JPWO2004054617A1 - Preventive and / or therapeutic agent for central diseases - Google Patents

Abstract

A prophylactic and / or therapeutic agent for central diseases containing a substance having a function-inhibiting action of G protein-coupled receptor protein 88 (GPR88; G-Protein coupled receptor 88) as an active ingredient, and formula (I) (wherein A Represents substituted or unsubstituted aryl, R1 represents a hydrogen atom, R2 represents -NR3R4, X represents -C (= O) R5, etc., Y represents a hydrogen atom, etc.) Or a pharmacologically acceptable salt thereof.

Description

  The present invention relates to a preventive and / or therapeutic agent for central diseases containing a substance having a function-inhibiting action of G protein-coupled receptor protein 88 (GPR88; G-Protein coupled receptor 88) as an active ingredient. The present invention also relates to an antisense oligonucleotide or pyrimidine derivative or a pharmacologically acceptable salt thereof which has a GPR88 function inhibitory action and is useful for the prevention and / or treatment of central diseases.

Parkinson's disease is a movement disorder that develops when the substantia nigra dopaminergic neurons are progressively degenerated and the nerve activity of the striatum, the projection site, becomes abnormal. Currently, levodopa, dopamine receptor agonists and the like are said to be able to improve symptoms. However, it is known that side effects such as involuntary movements and mental disorders and resistance to drug effects appear when taking levodopa and dopamine receptor agonists over a long period of 2-3 years. There is a need for therapeutic agents that have a mechanism of action and have few side effects.
In addition to Parkinson's disease, central diseases include mental disorders such as depression, anxiety, schizophrenia, etc. With regard to the therapeutic agents for these diseases, there are new therapeutic mechanisms with few side effects. Is required.
GPR88 is an orphan G protein-coupled receptor (GPCR) whose ligand and function are unknown. Human, rat and mouse GPR88 cDNAs have been isolated, the nucleotide sequences of each cDNA and the amino acids of each GPR88 encoded by them The sequence has been reported ("Genomics" 2000, 69, 314-321). GPR88 is expressed specifically in the striatum, nucleus accumbens and cerebellar olive nucleus, while expression in peripheral tissues is extremely low. Therefore, GPR88 has some motor and / or emotion regulation. There are no reports that have shown this experimentally so far.
On the other hand, a compound having a pyrimidine ring, a pyridine ring or a benzene ring in its structure is a therapeutic agent for inflammation (International Publication No. WO97 / 09315), a Syk tyrosine kinase inhibitor (International Publication No. WO99 / 31073, International Publication No. WO2000). / 75113), V-type phosphodiesterase inhibitor (International Publication No. WO2001 / 83460), antiviral agent (International Publication No. WO99 / 41253), antitumor agent (International Publication No. WO2000 / 39101), protein kinase C inhibition As an agent (International Publication No. WO2000 / 76980), a CD40 function inhibitor (Japanese Patent Laid-Open No. 2001-89452), a glycogen synthase kinase 3 inhibitor (International Publication No. WO2002 / 20495, International Publication No. WO99 / 65897), Each is known.

＜式中、Ａは置換もしくは非置換のアリール、置換もしくは非置換のアラルキル、置換もしくは非置換のアリールオキシアルキル、置換もしくは非置換のアラルキルオキシアルキル、置換もしくは非置換の複素環基、置換もしくは非置換の複素環アルキルまたはシクロアルキルアルキルを表し、
Ｒ^１は水素原子、置換もしくは非置換の低級アルキル、置換もしくは非置換のシクロアルキルまたは置換もしくは非置換のアラルキルを表すか、
またはＡとＲ^１が隣接する窒素原子と一緒になって置換もしくは非置換の複素環基を形成し、
Ｒ^２は水素原子、ヒドロキシ、ハロゲン、置換もしくは非置換の低級アルキル、置換もしくは非置換の低級アルケニル、置換もしくは非置換の低級アルキニル、置換もしくは非置換の低級アルコキシ、置換もしくは非置換の低級アルキルチオ、置換もしくは非置換のアリール、置換もしくは非置換のアラルキル、置換もしくは非置換の複素環基、置換もしくは非置換の複素環アルキルまたは−ＮＲ^３Ｒ^４（式中、Ｒ^３およびＲ^４は同一または異なって、水素原子、置換もしくは非置換の低級アルキル、置換もしくは非置換のシクロアルキル、置換もしくは非置換のシクロアルキルアルキル、置換もしくは非置換の低級アルケニル、置換もしくは非置換の低級アルキニル、置換もしくは非置換のアリール、置換もしくは非置換のアラルキル、置換もしくは非置換の複素環基または置換もしくは非置換の複素環アルキルを表すか、またはＲ^３とＲ^４が隣接する窒素原子と一緒になって置換もしくは非置換の複素環基を形成する）を表し、
Ｘは−Ｃ（＝Ｏ）Ｒ^５｛式中、Ｒ^５はヒドロキシ、置換もしくは非置換の低級アルコキシ、置換もしくは非置換のアリールオキシまたは−ＮＲ^６Ｒ^７〔式中、Ｒ^６およびＲ^７は同一または異なって、水素原子、置換もしくは非置換の低級アルキル、置換もしくは非置換のシクロアルキル、置換もしくは非置換のシクロアルキルアルキル、置換もしくは非置換の低級アルコキシ、置換もしくは非置換のアラルキル、置換もしくは非置換の複素環アルキル、ヒドロキシまたは−ＳＯ_２Ｒ^８（式中、Ｒ^８は置換もしくは非置換の低級アルキルまたは置換もしくは非置換のアリールを表す）を表すか、
またはＲ^６とＲ^７が隣接する窒素原子と一緒になって置換もしくは非置換の複素環基を形成する〕を表す｝、ニトロ、置換もしくは非置換のヒドロキシメチルまたは１，２，３，４−テトラゾール−５−イルを表し、
Ｙは、水素原子、ハロゲンまたは置換もしくは非置換の低級アルキルを表す＞で表されるピリミジン誘導体またはその薬理学的に許容される塩を有効成分として含有するＧＰＲ８８の機能抑制剤。
（１６） Ｙが水素原子である（１５）記載のＧＰＲ８８の機能抑制剤。
（１７） Ｒ^２が−ＮＲ^３Ｒ^４（式中、Ｒ^３およびＲ^４はそれぞれ前記と同義である）である（１５）または（１６）記載のＧＰＲ８８の機能抑制剤。
（１８） Ｘが１，２，３，４−テトラゾール−５−イルである（１５）〜（１７）のいずれかに記載のＧＰＲ８８の機能抑制剤。
（１９） Ｘが−Ｃ（＝Ｏ）ＮＲ^６Ｒ^７（式中、Ｒ^６およびＲ^７はそれぞれ前記と同義である）である（１５）〜（１７）のいずれかに記載のＧＰＲ８８の機能抑制剤。
（２０） Ｘが−Ｃ（＝Ｏ）Ｒ^５Ｂ（式中、Ｒ^５Ｂはヒドロキシまたは低級アルコキシを表す）である（１５）〜（１７）のいずれかに記載のＧＰＲ８８の機能抑制剤。
（２１） Ｘが置換もしくは非置換のヒドロキシメチルである（１５）〜（１７）のいずれかに記載のＧＰＲ８８の機能抑制剤。
（２２） Ａが置換もしくは非置換のアリール、置換もしくは非置換のアラルキルまたは置換もしくは非置換の複素環基である（１５）〜（２１）のいずれかに記載のＧＰＲ８８の機能抑制剤。
（２３） Ｒ^１が水素原子である（１５）〜（２２）のいずれかに記載のＧＰＲ８８の機能抑制剤。
（２４） Ｒ^３が置換もしくは非置換の低級アルキル、シクロアルキルアルキルまたは置換もしくは非置換のアラルキルであるか、またはＲ^３とＲ^４が隣接する窒素原子と一緒になって置換もしくは非置換の複素環基を形成する（１７）〜（２３）のいずれかに記載のＧＰＲ８８の機能抑制剤。
（２５） Ｒ^３が置換もしくは非置換の低級アルキル、シクロアルキルアルキルまたは置換もしくは非置換のアラルキルであり、Ｒ^４が水素原子または低級アルキルである（１７）〜（２３）のいずれかに記載のＧＰＲ８８の機能抑制剤。
（２６） 式（Ｉ）

（式中、Ａ、Ｒ^１、Ｒ^２、ＸおよびＹはそれぞれ前記と同義である）で表されるピリミジン誘導体またはその薬理学的に許容される塩を有効成分として含有する中枢疾患の予防および／または治療剤。
（２７） Ｙが水素原子である（２６）記載の中枢疾患の予防および／または治療剤。
（２８） Ｒ^２が−ＮＲ^３Ｒ^４（式中、Ｒ^３およびＲ^４はそれぞれ前記と同義である）である（２６）または（２７）記載の中枢疾患の予防および／または治療剤。
（２９） Ｘが１，２，３，４−テトラゾール−５−イルである（２６）〜（２８）のいずれかに記載の中枢疾患の予防および／または治療剤。
（３０） Ｘが−Ｃ（＝Ｏ）ＮＲ^６Ｒ^７（式中、Ｒ^６およびＲ^７はそれぞれ前記と同義である）である（２６）〜（２８）のいずれかに記載の中枢疾患の予防および／または治療剤。
（３１） Ｘが−Ｃ（＝Ｏ）Ｒ^５Ｂ（式中、Ｒ^５Ｂはヒドロキシまたは低級アルコキシを表す）である（２６）〜（２８）のいずれかに記載の中枢疾患の予防および／または治療剤。
（３２） Ｘが置換もしくは非置換のヒドロキシメチルである（２６）〜（２８）のいずれかに記載の中枢疾患の予防および／または治療剤。
（３３） Ａが置換もしくは非置換のアリール、置換もしくは非置換のアラルキルまたは置換もしくは非置換の複素環基である（２６）〜（３２）のいずれかに記載の中枢疾患の予防および／または治療剤。
（３４） Ｒ^１が水素原子である（２６）〜（３３）のいずれかに記載の中枢疾患の予防および／または治療剤。
（３５） Ｒ^３が置換もしくは非置換の低級アルキル、シクロアルキルアルキルまたは置換もしくは非置換のアラルキルであるか、またはＲ^３とＲ^４が隣接する窒素原子と一緒になって置換もしくは非置換の複素環基を形成する（２８）〜（３４）のいずれかに記載の中枢疾患の予防および／または治療剤。
（３６） Ｒ^３が置換もしくは非置換の低級アルキル、シクロアルキルアルキルまたは置換もしくは非置換のアラルキルであり、Ｒ^４が水素原子または低級アルキルである（２８）〜（３４）のいずれかに記載の中枢疾患の予防および／または治療剤。
（３７） 中枢疾患がパーキンソン病、うつ病および時差ぼけからなる群から選ばれる疾患である（２６）〜（３６）のいずれかに記載の予防および／または治療剤。
（３８） 式（ＩＡ）

