508




















TABLE 156







Ex
Syn
DAT




















350
18
ESI+: 576



351
286
ESI+: 473



352
286
ESI+: 422



353
12
ESI+: 531



354
64
ESI+: 545



355
286
ESI+: 597



356
356
ESI+: 476



357
356
ESI+: 446



358
356
ESI+: 446



359
356
ESI+: 460



360
356
ESI+: 473



361
356
ESI+: 486



362
356
ESI+: 432



363
356
ESI+: 418



364
356
ESI+: 432



365
356
ESI+: 418



366
356
ESI+: 466



367
356
ESI+: 478



368
356
ESI+: 460



369
356
ESI+: 466



370
356
ESI+: 378



371
356
ESI+: 444



372
356
ESI+: 473



373
375
ESI+: 459



374
375
ESI+: 433



375
375
ESI+: 419



376
356
ESI+: 458



377
356
ESI+: 447



378
356
ESI+: 471



379
356
ESI+: 490



380
356
ESI+: 417



381
375
ESI+: 403



382
356
ESI+: 459



383
356
ESI+: 501



384
356
ESI+: 519



385
356
ESI+: 477



386
356
ESI+: 459



387
356
ESI+: 476



388
286
ESI+: 517










INDUSTRIAL APPLICABILITY
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.
SEQUENCE LISTING FREE TEXT
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.

## Claims

1. A compound of formula (I) or a salt thereof:



wherein
X is N;
Y is CH;
L1 is ethylene or methylene-O—;
Z is CH;
each R1 is independently 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 from 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 -L2-R2 or R3;
L2 is an optionally substituted aromatic heterocyclic ring or an optionally substituted non-aromatic heterocyclic ring;
R2 is a non-aromatic heterocyclic group optionally substituted with lower alkyl, optionally substituted cycloalkyl, lower alkyl optionally substituted with at least one selected from the group consisting of —OH and —O-lower alkyl, —C(O)—R0, —C(O)-optionally substituted cycloalkyl, —NH—R0, —N(lower alkyl)-R0, an -L3-optionally substituted non-aromatic heterocyclic group, or H;
R0 is lower alkyl optionally substituted with —OH;
L3 is a bond, —NH—, —N(lower alkyl)-, or lower alkylene; and
R3 is:
a lower alkyl optionally substituted with at least one selected from the group consisting of —C(O)OH, —OH, —O—R0, amino optionally substituted with one or two R0, carbamoyl optionally substituted with one or two R0, 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 at least one selected from the group consisting of —C(O)OH, —OH, —O—R0, carbamoyl optionally substituted with one or two R0, an optionally substituted non-aromatic heterocyclic group, and a —C(O)-optionally substituted non-aromatic heterocyclic group),
—NH-(lower alkyl optionally substituted with at least one 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 R0),
—N(lower alkyl)-(lower alkyl optionally substituted with at least one 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 R0),
—C(O)OH,
—C(O)-optionally substituted non-aromatic heterocyclic group,
—O-(a non-aromatic heterocyclic group optionally substituted with lower alkyl), or carbamoyl optionally substituted with one or two R0.

2. The compound or salt thereof according to claim 1,
wherein
each R1 is independently —O-lower alkyl or halogen;
p is 2 or 4; and
ring W is an optionally substituted aromatic carbocyclic ring or an optionally substituted aromatic heterocyclic ring.

3. The compound or salt thereof according to claim 2,
wherein
p is 4; and
ring W is an optionally substituted benzene ring or optionally substituted pyrazole.

4. The compound or salt thereof according to claim 1,
wherein Q is -L2-R2;
L2 is an optionally substituted non-aromatic heterocyclic ring; and
R2 is lower alkyl optionally substituted with at least one 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.

5. The compound or salt thereof according to claim 4,
wherein p is 4;
L2 is piperazine optionally substituted with one or more methyl, piperidine optionally substituted with one or more methyl, or 3,9-diazaspiro[5.5]undecane; and
R2 is piperazine optionally substituted with methyl, piperidine optionally substituted with methyl, 2-hydroxyethylamino, or H.

6. The compound or salt thereof according to claim 5,
wherein each R1 is independently —O-methyl or F;
L1 is -methylene-O—;
ring W is a benzene ring optionally substituted with —O-methyl;
L2 is piperidine or 4-methylpiperazine; and
R2 is 4-methylpiperazine, 2-hydroxyethylamino, or H.

7. The compound or salt thereof according to claim 1,
wherein ring W is optionally substituted pyrazole;
Q is R3; and
R3 is lower alkyl substituted with at least one selected from the group consisting of —C(O)OH, carbamoyl optionally substituted with one or two R0, —OH, an optionally substituted non-aromatic heterocyclic group, and —C(O)-(an optionally substituted non-aromatic heterocyclic group).

8. The compound or salt thereof according to claim 7,
wherein p is 4; and
R3 is lower alkyl substituted with at least one 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).

9. The compound or salt thereof according to claim 8,
wherein each R1 is independently —O-methyl or F;
L1 is -methylene-O—;
ring W is pyrazole optionally substituted with methyl; and
R3 is 2-hydroxyethyl, 2,3-dihydroxypropyl, or 4-methylpiperazin-1-ylmethyl.

10. The compound or salt thereof according to claim 1, wherein the compound is selected from the group consisting of:
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]-1 H-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.

11. The compound or salt thereof according to claim 10, wherein the compound is
5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine.

12. The compound or salt thereof according to claim 10, wherein the compound is
2-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]ethanol.

13. The compound or salt thereof according to claim 10, wherein the compound is
(2R)-3-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)-1H-pyrazol-1-yl]propane-1,2-diol.

14. The compound or salt thereof according to claim 10, wherein the compound is
2-({1-[4-({5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]pyrimidin-2-yl}amino)phenyl]piperidin-4-yl}amino)ethanol.

15. The compound or salt thereof according to claim 10, wherein the compound is
5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-{1-methyl-5-[(4-methylpiperazin-1-yl)methyl]-1H-pyrazol-3-yl}pyrimidin-2-amine.

16. The compound or salt thereof according to claim 10, wherein the compound is
5-[(2,6-difluoro-3,5-dimethoxybenzyl)oxy]-N-[4-(4-methylpiperazin-1-yl)phenyl]pyrimidin-2-amine.

17. A pharmaceutical composition, comprising:
a compound or salt thereof according to claim 10 and
a pharmaceutically acceptable excipient.

18. The pharmaceutical composition according to claim 17, wherein the compound is suitable for treatment of mutant FGFR3-positive cancer.

19. A method of manufacturing a pharmaceutical composition, the method comprising:
manufacturing the pharmaceutical composition with the compound or salt thereof according to claim 10,
wherein the pharmaceutical composition is suitable for treatment of mutant FGFR3-positive cancer.

20. A method of treating mutant FGFR3-positive cancer, the method comprising:
administering an effective amount of the compound or salt thereof according to claim 10 to a subject in need thereof.