｛式中、ＡおよびＲ^１はそれぞれ前記と同義であり、Ｘ^Ａは１，２，３，４−テトラゾール−５−イル、−Ｃ（＝Ｏ）Ｒ^５Ａ［式中、Ｒ^５Ａはヒドロキシまたは−ＮＲ^６ＡＲ^７Ａ（式中、Ｒ^６Ａは水素原子または低級アルキルを表し、Ｒ^７Ａは水素原子、ヒドロキシ、ヒドロキシ低級アルキル、カルボキシ低級アルキル、ｐ−トルエンスルホニル、メタンスルホニルまたはトリフルオロメタンスルホニルを表す）を表す］または置換もしくは非置換のヒドロキシメチルを表し、（ｉ）Ｘ^Ａが１，２，３，４−テトラゾール−５−イルであるとき、Ｒ^３ＡおよびＲ^４Ａは、同一または異なって、水素原子、置換もしくは非置換の低級アルキル、置換もしくは非置換のシクロアルキル、置換もしくは非置換のシクロアルキルアルキルまたは置換もしくは非置換のアラルキルを表す（ただし、同時に水素原子にはならない）か、またはＲ^３ＡとＲ^４Ａが隣接する窒素原子と一緒になって置換もしくは非置換の複素環基を形成し、（ｉｉ）Ｘ^Ａが−Ｃ（＝Ｏ）Ｒ^５Ａまたは置換もしくは非置換のヒドロキシメチルであるとき、Ｒ^３ＡおよびＲ^４Ａは、同一または異なって、水素原子、置換もしくは非置換の低級アルキル、置換もしくは非置換のシクロアルキル、置換もしくは非置換のシクロアルキルアルキル、置換もしくは非置換の複素環アルキルまたは置換もしくは非置換のアラルキルを表す（ただし、同時に水素原子にはならない）か、またはＲ^３ＡとＲ^４Ａが隣接する窒素原子と一緒になって置換もしくは非置換のピペラジニル、４−メチルホモピペラジニル、４−メチルピペリジノ、４−ピペリジノピペリジノ、４−ベンジルピペリジノ、４，４−エチレンジオキシピペリジノまたはデカヒドロイソキノリン−１−イルを形成する｝で表されるピリミジン誘導体またはその薬理学的に許容される塩。
（３９） Ｘ^Ａが１，２，３，４−テトラゾール−５−イルである（３８）記載のピリミジン誘導体またはその薬理学的に許容される塩。
（４０） Ｘ^Ａが−Ｃ（＝Ｏ）Ｒ^５Ａ（式中、Ｒ^５Ａは前記と同義である）である（３８）記載のピリミジン誘導体またはその薬理学的に許容される塩。
（４１） Ｘ^Ａが−Ｃ（＝Ｏ）ＯＨである（３８）記載のピリミジン誘導体またはその薬理学的に許容される塩。
（４２） Ｘ^Ａがヒドロキシメチル、１−ヒドロキシエチルまたは１−ヒドロキシ−１−メチルエチルである（３８）記載のピリミジン誘導体またはその薬理学的に許容される塩。
（４３） Ａが置換もしくは非置換のアリール、置換もしくは非置換のアラルキル、置換もしくは非置換のアリールオキシアルキル、置換もしくは非置換の複素環基またはシクロアルキルアルキルであるか、またはＡとＲ^１が隣接する窒素原子と一緒になって置換もしくは非置換の複素環基を形成する（３８）〜（４２）のいずれかに記載のピリミジン誘導体またはその薬理学的に許容される塩。
（４４） Ａが置換もしくは非置換のアリール、アラルキル、複素環基またはシクロアルキルアルキルである（３８）〜（４２）のいずれかに記載のピリミジン誘導体またはその薬理学的に許容される塩。
（４５） Ａがベンジル、４−フェニル−ｎ−ブチル、３−フェニルプロピル、３−フェニル−１−メチルプロピル、２−フェニルプロピル、２−フェノキシエチル、２−フェノキシ−１−メチルエチル、１，４−ベンゾジオキサン−６−イル、２−ベンジルフェニル、２−フェノキシフェニル、３−フェノキシフェニル、４−フェノキシフェニル、４−ビフェニル、ビフェニル−４−イルメチル、２−インダニルおよびシクロプロピルメチルからなる群から選ばれる基である（３８）〜（４２）のいずれかに記載のピリミジン誘導体またはその薬理学的に許容される塩。
（４６） Ｒ^１が水素原子、低級アルキルまたはアラルキルである請求の範囲第３８〜４５項のいずれかに記載のピリミジン誘導体またはその薬理学的に許容される塩。
（４７） Ｒ^１が水素原子である（３８）〜（４５）のいずれかに記載のピリミジン誘導体またはその薬理学的に許容される塩。
（４８） Ｒ^３ＡおよびＲ^４Ａが同一または異なって、水素原子、置換もしくは非置換の低級アルキル、シクロアルキルアルキル、置換もしくは非置換のアラルキルまたは置換もしくは非置換の複素環アルキルであるか、またはＲ^３ＡとＲ^４Ａが隣接する窒素原子と一緒となって置換もしくは非置換の複素環基を形成する（３８）〜（４７）のいずれかに記載のピリミジン誘導体またはその薬理学的に許容される塩。
（４９） Ｒ^３ＡおよびＲ^４Ａが同一または異なって、水素原子、低級アルキル、シクロアルキルメチル、置換もしくは非置換のアラルキルまたは複素環アルキルである（３８）〜（４７）のいずれかに記載のピリミジン誘導体またはその薬理学的に許容される塩。
（５０） Ｒ^３Ａが低級アルキル、シクロアルキルメチルまたは置換もしくは非置換のアラルキルであり、Ｒ^４Ａが水素原子である（３８）〜（４７）のいずれかに記載のピリミジン誘導体またはその薬理学的に許容される塩。
（５１） Ｒ^３Ａが３−メトキシベンジル、３−クロロベンジル、３−フルオロベンジル、２，６−ジフルオロベンジル、２−メチルベンジル、３−トリフルオロベンジル、２−ピリジルメチル、３−ピリジルメチル、シクロヘキシルメチル、シクロヘプチル、１，５−ジメチル−ｎ−ヘキシルおよび２，３−ジヒドロベンゾ［１，４］ジオキシン−２−イルメチルからなる群から選ばれる基であり、Ｒ^４Ａが水素原子であるか、Ｒ^３ＡがベンジルでありＲ^４Ａがｎ−ブチルであるか、Ｒ^３Ａがｎ−ヘキシルでありＲ^４Ａがメチルであるか、またはＲ^３ＡとＲ^４Ａが隣接する窒素原子と一緒になって４−ベンジルピペリジノ、４，４−エチレンジオキシピペリジノもしくはデカヒドロイソキノリン−１−イルを形成する（３８）および（４０）〜（４７）のいずれかに記載のピリミジン誘導体またはその薬理学的に許容される塩。
（５２） Ｒ^３Ａが３−メトキシベンジル、３−クロロベンジル、３−フルオロベンジル、２，６−ジフルオロベンジル、２−メチルベンジル、３−トリフルオロベンジル、シクロヘキシルメチル、シクロヘプチルおよび１，５−ジメチル−ｎ−ヘキシルからなる群から選ばれる基であり、Ｒ^４Ａが水素原子であるか、Ｒ^３ＡがベンジルでありＲ^４Ａがｎ−ブチルであるか、Ｒ^３Ａがｎ−ヘキシルでありＲ^４Ａがメチルであるか、またはＲ^３ＡとＲ^４Ａが隣接する窒素原子と一緒になって４−ベンジルピペリジノ、４，４−エチレンジオキシピペリジノもしくはデカヒドロイソキノリン−１−イルを形成する（３９）記載のピリミジン誘導体またはその薬理学的に許容される塩。
（５３） 式（ＩＡ）で表される化合物が、４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボン酸、４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボヒドロキサム酸、［４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボニル］アミノ酢酸、｛［４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボニル］メチルアミノ｝酢酸、４−（２−ベンジルフェニルアミノ）−５−ヒドロキシメチル−２−（シクロヘキシルメチルアミノ）ピリミジン、Ｎ−［４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボニル］−４−メチルベンゼンスルホンアミド、Ｎ−［４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボニル］メタンスルホンアミド、［４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボニル］トリフルオロメタンスルホンアミド、２−（シクロヘキシルメチルアミノ）−４−（１−メチル−２−フェノキシエチルアミノ）ピリミジン−５−カルボン酸、２−（シクロヘキシルメチルアミノ）−４−（２−フェノキシエチルアミノ）ピリミジン−５−カルボン酸、２−（シクロヘキシルメチルアミノ）−４−（２−フェノキシフェニルアミノ）ピリミジン−５−カルボン酸、４−（４−フェニルピペリジン−１−イル）−２−（３−トリフルオロメチルベンジルアミノ）ピリミジン−５−カルボン酸、２−（シクロヘキシルメチルアミノ）−５−ヒドロキシメチル−４−（１−メチル−２−フェノキシエチルアミノ）ピリミジン、２−（シクロヘキシルメチルアミノ）−５−ヒドロキシメチル−４−（２−フェノキシエチルアミノ）ピリミジン、２−（ベンジルブチルアミノ）−５−ヒドロキシメチル−４−（２−フェノキシエチルアミノ）ピリミジン、５−ヒドロキシメチル−２−（オクタヒドロイソキノリン−２−イル）−４−（２−フェノキシエチルアミノ）ピリミジン、２−（シクロヘキシルメチルアミノ）−５−ヒドロキシメチル−４−（２−フェノキシフェニルアミノ）ピリミジン、５−ヒドロキシメチル−４−（２−フェニルプロピルアミノ）−２−（３−トリフルオロメチルベンジルアミノ）ピリミジン、５−ヒドロキシメチル−４−（４−フェニルピペリジン−１−イル）−２−（３−トリフルオロメチルベンジルアミノ）ピリミジン、４−（２−ベンジルフェニルアミノ）−２−（３−クロロベンジルアミノ）ピリミジン−５−カルボン酸、４−（２−ベンジルフェニルアミノ）−２−（３−フルオロベンジルアミノ）ピリミジン−５−カルボン酸、２−（ベンジルブチルアミノ）−４−（２，３−ジヒドロベンゾ［１，４］ジオキシン−６−イルアミノ）ピリミジン−５−カルボン酸、２−（３−メトキシベンジルアミノ）−４−（２−フェノキシフェニルアミノ）ピリミジン−５−カルボン酸、２−（２，６−ジフルオロベンジルアミノ）−４−（２−フェノキシフェニルアミノ）ピリミジン−５−カルボン酸、２−（２−メチルベンジルアミノ）−４−（２−フェノキシフェニルアミノ）ピリミジン−５−カルボン酸、４−（２−フェノキシフェニルアミノ）−２−（３−トリフルオロベンジルアミノ）ピリミジン−５−カルボン酸、２−（３−フルオロベンジルアミノ）−４−（４−フェニルブチルアミノ）ピリミジン−５−カルボン酸、２−（ベンジルブチルアミノ）−４−（４−ビフェニルメチルアミノ）ピリミジン−５−カルボン酸、４−（２，３−ジヒドロベンゾ［１，４］ジオキシン−６−イルアミノ）−５−ヒドロキシメチル−２−（３−トリフルオロメチルベンジルアミノ）ピリミジン、４−（２，３−ジヒドロベンゾ［１，４］ジオキシン−６−イルアミノ）−５−ヒドロキシメチル−２−（２−ピリジルメチルアミノ）ピリミジンおよび２−［ビス（２−メトキシエチル）アミノ］−４−（２，３−ジヒドロベンゾ［１，４］ジオキシン−６−イルアミノ）−５−ヒドロキシメチルピリミジン、２−（３−フルオロベンジルアミノ）−５−ヒドロキシメチル−４−（２−フェノキシエチルアミノ）ピリミジンからなる群から選択される化合物であるピリミジン誘導体またはその薬理学的に許容される塩。
（５４） （３８）〜（５３）のいずれかに記載のピリミジン誘導体またはその薬理学的に許容される塩を有効成分として含有する医薬。
（５５） （３８）〜（５３）のいずれかに記載のピリミジン誘導体またはその薬理学的に許容される塩を有効成分として含有するＧＰＲ８８の機能抑制剤。
（５６） （３８）〜（５３）のいずれかに記載のピリミジン誘導体またはその薬理学的に許容される塩を有効成分として含有する中枢疾患の予防および／または治療剤。
（５７） 中枢疾患がパーキンソン病、うつ病および時差ぼけからなる群から選ばれる疾患である（５６）記載の予防および／または治療剤。
（５８） 中枢疾患の予防および／または治療剤の製造のための、ＧＰＲ８８の機能抑制作用を有する物質の使用。
（５９） ＧＰＲ８８の機能抑制剤の製造のための、配列番号１、３または５で表される塩基配列の連続する１０塩基以上の配列に相補的または実質的に相補的な塩基配列を有するアンチセンスオリゴヌクレオチドの使用。
（６０） ＧＰＲ８８の機能抑制剤の製造のための、（１５）記載のピリミジン誘導体またはその薬理学的に許容される塩の使用。
（６１） 中枢疾患の予防および／または治療剤の製造のための、（１５）記載のピリミジン誘導体またはその薬理学的に許容される塩の使用。
（６２） ＧＰＲ８８の機能抑制作用を有する物質の有効量を投与することを特徴とする中枢疾患の予防および／または治療方法。
（６３） 配列番号１、３または５で表される塩基配列の連続する１０塩基以上の配列に相補的または実質的に相補的な塩基配列を有するアンチセンスオリゴヌクレオチドの有効量を投与することを特徴とするＧＰＲ８８の機能抑制方法。
（６４） （１５）記載のピリミジン誘導体またはその薬理学的に許容される塩の有効量を投与することを特徴とするＧＰＲ８８の機能抑制方法。
（６５） （１５）記載のピリミジン誘導体またはその薬理学的に許容される塩の有効量を投与することを特徴とする中枢疾患の予防および／または治療方法。
本発明において、ＧＰＲ８８としては、配列番号２で表されるアミノ酸配列を有するヒトＧＰＲ８８ポリペプチド、配列番号４で表されるアミノ酸配列を有するマウスＧＰＲ８８ポリペプチドおよび配列番号６で表されるアミノ酸配列を有するラットのＧＰＲ８８ポリペプチドが挙げられる。また、ヒトＧＰＲ８８ ｃＤＮＡのコード領域の配列である配列番号１で表される塩基配列の４３８〜１５９２番目の配列と相補的な塩基配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズするＤＮＡがコードする、上記のポリペプチドと実質的に同一な機能を有するポリペプチドも含まれる。本発明で用いるＧＰＲ８８としては、特に配列番号２で表されるアミノ酸配列を有するヒトＧＰＲ８８ポリペプチドが好ましい。
ストリンジェントな条件下でハイブリダイズするＤＮＡとは、配列番号１で表される塩基配列と相補的な配列を有するＤＮＡをプローブとして、コロニー・ハイブリダイゼーション法、プラーク・ハイブリダイゼーション法あるいはサザンブロットハイブリダイゼーション法等を用いることにより得られるＤＮＡを意味し、具体的には、コロニーあるいはプラーク由来のＤＮＡを固定化したフィルターを用いて、０．７〜１．０ｍｏｌ／ＬのＮａＣｌ存在下、６５℃でハイブリダイゼーションを行った後、０．１〜２倍濃度のＳＳＣ溶液（１倍濃度のＳＳＣ溶液の組成は、１５０ｍｍｏｌ／Ｌ ＮａＣｌ、１５ｍｍｏｌ／Ｌクエン酸ナトリウムよりなる）を用い、６５℃条件下でフィルターを洗浄することにより同定できるＤＮＡを挙げることができる。ハイブリダイゼーションは、Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ：Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ，３ｒｄ Ｅｄｉｔｉｏｎ，Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ（２００１）、Ｃｕｒｒｅｎｔ Ｐｒｏｔｏｃｏｌｓ ｉｎ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ，Ｊｏｈｎ Ｗｉｌｅｙ ＆ Ｓｏｎｓ（１９８７−２００１）、ＤＮＡ Ｃｌｏｎｉｎｇ １：Ｃｏｒｅ Ｔｅｃｈｎｉｑｕｅｓ，Ａ Ｐｒａｃｔｉｃａｌ Ａｐｐｒｏａｃｈ，Ｓｅｃｏｎｄ Ｅｄｉｔｉｏｎ，Ｏｘｆｏｒｄ Ｕｎｉｖｅｒｓｉｔｙ（１９９５）等に記載されている方法に準じて行うことができる。ハイブリダイズ可能なＤＮＡとして具体的には、配列番号１で表される塩基配列と少なくとも６０％以上の相同性を有するＤＮＡ、好ましくは７０％以上、より好ましくは８０％以上、さらに好ましくは９０％以上、特に好ましくは９５％以上、最も好ましくは９８％以上の相同性を有するＤＮＡを挙げることができる。
＜ＧＰＲ８８発現細胞を用いた、ＧＰＲ８８の構成活性阻害物質、中枢疾患の予防および／または治療のための作用を有する化合物のスクリーニング法＞
参考例５〜７に示す通り、ＧＰＲ８８は、細胞に過剰に発現させた際に、リガンドが存在しなくても活性化し、シグナル情報伝達を起こす構成活性型ＧＰＣＲであるため、ＧＰＲ８８を発現する細胞は、リガンドが存在しなくともＧＰＲ８８の活性化に伴う細胞応答を示す。ＧＰＲ８８を発現する細胞に任意の試験物質を添加して、該細胞と試験物質とを接触させた場合の該細胞の細胞応答と、試験物質を添加しない場合の該細胞の細胞応答をそれぞれ測定して比較し、試験物質を添加しなかった場合と比較して、試験物質を添加した場合の細胞応答が低下する試験物質を、ＧＰＲ８８の構成活性阻害物質として選択することができる。また、他の構成活性型ＧＰＣＲを発現させた細胞を以下に示すＧＰＲ８８発現細胞と同様にして作製し、上記の方法と同様にして、該細胞と上記の方法で選択された物質とを接触させた場合の細胞応答の低下を測定して、ＧＰＲ８８発現細胞での細胞応答の低下と比較することにより、選択された物質がＧＰＲ８８特異的に構成活性を阻害するかどうかを調べることができる。ＧＰＲ８８の構成活性阻害物質は、ＧＰＲ８８の機能を抑制する作用を有しており、ＧＰＲ８８の機能抑制剤となる。ＧＰＲ８８の構成活性阻害物質には、ＧＰＲ８８と結合してその構成活性を阻害するインバースアゴニスト、ＧＰＲ８８とＧ蛋白質とのカップリングを阻害する物質等のＧＰＲ８８が関与するシグナル伝達を阻害する物質が含まれる。後述するように、ＧＰＲ８８の機能抑制剤は、中枢疾患の予防および／または治療に有効であるので、上記の方法により中枢疾患治療薬または予防作用を有する化合物をスクリーニングすることができる。
ＧＰＲ８８を発現する細胞は、ＧＰＲ８８を天然に発現する組織から、あるいはＧＰＲ８８をコードするＤＮＡを含む発現ベクター（ＧＰＲ８８発現ベクター）を宿主細胞に導入することによって得ることができる。ＧＰＲ８８の構成活性を測定するためには、ＧＰＲ８８を大量に発現している細胞が適しているため、ＧＰＲ８８発現ベクターを宿主細胞に導入して得られる形質転換細胞が好ましい。
ＧＰＲ８８をコードするＤＮＡは、以下のようにして取得できる。ＧＰＲ８８ ｃＤＮＡの塩基配列、例えば配列番号１で表されるヒトＧＰＲ８８ ｃＤＮＡの塩基配列を元にして、該受容体をコードする領域を含む該ｃＤＮＡの領域を適当に選択する。選択した領域の塩基配列の５’端２０〜４０塩基の配列を３’端に含むＤＮＡ、選択した領域の塩基配列の３’端２０〜４０塩基と相補的な配列を３’端に含むＤＮＡをそれぞれＤＮＡ合成機で合成する。例えば、配列番号２６および２７で表される塩基配列をそれぞれ有するＤＮＡが例示される。組織や細胞から染色体ＤＮＡまたはｃＤＮＡを調製する。ｃＤＮＡの場合はＧＰＲ８８が発現している組織や細胞を用いる。調製した染色体ＤＮＡまたはｃＤＮＡを鋳型とし、２種類の合成ＤＮＡをプライマーとして用いたＰＣＲにより該受容体をコードするＤＮＡを増幅し単離することができる。組織や細胞からの染色体ＤＮＡまたはｃＤＮＡの調製、およびＰＣＲはＭｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ：Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ ３ｒｄ ｅｄ．，Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ（２００１）に記載の方法に従って行うことができる。
発現ベクターとしては、上記宿主細胞において自立複製が可能、または染色体中への組込みが可能で、導入する宿主細胞で機能するプロモーターを含有しているものを用いることができる。発現ベクター中のプロモーターの下流にＧＰＲ８８をコードするＤＮＡを挿入することにより、ＧＰＲ８８発現ベクターを構築することができる。
宿主細胞にＧＰＲ８８発現ベクターを導入する方法としては、宿主細胞にＤＮＡを導入する方法であればいずれも用いることができる。宿主細胞としては、酵母、昆虫細胞、動物細胞、植物細胞等を用いることができる。
酵母菌株を宿主細胞として用いる場合には、発現ベクターとして、例えば、ＹＥｐ１３（ＡＴＣＣ３７１１５）、ＹＥｐ２４（ＡＴＣＣ３７０５１）、ＹＣｐ５０（ＡＴＣＣ３７４１９）、ｐＨＳ１９、ｐＨＳ１５等を例示することができる。プロモーターとしては、酵母菌株中で発現できるものであればいかなるものでもよく、例えば、ＰＨＯ５プロモーター、ＰＧＫプロモーター、ＧＡＰプロモーター、ＡＤＨプロモーター、ｇａｌ１プロモーター、ｇａｌ１０プロモニター、ヒートショック蛋白質プロモーター、ＭＦα１プロモーター、ＣＵＰ１プロモーター等のプロモーターを挙げることができる。
宿主細胞としては、例えば、サッカロマイセス属、シゾサッカロマイセス属、クルイベロミセス属、トリコスポロン属、シワニオミセス属等に属する酵母菌株を挙げることができ、具体的には、Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ、Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓｐｏｍｂｅ、Ｋｌｕｙｖｅｒｏｍｙｃｅｓ ｌａｃｔｉｓ、Ｔｒｉｃｈｏｓｐｏｒｏｎ ｐｕｌｌｕｌａｎｓ、Ｓｃｈｗａｎｎｉｏｍｙｃｅｓａｌｌｕｖｉｕｓ等を挙げることができる。またＧＰＣＲの発現に適した変異株を用いることもできる〔Ｔｒｅｎｄｓ ｉｎ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ，１５，４８７（１９９７）、Ｍｏｌ．Ｃｅｌｌ．Ｂｉｏｌ．，１５，６１８８（１９９５）、Ｍｏｌ．Ｃｅｌｌ．Ｂｉｏｌ．，１６，４７００（１９９６）〕。
組換えベクターの導入方法としては、酵母にＤＮＡを導入する方法であればいずれも用いることができ、例えば、エレクトロポレーション法〔Ｍｅｔｈｏｄｓ．ｉｎ Ｅｎｚｙｍｏｌ．，１９４，１８２（１９９０）〕、スフェロプラスト法〔Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，８４，１９２９（１９７８）〕、酢酸リチウム法〔Ｊ．Ｂａｃｔｅｒｉｏｌ．，１５３，１６３（１９８３）〕、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，７５，１９２９（１９７８）記載の方法等を挙げることができる。
動物細胞を宿主細胞として用いる場合には、発現ベクターとして、例えば、ｐｃＤＮＡ３．１（＋）、ｐＣＤＭ８、ｐＡＧＥ１０７、ｐＲＥＰ４、ｐＡＧＥ１０３、ｐＡＭｏ、ｐＡＭｏＡ、ｐＡＳ３−３等を例示することができる。特に誘導発現用のベクター、例えば、Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ由来の転写因子Ｇａｌ４のＤＮＡ結合領域とエストロジェン受容体のリガンド結合領域のキメラ蛋白質（Ｇａｌ４−ＥＲ）〔Ｃｅｌｌ，５４，１９９（１９８８）、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，９０，１６５７（１９９３）〕を発現する宿主細胞に導入した場合に、１７β−エストラジオールの添加により発現誘導が可能となるｐＡＧａｌ９−ｄおよびｐＡＧａｌ９−ｎｄ等が好ましい。また、複製開始点として、エプスタイン・バー・ウイルスのｏｒｉＰを有するｐＡＭｏ、ｐＡＭｏＡ、ｐＡＧａｌ９−ｄ、ｐＡＧａｌ９−ｎｄ等のベクターは、Ｎａｍａｌｗａ細胞、Ｎａｍａｌｗａ ＫＪＭ−１細胞等のエプスタイン・バー・ウイルスのＥＢＮＡ−１遺伝子を発現するＢ細胞株を宿主細胞としたときに、染色体外にプラスミド状態で安定に存在するため、高い発現を示す形質転換株を容易に得ることができる。
プロモーターとしては、動物細胞中で発現できるものであればいずれも用いることができ、例えば、サイトメガロウイルス（ＣＭＶ）のＩＥ（ｉｍｍｅｄｉａｔｅ ｅａｒｌｙ）遺伝子のプロモーター、ＳＶ４０の初期プロモーター、モロニーネズミ白血病ウイルスのロング・ターミナル・リピート・プロモーター（Ｌｏｎｇ Ｔｅｒｍｉｎａｌ Ｒｅｐｅａｔ Ｐｒｏｍｏｔｅｒ）、レトロウイルスのプロモーター、ヒートショックプロモーター、ＳＲαプロモーター、あるいはメタロチオネインのプロモーター等を挙げることができる。また、ヒトＣＭＶのＩＥ遺伝子のエンハンサーをプロモーターと共に用いてもよい。
宿主細胞としては、例えば、マウス・ミエローマ細胞、ラット・ミエローマ細胞、マウス・ハイブリドーマ細胞、ＣＨＯ細胞、ＢＨＫ細胞、アフリカミドリザル腎臓細胞、Ｎａｍａｌｗａ細胞、Ｎａｍａｌｗａ ＫＪＭ−１細胞、ヒト胎児腎臓細胞、ヒト白血病細胞、ＨＢＴ５６３７（特開昭６３−２９９）、ヒト大腸癌細胞株、カエルの卵母細胞およびカエルのメラニン細胞等を挙げることができる。
具体的には、マウス・ミエローマ細胞としては、ＳＰ２／０、ＮＳＯ等、ラット・ミエローマ細胞としてはＹＢ２／０等、ヒト胎児腎臓細胞としてはＨＥＫ２９３等、ヒト白血病細胞としてはＢＡＬＬ−１等、アフリカミドリザル腎臓細胞としてはＣＯＳ−１、ＣＯＳ−７、ヒト大腸癌細胞株としてはＨＣＴ−１５等を挙げることができる。
組換えベクターの導入方法としては、動物細胞にＤＮＡを導入する方法であればいずれも用いることができ、例えば、エレクトロポレーション法〔Ｃｙｔｏｔｅｃｈｎｏｌｏｇｙ，３，１３３（１９９０）〕、リン酸カルシウム法（特開平２−２２７０７５）、リポフェクション法〔Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，８４，７４１３（１９８７）〕、Ｖｉｒｏｌｏｇｙ，５２，４５６（１９７３）に記載の方法等を挙げることができる。形質転換体の取得および培養は、特開平２−２２７０７５号公報あるいは特開平２−２５７８９１号公報に記載されている方法に準じて行なうことができる。
昆虫細胞を宿主細胞として用いる場合には、例えば、Ｂａｃｕｌｏｖｉｒｕｓ Ｅｘｐｒｅｓｓｉｏｎ Ｖｅｃｔｏｒｓ，Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ，Ｗ．Ｈ．Ｆｒｅｅｍａｎ ａｎｄ Ｃｏｍｐａｎｙ，Ｎｅｗ Ｙｏｒｋ（１９９２）、Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ：Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ ３ｒｄ ｅｄ．，Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ（２００１）、Ｂｉｏ／Ｔｅｃｈｎｏｌｏｇｙ，６，４７（１９８８）等に記載された方法によって、ＧＰＲ８８を発現する形質転換体を得ることができる。
即ち、組換え遺伝子導入ベクターおよびバキュロウイルスを昆虫細胞に共導入して昆虫細胞培養上清中に組換えウイルスを得た後、さらに組換えウイルスを昆虫細胞に感染させ、ポリペプチドを発現させることができる。
該方法において用いられる遺伝子導入ベクターとしては、例えば、ｐＶＬ１３９２、ｐＶＬ１３９３、ｐＢｌｕｅＢａｃＩＩＩ（すべてインビトロジェン社製）等を挙げることができる。
バキュロウイルスとしては、例えば、ヤガ科昆虫に感染するウイルスであるアウトグラファ・カリフォルニカ・ヌクレアー・ポリヘドロシス・ウイルス（Ａｕｔｏｇｒａｐｈａ ｃａｌｉｆｏｒｎｉｃａ ｎｕｃｌｅａｒ ｐｏｌｙｈｅｄｒｏｓｉｓ ｖｉｒｕｓ）等を用いることができる。
昆虫細胞としては、Ｓｐｏｄｏｐｔｅｒａ ｆｒｕｇｉｐｅｒｄａの卵巣細胞、Ｔｒｉｃｈｏｐｌｕｓｉａｎｉの卵巣細胞、カイコ卵巣由来の培養細胞等を用いることができる。Ｓｐｏｄｏｐｔｅｒａ ｆｒｕｇｉｐｅｒｄａの卵巣細胞としてはＳｆ９、Ｓｆ２１（バキュロウイルス・イクスプレッション・ベクターズ ア・ラボラトリー・マニュアル）等、Ｔｒｉｃｈｏｐｌｕｓｉａ ｎｉの卵巣細胞としてはＨｉｇｈ５（インビトロジェン社製）等、カイコ卵巣由来の培養細胞としてはＢｏｍｂｙｘｍｏｒｉ Ｎ４等を挙げることができる。
組換えウイルスを調製するための、昆虫細胞への上記組換え遺伝子導入ベクターと上記バキュロウイルスの共導入方法としては、例えば、リン酸カルシウム法（特開平２−２２７０７５）、リポフェクション法〔Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，８４，７４１３（１９８７）〕等を挙げることができる。また、動物細胞にＤＮＡを導入する方法と同様の方法、例えば、エレクトロポレーション法〔Ｃｙｔｏｔｅｃｈｎｏｌｏｇｙ，３，１３３（１９９０）〕、リン酸カルシウム法（特開平２−２２７０７５）、リポフェクション法〔Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，８４，７４１３（１９８７）〕等を用いて、昆虫細胞にＤＮＡを導入することもできる。
植物細胞を宿主細胞として用いる場合には、公知の方法〔組織培養，２０（１９９４）、組織培養，２１（１９９５）、Ｔｒｅｎｄｓ ｉｎ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ，１５，４５（１９９７）〕に準じてＧＰＲ８８を発現する形質転換体を得ることができる。
このとき用いられるプロモーターとしては、植物細胞中で発現できるものであればいずれも用いることができ、例えば、カリフラワーモザイクウイルスの３５Ｓプロモーター、イネアクチン１プロモーター等を挙げることができる。また、プロモーターと発現させる遺伝子の間に、トウモロコシのアルコール脱水素酵素遺伝子のイントロン１等を挿入することにより、遺伝子の発現効率を挙げることもできる。
このとき用いられる宿主細胞としては、例えば、ジャガイモ、タバコ、トウモロコシ、イネ、アブラナ、大豆、トマト、小麦、大麦、ライ麦、アルファルファ、亜麻等の植物細胞等を挙げることができる。
このとき用いられる組換えベクターの導入方法としては、植物細胞にＤＮＡを導入する方法であればいずれも用いることができ、例えば、アグロバクテリウム（Ａｇｒｏｂａｃｔｅｒｉｕｍ）（特開昭５９−１４０８８５、特開昭６０−７００８０、ＷＯ９４／００９７７）、エレクトロポレーション法〔Ｃｙｔｏｔｅｃｈｎｏｌｏｇｙ，３，１３３（１９９０）、特開昭６０−２５１８８７〕、パーティクルガン（遺伝子銃）を用いる方法（特許第２６０６８５６号、特許第２５１７８１３号）等を挙げることができる。
細胞の応答としては、例えば、アラキドン酸遊離、アセチルコリン遊離、細胞内Ｃａ^２＋遊離、細胞内ｃＡＭＰ生成、細胞内ｃＡＭＰ減少、細胞内ｃＧＭＰ生成、イノシトールリン酸産生、細胞膜電位変動、細胞内蛋白質（例えば、ＣＲＥＢ、ＳＴＡＴ１、ＳＴＡＴ２、ＳＴＡＴ３、ＳＴＡＴ４、ＳＴＡＴ５ａ、ＳＴＡＴ５ｂ、ＳＴＡＴ６、ＭＡＰＫ、ＡＴＦ−２、ｃ−Ｊｕｎ、ｃ−ｆｏｓ、Ｉκ−Ｂα、Ｉκ−Ｂβ、Ｓｍａｄ１、Ｓｍａｄ２、Ｓｍａｄ３、Ｓｍａｄ５、Ｓｍａｄ８等）のリン酸化、ｃ−ｆｏｓ活性化、ｐＨの変動、細胞増殖活性、あるいはレポーター遺伝子やマーカー遺伝子の発現量があげられ、これらを調べることにより細胞の応答を測定することができる〔Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，２７１，１８５７（１９９６）、Ｓｃｉｅｎｃｅ，２６８，９８（１９９５）、Ｊ．Ｐｈａｒｍａｃｏｌ．Ｅｘｐ．Ｔｈｅｒ．，２７５，１２７４（１９９５）、Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，２７２，１８２２（１９９７）、Ｊ．Ｒｅｃｅｐｔ．Ｓｉｇｎａｌ Ｔｒａｎｓｄｕｃｔ．Ｒｅｓ．，１７，５７（１９９７）、Ｅｎｄｏｃｒｉｎｏｌｏｇｙ１３８，１４００（１９９７）、Ｅｎｄｏｃｒｉｎｏｌｏｇｙ１３８，１４７１（１９９７）、Ｎａｔ．Ｂｉｏｔｅｃｈｎｏｌ．，１６，１３３４（１９９８）、Ｂｉｏｃｈｅｍ．Ｂｉｏｐｈｙｓ．Ｒｅｓ．Ｃｏｍｍｕｎ．，２５１，４７１（１９９８）、Ｂｒ．Ｊ．Ｐｈａｒｍａｃｏｌ．，１２５，１３８７（１９９８）、Ｔｒｅｎｄｓ Ｂｉｏｔｅｃｈｎｏｌ．，１５，４８７（１９９７）、Ａｎａｌ．Ｂｉｏｃｈｅｍ．，２５２，１１５（１９９７）、Ｎａｔｕｒｅ，３５８，３２５（１９９２）、Ｎａｔｕｒｅ，３９３，２７２（１９９８）、Ｃｅｌｌ，９２，５７３（１９９８）、Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，２７２，２７４９７（１９９７）、Ｔｒｅｎｄｓ Ｐｈａｒｍａｃｏｌ．Ｓｃｉ．，１８，４３０（１９９７）、Ｔｒｅｎｄｓ Ｐｈａｒｍａｃｏｌ．Ｓｃｉ．，２０，３７０（１９９９）、ＷＯ９８／４６９９５〕。
レポーター系を用いて細胞の応答をモニターする場合は、例えば、ＧＰＲ８８を発現させた細胞に、ＧＰＲ８８の活性化により発現が誘導されるような適当な転写因子の結合配列と基本プロモーターからなる人工プロモーター配列の下流に適当なレポーター遺伝子を連結したＤＮＡを導入することにより、ＧＰＲ８８の活性化をレポーター遺伝子の発現で測定することができる。転写因子の結合配列としては、Ｇ_ｓと共役するＧＰＣＲの活性化に伴い転写を活性化するＣＲＥ（ｃＡＭＰ ｒｅｓｐｏｎｓｉｖｅ ｅｌｅｍｅｎｔ）、Ｇ_ｑと共役するＧＰＣＲの活性化に伴い転写を活性化するＴＲＥ（ＴＰＡ ｒｅｓｐｏｎｓｉｖｅ ｅｌｅｍｅｎｔ）、Ｇ_ｉと共役するＧＰＣＲの活性化に伴い転写を活性化するＳＲＥ（ｓｅｒｕｍ ｒｅｓｐｏｎｓｉｖｅ ｅｌｅｍｅｎｔ）等が利用できる。また、Ｇα_ｓのＣ末端５アミノ酸をＧα_ｉおよびＧα_ｑのＣ末端５アミノ酸にそれぞれ置換したキメラＧα_ｓをＧＰＲ８８の発現細胞に発現させることにより、ＧＰＲ８８が共役するＧ蛋白質の種類が不明でＧｓ、Ｇｉ、Ｇｑのいずれであっても、ＧＰＲ８８の活性化に伴いＣＲＥを含む人工プロモーターからのレポーター遺伝子の発現が誘導される。
レポーター遺伝子としては、あらゆるレポーター遺伝子を使用可能であるが、例えば、ホタル・ルシフェラーゼ遺伝子、ウミシイタケ・ルシフェラーゼ遺伝子、クロラムフェニコール・アセチルトランスフェラーゼ遺伝子、β−グルクロニダーゼ遺伝子、β−ガラクトシダーゼ遺伝子、β−ラクタマーゼ遺伝子、エクオリン遺伝子およびグリーン・フルオレッセント・プロテイン遺伝子等が利用できる。
本発明で予防および／または治療される中枢疾患としては、例えば脳内におけるＧＰＲ８８の発現部位との関連が知られている疾患が挙げられ、具体的にはパーキンソン病、うつ病、不安、精神分裂病、アルツハイマー病、ハンチントン舞踏病、脊髄小脳変性症、脳卒中、不随意運動、注意欠陥障害、錐体外路症状、薬物中毒、アルコール中毒、時差ぼけ等が挙げられる。
本発明において、ＧＰＲ８８の機能抑制剤は、その性質のひとつとしてＧＰＲ８８の機能抑制作用を有している物質を有効成分として含有していればよく、その物質の構造は特に限定されない。ＧＰＲ８８の機能抑制作用を有する物質としては例えば、ＧＰＲ８８拮抗作用を有する化合物（例えば、インバースアゴニスト、アンタゴニスト）、抗体（例えば、中和抗体等）、アンチセンスオリゴヌクレオチド等の、ＧＰＲ８８の発現を低下させる物質、ＧＰＲ８８とＧ蛋白質とのカップリングを阻害するもの等シグナル伝達を阻害する物質等が挙げられる。
本発明で用いられるＧＰＲ８８遺伝子に対するアンチセンスオリゴヌクレオチドとしては、配列番号１、３または５の塩基配列の連続する１０塩基以上、好ましくは１０〜４０塩基、より好ましくは１５〜３０塩基の配列に相補的な、または実質的に相補的な塩基配列を有し、ＧＰＲ８８遺伝子の発現を特異的に抑制し得る作用を有するものであれば、いずれのアンチセンスオリゴヌクレオチドであってもよい。特にヒトＧＰＲ８８ｃＤＮＡの配列である配列番号１の塩基配列の連続する１０塩基以上、好ましくは１０〜４０塩基、より好ましくは１５〜３０塩基の配列に相補的な、または実質的に相補的な塩基配列を有するアンチセンスオリゴヌクレオチドが好ましい。ＧＰＲ８８遺伝子の発現を抑制し得る作用を有するためには、翻訳開始点（配列番号１に示す塩基配列の４３８番目の塩基に相当する）を含む領域あるいは翻訳開始点近傍の領域と相補的な配列を含む塩基配列からなるオリゴヌクレオチドが好ましい。実質的に相補的な塩基配列とは、配列番号１、３または５の塩基配列の連続する１０〜４０塩基の配列と９０％以上の相同性を有し、配列番号１の塩基配列からなるＤＮＡとストリンジェントな条件下でハイブリダイズするような塩基配列をいう。ヌクレアーゼ等の加水分解酵素による分解を防ぐために、アンチセンスＤＮＡを構成する各ヌクレオチドのリン酸残基は例えば、ホスホロチオエート、メチルホスホネート、ホスホロジチオネート等の化学修飾リン酸残基に置換されていてもよい。これらのアンチセンスオリゴヌクレオチドは、公知のＤＮＡ合成装置、ＷＯ９３／２００９５、ＷＯ０２／１０１８５に記載の方法等を用いて製造することができる。
本発明で用いられるＧＰＲ８８に対する抗体は、以下のようにして作製することができる。
ＧＰＲ８８ポリペプチドまたは部分ペプチドを抗原として用い、動物に投与することによりポリクローナル抗体を作製することができる。該抗原を投与する動物としては、ウサギ、ヤギ、ラット、マウス、ハムスター等を挙げることができる。該抗原の投与量は動物１匹当たり５０〜１００μｇが好ましい。抗原としてペプチドを用いる場合は、ペプチドをキーホール・リンペット・ヘモシアニンや牛チログロブリン等のキャリア蛋白に共有結合させたものを用いることができる。ＧＰＲ８８ポリペプチドは、上記のＧＰＲ８８を発現する細胞から、通常の蛋白質の精製法により得ることができる。また、ＧＰＲ８８を発現する細胞あるいは、その膜画分を抗原とすることもできる。ＧＰＲ８８の部分ペプチドは、ペプチド合成機によって合成することができる。
該抗原の投与は、１回目の投与の後１〜２週間おきに３〜１０回行う。各投与後、３〜７日目に眼底静脈叢より採血し、該血清が免疫に用いた抗原と反応することを酵素免疫測定法〔酵素免疫測定法：医学書院刊（１９７６年）、Ａｎｔｉｂｏｄｉｅｓ−Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ，Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ（１９８８）〕等で確認する。
免疫に用いた抗原に対し、その血清が充分な抗体価を示した上記動物より血清を取得し、該血清を分離、精製することによりポリクローナル抗体を取得することができる。
分離、精製する方法としては、遠心分離、４０〜５０％飽和硫酸アンモニウムによる塩析、カプリル酸沈殿〔Ａｎｔｉｂｏｄｉｅｓ−Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ，Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ，（１９８８）〕、またはＤＥＡＥ−セファロースカラム、陰イオン交換カラム、プロテインＡまたはＧ−カラムあるいはゲル濾過カラム等を用いるクロマトグラフィー等を、単独または組み合わせて処理する方法が挙げられる。
さらに、ＧＰＲ８８に対するモノクローナル抗体を以下のようにして作製することができる。
免疫に用いた抗原に対し、その血清が十分な抗体価を示した上記動物に抗原物質を最終投与した後３〜７日目に、脾臓を摘出する。該脾臓をＭＥＭ培地（日水製薬社製）中で細断し、ピンセットでほぐし、１，２００ｒｐｍで５分間遠心分離した後、上清を捨てる。得られた沈殿画分の脾細胞をトリスリン酸−塩化アンモニウム緩衝液（ｐＨ７．６５）で１〜２分間処理し赤血球を除去した後、ＭＥＭ培地で３回洗浄し、得られた脾細胞を抗体産生細胞として用いる。
骨髄腫細胞としては、マウスまたはラットから取得した株化細胞を使用する。例えば、８−アザグアニン耐性マウス（ＢＡＬＢ／ｃ由来）骨髄腫細胞株Ｐ３−Ｘ６３Ａｇ８−Ｕ１〔Ｃｕｒｒ．Ｔｏｐｉｃｓ．Ｍｉｃｒｏｂｉｏｌ．Ｉｍｍｕｎｏｌ．，８１，１（１９７８）、Ｅｕｒ．Ｊ．Ｉｍｍｕｎｏｌ．，６，５１１（１９７６）〕、ＳＰ２／０−Ａｇ１４〔Ｎａｔｕｒｅ，２７６，２６９（１９７８）〕、Ｐ３−Ｘ６３−Ａｇ８６５３〔Ｊ．Ｉｍｍｕｎｏｌ．，１２３，１５４８（１９７９）］、Ｐ３−Ｘ６３−Ａｇ８〔Ｎａｔｕｒｅ，２５６，４９５（１９７５）〕等を用いることができる。これらの細胞株は、８−アザグアニン培地〔ＲＰＭＩ １６４０培地に１．５ｍｍｏｌ／Ｌグルタミン、５０μｍｏｌ／Ｌ２−メルカプトエタノール、１０μｇ／ｍＬゲンタマイシンおよび１０％ウシ胎児血清を加えた培地（以下、正常培地という）に、さらに１５μｇ／ｍＬ８−アザグアニンを加えた培地〕で継代するが、細胞融合の３〜４日前に正常培地で培養し、融合には該細胞を２×１０^７個以上用いる。
上記の抗体産生細胞と骨髄腫細胞をＭＥＭ培地またはＰＢＳ（１．８３ｇ／Ｌリン酸二ナトリウム、０．２１ｇ／Ｌリン酸一カリウム、７．６５ｇ／Ｌ ＮａＣｌ、ｐＨ７．２）でよく洗浄し、細胞数が、抗体産生細胞：骨髄腫細胞＝５〜１０：１になるよう混合し、１，２００ｒｐｍで５分間遠心分離した後、上清を捨てる。得られた沈澱画分の細胞群をよくほぐし、該細胞群に、攪拌しながら、３７℃で、１０^８抗体産生細胞あたり、ポリエチレングリコール−１０００ ２ｇ、ＭＥＭ培地２ｍＬおよびジメチルスルホキシド０．７ｍＬを混合した溶液を０．２〜１ｍＬ添加し、更に１〜２分間毎にＭＥＭ培地１〜２ｍＬを数回添加する。添加後、ＭＥＭ培地を加えて全量が５０ｍＬになるように調製する。
該調製液を９００ｒｐｍで５分間遠心分離後、上清を捨てる。得られた沈殿画分の細胞を、ゆるやかにほぐした後、メスピペットによる吸込み、吹出しでゆるやかにＨＡＴ培地〔正常培地に０．１ｍｍｏｌ／Ｌヒポキサンチン、１５μｍｏｌ／Ｌチミジンおよび０．４μｍｏｌ／Ｌアミノプテリンを加えた培地〕１００ｍＬ中に懸濁する。該懸濁液を９６ウェル培養用プレートに１００μＬ／ウェルずつ分注し、５％ＣＯ_２インキュベーター中、３７℃で７〜１４日間培養する。培養後、培養上清の一部をとりＡｎｔｉｂｏｄｉｅｓ−Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ，Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ，（１９８８）等に記載されている酵素免疫測定法により、免疫に用いた抗原に特異的に反応するハイブリドーマを選択する。
酵素免疫測定法の具体的例として、以下の方法を挙げることができる。免疫の際、抗原に用いたＧＰＲ８８ポリペプチドまたはＧＰＲ８８の部分ペプチドをプレートにコートし、ハイブリドーマ培養上清を第一抗体として反応させる。さらに第二抗体としてペルオキシダーゼ等の酵素で標識した抗イムノグロブリン抗体（免疫の際、抗原を投与した動物のイムノグロブリンに対する抗体、例えばマウスを免疫した場合は抗マウスイムノグロブリン抗体を用いる）を反応させた後に、酵素により発色する試薬を添加して反応を行ない、その培養上清が特異的に反応したものをＧＰＲ８８に対するモノクローナル抗体を生産するハイブリドーマとして選択する。
選択したハイブリドーマを用いて、限界希釈法によりクローニングを２回繰り返し〔１回目は、ＨＴ培地（ＨＡＴ培地からアミノプテリンを除いた培地）、２回目は、正常培地を使用する〕、安定して強い抗体価の認められたものをＧＰＲ８８に対する抗体の産生ハイブリドーマ株として選択する。
プリスタン（２，６，１０，１４−テトラメチルペンタデカン）０．５ｍＬを腹腔内投与し、２週間飼育した８〜１０週令のマウスまたはヌードマウスに、取得したＧＰＲ８８に対する抗体の産生ハイブリドーマ株５〜２０×１０^６細胞／匹を腹腔内に注射する。１０〜２１日間でハイブリドーマは腹水癌化する。該腹水癌化したマウスから腹水を採取し、３，０００ｒｐｍで５分間遠心分離して固形分を除去する。得られた上清より、ポリクローナル抗体の作製で用いた方法と同様の方法でモノクローナル抗体を精製、取得することができる。
以上のようにして得られた、ポリクローナル抗体またはモノクローナル抗体を試験物質として、上記のＧＰＲ８８の構成活性阻害物質のスクリーニング法を行い、該抗体から、ＧＰＲ８８の構成活性を抑制する抗体を選択することができる。
後述の試験例２および３に記載されているように、ＧＰＲ８８遺伝子に対するアンチセンスオリゴヌクレオチドの投与によりドパミン受容体拮抗薬により誘発される運動不全状態の改善、レセルピン投与により誘発される中枢機能不全状態の改善が認められたことから、ＧＰＲ８８の機能を抑制する化合物もしくはその塩またはＧＰＲ８８の機能を抑制するＧＰＲ８８に対する抗体も同様の作用を示すと考えられる。したがって、ＧＰＲ８８遺伝子に対するアンチセンスオリゴヌクレオチド、ＧＰＲ８８の機能を抑制する化合物もしくはその塩またはＧＰＲ８８の機能を抑制するＧＰＲ８８に対する抗体を含有する医薬は、ドパミン神経系の異常に基づく疾患、およびカテコールアミン神経系の異常に基づく疾患、例えば、パーキンソン病、不随意運動、錘体外路症、ハンチントン舞踏病、脊髄小脳変性症、脳卒中等の運動障害を伴う疾患の予防および／または治療剤として使用することができる。またうつ病、精神分裂病、不安、アルツハイマー病、注意欠陥障害、薬物中毒、アルコール中毒等の精神症状を改善する作用も期待できる。
本発明のアンチセンスオリゴヌクレオチドを含有する医薬は、ＧＰＲ８８ ｍＲＮＡの一部、好ましくは翻訳開始点を含む領域あるいはその近傍の領域に相補的な配列を有し、ＧＰＲ８８遺伝子の発現を抑制することができるアンチセンスオリゴヌクレオチドは、低毒性であり、生体内におけるＧＰＲ８８の機能を抑制することができるので、例えば、パーキンソン病、不随意運動、錘体外路症、ハンチントン舞踏病、脊髄小脳変性症、脳卒中等の運動障害を伴う疾患の予防および／または治療剤として使用することができる。またうつ病、精神分裂病、不安、アルツハイマー病、注意欠陥障害、薬物中毒、アルコール中毒等の精神症状の予防および／または治療剤として使用することができる。上記アンチセンスヌクレオチドを上記の予防および／または治療剤として使用する場合、自体公知の方法に従って製剤化し、投与することができる。例えば、該アンチセンスヌクレオチドを用いる場合、該アンチセンスヌクレオチドを単独あるいはレトロウイルスベクター、アデノウイルスベクター、アデノウイルスアソシエーテッドウイルスベクター等の適当なベクターに挿入した後、常套手段に従って、ヒトまたは哺乳動物（ラット、マウス、ウサギ、ヒツジ、ブタ、サル、イヌ、ウシ、ネコ等）に対して経口的または非経口的に投与することができる。該アンチセンスヌクレオチドは、そのままで、あるいは摂取促進のために補助剤等の生理学的に認められる担体とともに製剤化し、遺伝子銃やカテーテルによっても投与できる。また、脳室内、あるいは側座核、小脳のオリーブ核または線条体内注入によっても投与できる。
ＧＰＲ８８の機能抑制作用を有する化合物としては、例えば式（Ｉ）および（ＩＡ）で表される化合物が挙げられる。以下、式（Ｉ）および（ＩＡ）で表される化合物をそれぞれ化合物（Ｉ）および（ＩＡ）という。他の式番号の化合物についても同様である。
化合物（Ｉ）および化合物（ＩＡ）の各基の定義において、以下の例示が挙げられる。
（ｉ）低級アルキル、低級アルコキシおよび低級アルキルチオの低級アルキル部分としては、例えば直鎖または分岐状の炭素数１〜１０のアルキルが挙げられ、具体的にはメチル、エチル、プロピル、イソプロピル、ブチル、イソブチル、ｓｅｃ−ブチル、ｔｅｒｔ−ブチル、ペンチル、イソペンチル、ネオペンチル、ヘキシル、ヘプチル、オクチル、１，５−ジメチルヘキシル、イソオクチル、ノニル、デシル等が挙げられる。
（ｉｉ）シクロアルキルおよびシクロアルキルアルキルのシクロアルキル部分としては、例えば炭素数３〜８のシクロアルキルが挙げられ、具体的にはシクロプロピル、シクロブチル、シクロペンチル、シクロヘキシル、シクロヘプチル、シクロオクチル等が挙げられる。
（ｉｉｉ）低級アルケニルとしては、例えば直鎖、分岐または環状の炭素数２〜８のアルケニルが挙げられ、具体的にはビニル、アリル、１−プロペニル、ブテニル、ペンテニル、ヘキセニル、ヘプテニル、オクテニル、シクロヘキセニル、２，６−オクタジエニル等が挙げられる。
（ｉｖ）低級アルキニルとしては、例えば直鎖または分岐状の炭素数２〜８のアルキニルが挙げられ、具体的にはエチニル、プロピニル、ブチニル、ペンチニル、ヘキシニル、ヘプチニル、オクチニル等が挙げられる。
（ｖ）ハロゲンは、フッ素、塩素、臭素およびヨウ素の各原子を表す。
（ｖｉ）アリール、アリールオキシおよびアリールオキシアルキルのアリール部分としては、例えば炭素数６〜１４の単環式、二環式または三環式のアリールが挙げられ、具体的にはフェニル、ナフチル、インデニル、アントラニル等が挙げられる。
（ｖｉｉ）アラルキル、複素環アルキル、シクロアルキルアルキル、ヒドロキシ低級アルキル、カルボキシ低級アルキルおよびアリールオキシアルキルのアルキレン部分ならびにアラルキルオキシアルキルの２つのアルキレン部分は、前記低級アルキル（ｉ）の定義で挙げた基から水素を一つ除いたものと同義である。アラルキルオキシアルキルの２つのアルキレン部分は、同一でも異なっていてもよい。
（ｖｉｉｉ）アラルキルおよびアラルキルオキシアルキルのアリール部分としては、前記アリール（ｖｉ）の定義で挙げた基に加え、例えば前記アリール（ｖｉ）の定義で挙げた基がシクロアルキルと縮合した多環性縮合環基が挙げられ、具体的にはインダニル、１，２，３，４−テトラヒドロナフチル、６，７，８，９−テトラヒドロ−５Ｈ−ベンゾシクロヘプチル等が挙げられる。
（ｉｘ）複素環基および複素環アルキルの複素環基部分としては、例えば窒素原子、酸素原子および硫黄原子から選ばれる少なくとも１個の原子を含む５員または６員の単環性複素環基、３〜８員の環が縮合した二環または三環式であって窒素原子、酸素原子および硫黄原子から選ばれる少なくとも１個の原子を含む縮環性複素環基等が挙げられ、具体的にはピリジル、ピラジニル、ピリミジニル、ピリダジニル、ベンゾイミダゾリル、２−オキソベンゾイミダゾリル、ベンゾトリアゾリル、ベンゾフリル、ベンゾチエニル、プリニル、ベンゾオキサゾリル、ベンゾチアゾリル、ベンゾジオキソリル、インダゾリル、インドリル、イソインドリル、キノリル、イソキノリル、フタラジニル、ナフチルリジニル、キノキサリニル、キナゾリニル、シンノリニル、ピロリル、ピラゾリル、トリアゾリル、テトラゾリル、イミダゾリル、オキサゾリル、イソオキサゾリル、チアゾリル、イソチアゾリル、チエニル、フリル、ピロリジニル、２，５−ジオキソピロリジニル、チアゾリジニル、オキサゾリジニル、ピペリジル、ピペリジノ、ピペラジニル、ホモピペラジニル、ホモピペリジル、ホモピペリジノ、モルホリニル、モルホリノ、チオモルホリニル、チオモルホリノ、ピラニル、テトラヒドロピリジル、テトラヒドロピラニル、テトラヒドロフラニル、テトラヒドロキノリル、テトラヒドロイソキノリル、オクタヒドロキノリル、インドリニル、２，３−ジヒドロベンゾ［１，４］ジオキシニル等が挙げられる。
（ｘ）隣接する窒素原子と一緒になって形成される複素環基としては、例えば少なくとも１個の窒素原子を含む５員または６員の単環性複素環基（該単環性複素環基は、他の窒素原子、酸素原子または硫黄原子を含んでいてもよい）、３〜８員の環が縮合した二環または三環式で少なくとも１個の窒素原子を含む縮環性複素環基（該縮環性複素環基は、他の窒素原子、酸素原子または硫黄原子を含んでいてもよい）等が挙げられ、具体的にはピリジル、テトラヒドロピリジル、インドリニル、イソインドリニル、ピロリジニル、チアゾリジニル、オキサゾリジニル、ピペリジノ、ホモピペリジノ、ピペラジニル、ホモピペラジニル、モルホリノ、チオモルホリノ、ペルヒドロアゼピニル、ペルヒドロアゾシニル、テトラヒドロキノリル、テトラヒドロイソキノリル、オクタヒドロキノリル、デカヒドロイソキノリニル、ベンゾイミダゾリル、インダゾリル、インドリル、イソインドリル、プリニル、ジヒドロインドリル、ピロリル、ピラゾリル、トリアゾリル、テトラゾリル、イミダゾリル等が挙げられる。
（ｘｉ）ハロゲン化低級アルキルは、ハロゲン（該ハロゲンは、前記ハロゲン（ｖ）と同義である）で置換された低級アルキル（該低級アルキルは前記低級アルキル（ｉ）と同義である）を意味し、置換基であるハロゲンは同一または異なって例えば置換数１〜３である。
（ｘｉｉ）置換低級アルキル、置換低級アルコキシおよび置換低級アルキルチオにおける置換基としては、同一または異なって、例えば置換数１〜３の、低級アルカノイル、低級アルコキシ、アリールオキシ、置換アリールオキシ、アラルキルオキシ、置換アラルキルオキシ、低級アルカノイルオキシ、低級アルコキシカルボニル、ハロゲン、シアノ、ニトロ、ヒドロキシ、カルボキシ、アミノ、低級アルキルチオ、モノまたはジ低級アルキルアミノ、低級アルキルスルホニル、低級アルキルスルフィニル、低級アルコキシカルボニルアミノ、低級アルカノイルアミノ、（低級アルカノイル）（低級アルキル）アミノ、モノまたはジ低級アルキルアミノカルボニル、モノまたはジ低級アルキルアミノカルボニルオキシ等が挙げられる。
ここで示した置換アリールオキシおよび置換アラルキルオキシにおける置換基（ｘｉｉｉ）としては、同一または異なって、例えば置換数１〜３の、低級アルキル、低級アルコキシ、低級アルコキシカルボニル、アリール、複素環基、オキソ、ハロゲン、シアノ、ニトロ、ヒドロキシ、カルボキシ、アミノ等が挙げられ、低級アルキル、低級アルコキシおよび低級アルコキシカルボニルの低級アルキル部分、アリール、複素環基は、それぞれ前記低級アルキル（ｉ）、アリール（ｖｉ）、複素環基（ｉｘ）と同義であり、ハロゲンは前記ハロゲン（ｖ）と同義である。
また、ここで示したアリールオキシおよびアラルキルオキシのアリール部分、ハロゲンならびに低級アルカノイル、低級アルコキシ、低級アルカノイルオキシ、低級アルコキシカルボニル、低級アルキルチオ、モノまたはジ低級アルキルアミノ、低級アルキルスルホニル、低級アルキルスルフィニル、低級アルコキシカルボニルアミノ、低級アルカノイルアミノ、（低級アルカノイル）（低級アルキル）アミノ、モノまたはジ低級アルキルアミノカルボニルおよびモノまたはジ低級アルキルアミノカルボニルオキシの低級アルキル部分は、それぞれ前記アリール（ｖｉ）、ハロゲン（ｖ）および低級アルキル（ｉ）と同義であり、アラルキルオキシのアルキレン部分は、前記アラルキルのアルキレン部分（ｖｉｉ）と同義である。
また、ここで示したジ低級アルキルアミノ、（低級アルカノイル）（低級アルキル）アミノ、ジ低級アルキルアミノカルボニルおよびジ低級アルキルアミノカルボニルオキシにおける２つの低級アルキル部分は、同一でも異なっていてもよい。
（ｘｉｖ）置換アリール、置換アラルキル、置換シクロアルキル、置換シクロアルキルアルキル、置換低級アルケニル、置換低級アルキニル、置換アリールオキシ、置換複素環基、置換複素環アルキル、置換アリールオキシアルキル、置換アラルキルオキシアルキル、置換ピペラジニルおよび隣接する窒素原子と一緒になって形成される置換複素環基における置換基としては、前記置換低級アルキルにおける置換基（ｘｉｉ）の定義で挙げた基に加え、例えば低級アルキル、置換低級アルキル、低級アルケニル、低級アルキニル、シクロアルキル、置換シクロアルキル、アリール、置換アリール、アラルキル、置換アラルキル、アリールカルボニル、置換アリールカルボニル、複素環基、置換複素環基、複素環アルキル、置換複素環アルキル、エチレンジオキシ等が挙げられる。
ここで示した置換低級アルキル、置換シクロアルキル、置換アリール、置換アラルキル、置換アリールカルボニル、置換複素環基および置換複素環アルキルにおける置換基は、前記置換アリールオキシおよび置換アラルキルオキシにおける置換基（ｘｉｉｉ）と同義である。
また、ここで示した低級アルキル、シクロアルキル、低級アルケニル、低級アルキニル、アリールおよびアリールカルボニルのアリール部分、複素環基および複素環アルキルの複素環基部分、アラルキルおよび複素環アルキルのアルキレン部分ならびにアラルキルのアリール部分は、それぞれ前記低級アルキル（ｉ）、シクロアルキル（ｉｉ）、低級アルケニル（ｉｉｉ）、低級アルキニル（ｉｖ）、アリール（ｖｉ）、複素環基（ｉｘ）、アラルキルのアルキレン部分（ｖｉｉ）およびアラルキルのアリール部分（ｖｉｉｉ）と同義である。
（ｘｖ）置換ヒドロキシメチルの置換基としては、同一または異なって、例えば置換数１または２の低級アルキル等が挙げられる。
ここで示した低級アルキルは前記低級アルキル（ｉ）と同義である。
化合物（Ｉ）および化合物（ＩＡ）の薬理学的に許容される塩としては、毒性のない、水溶性のものが好ましく、例えば塩酸塩、臭化水素酸塩、硝酸塩、硫酸塩、リン酸塩等の無機酸塩、ベンゼンスルホン酸塩、安息香酸塩、クエン酸塩、フマル酸塩、グルコン酸塩、乳酸塩、マレイン酸塩、リンゴ酸塩、シュウ酸塩、メタンスルホン酸塩、酒石酸塩等の有機酸塩等の酸付加塩、ナトリウム塩、カリウム塩等のアルカリ金属塩、マグネシウム塩、カルシウム塩等のアルカリ土類金属塩、アルミニウム塩、亜鉛塩等の金属塩、アンモニウム、テトラメチルアンモニウム等のアンモニウム塩、モルホリン付加塩、ピペリジン付加塩等の有機アミン付加塩、グリシン付加塩、フェニルアラニン付加塩、リジン付加塩、アスパラギン酸付加塩、グルタミン酸付加塩等のアミノ酸付加塩等が挙げられる。
次に化合物（Ｉ）の製造法について説明する。
なお、以下に示した製造法において、定義した基が反応条件下変化するか、または方法を実施するのに不適切な場合、有機合成化学で常用される方法、例えば官能基の保護、脱保護等［例えば、プロテクティブ・グループス・イン・オーガニック・シンセシス第三版（Ｐｒｏｔｅｃｔｉｖｅ Ｇｒｏｕｐｓ ｉｎ Ｏｒｇａｎｉｃ Ｓｙｎｔｈｅｓｉｓ，ｔｈｅ ｔｈｉｒｄ ｅｄｉｔｉｏｎ）、グリーン（Ｔ．Ｗ．Ｇｒｅｅｎｅ）、ワッツ（Ｐｅｔｅｒ Ｇ．Ｍ．Ｗｕｔｓ）著、ジョン・ワイリー・アンド・サンズ・インコーポレーティッド（Ｊｏｈｎ Ｗｉｌｅｙ ＆ Ｓｏｎｓ Ｉｎｃ．）（１９９９年）］の手段に付すことにより容易に製造を実施することができる。また、必要に応じて置換基導入等の反応工程の順序を変えることもできる。
化合物（Ｉ）のうちＹが水素原子であり、Ｘが１，２，３，４−テトラゾール−５−イルであり、Ｒ^２が−ＮＲ^３Ｒ^４（式中、Ｒ^３およびＲ^４はそれぞれ前記と同義である）である化合物（Ｉ−ｉ）は、例えば以下に示す製造法１によって得ることができる。
製造法１：

（式中、Ａ、Ｒ^１、Ｒ^３およびＲ^４はそれぞれ前記と同義であり、ｍは１または２を表す）
［工程１］
２−エトキシメチレン−２−シアノ酢酸エチル〔化合物（Ａ）〕を、溶媒中、アルカリ存在下、０．５当量〜大過剰量、好ましくは０．５当量〜２当量の硫酸メチルイソチオ尿素〔化合物（Ｂ）〕と反応させることにより、化合物（Ｃ）を得ることができる。
溶媒としては、例えばメタノール、エタノール、２−プロパノール、テトラヒドロフラン、ジオキサン等を単独でまたはそれらを混合して用いることができ、中でもエタノールが好ましい。
アルカリとしては、例えば水酸化リチウム、水酸化カリウム、水酸化ナトリウム、水酸化マグネシウムまたは水酸化カルシウム等のアルカリ水溶液、カリウムｔｅｒｔ−ブトキシドの水溶液、テトラヒドロフラン溶液もしくは２−メチル−２−プロパノール溶液、ナトリウムメトキシドの水溶液もしくはメタノール溶液等を用いることができ、中でも水酸化ナトリウム水溶液が好ましい。
反応は、０℃〜５０℃の間の温度、好ましくは０℃〜１５℃の間の温度で行われ、通常１時間〜４８時間で終了する。
［工程２］
工程１で得られる化合物（Ｃ）を、反応に不活性な溶媒中または無溶媒で、１当量〜大過剰量の塩基の存在下または非存在下、１当量〜大過剰量の塩素化剤と反応させることにより化合物（Ｄ）を得ることができる。
塩素化剤としては、例えばオキシ塩化リン等が用いられる。
反応に不活性な溶媒は、反応に不活性なものであればいずれでもよく、特に限定されないが、例えば１，２−ジクロロエタン、テトラヒドロフラン、ジオキサン、クロロホルム、ベンゼン、トルエン、キシレン、酢酸エチル等を単独でまたはそれらを混合して用いることができる。塩基としては、例えばトリエチルアミン、ピリジン、Ｎ，Ｎ−ジメチルアニリン等が挙げられる。
反応は、０℃〜溶媒の沸点の間の温度、好ましくは５０℃〜溶媒の沸点の間の温度で行われ、通常１時間〜４８時間で終了する。
なお、本工程で得られる化合物（Ｄ）は上記の方法以外に、ジャーナル・オブ・ヘテロサイクリック・ケミストリー（Ｊ．Ｈｅｔｅｒｏｃｙｃｌ．Ｃｈｅｍ．）、８（３）巻、４４５頁（１９７１年）、ＷＯ９９／６１４４４等に記載の方法でまたはそれらに準じた方法によっても得ることができる。
［工程３］
工程２で得られる化合物（Ｄ）を、無溶媒または反応に不活性な溶媒中、１当量〜大過剰量、好ましくは１当量〜３当量の化合物（Ｅ）と反応させることにより、化合物（Ｆ）を得ることができる。
反応に不活性な溶媒は、反応に不活性なものであればいずれでもよく、特に限定されないが、例えばテトラヒドロフラン、ジオキサン、ジエチルエーテル、ジイソプロピルエーテル、ベンゼン、トルエン、キシレン、酢酸エチル、アセトニトリル、ジクロロメタン、クロロホルム、１，２−ジクロロエタン、Ｎ，Ｎ−ジメチルホルムアミド、ジメチルアセトアミド、Ｎ−メチルピロリドン、ジメチルスルホキシド等を単独でまたはそれらを混合して用いることができ、中でもテトラヒドロフラン、クロロホルムまたはそれらの混合溶媒が好ましい。
反応は、０℃〜１００℃の間の温度、好ましくは０℃〜５０℃の間の温度で行われ、通常１０分間〜４８時間で終了する。
また、この反応は必要に応じて、１当量〜大過剰量、好ましくは１当量〜１０当量の塩基を添加して行ってもよい。
塩基としては、例えばトリエチルアミン、Ｎ，Ｎ−ジイソプロピルエチルアミン、１，８−ジアザビシクロ［５．４．０］−ウンデク−７−エン（ＤＢＵ）、Ｎ，Ｎ−ジメチルアニリン、ピリジン、キノリン等の有機塩基、炭酸カリウム、炭酸ナトリウム、炭酸リチウム、炭酸水素ナトリウム、水酸化カリウム、水酸化ナトリウム、水酸化リチウム、カリウムｔｅｒｔ−ブトキシド等の無機塩基、アンバーリストＡ−２１（ロームアンドハース社製）、ＡＧ１−Ｘ８（バイオラッド社製）等の塩基性アニオン交換レジン、ポリビニルピリジン、モルホリノメチルポリスチレン等の固相に担持された塩基等を用いることができ、並列合成法（コンビナトリアル・ケミストリー）で反応を行う場合には、中でもモルホリノメチルポリスチレンが好ましい。
化合物（Ｅ）は、市販品としてまたはコンプリヘンシブ・オーガニック・トランスフォーメーションズ第二版（Ｃｏｍｐｒｅｈｅｎｓｉｖｅ Ｏｒｇａｎｉｃ Ｔｒａｎｓｆｏｒｍａｔｉｏｎｓ，ｔｈｅ ｓｅｃｏｎｄ ｅｄｉｔｉｏｎ）、ラロック（Ｒ．Ｃ．Ｌａｒｏｃｋ）著、ジョン・ワイリー・アンド・サンズ・インコーポレイテッド（Ｊｏｈｎ Ｗｉｌｅｙ ＆ Ｓｏｎｓ Ｉｎｃ．）（１９９９年）等に記載の方法に準じて得ることができる。
［工程４］
工程３で得られる化合物（Ｆ）を、反応に不活性な溶媒中、１当量〜大過剰量、好ましくは１当量〜５当量の酸化剤で処理することにより、化合物（Ｇ）を得ることができる。
反応に不活性な溶媒は、反応に不活性なものであればいずれでもよく、特に限定されないが、例えばジクロロメタン、クロロホルム、１，２−ジクロロエタン、テトラヒドロフラン、ジオキサン、ジエチルエーテル、ジイソプロピルエーテル、メタノール、エタノール、イソプロピルアルコール、ベンゼン、トルエン、キシレン、酢酸エチル、アセトニトリル、水等を単独でまたはそれらを混合して用いることができ、中でもジクロロメタンが好ましい。
酸化剤としては、例えばメタクロ濾過安息香酸、過酸化ベンゾイル、過酢酸、過酸化水素水、過ヨウ素酸ナトリウム等を用いることができ、中でもメタクロ濾過安息香酸が好ましい。
反応は０℃〜１００℃の間の温度、好ましくは０℃〜５０℃の間の温度で行われ、通常１０分間〜２４時間で終了する。
なお、化合物（Ｇ）において、ｍが１である化合物とｍが２である化合物は、それぞれ例えば酸化剤の当量数、反応の温度等の条件等を調節することにより得られ、それらが混在することもある。混在する場合、その比率は特に限定されず、いずれの場合もそのまま次工程に使用することができる。
［工程５］
工程４で得られる化合物（Ｇ）を、無溶媒または反応に不活性な溶媒中、１当量〜大過剰量の、好ましくは１当量〜５当量の化合物（Ｈ）と反応させることにより、化合物（Ｊ）を得ることができる。
反応に不活性な溶媒は、反応に不活性なものであればいずれでもよく、特に限定されないが、例えばジクロロメタン、クロロホルム、１，２−ジクロロエタン、Ｎ，Ｎ−ジメチルホルムアミド、ジメチルアセトアミド、Ｎ−メチルピロリドン、ジメチルスルホキシド、テトラヒドロフラン、ジオキサン、ジエチルエーテル、ジイソプロピルエーテル、ベンゼン、トルエン、キシレン、酢酸エチル、アセトニトリル等を単独でまたはそれらを混合して用いることができ、中でもテトラヒドロフランが好ましい。
反応は０℃〜１００℃の間の温度、好ましくは２０℃〜６０℃の間の温度で行われ、通常１時間〜７２時間で終了する。
化合物（Ｈ）は、市販品としてまたはコンプリヘンシブ・オーガニック・トランスフォーメーションズ第二版（Ｃｏｍｐｒｅｈｅｎｓｉｖｅ Ｏｒｇａｎｉｃ Ｔｒａｎｓｆｏｒｍａｔｉｏｎｓ，ｔｈｅ ｓｅｃｏｎｄ ｅｄｉｔｉｏｎ）、ラロック（Ｒ．Ｃ．Ｌａｒｏｃｋ）著、ジョン・ワイリー・アンド・サンズ・インコーポレイテッド（Ｊｏｈｎ Ｗｉｌｅｙ ＆ Ｓｏｎｓ Ｉｎｃ．）（１９９９年）等に記載の方法に準じて得ることができる。
［工程６］
工程５で得られる化合物（Ｊ）を、反応に不活性な溶媒中、１当量〜１０当量のアジ化ナトリウムまたはアジ化アンモニウムと反応させることにより、化合物（Ｉ−ｉ）を得ることができる。
反応に不活性な溶媒は、反応に不活性なものであればいずれでもよく、特に限定されないが、例えばクロロホルム、１，２−ジクロロエタン、Ｎ，Ｎ−ジメチルホルムアミド、ジメチルアセトアミド、Ｎ−メチルピロリドン、ジメチルスルホキシド、テトラヒドロフラン、ジオキサン、ジイソプロピルエーテル、ベンゼン、トルエン、キシレン、酢酸エチル、アセトニトリル等を単独でまたはそれらを混合して用いることができ、中でもＮ，Ｎ−ジメチルホルムアミドが好ましい。また、反応を加速する目的で、反応系中へ塩化アンモニウム、塩化エチルアンモニウム等を１当量〜大過剰量、好ましくは１当量〜１０当量添加することもできる。
反応は０℃〜１８０℃の間の温度、好ましくは５０℃〜１２０℃の間の温度で行われ、通常１時間〜７２時間で終了する。
化合物（Ｉ）のうち、Ｘが−Ｃ（＝Ｏ）Ｒ^５であり、Ｒ^２が−ＮＲ^３Ｒ^４（式中、Ｒ^３およびＲ^４はそれぞれ前記と同義である）である化合物（Ｉ−ｉｉ）は、例えば以下に示す製造法２によって得ることができる。
製造法２：

（式中、Ａ、Ｒ^１、Ｒ^３、Ｒ^４、Ｒ^５、Ｙおよびｍはそれぞれ前記と同義である。）
［工程７］
化合物（Ｋ）を、無溶媒または反応に不活性な溶媒中、１当量〜大過剰量、好ましくは１当量〜３当量の化合物（Ｅ）と反応させることにより、化合物（Ｌ）を得ることができる。
反応に不活性な溶媒は、反応に不活性なものであればいずれでもよく、特に限定されないが、例えばテトラヒドロフラン、ジオキサン、１，２−ジメトキシエタン、ジイソプロピルエーテル、ベンゼン、トルエン、キシレン、酢酸エチル、アセトニトリル、ジクロロメタン、クロロホルム、１，２−ジクロロエタン、Ｎ，Ｎ−ジメチルホルムアミド、ジメチルアセトアミド、Ｎ−メチルピロリドン、ジメチルスルホキシド等を単独でまたはそれらを混合して用いることができ、中でもテトラヒドロフラン、クロロホルム、１，２−ジメトキシエタンまたはそれらの混合溶媒が好ましい。
反応は、０℃〜１５０℃の間の温度、好ましくは５０℃〜１２０℃の間の温度で行われ、通常１時間〜９６時間で終了する。
また、この反応は必要に応じて、１当量〜大過剰量、好ましくは１当量〜１０当量の塩基を添加して行ってもよい。
塩基としては、例えばトリエチルアミン、Ｎ，Ｎ−ジイソプロピルエチルアミン、ＤＢＵ、Ｎ，Ｎ−ジメチルアニリン、ピリジン、キノリン等の有機塩基、炭酸カリウム、炭酸ナトリウム、炭酸リチウム、炭酸水素ナトリウム、水酸化カリウム、水酸化ナトリウム、水酸化リチウム、カリウムｔｅｒｔ−ブトキシド等の無機塩基、アンバーリストＡ−２１（ロームアンドハース社製）、ＡＧ１−Ｘ８（バイオラッド社製）等の塩基性アニオン交換レジン、ポリビニルピリジン、モルホリノメチルポリスチレン等の固相に担持された塩基等を用いることができ、中でもトリエチルアミン、Ｎ，Ｎ−ジイソプロピルエチルアミンまたはＤＢＵが好ましい。
化合物（Ｅ）は、市販品としてまたはコンプリヘンシブ・オーガニック・トランスフォーメーションズ第二版（Ｃｏｍｐｒｅｈｅｎｓｉｖｅ Ｏｒｇａｎｉｃ Ｔｒａｎｓｆｏｒｍａｔｉｏｎｓ，ｔｈｅ ｓｅｃｏｎｄ ｅｄｉｔｉｏｎ）、ラロック（Ｒ．Ｃ．Ｌａｒｏｃｋ）著、ジョン・ワイリー・アンド・サンズ・インコーポレイテッド（Ｊｏｈｎ Ｗｉｌｅｙ ＆ Ｓｏｎｓ Ｉｎｃ．）（１９９９年）等に記載の方法に準じて得ることができる。
化合物（Ｋ）は、市販品としてまたは例えばＷＯ００／７６９８０、ＷＯ００／２７８２６、ＷＯ０１／８３４６０、ＷＯ９７／０９３１５、特開２００１−８９４５２号公報、ＷＯ９９／３１０７３等に記載の方法に準じて得ることができる。
［工程８］
工程７で得られる化合物（Ｌ）を、反応に不活性な溶媒中、１当量〜大過剰量、好ましくは１当量〜５当量の酸化剤で処理することにより、化合物（Ｍ）を得ることができる。
反応に不活性な溶媒は、反応に不活性なものであればいずれでもよく、特に限定されないが、例えばジクロロメタン、クロロホルム、１，２−ジクロロエタン、テトラヒドロフラン、ジオキサン、ジエチルエーテル、ジイソプロピルエーテル、メタノール、エタノール、イソプロピルアルコール、ベンゼン、トルエン、キシレン、酢酸エチル、アセトニトリル、水等を単独でまたはそれらを混合して用いることができ、中でもジクロロメタンが好ましい。
酸化剤としては、例えばメタクロ濾過安息香酸、過酸化ベンゾイル、過酢酸、過酸化水素水、過ヨウ素酸ナトリウム等を用いることができ、中でもメタクロ濾過安息香酸が好ましい。
反応は０℃〜１００℃の間の温度、好ましくは０℃〜５０℃の間の温度で行われ、通常１０分間〜２４時間で終了する。
なお、化合物（Ｍ）において、ｍが１である化合物とｍが２である化合物は、それぞれ例えば酸化剤の当量数、反応の温度等の条件等を調節することにより得られ、それらが混在することもある。混在する場合、その比率は特に限定されず、いずれの場合もそのまま次工程に使用することができる。
［工程９］
工程８で得られる化合物（Ｍ）を、反応に不活性な溶媒中、１当量〜５当量の化合物（Ｈ）と反応させることにより、化合物（Ｉ−ｉｉ）を得ることができる。
反応に不活性な溶媒は、反応に不活性なものであればいずれでもよく、特に限定されないが、例えばジクロロメタン、クロロホルム、１，２−ジクロロエタン、Ｎ，Ｎ−ジメチルホルムアミド、ジメチルアセトアミド、Ｎ−メチルピロリドン、ジメチルスルホキシド、テトラヒドロフラン、ジオキサン、１，２−ジメトキシエタン、ジイソプロピルエーテル、ベンゼン、トルエン、キシレン、酢酸エチル、アセトニトリル等を単独でまたはそれらを混合して用いることができ、中でもテトラヒドロフランまたは１，２−ジメトキシエタンが好ましい。
反応は２０℃〜１５０℃の間の温度、好ましくは５０℃〜１２０℃の間の温度で行われ、通常１時間〜７日間で終了する。
化合物（Ｈ）は、市販品としてまたはコンプリヘンシブ・オーガニック・トランスフォーメーションズ第二版（Ｃｏｍｐｒｅｈｅｎｓｉｖｅ Ｏｒｇａｎｉｃ Ｔｒａｎｓｆｏｒｍａｔｉｏｎｓ，ｔｈｅ ｓｅｃｏｎｄ ｅｄｉｔｉｏｎ）、ラロック（Ｒ．Ｃ．Ｌａｒｏｃｋ）著、ジョン・ワイリー・アンド・サンズ・インコーポレイテッド（Ｊｏｈｎ Ｗｉｌｅｙ ＆ Ｓｏｎｓ Ｉｎｃ．）（１９９９年）等に記載の方法に準じて得ることができる。
また、化合物（Ｉ−ｉｉ）のうち、Ｒ^５がヒドロキシである化合物（Ｉ−ｉｉｂ）は、化合物（Ｉ−ｉｉ）のうち、Ｒ^５がＲ^５ａ（式中、Ｒ^５ａはＲ^５の定義のうち置換もしくは非置換の低級アルコキシを表す）である化合物（Ｉ−ｉｉａ）から、以下に示す工程１０によっても得ることができる。
［工程１０］

（式中、Ｒ^１、Ａ、Ｒ^３、Ｒ^４、Ｒ^５ａおよびＹはそれぞれ前記と同義である）
工程９で得られる化合物（Ｉ−ｉｉａ）を、溶媒中、１当量〜１０当量、好ましくは１当量〜５当量の塩基で処理することにより、化合物（Ｉ−ｉｉｂ）を得ることができる。
溶媒は、特に限定されないが、例えばメタノール、エタノール、プロパノール、ブタノール等のプロトン性溶媒と水との混合溶媒、またはテトラヒドロフラン、ジオキサン等の非プロトン性溶媒と水との混合溶媒があげられ、これらを混合して用いることもでき、中でもエタノールと水の混合溶媒が好ましい。
塩基としては、例えば水酸化ナトリウム、水酸化リチウム、水酸化カリウム、炭酸ナトリウム、炭酸カリウム、炭酸リチウム等の無機塩基、アンバーリストＡ−２１（ロームアンドハース社製）、ＡＧ１−Ｘ８（バイオラッド社製）等の塩基性アニオン交換レジン等を用いることができる。
反応は０℃〜１５０℃の間の温度、好ましくは２０℃〜１００℃の間の温度で行われ、通常１時間〜４８時間で終了する。
また、化合物（Ｉ−ｉｉ）のうち、Ｒ^５が−ＮＲ^６Ｒ^７である化合物（Ｉ−ｉｉｃ）は、例えば以下に示す製造法３によっても得ることができる。
製造法３：

（式中、Ｒ^１、Ａ、Ｒ^３、Ｒ^４、Ｒ^６、Ｒ^７およびＹはそれぞれ前記と同義である）
［工程１１］
製造法２の工程１０で得られる化合物（Ｉ−ｉｉｂ）を、反応に不活性な溶媒中、１当量〜１０当量の縮合剤の存在下、１当量〜５当量、好ましくは１当量〜３当量の１−ヒドロキシベンゾトリアゾールと反応させることにより、化合物（Ｎ）を得ることができる。
反応に不活性な溶媒は、反応に不活性なものであればいずれでもよく、特に限定されないが、例えばジクロロメタン、クロロホルム、１，２−ジクロロエタン、Ｎ，Ｎ−ジメチルホルムアミド、ジメチルアセトアミド、Ｎ−メチルピロリドン、ジメチルスルホキシド、テトラヒドロフラン、ジオキサン、ジエチルエーテル、ジイソプロピルエーテル、ベンゼン、トルエン、キシレン、酢酸エチル、アセトニトリル、水等を単独でまたはそれらを混合して用いることができ、中でもクロロホルム、テトラヒドロフランまたはそれらの混合溶媒が好ましい。
縮合剤としては、例えばジシクロヘキシルカルボジイミド、ジイソプロピルカルボジイミド、Ｎ−エチル−Ｎ’−（３−ジメチルアミノプロピル）カルボジイミドまたはその塩酸塩、ポリスチレンに担持されたＮ−エチル−Ｎ’−（３−ジメチルアミノプロピル）カルボジイミド、ポリスチレンに担持されたＮ−ベンジル−Ｎ’−シクロヘキシルカルボジイミド、ベンゾトリアゾール−１−イル−トリス（ジメチルアミノ）ホスホニウムヘキサフルオロリン化物塩、ジフェニルホスホリルアジド等を用いることができ、中でもＮ−エチル−Ｎ’−（３−ジメチルアミノプロピル）カルボジイミドまたはその塩酸塩またはポリスチレンに担持されたＮ−エチル−Ｎ’−（３−ジメチルアミノプロピル）カルボジイミドが好ましい。
反応は０℃〜１００℃の間の温度、好ましくは０℃〜６０℃の間の温度で行われ、通常１時間〜１２０時間で終了する。
［工程１２］
工程１１で得られる化合物（Ｎ）を無溶媒または反応に不活性な溶媒中、１当量〜大過剰量、好ましくは１当量〜５当量のＨＮＲ^６Ｒ^７（式中、Ｒ^６およびＲ^７はそれぞれ前記と同義である）と反応させることにより、化合物（Ｉ−ｉｉｃ）を得ることができる。
反応に不活性な溶媒は、反応に不活性なものであればいずれでもよく、特に限定されないが、例えばテトラヒドロフラン、ジオキサン、１，２−ジメトキシエタン、ジイソプロピルエーテル、ベンゼン、トルエン、キシレン、酢酸エチル、アセトニトリル、ジクロロメタン、クロロホルム、１，２−ジクロロエタン、Ｎ，Ｎ−ジメチルホルムアミド、ジメチルアセトアミド、Ｎ−メチルピロリドン、ジメチルスルホキシド、水等を単独でまたはそれらを混合して用いることができ、中でもテトラヒドロフランと水の混合溶媒が好ましい。
反応は０℃〜１００℃の間の温度、好ましくは２０℃〜６０℃の間の温度で行われ、通常１時間〜９６時間で終了する。
また、この反応は必要に応じて、１当量〜１０当量、好ましくは１当量〜５当量の塩基を添加して行ってもよい。
塩基としては、例えばトリエチルアミン、Ｎ，Ｎ−ジイソプロピルエチルアミン、ＤＢＵ、Ｎ，Ｎ−ジメチルアニリン、ピリジン、キノリン等の有機塩基、炭酸カリウム、炭酸ナトリウム、炭酸リチウム、炭酸水素ナトリウム、水酸化カリウム、水酸化ナトリウム、水酸化リチウム、カリウムｔｅｒｔ−ブトキシド等の無機塩基、アンバーリストＡ−２１（ロームアンドハース社製）、ＡＧ１−Ｘ８（バイオラッド社製）等の塩基性アニオン交換レジン、ポリビニルピリジン、モルホリノメチルポリスチレン等の固相に担持された塩基等を用いることができ、中でも炭酸カリウムまたはＤＢＵが好ましい。
［工程１３］
また、製造法３で得られる化合物（Ｉ−ｉｉｃ）のうち、Ｒ^７にカルボキシ低級アルキル（該カルボキシ低級アルキルは前記と同義である）を有する化合物（Ｉ−ｉｉｃａ）は、製造法２の工程１０と同様にして、化合物（Ｉ−ｉｉｃ）のうち、Ｒ^７に低級アルコキシカルボニル低級アルキル（該低級アルコキシカルボニル低級アルキルは前記と同義である）を有する化合物（Ｉ−ｉｉｃｂ）を、加水分解処理することによっても得ることができる。
また、化合物（Ｉ−ｉｉｃｂ）のうち、低級アルコキシカルボニル低級アルキルの低級アルコキシ部分がｔｅｒｔ−ブトキシである化合物（Ｉ−ｉｉｃｃ）を、溶媒中、１当量〜大過剰量の酸で処理することにより化合物（Ｉ−ｉｉｃａ）を得ることもできる。
溶媒としては、反応に不活性なものであればいずれでもよく、特に限定されないが、例えばジクロロメタン、クロロホルム、１，２−ジクロロエタン、Ｎ，Ｎ−ジメチルホルムアミド、ジメチルアセトアミド、Ｎ−メチルピロリドン、ジメチルスルホキシド、テトラヒドロフラン、ジオキサン、ジエチルエーテル、ジイソプロピルエーテル、ベンゼン、トルエン、キシレン、酢酸エチル、アセトニトリル、水等を単独でまたは混合して用いることができ、中でもジクロロメタンが好ましい。
酸としては、例えばトリフルオロ酢酸等のカルボン酸、塩酸等の鉱酸、トリフルオロメタンスルホン酸、ベンゼンスルホン酸等のスルホン酸等を用いることができ、中でもトリフルオロ酢酸が好ましい。
反応は０℃〜１５０℃の間の温度、好ましくは０℃〜５０℃の間の温度で行われ、通常１時間〜４８時間で終了する。
化合物（Ｉ）のうち、Ｘがヒドロキシメチルであり、Ｒ^２が−ＮＲ^３Ｒ^４（式中、Ｒ^３およびＲ^４はそれぞれ前記と同義である）である化合物（Ｉ−ｉｉｉａ）は、例えば以下に示す製造法４によって得ることができる。
製造法４：

（式中、Ａ、ｍ、Ｒ^１、Ｒ^３、Ｒ^４、Ｒ^５ａおよびＹはそれぞれ前記と同義である）
［工程１４］
製造法２の工程７で得られる化合物（Ｌ）のうち、Ｒ^５がＲ^５ａ（式中、Ｒ^５ａは前記と同義である）である化合物（Ｌａ）を、反応に不活性な溶媒中、２当量〜１０当量、好ましくは２当量〜６当量の還元剤で処理することにより、化合物（Ｏ）を得ることができる。
反応に不活性な溶媒は、反応に不活性なものであればいずれでもよく、特に限定されないが、例えば、ジエチルエーテル、テトラヒドロフラン、ジメトキシエタン、ジオキサン、ベンゼン、トルエン、キシレン等を単独でまたはそれらを混合して用いることができる。
還元剤としては、例えば水素化リチウムアルミニウム、水素化ジイソブチルアルミニウム等を用いることができ、中でも水素化リチウムアルミニウムが好ましい。
反応は−７８℃〜溶媒の沸点の間の温度、好ましくは０℃〜５０℃の間の温度で行われ、通常１分間〜１０時間で終了する。
［工程１５］
製造法１の工程４に示した方法と同様にして、工程１４で得られた化合物（Ｏ）を、反応に不活性な溶媒中、１当量〜大過剰量、好ましくは１当量〜５当量の酸化剤で処理することにより、化合物（Ｐ）を得ることができる。
溶媒、酸化剤、反応温度、反応時間等の各種条件は工程４に記載の条件と同様である。
［工程１６］
工程１５で得られる化合物（Ｐ）を、無溶媒または反応に不活性な溶媒中、１当量〜大過剰量、好ましくは１当量〜１０当量の塩基の存在下または非存在下、１当量〜５当量の化合物（Ｈ）と反応させることにより、化合物（Ｉ−ｉｉｉａ）を得ることができる。
反応に不活性な溶媒は、反応に不活性なものであればいずれでもよく、特に限定されないが、例えばジクロロメタン、クロロホルム、１，２−ジクロロエタン、Ｎ，Ｎ−ジメチルホルムアミド、ジメチルアセトアミド、Ｎ−メチルピロリドン、ジメチルスルホキシド、テトラヒドロフラン、ジオキサン、１，２−ジメトキシエタン、ジイソプロピルエーテル、ベンゼン、トルエン、キシレン、酢酸エチル、アセトニトリル等を単独でまたはそれらを混合して用いることができ、中でもジオキサンまたは１，２−ジメトキシエタンが好ましい。
塩基としては、例えばトリエチルアミン、Ｎ，Ｎ−ジイソプロピルエチルアミン、Ｎ−メチルモルホリン、モルホリノメチルポリスチレン等の固相に担持された塩基等を用いることができ、中でもＮ，Ｎ−ジイソプロピルエチルアミンが好ましい。
反応は２０℃〜１５０℃の間の温度、好ましくは８０℃〜１２０℃の間の温度で行われ、通常１時間〜９６時間で終了する。
化合物（Ｉ）のうちＸが置換ヒドロキシメチルであり、Ｒ^２が−ＮＲ^３Ｒ^４（式中、Ｒ^３およびＲ^４はそれぞれ前記と同義である）である化合物（Ｉ−ｉｉｉｂａ）は、例えば以下に示す製造法５によって得ることができる。
製造法５：

（式中、Ａ、Ｒ^１、Ｒ^３、Ｒ^４およびＹはそれぞれ前記と同義であり、Ｒ^８は低級アルキルを表し、該低級アルキルは前記低級アルキル（ｉ）と同義である）
［工程１７］
製造法４の工程１４で得られる化合物（Ｏ）を溶媒中、酸化剤で処理することにより、化合物（Ｑ）を得ることができる。
酸化剤としては、アルコールの酸化反応に用いられる各種試薬を任意に用いることができ、例えば二酸化マンガン、クロロクロム酸ピリジニウム（ＰＣＣ）、二クロム酸ピリジニウム（ＰＤＣ）、酸化クロム（ＩＶ）−ピリジシ錯体、重クロム酸カリウム、重クロム酸ナトリウム、硝酸セリウム（ＩＶ）アンモニウム等が挙げられるが、中でも二酸化マンガンが好ましい。
溶媒は反応に不活性なものであればいずれでもよく、特に限定されないが、例えばジクロロメタン、クロロホルム、１，２−ジクロロエタン、Ｎ，Ｎ−ジメチルホルムアミド、ジメチルアセトアミド、Ｎ−メチルピロリドン、ジメチルスルホキシド、テトラヒドロフラン、ジオキサン、１，２−ジメトキシエタン、ジイソプロピルエーテル、ベンゼン、トルエン、キシレン、酢酸エチル、アセトニトリル、ピリジン、水等を単独でまたはそれらを混合して用いることができ、中でもクロロホルムが好ましい。
反応は、−７８℃〜溶媒の沸点の間の温度、好ましくは０℃〜５０℃の間の温度で行われ、通常１時間〜９６時間で終了する。
［工程１８］
工程１７で得られる化合物（Ｑ）を、反応に不活性な溶媒中、１当量〜５当量、好ましくは２当量〜３当量の対応するアルキル化剤と反応させることにより、化合物（Ｒ）を得ることができる。
対応するアルキル化剤としては、例えばＲ^８ＭｇＢｒ（式中、Ｒ^８は前記と同義である）、Ｒ^８Ｌｉ（式中、Ｒ^８は前記と同義である）等が用いられる。より具体的には、Ｒ^８がメチルの場合、例えば臭化メチルマグネシウム、メチルリチウム等を用いることができ、中でも臭化メチルマグネシウムが好ましい。
反応に不活性な溶媒は、反応に不活性であればいずれでもよく、例えば、テトラヒドロフラン、ジオキサン、１，２−ジメトキシエタン、ジイソプロピルエーテル等を単独でまたはそれらを混合して用いることができ、中でもテトラヒドロフランが好ましい。
反応は−７８℃〜溶媒の沸点の間の温度、好ましくは−４０℃〜２０℃の間の温度で行われ、通常１時間〜９６時間で終了する。
［工程１９］
工程１８で得られる化合物（Ｒ）から、製造法４の工程１５および工程１６と同様にして、化合物（Ｉ−ｉｉｉｂ）のうち、Ｘ^Ａがモノ置換ヒドロキシメチルである化合物（Ｉ−ｉｉｉｂａ）を得ることができる。
［工程２０］
また、化合物（Ｉ−ｉｉｉｂ）のうち、Ｘ^Ａがジ置換ヒドロキシメチルである化合物（Ｉ−ｉｉｉｂｂ）は、工程１８で得られる化合物から、工程１７、工程１８次いで工程１９と同様にして処理することにより得ることができる。
製造法６：
製造法４に記載した方法に加え、下記の製造法に従っても化合物（Ｉ−ｉｉｉａ）を得ることができる。

（式中、Ａ、Ｒ^１、Ｒ^３、Ｒ^４、Ｒ^５ａおよびＹはそれぞれ前記と同義である）
［工程２１］
製造法２の工程９で得られる化合物（Ｉ−ｉｉａ）を、溶媒中、１当量〜１０当量、好ましくは１．５当量〜３当量の還元剤で処理することにより、化合物（Ｉ−ｉｉｉａ）を得ることができる。
溶媒は反応に不活性なものであればいずれでもよく、特に限定されないが、例えばペンタン、ヘキサン、ベンゼン、トルエン、キシレン、ジエチルエーテル、テトラヒドロフラン、ジメトキシエタン、ジオキサン等を単独でまたはそれらを混合して用いることができる。
還元剤としては、例えば水素化リチウムアルミニウム、水素化ジイソブチルアルミニウム、水素化ビス（メトキシエトキシ）アルミニウム等を用いることができ、中でも水素化ビス（メトキシエトキシ）アルミニウムが好ましい。
反応は−７８℃〜溶媒の沸点の間の温度、好ましくは０℃〜３０℃の間の温度で行われ、通常１０分間〜２４時間で終了する。
製造法７：
化合物（Ｉ）は上記で示した製造法の他に、例えばＷＯ９２／０１６７５、ＷＯ９７／０９３１５、ＷＯ９９／６５８９７、特開２００１−８９４５２等に記載の方法でまたはそれらに準じて得ることができる。また、例えばＷＯ９２／０１６７５、ＷＯ９７／０９３１５、ＷＯ９９／６５８９７、特開２００１−８９４５２、イー・キリングスバーグ（Ｅ．Ｋｌｉｎｇｓｂｅｒｇ）編、ザ・ケミストリー・オブ・ヘテロサイクリック・コンパウンズ（Ｔｈｅ Ｃｈｅｍｉｓｔｒｙ ｏｆ Ｈｅｔｅｒｏｃｙｃｌｉｃ Ｃｏｍｐｏｕｎｄｓ）、ｖｏｌ．１４、ピリジン・アンド・イッツ・デリバティブス（Ｐｙｒｉｄｉｎｅ ａｎｄ Ｉｔｓ Ｄｅｒｉｖａｔｉｖｅｓ）、パート１、ニューヨーク、インターサイエンス（Ｉｎｔｅｒｓｃｉｅｎｃｅ）（１９６０年）、同パート２（１９６１年）、同パート３（１９６２年）、同パート４（１９６４年）、ジー・アール・ニュウコム（Ｇ．Ｒ．Ｎｅｗｋｏｍｅ）編、ザ・ケミストリー・オブ・ヘテロサイクリック・コンパウンズ（Ｔｈｅ Ｃｈｅｍｉｓｔｒｙ ｏｆ Ｈｅｔｅｒｏｃｙｃｌｉｃ Ｃｏｍｐｏｕｎｄｓ）、ｖｏｌ．１４、ピリジン・アンド・イッツ・デリバティブス（Ｐｙｒｉｄｉｎｅ ａｎｄ Ｉｔｓ Ｄｅｒｉｖａｔｉｖｅｓ）、パート５（１９８４年）等に記載の方法でまたはそれらに準じて得られる化合物を中間体として、例えばシンレット（Ｓｙｎｌｅｔｔ）、２０００年、ｐ．６２５−６２６、ジャーナル・オブ・ケミカル・ソサエティ（Ｊｏｕｒｎａｌ ｏｆ Ｃｈｅｍｉｃａｌ Ｓｏｃｉｅｔｙ）、１９５２年、ｐ．８００−８０３、テトラヘドロン（Ｔｅｔｒａｈｅｄｏｒｏｎ）、１９８７年、第４３巻、ｐ．２５５７−２５６４、ジャーナル・オブ・ヘテロサイクリック・ケミストリー（Ｊｏｕｒｎａｌ ｏｆ Ｈｅｔｅｒｏｃｙｃｌｉｃ Ｃｈｅｍｉｓｔｒｙ）、１９８７年、第２４巻、ｐ．８５−８９、ジャーナル・オブ・オーガニック・ケミストリー（Ｊｏｕｒｎａｌ ｏｆ Ｏｒｇａｎｉｃ Ｃｈｅｍｉｓｔｒｙ）、１９９５年、第６０巻、ｐ．１８７５−１８７７等に記載の方法でまたはそれらに準じて官能基変換を行うことにより、目的とする化合物を得ることができる。
化合物（Ｉ）、原料化合物および中間体における各官能基の変換および置換基に含まれる官能基の変換は、公知の方法［例えば、コンプリヘンシブ・オーガニック・トランスフォーメーションズ第二版（Ｃｏｍｐｒｅｈｅｎｓｉｖｅ Ｏｒｇａｎｉｃ Ｔｒａｎｓｆｏｒｍａｔｉｏｎｓ，ｔｈｅ ｓｅｃｏｎｄ ｅｄｉｔｉｏｎ）、ラロック（Ｒ．Ｃ．Ｌａｒｏｃｋ）著、ジョン・ワイリー・アンド・サンズ・インコーポレーティッド（Ｊｏｈｎ Ｗｉｌｅｙ ＆ Ｓｏｎｓ Ｉｎｃ．）（１９９９年）に記載の方法］等によって行うことができる。
上記の方法等を適宜組み合わせて実施することにより、所望の位置に所望の官能基を有する化合物（Ｉ）を得ることができる。
上記製造法における中間体および目的化合物の単離・精製は、通常の有機合成で用いられる方法、例えば濾過、抽出、洗浄、乾燥、濃縮、結晶化、各種クロマトグラフィー等を適宜組み合わせて行うことができる。さらに一般的な並列合成法で常用される精製法、例えば、スカベンジャーレジン、イオン交換レジン等を用いた精製法によって行うことができる。また、中間体においては、特に精製することなく次の反応に供することもできる。
化合物（Ｉ）および化合物（ＩＡ）には、位置異性体、幾何異性体、光学異性体または互変異性体のような異性体が存在し得るものもあるが、これらを含め可能な全ての異性体および該異性体のいかなる比率における混合物も本発明の治療剤に用いられるか、本発明に包含される。
化合物（Ｉ）または化合物（ＩＡ）の塩を取得したい場合には、化合物（Ｉ）または化合物（ＩＡ）の塩が得られるときはそのまま精製すればよく、化合物（Ｉ）または化合物（ＩＡ）が遊離の形で得られるときは化合物（Ｉ）または化合物（ＩＡ）を適当な溶媒に溶解または懸濁し、酸または塩基を加えて単離・精製すればよい。
また、化合物（Ｉ）、化合物（ＩＡ）またはそれらの薬理学的に許容される塩は、水または各種溶媒との付加物の形で存在することもあるが、これらの付加物も本発明の治療剤に用いられるか、本発明に包含される。
以下、本発明によって得られる化合物（Ｉ）の具体例を第１表〜第６表に示す。

次に、代表的な化合物（Ｉ）の薬理作用について試験例により具体的に説明する。
試験例１：ＧＰＲ８８の機能抑制作用（ＧＰＲ８８特異的な構成活性阻害作用）
参考例５で構築したＧＰＲ８８アッセイ細胞１を白色プレートに１ウェル当たり１０^５個ずつ播種し、ＧＰＲ８８を誘導発現するため、反応液中１０ｎｍｏｌ／Ｌになるように１７β−エストラジオール（１７β−ｅｓｔｒａｄｉｏｌ、以下Ｅ２と略する；Ｓｉｇｍａ−Ａｌｄｒｉｃｈ社製）を培地で希釈したものと試験化合物とを加え、５％ＣＯ_２インキュベーター中、３７℃で６時間反応させた。その後、Ｓｔｅａｄｙ Ｇｌｏ Ｌｕｃｉｆｅｒａｓｅ Ａｓｓａｙ Ｓｙｓｔｅｍ（Ｐｒｏｍｅｇａ社製）溶液を加え反応を停止し、トップカウント（Ｐａｃｋａｒｄ社製）で１秒間の発光量を測定した。
試験化合物の活性（拮抗作用）は、下の式に示す通りＥ２添加時と非添加時の１秒あたりのカウント数をもとに算出した阻害率で表した。
阻害率（％）＝［１−｛（Ａ−Ｂ）／（Ｃ−Ｂ）｝］ｘ１００
式中、Ａ、Ｂ、Ｃは各々以下の意味を表す。
Ａ：Ｅ２および試験化合物添加時の１秒あたりのカウント数
Ｂ：Ｅ２および試験化合物ともに非添加時の１秒あたりのカウント数
Ｃ：Ｅ２のみ添加時の１秒あたりのカウント数
ＩＣ_５０値は、阻害率からＬｏｇｉｔ−Ｌｏｇ変換法の線形近似解析法によって算出した。その結果を第７表に示す。

また、細胞を試験化合物の最終濃度が３μｍｏｌ／Ｌになる条件で処理したときの阻害率（％）を第８表に示す。

以上の結果より、化合物（Ｉ）はＧＰＲ８８の機能抑制作用（ＧＰＲ８８拮抗作用）を有することが示された。
また、ＷＯ０３／０８７３６６に記載の方法に従って、他の構成活性型ＧＰＣＲ（例えば、ＧＰＲ３、ＧＰＲ４、ＧＰＲ６またはＧＰＲ１２）を誘導発現するアッセイ細胞を用いて同様のアッセイを行った際には、化合物（Ｉ）は阻害活性を示さなかった。このことから、化合物（Ｉ）はＧＰＲ８８の構成活性を特異的に阻害することが示された。
なお、参考例６および７でそれぞれ構築したＧＰＲ８８アッセイ細胞２および３も、ＧＰＲ８８アッセイ細胞１と同様に、ＧＰＲ８８特異的な構成活性阻害物質（ＧＰＲ８８の機能抑制作用を有する物質）の探索に用いることができる。
試験例２：ＧＰＲ８８遺伝子に対するアンチセンスオリゴヌクレオチドの脳室内投与による運動機能改善（ドパミン受容体拮抗薬によるモデル動物での評価）
配列番号７に示す塩基配列を有するＧＰＲ８８遺伝子に対するホスホロチオエート型アンチセンスオリゴヌクレオチド（Ｓｔｒｇ−ＡＳ１：ジェンセット社に合成を依頼した）（１０μｇ／２０μＬ）を、生理食塩液（大塚製薬社製）に溶解し、この溶液を、後述のドパミン受容体拮抗薬ＳＣＨ２３３９０投与の３日前から、２段針を用いて、１２時間ごとに６回、マウスの脳室内へ繰り返し投与した。別のマウスに同様の手法でクマジー液を脳室内投与し、脳を摘出し投与液が脳内に拡散していることを確認した。Ｓｔｒｇ−ＡＳ１（１０μｇ／２０μＬ）の最終脳室内投与の３時間後に、ドパミン受容体拮抗薬ＳＣＨ２３３９０（０．５ｍｇ／ｋｇ；シグマ社製を生理食塩水で０．０５ｍｇ／ｍＬに希釈）を皮下注射して、運動不全状態を作製した。ＳＣＨ２３３９０の皮下投与の３０分後に、１匹ずつ高さ４．５ｃｍ、幅１．０ｃｍの垂直に立てたアクリル製の台にマウスの両後肢を掛け、１分間を上限に動物が動き出すまでの時間（不動時間）を測定した。この不動時間を指標として運動機能不全の程度を表わした。なお、Ｓｔｒｇ−ＡＳ１を投与せずＳＣＨ２３３９０のみを皮下投与した群を、陰性対照群とした。
その結果、ＳＣＨ２３３９０のみを皮下投与した陰性対照群における不動時間の平均は５８秒間であった。一方、Ｓｔｒｇ−ＡＳ１を脳室内投与した群における不動時間は３４．４秒間と減少し、運動機能を改善する作用が認められた。
つまり、Ｓｔｒｇ−ＡＳ１の繰り返し投与により、ドパミン受容体拮抗薬により誘発される運動不全状態は改善されたことから、ＧＰＲ８８遺伝子がドパミン神経系の運動調節に関係していることが明らかとなり、ＧＰＲ８８遺伝子を標的とした薬物は、脳内ドパミン神経系の異常に基づく疾患（例えばパーキンソン病、不随意運動、錐体外路症、うつ病、精神分裂病等）の治療および／または予防に有効であることが示唆された。
なお、Ｓｔｒｇ−ＡＳ１はマウスＧＰＲ８８ｍＲＮＡの翻訳開始点を含む、配列番号５に示す塩基配列の４０１〜４１８番目の配列と相補的な配列からなるホスホロチオエート・オリゴＤＮＡである。また、Ｓｔｒｇ−ＡＳ１の塩基配列は、マウスＧＰＲ８８ｍＲＮＡだけでなく、ヒトＧＰＲ８８ｍＲＮＡの翻訳開始点を含む、配列番号４に示す塩基配列の４２５〜４４２番目の配列とも相補的な配列であるため、ヒトに投与した場合もＧＰＲ８８遺伝子の発現を抑制し、下記および試験例２で示されるマウスに対する作用と同様の作用を示すと考えられる。
試験例３：ＧＰＲ８８遺伝子に対するアンチセンスオリゴヌクレオチドの脳室内投与による、中枢機能改善（レセルピン処置動物での評価）
後述のレセルピン投与の３日前から、２段針を用いて、生理食塩液に溶解したＳｔｒｇ−ＡＳ１（１０μｇ／２０μＬ）を、１２時間ごとに６回マウスの脳室内へ繰り返し投与した。別のマウスに同様の手法でクマジー液を脳室内投与し、脳を摘出し投与液が脳内に拡散していることを確認した。Ｓｔｒｇ−ＡＳ１（１０μｇ／２０μＬ）の最終脳室内投与の２０時間前に、レセルピン（２．５ｍｇ／ｋｇ：アポプロン注；第一製薬を蒸留水で０．２５ｍｇ／ｍＬに希釈）を皮下注射して、中枢機能不全状態を作製した。レセルピン投与の２４時間後に、試験例２と同様の方法により測定を１匹ずつ行なって、１分間を上限にマウスが動き出すまでの時間（不動時間）を測定した。この不動時間を指標として中枢機能不全の程度を表わした。なお、Ｓｔｒｇ−ＡＳ１を投与せずレセルピンのみを皮下投与した群を、陰性対照群とした。
その結果、レセルピンのみを皮下投与した陰性対照群における不動時間の平均は６０秒間であった。一方、Ｓｔｒｇ−ＡＳ１を脳室内投与した群における不動時間は３９．６秒間と減少し、中枢機能を改善する作用が認められた。
つまり、ＧＰＲ８８遺伝子に対するホスホロチオエート型アンチセンスオリゴヌクレオチドであるＳｔｒｇ−ＡＳ１の繰り返し投与により、レセルピン投与により誘発される中枢機能不全状態は改善された。すなわち、ＧＰＲ８８遺伝子を標的とした薬物は、脳内カテコールアミン神経系の異常に基づく（カテコールアミン枯渇に基づく）疾患（例えばパーキンソン病、不随意運動、錐体外路症、うつ病、精神分裂病等）の治療および／または予防に有効であることが示唆された。
試験例４：ドパミン受容体拮抗薬ＳＣＨ２３３９０誘発カタレプシーに対する作用
５週齢の雄性ｄｄＹマウス（体重２２〜２５ｇ、日本エスエルシー）を１群１０匹用いて実験を行った。ＳＣＨ２３３９０（シグマ社製）を生理食塩液（大塚製薬社製）に溶解し、マウスに０．５ｍｇ／ｋｇで皮下注射した。試験化合物（化合物３７）は蒸留水（大塚製薬社製）で懸濁して用いた。ＳＣＨ２３３９０を皮下注射する３０分前に、化合物３７を含む懸濁液または蒸留水のみ（対照）をそれぞれ経口投与した（マウス体重１０ｇあたり０．１ｍＬ）。化合物３７投与の１時間後に１匹ずつ高さ４．５ｃｍ、幅１．０ｃｍの垂直に立てたアクリル製の台にマウスの両前肢のみ、両後肢のみを順次懸け、カタレプシーを測定した。第９表にカタレプシースコアの判定基準を示す。

作用の強度は、１群１０匹のカタレプシースコアの平均値として表した（満点５点）。その結果を図１に示す。
図１より、化合物３７の投与によりドパミン受容体拮抗薬の投与により誘発されるカタレプシー症状の改善（運動機能の改善）が認められ、化合物（Ｉ）がドパミン神経系の異常に基づく疾患（例えばパーキンソン病、不随意運動、錐体外路症、うつ病、精神分裂病等）の治療および／または予防に有効であることが示唆された。
試験例５：レセルピン誘発カタレプシーに対する作用
レセルピンのような抗精神病薬の投与で誘発されるカタレプシーは、パーキンソン病やうつ病の症候モデルとされている［ジャーナル・オブ・ニューラル・トランスミッション（Ｊｏｕｒｎａｌ ｏｆ Ｎｅｕｒａｌ Ｔｒａｎｓｍｉｓｓｉｏｎ）、８巻、３９−７１頁（１９９４年）］。
５週齢の雄性ｄｄＹマウス（体重２２〜２５ｇ、日本エスエルシー）を１群１０匹用いて実験を行った。予備飼育期間中は、室温２２±３℃、湿度５０±２０％の動物室内で飼育し、餌、水は自由に摂取させた。レセルピン（５ｍｇ／ｋｇ：アポプロン注：第一製薬社製を蒸留水で希釈）を皮下投与して１８時間後よりカタレプシー惹起作用を観察し、カタレプシースコアが５（試験例４第４表判定基準）を示すマウスを選別して実験に供した。試験化合物（化合物３７）は、注射用蒸留水（大塚製薬社製）で懸濁液として用い、化合物３７を含む懸濁液または注射用蒸留水のみ（対照）をそれぞれ経口投与（マウス体重１０ｇあたり０．１ｍＬ）し、化合物３７の投与１時間後に１匹ずつ高さ４．５ｃｍ、幅１．０ｃｍの台にマウス両前肢のみ、両後肢のみを順次懸け、カタレプシーを測定した。
作用の強度は、１群１０匹のカタレプシースコアの平均値として表した（満点５点）。その結果を図２に示す。
図２より、化合物３７の投与によりレセルピン投与により誘発されるカタレプシーの改善（中枢機能の改善）が認められ、化合物（Ｉ）は、脳内カテコールアミン神経系の異常に基づく（カテコールアミン枯渇に基づく）疾患（例えばパーキンソン病、不随意運動、錐体外路症、うつ病、精神分裂病等）の治療および／または予防に有効であることが示唆された。
試験例６：自発運動量の増大作用
５週齢の雄性ｄｄＹマウス（体重２２〜２５ｇ、日本エスエルシー）を１群１０匹用いて実験を行った。化合物２６３を投与する一時間前に、マウスを幅４０ｃｍ、奥行き４０ｃｍ、高さ４０ｃｍのアクリル製の箱に静置し環境に馴化させた。化合物２６３は０．５％カルボキシメチルセルロース（ＣＭＣ；大塚製薬社製）を含む蒸留水で懸濁して用いた。化合物２６３（２．５ｍｇ／ｍＬ）を含む懸濁液または溶媒のみをそれぞれ腹腔内投与した（マウス体重１０ｇあたり０．１ｍＬ）。自発運動量測定装置（ＳＣＡＮＥＴ ＭＶ−１０ＭＴ、東洋産業株式会社医薬機器事業部）を用いて、身づくろい行動、歩行行動および後ろ足立ち行動の３項目について、投与直後から４時間測定した。明期の運動は午前１０時から、暗期の運動は午後７時から測定した。
その結果、明期においては、溶媒を投与した陰性対照群および化合物２６３を投与した群における身づくろい行動はそれぞれ１１０１３カウントおよび１７８３０カウント、歩行行動はそれぞれ６７４５カウントおよび１３６１３カウント、後ろ足立ち行動はそれぞれ１９カウントおよび５２カウント、と化合物２６３投与群でいずれも増大していた。
化合物２６３投与によって自発運動量が増大したことから、ＧＰＲ８８の機能抑制剤は運動機能障害を伴う疾患、例えばパーキンソン病、不随意運動、錐体外路症候群、ハンチントン舞踏病、脳血管疾患、脊髄小脳変性症等の治療および／または予防に有効であることが示唆された。
一方、暗期においては、溶媒を投与した陰性対照群および化合物２６３を投与した群における身づくろい行動はそれぞれ２０１５６４カウントおよび１０８４１１カウント、歩行行動はそれぞれ９８２５４カウントおよび８６６７７カウント、後ろ足立ち行動はそれぞれ１４３９カウントおよび５４１カウント、と化合物２６３投与群でいずれも減少していた。
以上のように、夜行性動物であるマウスにおいて、化合物２６３の投与によって明期の運動量が増大し暗期の運動量が減少した。このことは、体内時計が外界の明暗環境の周期に合わせる光同調を、ＧＰＲ８８が修飾していることを示唆している。したがってＧＰＲ８８の機能制御剤はリズム障害、例えば時差ぼけや季節性感情障害等の治療および／または予防に有効であることが示唆された。
化合物（Ｉ）またはその薬理学的に許容される塩は、そのまま単独で投与することも可能であるが、通常各種の医薬製剤として提供するのが望ましい。また、それら医薬製剤は、動物および人に使用されるものである。
本発明に係わる医薬製剤は、活性成分としてＧＰＲ８８に対する抗体もしくは化合物（Ｉ）またはその薬理学的に許容される塩を単独で、あるいは任意の他の治療のための有効成分との混合物として含有することができる。また、それら医薬製剤は、活性成分を薬理学的に許容される一種もしくはそれ以上の担体と一緒に混合し、製剤学の技術分野においてよく知られている任意の方法により製造される。
投与経路としては、治療に際し最も効果的なものを使用するのが望ましく、経口投与または静脈内等の非経口投与をあげることができる。
投与形態としては、錠剤、注射剤等が例示される。
錠剤の調製にあたっては、例えば乳糖、マンニット等の賦形剤、デンプン等の崩壊剤、ステアリン酸マグネシウム等の滑沢剤、ヒドロキシプロピルセルロース等の結合剤、脂肪酸エステル等の界面活性剤、グリセリン等の可塑剤等を常法に従って用いればよい。
注射剤の調製にあたっては、水、生理食塩水、植物油、可溶化剤、保存剤、抗酸化剤等を常法に従って用いればよい。
化合物（Ｉ）またはその薬理学的に許容される塩の有効量および投与回数は、投与形態、患者の年齢、体重、治療すべき症状の性質もしくは重篤度等により異なるが、通常、例えば、成人一日当り０．１〜１００ｍｇ／ｋｇを３〜４回に分けて投与するのが好ましい。しかしながら、これら投与量および投与回数は前述の種々の条件等により変動する  An object of the present invention is to provide (A) a preventive and / or therapeutic agent for central diseases containing a substance having a GPR88 function inhibitory action as an active ingredient, and (B) useful for the prevention and / or treatment of central diseases. It is to provide a novel GPR88 antisense nucleotide or pyrimidine derivative or a pharmacologically acceptable salt thereof.
  The present invention relates to the following (1) to (65).
  (1) A prophylactic and / or therapeutic agent for central diseases comprising, as an active ingredient, a substance having a function-inhibiting action of G protein-coupled receptor protein 88 (GPR88; G-Protein coupled receptor 88).
  (2) The preventive and / or therapeutic agent according to (1), wherein the central disease is a disease selected from the group consisting of Parkinson's disease, depression and jet lag.
  (3) (a) contacting a test compound with a GPR88-expressing cell and measuring the cellular response; and (b) contacting the test compound as compared with the cellular response of the GPR88-expressing cell when the test compound is not added. And a method for screening a compound having a preventive and / or therapeutic action for a central disease, comprising the step of confirming that the cellular response is lowered.
  (4) The method according to (3), wherein the central disease is a disease selected from the group consisting of Parkinson's disease, depression and jet lag.
  (5) An antisense oligo having a base sequence complementary or substantially complementary to a sequence having 10 or more consecutive base sequences represented by SEQ ID NO: 1, 3 or 5 in which a substance having a GPR88 function inhibitory action The preventive and / or therapeutic agent for central diseases according to (1), which is a nucleotide.
  (6) The preventive and / or therapeutic agent for central diseases according to (1), wherein the substance having a GPR88 function inhibitory action is an antibody against GPR88.
  (7) A prophylactic and / or therapeutic agent for central diseases comprising, as an active ingredient, a compound that reduces the cellular response of GPR88 or a pharmacologically acceptable salt thereof that has been confirmed by the screening method described in (3).
  (8) The preventive and / or therapeutic agent according to any one of (5) to (7), wherein the central disease is a disease selected from the group consisting of Parkinson's disease, depression and jet lag.
  (9) GPR88 comprising, as an active ingredient, an antisense oligonucleotide having a base sequence complementary or substantially complementary to a sequence of 10 or more consecutive base sequences represented by SEQ ID NO: 1, 3 or 5 Function inhibitor.
  (10) An antisense oligonucleotide having the sequence represented by SEQ ID NO: 8.
  (11) The antisense oligonucleotide according to (10), wherein the antisense oligonucleotide is a phosphorothioate oligodeoxynucleotide.
  (12) A GPR88 function inhibitor comprising the antisense oligonucleotide according to (10) or (11) as an active ingredient.
  (13) A preventive and / or therapeutic agent for central diseases comprising the antisense oligonucleotide according to (10) or (11) as an active ingredient.
  (14) The preventive and / or therapeutic agent according to (13), wherein the central disease is a disease selected from the group consisting of Parkinson's disease, depression and jet lag.
  (15) Formula (I)

<In the formula, A is substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted aryloxyalkyl, substituted or unsubstituted aralkyloxyalkyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted Represents a substituted heterocyclic alkyl or cycloalkylalkyl;
R¹Represents a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl or substituted or unsubstituted aralkyl,
Or A and R¹Together with the adjacent nitrogen atom to form a substituted or unsubstituted heterocyclic group,
R²Is a hydrogen atom, hydroxy, halogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted lower alkoxy, substituted or unsubstituted lower alkylthio, substituted or Unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted heterocyclic alkyl or —NR³R⁴(Wherein R³And R⁴Are the same or different and are a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, Represents substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heterocyclic group or substituted or unsubstituted heterocyclic alkyl, or R³And R⁴Together with the adjacent nitrogen atom form a substituted or unsubstituted heterocyclic group)
X is -C (= O) R⁵{Where R is⁵Is hydroxy, substituted or unsubstituted lower alkoxy, substituted or unsubstituted aryloxy or —NR⁶R⁷[In the formula, R⁶And R⁷Are the same or different, hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted lower alkoxy, substituted or unsubstituted aralkyl, substituted Or unsubstituted heterocyclic alkyl, hydroxy or -SO₂R⁸(Wherein R⁸Represents substituted or unsubstituted lower alkyl or substituted or unsubstituted aryl), or
Or R⁶And R⁷Represents, together with the adjacent nitrogen atom, form a substituted or unsubstituted heterocyclic group], nitro, substituted or unsubstituted hydroxymethyl or 1,2,3,4-tetrazol-5-yl Represent,
Y represents a hydrogen atom, a halogen, or a substituted or unsubstituted lower alkyl, a pyrimidine derivative represented by> or a pharmacologically acceptable salt thereof as a GPR88 function inhibitor.
  (16) The function inhibitor of GPR88 according to (15), wherein Y is a hydrogen atom.
  (17) R²-NR³R⁴(Wherein R³And R⁴Are the same as defined above, respectively, and the GPR88 function inhibitor according to (15) or (16).
  (18) The function inhibitor of GPR88 according to any one of (15) to (17), wherein X is 1,2,3,4-tetrazol-5-yl.
  (19) X is -C (= O) NR⁶R⁷(Wherein R⁶And R⁷Are the same as defined above, respectively, and the GPR88 function inhibitor according to any one of (15) to (17).
  (20) X is -C (= O) R^5B(Wherein R^5BIs a hydroxy or lower alkoxy), GPR88 function inhibitor according to any one of (15) to (17).
  (21) The function inhibitor of GPR88 according to any one of (15) to (17), wherein X is substituted or unsubstituted hydroxymethyl.
  (22) The function inhibitor of GPR88 according to any one of (15) to (21), wherein A is substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, or a substituted or unsubstituted heterocyclic group.
  (23) R¹The function inhibitor of GPR88 according to any one of (15) to (22), wherein is a hydrogen atom.
  (24) R³Is substituted or unsubstituted lower alkyl, cycloalkylalkyl or substituted or unsubstituted aralkyl, or R³And R⁴The function inhibitor of GPR88 according to any one of (17) to (23), wherein is combined with an adjacent nitrogen atom to form a substituted or unsubstituted heterocyclic group.
  (25) R³Is substituted or unsubstituted lower alkyl, cycloalkylalkyl or substituted or unsubstituted aralkyl, and R⁴The function inhibitor of GPR88 according to any one of (17) to (23), wherein is a hydrogen atom or lower alkyl.
  (26) Formula (I)

(Where A, R¹, R², X and Y have the same meanings as described above), and a preventive and / or therapeutic agent for a central disease containing as an active ingredient a pyrimidine derivative represented by the formula or a pharmacologically acceptable salt thereof.
  (27) The preventive and / or therapeutic agent for central disease according to (26), wherein Y is a hydrogen atom.
  (28) R²-NR³R⁴(Wherein R³And R⁴Are the same as defined above, respectively, for preventing and / or treating central diseases according to (26) or (27).
  (29) The preventive and / or therapeutic agent for central disease according to any one of (26) to (28), wherein X is 1,2,3,4-tetrazol-5-yl.
  (30) X is -C (= O) NR⁶R⁷(Wherein R⁶And R⁷Are the same as defined above, respectively, and the preventive and / or therapeutic agent for central diseases according to any one of (26) to (28).
  (31) X is -C (= O) R^5B(Wherein R^5BRepresents hydroxy or lower alkoxy). The preventive and / or therapeutic agent for central diseases according to any of (26) to (28).
  (32) The agent for preventing and / or treating a central disease according to any one of (26) to (28), wherein X is substituted or unsubstituted hydroxymethyl.
  (33) Prevention and / or treatment of a central disease according to any of (26) to (32), wherein A is substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, or a substituted or unsubstituted heterocyclic group Agent.
  (34) R¹The prophylactic and / or therapeutic agent for central diseases according to any one of (26) to (33), wherein is a hydrogen atom.
  (35) R³Is substituted or unsubstituted lower alkyl, cycloalkylalkyl or substituted or unsubstituted aralkyl, or R³And R⁴The prophylactic and / or therapeutic agent for central diseases according to any one of (28) to (34), wherein, together with the adjacent nitrogen atom, forms a substituted or unsubstituted heterocyclic group.
  (36) R³Is substituted or unsubstituted lower alkyl, cycloalkylalkyl or substituted or unsubstituted aralkyl, and R⁴The prophylactic and / or therapeutic agent for central diseases according to any one of (28) to (34), wherein is a hydrogen atom or lower alkyl.
  (37) The preventive and / or therapeutic agent according to any one of (26) to (36), wherein the central disease is a disease selected from the group consisting of Parkinson's disease, depression and jet lag.
  (38) Formula (IA)

{Where A and R¹Are as defined above and X^AIs 1,2,3,4-tetrazol-5-yl, —C (═O) R^5A[Wherein R^5AIs hydroxy or -NR^6AR^7A(Wherein R^6ARepresents a hydrogen atom or lower alkyl, R^7ARepresents a hydrogen atom, hydroxy, hydroxy lower alkyl, carboxy lower alkyl, p-toluenesulfonyl, methanesulfonyl or trifluoromethanesulfonyl)] or a substituted or unsubstituted hydroxymethyl, (i) X^AIs 1,2,3,4-tetrazol-5-yl, R^3AAnd R^4AAre the same or different and each represents a hydrogen atom, a substituted or unsubstituted lower alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted cycloalkylalkyl, or a substituted or unsubstituted aralkyl. Or R)^3AAnd R^4ATogether with the adjacent nitrogen atom form a substituted or unsubstituted heterocyclic group, (ii) X^AIs -C (= O) R^5AOr when substituted or unsubstituted hydroxymethyl, R^3AAnd R^4AAre the same or different and are a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted heterocyclic alkyl, or substituted or unsubstituted aralkyl. (But not simultaneously hydrogen atoms) or R^3AAnd R^4ASubstituted or unsubstituted piperazinyl, 4-methylhomopiperazinyl, 4-methylpiperidino, 4-piperidinopiperidino, 4-benzylpiperidino, 4,4-ethylene together with the adjacent nitrogen atom Forming dioxypiperidino or decahydroisoquinolin-1-yl}, or a pharmaceutically acceptable salt thereof.
  (39) X^AThe pyrimidine derivative or a pharmacologically acceptable salt thereof according to (38), wherein is 1,2,3,4-tetrazol-5-yl.
  (40) X^AIs -C (= O) R^5A(Wherein R^5AIs as defined above), or a pharmacologically acceptable salt thereof.
  (41) X^AThe pyrimidine derivative or a pharmaceutically acceptable salt thereof according to (38), wherein is -C (= O) OH.
  (42) X^AThe pyrimidine derivative or a pharmaceutically acceptable salt thereof according to (38), wherein is hydroxymethyl, 1-hydroxyethyl or 1-hydroxy-1-methylethyl.
  (43) A is substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted aryloxyalkyl, substituted or unsubstituted heterocyclic group or cycloalkylalkyl, or A and R¹The pyrimidine derivative or a pharmaceutically acceptable salt thereof according to any one of (38) to (42), wherein, together with the adjacent nitrogen atom, forms a substituted or unsubstituted heterocyclic group.
  (44) The pyrimidine derivative or a pharmaceutically acceptable salt thereof according to any one of (38) to (42), wherein A is substituted or unsubstituted aryl, aralkyl, a heterocyclic group, or cycloalkylalkyl.
  (45) A is benzyl, 4-phenyl-n-butyl, 3-phenylpropyl, 3-phenyl-1-methylpropyl, 2-phenylpropyl, 2-phenoxyethyl, 2-phenoxy-1-methylethyl, 1, From the group consisting of 4-benzodioxan-6-yl, 2-benzylphenyl, 2-phenoxyphenyl, 3-phenoxyphenyl, 4-phenoxyphenyl, 4-biphenyl, biphenyl-4-ylmethyl, 2-indanyl and cyclopropylmethyl The pyrimidine derivative or the pharmaceutically acceptable salt thereof according to any one of (38) to (42), which is a selected group.
  (46) R¹The pyrimidine derivative or a pharmaceutically acceptable salt thereof according to any one of claims 38 to 45, wherein is a hydrogen atom, lower alkyl or aralkyl.
  (47) R¹The pyrimidine derivative or a pharmaceutically acceptable salt thereof according to any one of (38) to (45), wherein is a hydrogen atom.
  (48) R^3AAnd R^4AAre the same or different and are a hydrogen atom, substituted or unsubstituted lower alkyl, cycloalkylalkyl, substituted or unsubstituted aralkyl or substituted or unsubstituted heterocyclic alkyl, or R^3AAnd R^4AThe pyrimidine derivative or the pharmaceutically acceptable salt thereof according to any one of (38) to (47), wherein A forms a substituted or unsubstituted heterocyclic group together with an adjacent nitrogen atom.
  (49) R^3AAnd R^4AAre the same or different and are a hydrogen atom, lower alkyl, cycloalkylmethyl, substituted or unsubstituted aralkyl or heterocyclic alkyl, or a pyrimidine derivative according to any one of (38) to (47) or a pharmaceutically acceptable salt thereof Salt.
  (50) R^3AIs lower alkyl, cycloalkylmethyl or substituted or unsubstituted aralkyl, R^4AThe pyrimidine derivative or the pharmaceutically acceptable salt thereof according to any one of (38) to (47), wherein is a hydrogen atom.
  (51) R^3AIs 3-methoxybenzyl, 3-chlorobenzyl, 3-fluorobenzyl, 2,6-difluorobenzyl, 2-methylbenzyl, 3-trifluorobenzyl, 2-pyridylmethyl, 3-pyridylmethyl, cyclohexylmethyl, cycloheptyl, A group selected from the group consisting of 1,5-dimethyl-n-hexyl and 2,3-dihydrobenzo [1,4] dioxin-2-ylmethyl;^4AIs a hydrogen atom or R^3AIs benzyl and R^4AIs n-butyl or R^3AIs n-hexyl and R^4AIs methyl or R^3AAnd R^4ATogether with the adjacent nitrogen atom form 4-benzylpiperidino, 4,4-ethylenedioxypiperidino or decahydroisoquinolin-1-yl (38) and (40)-(47) Any one of the pyrimidine derivatives or a pharmacologically acceptable salt thereof.
  (52) R^3AFrom 3-methoxybenzyl, 3-chlorobenzyl, 3-fluorobenzyl, 2,6-difluorobenzyl, 2-methylbenzyl, 3-trifluorobenzyl, cyclohexylmethyl, cycloheptyl and 1,5-dimethyl-n-hexyl A group selected from the group consisting of R^4AIs a hydrogen atom or R^3AIs benzyl and R^4AIs n-butyl or R^3AIs n-hexyl and R^4AIs methyl or R^3AAnd R^4AForms a 4-benzylpiperidino, 4,4-ethylenedioxypiperidino or decahydroisoquinolin-1-yl together with an adjacent nitrogen atom, or a pyrimidine derivative according to (39) or a pharmacological thereof Acceptable salt.
  (53) The compound represented by the formula (IA) is 4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carboxylic acid, 4- (2-benzylphenylamino) -2- (Cyclohexylmethylamino) pyrimidine-5-carbohydroxamic acid, [4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carbonyl] aminoacetic acid, {[4- (2-benzylphenylamino) ) -2- (cyclohexylmethylamino) pyrimidine-5-carbonyl] methylamino} acetic acid, 4- (2-benzylphenylamino) -5-hydroxymethyl-2- (cyclohexylmethylamino) pyrimidine, N- [4- ( 2-Benzylphenylamino) -2- (cyclohexylmethylamino) pyrim -5-carbonyl] -4-methylbenzenesulfonamide, N- [4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carbonyl] methanesulfonamide, [4- (2- Benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carbonyl] trifluoromethanesulfonamide, 2- (cyclohexylmethylamino) -4- (1-methyl-2-phenoxyethylamino) pyrimidine-5-carboxylic acid 2- (cyclohexylmethylamino) -4- (2-phenoxyethylamino) pyrimidine-5-carboxylic acid, 2- (cyclohexylmethylamino) -4- (2-phenoxyphenylamino) pyrimidine-5-carboxylic acid, 4 -(4-Phenylpiperidin-1-yl)- 2- (3-trifluoromethylbenzylamino) pyrimidine-5-carboxylic acid, 2- (cyclohexylmethylamino) -5-hydroxymethyl-4- (1-methyl-2-phenoxyethylamino) pyrimidine, 2- (cyclohexyl) Methylamino) -5-hydroxymethyl-4- (2-phenoxyethylamino) pyrimidine, 2- (benzylbutylamino) -5-hydroxymethyl-4- (2-phenoxyethylamino) pyrimidine, 5-hydroxymethyl-2 -(Octahydroisoquinolin-2-yl) -4- (2-phenoxyethylamino) pyrimidine, 2- (cyclohexylmethylamino) -5-hydroxymethyl-4- (2-phenoxyphenylamino) pyrimidine, 5-hydroxymethyl -4- (2-phenylpropylamino)- -(3-trifluoromethylbenzylamino) pyrimidine, 5-hydroxymethyl-4- (4-phenylpiperidin-1-yl) -2- (3-trifluoromethylbenzylamino) pyrimidine, 4- (2-benzylphenyl) Amino) -2- (3-chlorobenzylamino) pyrimidine-5-carboxylic acid, 4- (2-benzylphenylamino) -2- (3-fluorobenzylamino) pyrimidine-5-carboxylic acid, 2- (benzylbutyl) Amino) -4- (2,3-dihydrobenzo [1,4] dioxin-6-ylamino) pyrimidine-5-carboxylic acid, 2- (3-methoxybenzylamino) -4- (2-phenoxyphenylamino) pyrimidine -5-carboxylic acid, 2- (2,6-difluorobenzylamino) -4- (2-phenoxyphenyla) C) pyrimidine-5-carboxylic acid, 2- (2-methylbenzylamino) -4- (2-phenoxyphenylamino) pyrimidine-5-carboxylic acid, 4- (2-phenoxyphenylamino) -2- (3- Trifluorobenzylamino) pyrimidine-5-carboxylic acid, 2- (3-fluorobenzylamino) -4- (4-phenylbutylamino) pyrimidine-5-carboxylic acid, 2- (benzylbutylamino) -4- (4 -Biphenylmethylamino) pyrimidine-5-carboxylic acid, 4- (2,3-dihydrobenzo [1,4] dioxin-6-ylamino) -5-hydroxymethyl-2- (3-trifluoromethylbenzylamino) pyrimidine 4- (2,3-dihydrobenzo [1,4] dioxin-6-ylamino) -5-hydroxymethyl-2- (2 -Pyridylmethylamino) pyrimidine and 2- [bis (2-methoxyethyl) amino] -4- (2,3-dihydrobenzo [1,4] dioxin-6-ylamino) -5-hydroxymethylpyrimidine, 2- ( A pyrimidine derivative which is a compound selected from the group consisting of 3-fluorobenzylamino) -5-hydroxymethyl-4- (2-phenoxyethylamino) pyrimidine or a pharmaceutically acceptable salt thereof.
  (54) A medicament comprising the pyrimidine derivative according to any one of (38) to (53) or a pharmacologically acceptable salt thereof as an active ingredient.
  (55) A GPR88 function inhibitor comprising the pyrimidine derivative according to any one of (38) to (53) or a pharmacologically acceptable salt thereof as an active ingredient.
  (56) A preventive and / or therapeutic agent for central diseases comprising the pyrimidine derivative according to any one of (38) to (53) or a pharmacologically acceptable salt thereof as an active ingredient.
  (57) The preventive and / or therapeutic agent according to (56), wherein the central disease is a disease selected from the group consisting of Parkinson's disease, depression and jet lag.
  (58) Use of a substance having a GPR88 function-suppressing action for the manufacture of a preventive and / or therapeutic agent for central diseases.
  (59) An anti-antibody having a base sequence complementary or substantially complementary to a sequence of 10 bases or more of the base sequence represented by SEQ ID NO: 1, 3 or 5 for the production of a GPR88 function inhibitor Use of sense oligonucleotides.
  (60) Use of the pyrimidine derivative according to (15) or a pharmacologically acceptable salt thereof for the manufacture of a GPR88 function inhibitor.
  (61) Use of the pyrimidine derivative or a pharmaceutically acceptable salt thereof according to (15) for the manufacture of a preventive and / or therapeutic agent for central diseases.
  (62) A method for preventing and / or treating a central disease, which comprises administering an effective amount of a substance having a GPR88 function inhibitory action.
  (63) administering an effective amount of an antisense oligonucleotide having a base sequence complementary or substantially complementary to a sequence of 10 or more consecutive base sequences represented by SEQ ID NO: 1, 3 or 5 A method for suppressing GPR88 function.
  (64) A method for inhibiting the function of GPR88, comprising administering an effective amount of the pyrimidine derivative or the pharmaceutically acceptable salt thereof according to (15).
  (65) A method for preventing and / or treating a central disease, which comprises administering an effective amount of the pyrimidine derivative or a pharmacologically acceptable salt thereof according to (15).
  In the present invention, GPR88 includes the human GPR88 polypeptide having the amino acid sequence represented by SEQ ID NO: 2, the mouse GPR88 polypeptide having the amino acid sequence represented by SEQ ID NO: 4, and the amino acid sequence represented by SEQ ID NO: 6. And rat GPR88 polypeptide. In addition, a DNA that hybridizes under stringent conditions with a DNA having a base sequence complementary to the 438th to 1592th sequences of the base sequence represented by SEQ ID NO: 1 which is the sequence of the coding region of human GPR88 cDNA is encoded. In addition, a polypeptide having substantially the same function as the above-mentioned polypeptide is also included. As GPR88 used in the present invention, human GPR88 polypeptide having the amino acid sequence represented by SEQ ID NO: 2 is particularly preferable.
  The DNA that hybridizes under stringent conditions is a colony hybridization method, plaque hybridization method, or Southern blot hybridization using a DNA having a sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1 as a probe. DNA obtained by using a method or the like, specifically, at 65 ° C. in the presence of 0.7 to 1.0 mol / L NaCl using a filter on which colony or plaque-derived DNA is immobilized. After hybridization, 0.1 to 2 fold concentration SSC solution (composition of 1 fold concentration SSC solution consists of 150 mmol / L NaCl, 15 mmol / L sodium citrate) was used under the conditions of 65 ° C. List DNA that can be identified by washing the filter Can. Hybridization: Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press (2001), Current Protocols in Molecular Biology, John Wis. It can be performed according to the method described in Second Edition, Oxford University (1995) and the like. Specifically, the DNA capable of hybridizing is DNA having at least 60% homology with the base sequence represented by SEQ ID NO: 1, preferably 70% or more, more preferably 80% or more, and still more preferably 90%. As mentioned above, DNA having homology of 95% or more, most preferably 98% or more is particularly preferable.
<Method of screening for GPR88 constitutive activity inhibitor using GPR88-expressing cells, compound having action for prevention and / or treatment of central diseases>
  As shown in Reference Examples 5 to 7, GPR88 is a constitutively active GPCR that activates even in the absence of a ligand and causes signal information transmission when overexpressed in cells. Therefore, cells that express GPR88 Shows the cellular response associated with activation of GPR88 even in the absence of ligand. An arbitrary test substance is added to a cell expressing GPR88, and the cell response of the cell when the cell is brought into contact with the test substance and the cell response of the cell when no test substance is added are measured. Compared with the case where the test substance is not added, the test substance that decreases the cellular response when the test substance is added can be selected as the GPR88 constitutive activity inhibitor. In addition, a cell expressing another constitutively active GPCR is prepared in the same manner as the GPR88-expressing cell shown below, and the cell is contacted with a substance selected by the above method in the same manner as described above. It is possible to examine whether the selected substance specifically inhibits the constitutive activity by measuring the decrease in the cellular response when compared with the decrease in the cellular response in cells expressing GPR88. GPR88 constitutive activity inhibitor has an action of suppressing the function of GPR88, and becomes a GPR88 function inhibitor. GPR88 constitutive activity inhibitors include substances that inhibit GPR88-related signaling, such as inverse agonists that bind to GPR88 and inhibit its constitutive activity, and substances that inhibit the coupling of GPR88 and G protein. . As will be described later, since the GPR88 function inhibitor is effective for the prevention and / or treatment of central diseases, compounds having a therapeutic or preventive action for central diseases can be screened by the above methods.
  A cell that expresses GPR88 can be obtained from a tissue that naturally expresses GPR88, or by introducing an expression vector (GPR88 expression vector) containing DNA encoding GPR88 into a host cell. In order to measure the constitutive activity of GPR88, cells that express GPR88 in a large amount are suitable. Therefore, transformed cells obtained by introducing a GPR88 expression vector into a host cell are preferred.
  DNA encoding GPR88 can be obtained as follows. Based on the base sequence of GPR88 cDNA, for example, the base sequence of human GPR88 cDNA represented by SEQ ID NO: 1, the region of the cDNA including the region encoding the receptor is appropriately selected. DNA containing 5 'end 20-40 base sequences of the selected region at the 3' end, and DNA containing 3 'end 20-40 bases complementary to the selected region at the 3' end Are synthesized with a DNA synthesizer. For example, DNAs having the base sequences represented by SEQ ID NOs: 26 and 27 are exemplified. Prepare chromosomal DNA or cDNA from tissues or cells. In the case of cDNA, tissues or cells in which GPR88 is expressed are used. The DNA encoding the receptor can be amplified and isolated by PCR using the prepared chromosomal DNA or cDNA as a template and two kinds of synthetic DNAs as primers. Preparation of chromosomal DNA or cDNA from tissues and cells, and PCR are described in Molecular Cloning: A Laboratory Manual 3rd ed. , Cold Spring Harbor Laboratory Press (2001).
  As the expression vector, a vector that can replicate autonomously in the host cell or can be integrated into a chromosome and contains a promoter that functions in the host cell to be introduced can be used. A GPR88 expression vector can be constructed by inserting a DNA encoding GPR88 downstream of the promoter in the expression vector.
  As a method for introducing a GPR88 expression vector into a host cell, any method can be used as long as it is a method for introducing DNA into a host cell. As host cells, yeast, insect cells, animal cells, plant cells and the like can be used.
  When yeast strains are used as host cells, examples of expression vectors include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15 and the like. Any promoter can be used as long as it can be expressed in yeast strains. For example, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promonitor, heat shock protein promoter, MFα1 promoter, CUP1 promoter And the like.
  Examples of host cells include yeast strains belonging to the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Siwaniomyces, and the like.Saccharomyces cerevisiae,Schizosaccharomycespombe,Kluyveromyces lactis,Trichosporon pullulans,SchwanniomycesallluviusEtc. Mutants suitable for GPCR expression can also be used [Trends in Biotechnology,15487 (1997), Mol. Cell. Biol. ,156188 (1995), Mol. Cell. Biol. ,164700 (1996)].
  As a method for introducing a recombinant vector, any method for introducing DNA into yeast can be used. For example, electroporation [Methods. in Enzymol. ,194, 182 (1990)], spheroplast method [Proc. Natl. Acad. Sci. USA,84, 1929 (1978)], lithium acetate method [J. Bacteriol. ,153, 163 (1983)], Proc. Natl. Acad. Sci. USA,751929 (1978).
  When animal cells are used as host cells, examples of expression vectors include pcDNA3.1 (+), pCDM8, pAGE107, pREP4, pAGE103, pAMo, pAMoA, pAS3-3, and the like. In particular vectors for inducible expression, for exampleSaccharomyces cerevisiaeChimeric protein (Gal4-ER) [Cell, a DNA binding region of transcription factor Gal4 derived from a ligand binding region of estrogen receptor54, 199 (1988), Proc. Natl. Acad. Sci. USA,90, 1657 (1993)], pAGal9-d, pAGal9-nd, and the like that can be induced by addition of 17β-estradiol are preferred. In addition, vectors such as pAMo, pAMoA, pAGal9-d, and pAGal9-nd having Epstein-Barr virus oriP as replication origins are Epstein-Barr virus EBNA- such as Namalwa cells and Namalwa KJM-1 cells. When a B cell strain that expresses one gene is used as a host cell, it stably exists in the form of a plasmid outside the chromosome, so that a transformant exhibiting high expression can be easily obtained.
  Any promoter can be used as long as it can be expressed in animal cells. For example, cytomegalovirus (CMV) IE (immediate early) gene promoter, SV40 early promoter, Moloney murine leukemia virus long promoter Examples thereof include a terminal terminal repeat promoter, a retrovirus promoter, a heat shock promoter, an SRα promoter, and a metallothionein promoter. In addition, an enhancer of human CMV IE gene may be used together with a promoter.
  Examples of host cells include mouse myeloma cells, rat myeloma cells, mouse hybridoma cells, CHO cells, BHK cells, African green monkey kidney cells, Namalwa cells, Namalwa KJM-1 cells, human fetal kidney cells, human leukemia cells HBT5637 (Japanese Patent Laid-Open No. 63-299), human colon cancer cell lines, frog oocytes, frog melanocytes, and the like.
  Specifically, mouse myeloma cells are SP2 / 0, NSO, etc., rat myeloma cells are YB2 / 0, human fetal kidney cells are HEK293, human leukemia cells are BALL-1, etc., Africa Examples of the monkey kidney cells include COS-1 and COS-7, and examples of human colon cancer cell lines include HCT-15.
  As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into animal cells. For example, an electroporation method [Cytotechnology,3133 (1990)], calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), lipofection method [Proc. Natl. Acad. Sci. USA,847413 (1987)], Virology,52, 456 (1973). The transformant can be obtained and cultured according to the method described in JP-A-2-227075 or JP-A-2-257891.
  When insect cells are used as host cells, see, for example, Baculovirus Expression Vectors, A Laboratory Manual, W. et al. H. Freeman and Company, New York (1992), Molecular Cloning: A Laboratory Manual 3rd ed. , Cold Spring Harbor Laboratory Press (2001), Bio / Technology,6, 47 (1988) and the like, a transformant expressing GPR88 can be obtained.
  That is, a recombinant gene transfer vector and a baculovirus are co-introduced into insect cells to obtain a recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected into insect cells to express the polypeptide. Can do.
  Examples of the gene transfer vector used in the method include pVL1392, pVL1393, pBlueBacIII (all manufactured by Invitrogen) and the like.
  As the baculovirus, for example, Autographa californica nucleopolyhydrovirus virus, which is a virus that infects serpentine insects, can be used.
  As an insect cell,Spodoptera frugiperdaOvary cells,TrichoplusianiOvary cells, cultured cells derived from silkworm ovary, and the like can be used.Spodoptera frugiperdaAs ovary cells, Sf9, Sf21 (Baculovirus Expression Vectors A Laboratory Manual), etc.Trichoplusia niAs ovary cells of the above, High5 (manufactured by Invitrogen), etc., as cultured cells derived from silkworm ovaryBombyxmori  N4 etc. can be mentioned.
  Examples of the method for co-introducing the recombinant gene introduction vector and the baculovirus into insect cells for preparing a recombinant virus include, for example, the calcium phosphate method (Japanese Patent Laid-Open No. 227075), the lipofection method [Proc. Natl. Acad. Sci. USA,847413 (1987)]. In addition, a method similar to the method for introducing DNA into animal cells, for example, electroporation [Cytotechnology,3133 (1990)], calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), lipofection method [Proc. Natl. Acad. Sci. USA,84, 7413 (1987)] and the like can be used to introduce DNA into insect cells.
  When plant cells are used as host cells, known methods [tissue culture, 20 (1994), tissue culture, 21 (1995), Trends in Biotechnology,15, 45 (1997)], a transformant expressing GPR88 can be obtained.
  Any promoter can be used as long as it can be expressed in plant cells. Examples thereof include cauliflower mosaic virus 35S promoter and rice actin 1 promoter. Further, by inserting intron 1 of a corn alcohol dehydrogenase gene between a promoter and a gene to be expressed, gene expression efficiency can be increased.
  Examples of host cells used at this time include plant cells such as potato, tobacco, corn, rice, rape, soybean, tomato, wheat, barley, rye, alfalfa, and flax.
  As a method for introducing the recombinant vector used at this time, any method can be used as long as it is a method for introducing DNA into plant cells. For example, Agrobacterium (Agrobacterium) (JP 59-140885, JP 60-70080, WO 94/00977), Electroporation [Cytotechnology,3133 (1990), Japanese Patent Laid-Open No. Sho 60-251887], a method using a particle gun (gene gun) (Japanese Patent No. 2606856, Japanese Patent No. 2517813), and the like.
  Examples of cellular responses include arachidonic acid release, acetylcholine release, intracellular Ca²⁺Release, intracellular cAMP production, intracellular cAMP reduction, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein (eg, CREB, STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, STAT6, MAPK, ATF-2, c-Jun, c-fos, Iκ-Bα, Iκ-Bβ, Smad1, Smad2, Smad3, Smad5, Smad8, etc.) phosphorylation, c-fos activation, pH change, cell proliferation activity, or The expression level of the reporter gene and marker gene can be increased, and by examining these, the cell response can be measured [J. Biol. Chem. ,271, 1857 (1996), Science,268, 98 (1995), J.A. Pharmacol. Exp. Ther. ,275, 1274 (1995), J.A. Biol. Chem. ,272, 1822 (1997), J. Am. Receive. Signal Transduct. Res. ,17, 57 (1997), Endocrinology138, 1400 (1997), Endocrinology138, 1471 (1997), Nat. Biotechnol. ,16, 1334 (1998), Biochem. Biophys. Res. Commun. ,251, 471 (1998), Br. J. et al. Pharmacol. ,125, 1387 (1998), Trends Biotechnol. ,15487 (1997), Anal. Biochem. ,252, 115 (1997), Nature,358, 325 (1992), Nature,393272 (1998), Cell,92573 (1998); Biol. Chem. ,272, 27497 (1997), Trends Pharmacol. Sci. ,18, 430 (1997), Trends Pharmacol. Sci. ,20370 (1999), WO 98/46995].
  When monitoring a cell response using a reporter system, for example, an artificial promoter comprising a binding sequence of a suitable transcription factor and a basic promoter that induces expression by activation of GPR88 in cells expressing GPR88. By introducing DNA linked with an appropriate reporter gene downstream of the sequence, activation of GPR88 can be measured by expression of the reporter gene. As a transcription factor binding sequence, G_sCRE (cAMP responsible element) that activates transcription with the activation of GPCR coupled to G, G_qTRE (TPA reactive element), which activates transcription in association with activation of GPCR coupled to G,_iAn SRE (serum response element) that activates transcription in association with activation of a GPCR that is conjugated to the ribosome can be used. Gα_sOf the C-terminal 5 amino acids of Gα_iAnd Gα_qChimera Gα each substituted with 5 amino acids of C-terminal_sIs expressed in GPR88-expressing cells, the type of G protein to which GPR88 is conjugated is unknown, and any of Gs, Gi, and Gq can be used to generate a reporter gene from an artificial promoter containing CRE upon activation of GPR88. Expression is induced.
  Any reporter gene can be used as the reporter gene. Aequorin gene and green fluorescein protein gene can be used.
  Examples of central diseases that can be prevented and / or treated according to the present invention include diseases that are known to be associated with GPR88 expression sites in the brain. Specifically, Parkinson's disease, depression, anxiety, schizophrenia, and the like. Diseases, Alzheimer's disease, Huntington's chorea, spinocerebellar degeneration, stroke, involuntary movement, attention deficit disorder, extrapyramidal symptoms, drug addiction, alcohol addiction, jet lag, etc.
  In the present invention, the GPR88 function inhibitor only needs to contain, as one of its properties, a substance having a GPR88 function inhibitory action as an active ingredient, and the structure of the substance is not particularly limited. Examples of substances that have GPR88 function-suppressing action reduce GPR88 expression, such as GPR88 antagonistic compounds (eg, inverse agonists, antagonists), antibodies (eg, neutralizing antibodies), antisense oligonucleotides, and the like. And substances that inhibit signal transduction, such as substances that inhibit the coupling between GPR88 and G protein.
  The antisense oligonucleotide for the GPR88 gene used in the present invention is complementary to a sequence of 10 bases or more, preferably 10 to 40 bases, more preferably 15 to 30 bases of the base sequence of SEQ ID NO: 1, 3 or 5. Any antisense oligonucleotide may be used as long as it has a specific or substantially complementary nucleotide sequence and has an action capable of specifically suppressing the expression of the GPR88 gene. In particular, a base sequence complementary to or substantially complementary to a sequence of 10 bases or more, preferably 10 to 40 bases, more preferably 15 to 30 bases of the base sequence of SEQ ID NO: 1, which is the sequence of human GPR88 cDNA Antisense oligonucleotides having are preferred. In order to have an action capable of suppressing the expression of the GPR88 gene, a sequence complementary to a region containing a translation start point (corresponding to the 438th base of the base sequence shown in SEQ ID NO: 1) or a region near the translation start point An oligonucleotide having a base sequence containing is preferred. The substantially complementary base sequence is a DNA having a base sequence of SEQ ID NO: 1 having a homology of 90% or more with a continuous 10-40 base sequence of the base sequence of SEQ ID NO: 1, 3 or 5 And a base sequence that hybridizes under stringent conditions. In order to prevent degradation by a hydrolase such as nuclease, the phosphate residue of each nucleotide constituting the antisense DNA is substituted with a chemically modified phosphate residue such as phosphorothioate, methylphosphonate, phosphorodithionate, etc. Also good. These antisense oligonucleotides can be produced using a known DNA synthesizer, a method described in WO93 / 20095, WO02 / 10185, or the like.
  The antibody against GPR88 used in the present invention can be prepared as follows.
  Polyclonal antibodies can be produced by using GPR88 polypeptide or partial peptide as an antigen and administering it to animals. Examples of animals to which the antigen is administered include rabbits, goats, rats, mice, hamsters and the like. The dose of the antigen is preferably 50 to 100 μg per animal. When a peptide is used as an antigen, a peptide obtained by covalently binding the peptide to a carrier protein such as keyhole / limpet / hemocyanin or bovine thyroglobulin can be used. The GPR88 polypeptide can be obtained from the above-described cells expressing GPR88 by an ordinary protein purification method. Further, cells expressing GPR88 or a membrane fraction thereof can be used as an antigen. A partial peptide of GPR88 can be synthesized by a peptide synthesizer.
  The antigen is administered 3 to 10 times every 1 to 2 weeks after the first administration. On the 3rd to 7th day after each administration, blood was collected from the fundus venous plexus, and it was confirmed that the serum reacted with the antigen used for immunization. A Laboratory Manual, Cold Spring Harbor Laboratory (1988)].
  Polyclonal antibodies can be obtained by obtaining serum from the above animals whose serum showed a sufficient antibody titer against the antigen used for immunization, and separating and purifying the serum.
  Separation and purification methods include centrifugation, salting out with 40-50% saturated ammonium sulfate, precipitation of caprylic acid (Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, (1988)), or DEAE-Sepharose column, anion exchange. Examples include a method of treating a column, a protein A or G-column, a chromatography using a gel filtration column, etc. alone or in combination.
  Furthermore, a monoclonal antibody against GPR88 can be prepared as follows.
  The spleen is removed 3 to 7 days after the final administration of the antigenic substance to the animal whose serum showed a sufficient antibody titer against the antigen used for immunization. The spleen is shredded in MEM medium (Nissui Pharmaceutical Co., Ltd.), loosened with tweezers, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded. The spleen cells of the obtained precipitate fraction were treated with trisphosphate-ammonium chloride buffer (pH 7.65) for 1 to 2 minutes to remove erythrocytes, and then washed 3 times with MEM medium. Used as production cells.
  As myeloma cells, cell lines obtained from mice or rats are used. For example, 8-azaguanine resistant mice (BALB / c-derived) myeloma cell line P3-X63Ag8-U1 [Curr. Topics. Microbiol. Immunol. ,81, 1 (1978), Eur. J. et al. Immunol. ,6, 511 (1976)], SP2 / 0-Ag14 [Nature,276, 269 (1978)], P3-X63-Ag8653 [J. Immunol. ,123, 1548 (1979)], P3-X63-Ag8 [Nature,256, 495 (1975)]. These cell lines consist of 8-azaguanine medium [RPMI 1640 medium supplemented with 1.5 mmol / L glutamine, 50 μmol / L2-mercaptoethanol, 10 μg / mL gentamicin and 10% fetal bovine serum (hereinafter referred to as normal medium). In a medium supplemented with 15 μg / mL 8-azaguanine], the cells were cultured in a normal medium 3 to 4 days before cell fusion, and the cells were cultured at 2 × 10 2 for fusion.⁷Use more than one.
  The above antibody-producing cells and myeloma cells are thoroughly washed with MEM medium or PBS (1.83 g / L disodium phosphate, 0.21 g / L monopotassium phosphate, 7.65 g / L NaCl, pH 7.2). The cells are mixed so that the number of cells is antibody-producing cells: myeloma cells = 5 to 10: 1, centrifuged at 1,200 rpm for 5 minutes, and then the supernatant is discarded. Thoroughly loosen the cell group of the obtained precipitate fraction and stir the cell group at 37 ° C. with stirring.⁸0.2-1 mL of a solution prepared by mixing 2 g of polyethylene glycol-1000, 2 mL of MEM medium and 0.7 mL of dimethyl sulfoxide is added per antibody-producing cell, and 1-2 mL of MEM medium is added several times every 1-2 minutes. . After the addition, MEM medium is added to prepare a total volume of 50 mL.
  The preparation is centrifuged at 900 rpm for 5 minutes, and the supernatant is discarded. The cells of the obtained precipitate fraction are loosened gently, and then sucked with a pipette and blown out gently to give a HAT medium [0.1 mmol / L hypoxanthine, 15 μmol / L thymidine and 0.4 μmol / L amino in normal medium. Medium supplemented with pterin] Suspend in 100 mL. The suspension was dispensed into a 96-well culture plate at 100 μL / well, and 5% CO 2 was added.₂Incubate in an incubator at 37 ° C for 7-14 days. After culturing, a hybridoma that specifically reacts with the antigen used for immunization is obtained by removing a part of the culture supernatant and performing an enzyme immunoassay described in Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, (1988). select.
  Specific examples of the enzyme immunoassay include the following methods. At the time of immunization, the GPR88 polypeptide used as an antigen or a partial peptide of GPR88 is coated on a plate, and the hybridoma culture supernatant is reacted as a first antibody. In addition, an anti-immunoglobulin antibody labeled with an enzyme such as peroxidase is reacted as a second antibody (an antibody against the immunoglobulin of the animal to which the antigen was administered during immunization, for example, an anti-mouse immunoglobulin antibody is used when a mouse is immunized). After that, a reagent that develops color with an enzyme is added to carry out the reaction, and the culture supernatant specifically reacted is selected as a hybridoma that produces a monoclonal antibody against GPR88.
  Using the selected hybridoma, cloning was repeated twice by the limiting dilution method (first time was HT medium (medium obtained by removing aminopterin from HAT medium), second time normal medium was used), stable and strong Those having an antibody titer are selected as a hybridoma strain producing an antibody against GPR88.
  Production of antibodies against GPR88 obtained from hybridoma strain 5 to 8-10 weeks old mice or nude mice fed intraperitoneally with pristane (2,6,10,14-tetramethylpentadecane) 0.5 mL 20x10⁶Cells / animals are injected intraperitoneally. The hybridoma becomes ascites tumor in 10 to 21 days. Ascites is collected from the mouse with ascites tumor, and centrifuged at 3,000 rpm for 5 minutes to remove solids. From the obtained supernatant, the monoclonal antibody can be purified and obtained by the same method as used in the production of the polyclonal antibody.
  Using the polyclonal antibody or the monoclonal antibody obtained as described above as a test substance, the screening method for the GPR88 constitutive activity inhibitor is performed, and an antibody that suppresses the GPR88 constitutive activity is selected from the antibody. it can.
  As described in Test Examples 2 and 3 to be described later, improvement of the dyskinetic state induced by dopamine receptor antagonist by administration of antisense oligonucleotide to GPR88 gene, central dysfunction state induced by reserpine administration Therefore, it is considered that a compound or a salt thereof that suppresses the function of GPR88 or an antibody against GPR88 that suppresses the function of GPR88 also exhibits the same action. Therefore, a medicament containing an antisense oligonucleotide for the GPR88 gene, a compound or a salt thereof that suppresses the function of GPR88, or an antibody against GPR88 that suppresses the function of GPR88 is used for diseases caused by abnormalities in the dopamine nervous system and catecholamine nervous system. It can be used as a prophylactic and / or therapeutic agent for diseases based on abnormalities, for example, diseases associated with movement disorders such as Parkinson's disease, involuntary movement, extrapyramidal dysfunction, Huntington's chorea, spinocerebellar degeneration, and stroke. In addition, it can be expected to improve mental symptoms such as depression, schizophrenia, anxiety, Alzheimer's disease, attention deficit disorder, drug addiction and alcoholism.
  The medicament containing the antisense oligonucleotide of the present invention has a sequence complementary to a part of GPR88 mRNA, preferably a region including the translation initiation site or a region in the vicinity thereof, and suppresses the expression of the GPR88 gene. Antisense oligonucleotides that can be produced have low toxicity and can suppress the function of GPR88 in vivo. For example, Parkinson's disease, involuntary movement, extrapyramidal disease, Huntington's chorea, spinocerebellar degeneration, stroke It can be used as a preventive and / or therapeutic agent for diseases associated with movement disorders such as. Moreover, it can be used as a preventive and / or therapeutic agent for mental symptoms such as depression, schizophrenia, anxiety, Alzheimer's disease, attention deficit disorder, drug addiction and alcoholism. When the antisense nucleotide is used as the prophylactic and / or therapeutic agent, it can be formulated and administered according to a method known per se. For example, when the antisense nucleotide is used, the antisense nucleotide is inserted alone or into an appropriate vector such as a retrovirus vector, adenovirus vector, adenovirus associated virus vector, etc., and then human or mammal ( Rats, mice, rabbits, sheep, pigs, monkeys, dogs, cows, cats, etc.) orally or parenterally. The antisense nucleotide can be prepared as it is or with a physiologically recognized carrier such as an adjuvant for promoting ingestion and administered by a gene gun or a catheter. It can also be administered by intraventricular, or nucleus accumbens, cerebellar olive nucleus or intrastriatal injection.
  Examples of the compound having GPR88 function-inhibiting action include compounds represented by formulas (I) and (IA). Hereinafter, the compounds represented by formulas (I) and (IA) are referred to as compounds (I) and (IA), respectively. The same applies to the compounds of other formula numbers.
  In the definition of each group of compound (I) and compound (IA), the following examples are given.
(I) Lower alkyl part of lower alkyl, lower alkoxy and lower alkylthio includes, for example, linear or branched alkyl having 1 to 10 carbon atoms, specifically, methyl, ethyl, propyl, isopropyl, butyl, Examples include isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, 1,5-dimethylhexyl, isooctyl, nonyl, decyl and the like.
(Ii) Examples of cycloalkyl and the cycloalkyl part of cycloalkylalkyl include cycloalkyl having 3 to 8 carbon atoms, and specific examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like. It is done.
(Iii) Examples of lower alkenyl include linear, branched or cyclic alkenyl having 2 to 8 carbon atoms, specifically vinyl, allyl, 1-propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, cyclo Hexenyl, 2,6-octadienyl and the like can be mentioned.
(Iv) Examples of lower alkynyl include linear or branched alkynyl having 2 to 8 carbon atoms, and specific examples include ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl and the like.
(V) Halogen represents each atom of fluorine, chlorine, bromine and iodine.
(Vi) The aryl moiety of aryl, aryloxy and aryloxyalkyl includes, for example, monocyclic, bicyclic or tricyclic aryl having 6 to 14 carbon atoms, specifically phenyl, naphthyl and indenyl. , Anthranyl and the like.
(Vii) the alkylene part of aralkyl, heterocyclic alkyl, cycloalkylalkyl, hydroxy lower alkyl, carboxy lower alkyl and aryloxyalkyl and the two alkylene parts of aralkyloxyalkyl are the groups mentioned in the definition of lower alkyl (i). It is synonymous with one hydrogen removed from The two alkylene moieties of aralkyloxyalkyl may be the same or different.
(Viii) As the aryl moiety of aralkyl and aralkyloxyalkyl, in addition to the groups mentioned in the definition of aryl (vi), for example, the polycyclic condensation in which the group mentioned in the definition of aryl (vi) is condensed with cycloalkyl Examples thereof include a ring group, specifically, indanyl, 1,2,3,4-tetrahydronaphthyl, 6,7,8,9-tetrahydro-5H-benzocycloheptyl and the like.
(Ix) As the heterocyclic group part of the heterocyclic group and the heterocyclic alkyl, for example, a 5-membered or 6-membered monocyclic heterocyclic group containing at least one atom selected from a nitrogen atom, an oxygen atom and a sulfur atom, And a condensed heterocyclic group containing at least one atom selected from a nitrogen atom, an oxygen atom and a sulfur atom, which is a bicyclic or tricyclic condensed 3- to 8-membered ring. Is pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, benzoimidazolyl, 2-oxobenzoimidazolyl, benzotriazolyl, benzofuryl, benzothienyl, purinyl, benzoxazolyl, benzothiazolyl, benzodioxolyl, indazolyl, indolyl, isoindolyl, quinolyl, isoquinolyl, Phthalazinyl, naphthylridinyl, quinoxalinyl, quinazolinyl Cinnolinyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, thienyl, furyl, pyrrolidinyl, 2,5-dioxopyrrolidinyl, thiazolidinyl, oxazolidinyl, piperidyl, piperidino, homopiperazinyl, homopiperazinyl, homopiperazinyl , Homopiperidino, morpholinyl, morpholino, thiomorpholinyl, thiomorpholino, pyranyl, tetrahydropyridyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydroquinolyl, tetrahydroisoquinolyl, octahydroquinolyl, indolinyl, 2,3-dihydrobenzo [1,4] And dioxinyl.
(X) Examples of the heterocyclic group formed together with the adjacent nitrogen atom include a 5-membered or 6-membered monocyclic heterocyclic group containing at least one nitrogen atom (the monocyclic heterocyclic group). May contain another nitrogen atom, oxygen atom or sulfur atom), a condensed bicyclic or tricyclic condensed ring having 3 to 8 members and containing at least one nitrogen atom (The condensed heterocyclic group may contain other nitrogen atom, oxygen atom or sulfur atom) and the like, specifically, pyridyl, tetrahydropyridyl, indolinyl, isoindolinyl, pyrrolidinyl, thiazolidinyl, oxazolidinyl Piperidino, homopiperidino, piperazinyl, homopiperazinyl, morpholino, thiomorpholino, perhydroazepinyl, perhydroazosinyl, tetrahydroquinolyl, teto Tetrahydroisoquinoline isoquinolylmethyl, octa tetrahydroquinolyl, decahydroisoquinolinyl, benzimidazolyl, indazolyl, indolyl, isoindolyl, purinyl, dihydroindolyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, imidazolyl, and the like.
(Xi) Halogenated lower alkyl means lower alkyl substituted with halogen (wherein the halogen is as defined above for halogen (v)) (wherein the lower alkyl is as defined above for lower alkyl (i)). The halogens as substituents are the same or different and have, for example, 1 to 3 substituents.
(Xii) Substituents in substituted lower alkyl, substituted lower alkoxy and substituted lower alkylthio are the same or different, for example, from 1 to 3 lower alkanoyl, lower alkoxy, aryloxy, substituted aryloxy, aralkyloxy, substituted Aralkyloxy, lower alkanoyloxy, lower alkoxycarbonyl, halogen, cyano, nitro, hydroxy, carboxy, amino, lower alkylthio, mono- or di-lower alkylamino, lower alkylsulfonyl, lower alkylsulfinyl, lower alkoxycarbonylamino, lower alkanoylamino, (Lower alkanoyl) (lower alkyl) amino, mono- or di-lower alkylaminocarbonyl, mono- or di-lower alkylaminocarbonyloxy and the like.
  The substituents (xiii) in the substituted aryloxy and substituted aralkyloxy shown here are the same or different, for example, lower alkyl, lower alkoxy, lower alkoxycarbonyl, aryl, heterocyclic group, oxo having 1 to 3 substituents. , Halogen, cyano, nitro, hydroxy, carboxy, amino and the like. Lower alkyl part of lower alkyl, lower alkoxy and lower alkoxycarbonyl, aryl and heterocyclic group are the lower alkyl (i) and aryl (vi), respectively. , Is the same as the heterocyclic group (ix), and the halogen is the same as the halogen (v).
  In addition, the aryl moiety of aryloxy and aralkyloxy shown here, halogen and lower alkanoyl, lower alkoxy, lower alkanoyloxy, lower alkoxycarbonyl, lower alkylthio, mono- or di-lower alkylamino, lower alkylsulfonyl, lower alkylsulfinyl, lower The lower alkyl part of alkoxycarbonylamino, lower alkanoylamino, (lower alkanoyl) (lower alkyl) amino, mono- or di-lower alkylaminocarbonyl and mono- or di-lower alkylaminocarbonyloxy is the aryl (vi), halogen (v ) And lower alkyl (i), and the alkylene part of aralkyloxy has the same meaning as the alkylene part (vii) of the aralkyl.
  The two lower alkyl moieties in the di-lower alkylamino, (lower alkanoyl) (lower alkyl) amino, di-lower alkylaminocarbonyl and di-lower alkylaminocarbonyloxy shown here may be the same or different.
(Xiv) substituted aryl, substituted aralkyl, substituted cycloalkyl, substituted cycloalkylalkyl, substituted lower alkenyl, substituted lower alkynyl, substituted aryloxy, substituted heterocyclic group, substituted heterocyclic alkyl, substituted aryloxyalkyl, substituted aralkyloxyalkyl, Examples of the substituent in the substituted heterocyclic group formed together with the substituted piperazinyl and the adjacent nitrogen atom include, for example, lower alkyl, substituted lower group, in addition to the groups mentioned in the definition of the substituent (xii) in the substituted lower alkyl. Alkyl, lower alkenyl, lower alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, arylcarbonyl, substituted arylcarbonyl, heterocyclic group, substituted heterocyclic group, heterocyclic alkyl, substituted heterocyclic alkyl Ethylenedioxy, and the like.
  The substituent in the substituted lower alkyl, substituted cycloalkyl, substituted aryl, substituted aralkyl, substituted arylcarbonyl, substituted heterocyclic group and substituted heterocyclic alkyl shown here is the substituent in the substituted aryloxy and substituted aralkyloxy (xiii) It is synonymous with.
  In addition, the lower alkyl, cycloalkyl, lower alkenyl, lower alkynyl, aryl moiety of aryl and arylcarbonyl, the heterocyclic group moiety of the heterocyclic group and heterocyclic alkyl, the alkylene moiety of aralkyl and heterocyclic alkyl, and the aralkyl group shown here. The aryl moiety is the lower alkyl (i), cycloalkyl (ii), lower alkenyl (iii), lower alkynyl (iv), aryl (vi), heterocyclic group (ix), aralkyl alkylene part (vii) and It is synonymous with the aryl part (viii) of aralkyl.
(Xv) Substituents of substituted hydroxymethyl are the same or different and include, for example, lower alkyl having 1 or 2 substituents.
  The lower alkyl shown here has the same meaning as the lower alkyl (i).
  The pharmacologically acceptable salts of compound (I) and compound (IA) are preferably non-toxic and water-soluble salts, such as hydrochloride, hydrobromide, nitrate, sulfate, phosphate. Inorganic salts such as benzene sulfonate, benzoate, citrate, fumarate, gluconate, lactate, maleate, malate, oxalate, methanesulfonate, tartrate, etc. Acid addition salts such as organic acid salts, alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, metal salts such as aluminum salt and zinc salt, ammonium, tetramethylammonium and the like Ammonium salt, morpholine addition salt, organic amine addition salt such as piperidine addition salt, glycine addition salt, phenylalanine addition salt, lysine addition salt, aspartic acid addition salt, glutamine Amino acid addition salts such as phosphate addition salts.
  Next, the manufacturing method of compound (I) is demonstrated.
  In the production method shown below, when the defined group changes under the reaction conditions or is inappropriate for carrying out the method, a method commonly used in organic synthetic chemistry, for example, functional group protection and deprotection. [For example, Protective Groups in Organic Synthesis, the third edition, by TW Greene, Peter GM Wuts, John. Manufacturing can be easily carried out by attaching to the means of John Wiley & Sons Inc. (1999)]. Further, the order of reaction steps such as introduction of substituents can be changed as necessary.
  In compound (I), Y is a hydrogen atom, X is 1,2,3,4-tetrazol-5-yl, R²-NR³R⁴(Wherein R³And R⁴Can be obtained by, for example, Production Method 1 shown below.
Production method 1:

(Where A, R¹, R³And R⁴Are as defined above, and m represents 1 or 2.
[Step 1]
  2-Ethoxymethylene-2-cyanoacetic acid ethyl [compound (A)] is added in a solvent in the presence of an alkali in an amount of 0.5 equivalents to a large excess, preferably 0.5 equivalents to 2 equivalents of methylisothiourea sulfate [compound ( Compound (C) can be obtained by reacting with (B)].
  As the solvent, for example, methanol, ethanol, 2-propanol, tetrahydrofuran, dioxane and the like can be used alone or as a mixture thereof, and ethanol is particularly preferable.
  Examples of the alkali include alkaline aqueous solutions such as lithium hydroxide, potassium hydroxide, sodium hydroxide, magnesium hydroxide and calcium hydroxide, aqueous solutions of potassium tert-butoxide, tetrahydrofuran solutions or 2-methyl-2-propanol solutions, sodium methoxy An aqueous solution of methanol or a methanol solution can be used, and among them, an aqueous sodium hydroxide solution is preferable.
  The reaction is carried out at a temperature between 0 ° C. and 50 ° C., preferably at a temperature between 0 ° C. and 15 ° C., and is usually completed in 1 hour to 48 hours.
[Step 2]
  Compound (C) obtained in Step 1 is reacted with 1 equivalent to a large excess of chlorinating agent in the presence or absence of 1 equivalent to a large excess of base in a solvent inert to the reaction or without solvent. Compound (D) can be obtained by reacting.
  As the chlorinating agent, for example, phosphorus oxychloride is used.
  The solvent inert to the reaction may be any solvent as long as it is inert to the reaction and is not particularly limited. For example, 1,2-dichloroethane, tetrahydrofuran, dioxane, chloroform, benzene, toluene, xylene, ethyl acetate and the like are used alone. Or a mixture of them. Examples of the base include triethylamine, pyridine, N, N-dimethylaniline and the like.
  The reaction is carried out at a temperature between 0 ° C. and the boiling point of the solvent, preferably at a temperature between 50 ° C. and the boiling point of the solvent, and is usually completed in 1 hour to 48 hours.
  In addition to the above method, the compound (D) obtained in this step may be used in Journal of Heterocyclic Chemistry (J. Heterocycl. Chem.), 8 (3), 445 (1971), WO99. / 61444 etc., or a method according to them.
[Step 3]
  The compound (F) obtained by reacting the compound (D) obtained in step 2 with 1 equivalent to a large excess, preferably 1 equivalent to 3 equivalents, of the compound (E) in a solvent-free or inert solvent is used. ) Can be obtained.
  The solvent inert to the reaction may be any solvent as long as it is inert to the reaction, and is not particularly limited. For example, tetrahydrofuran, dioxane, diethyl ether, diisopropyl ether, benzene, toluene, xylene, ethyl acetate, acetonitrile, dichloromethane, Chloroform, 1,2-dichloroethane, N, N-dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide and the like can be used alone or in admixture thereof, among which tetrahydrofuran, chloroform or a mixed solvent thereof is used. preferable.
  The reaction is carried out at a temperature between 0 ° C. and 100 ° C., preferably at a temperature between 0 ° C. and 50 ° C., and is usually completed in 10 minutes to 48 hours.
  Further, this reaction may be carried out by adding 1 equivalent to a large excess amount, preferably 1 equivalent to 10 equivalents of a base, if necessary.
  Examples of the base include organic bases such as triethylamine, N, N-diisopropylethylamine, 1,8-diazabicyclo [5.4.0] -undec-7-ene (DBU), N, N-dimethylaniline, pyridine and quinoline. Inorganic bases such as potassium carbonate, sodium carbonate, lithium carbonate, sodium hydrogen carbonate, potassium hydroxide, sodium hydroxide, lithium hydroxide, potassium tert-butoxide, Amberlyst A-21 (manufactured by Rohm and Haas), AG1- When a basic anion exchange resin such as X8 (manufactured by Bio-Rad), a base supported on a solid phase such as polyvinyl pyridine or morpholinomethyl polystyrene can be used, and the reaction is performed by a parallel synthesis method (combinatorial chemistry) Among these, morpholinomethyl polystyrene is preferred. Arbitrariness.
  Compound (E) can be obtained commercially or by Comprehensive Organic Transformations, 2nd edition (by Comprehensive Organic Transformations, the second edition), R. C. Larock, John Wiley and Sons. Incorporated (John Wiley & Sons Inc.) (1999) etc. can obtain.
[Step 4]
  Compound (G) can be obtained by treating compound (F) obtained in step 3 with an oxidant in an amount of 1 equivalent to a large excess, preferably 1 equivalent to 5 equivalents, in a solvent inert to the reaction. it can.
  The solvent inert to the reaction may be any solvent as long as it is inert to the reaction, and is not particularly limited. For example, dichloromethane, chloroform, 1,2-dichloroethane, tetrahydrofuran, dioxane, diethyl ether, diisopropyl ether, methanol, ethanol , Isopropyl alcohol, benzene, toluene, xylene, ethyl acetate, acetonitrile, water and the like can be used alone or in admixture thereof, with dichloromethane being preferred.
  As the oxidizing agent, for example, methacrofiltered benzoic acid, benzoyl peroxide, peracetic acid, hydrogen peroxide, sodium periodate, etc. can be used.
  The reaction is carried out at a temperature between 0 ° C. and 100 ° C., preferably at a temperature between 0 ° C. and 50 ° C., and is usually completed in 10 minutes to 24 hours.
  In addition, in the compound (G), the compound in which m is 1 and the compound in which m is 2 are obtained, for example, by adjusting conditions such as the number of equivalents of the oxidizing agent and the temperature of the reaction. Sometimes. When mixed, the ratio is not particularly limited, and in any case, the ratio can be used as it is in the next step.
[Step 5]
  The compound (G) obtained in step 4 is reacted with 1 equivalent to a large excess, preferably 1 equivalent to 5 equivalents, of compound (H) in a solventless or inert solvent. J) can be obtained.
  The solvent inert to the reaction is not particularly limited as long as it is inert to the reaction. For example, dichloromethane, chloroform, 1,2-dichloroethane, N, N-dimethylformamide, dimethylacetamide, N-methyl Pyrrolidone, dimethyl sulfoxide, tetrahydrofuran, dioxane, diethyl ether, diisopropyl ether, benzene, toluene, xylene, ethyl acetate, acetonitrile and the like can be used alone or in combination, and tetrahydrofuran is preferred.
  The reaction is carried out at a temperature between 0 ° C. and 100 ° C., preferably at a temperature between 20 ° C. and 60 ° C., and is usually completed in 1 hour to 72 hours.
  Compound (H) can be obtained commercially or by Comprehensive Organic Transformations, 2nd edition (Comprehensive Organic Transformations, the second edition), by John Lay, RC Wyland and Sons. Incorporated (John Wiley & Sons Inc.) (1999) etc. can obtain.
[Step 6]
  Compound (Ii) can be obtained by reacting compound (J) obtained in step 5 with 1 to 10 equivalents of sodium azide or ammonium azide in a solvent inert to the reaction.
  The solvent inert to the reaction is not particularly limited as long as it is inert to the reaction. Examples thereof include chloroform, 1,2-dichloroethane, N, N-dimethylformamide, dimethylacetamide, N-methylpyrrolidone, Dimethyl sulfoxide, tetrahydrofuran, dioxane, diisopropyl ether, benzene, toluene, xylene, ethyl acetate, acetonitrile and the like can be used alone or as a mixture thereof. Among them, N, N-dimethylformamide is preferable. For the purpose of accelerating the reaction, ammonium chloride, ethylammonium chloride and the like can be added to the reaction system in an amount of 1 equivalent to a large excess, preferably 1 equivalent to 10 equivalents.
  The reaction is carried out at a temperature between 0 ° C. and 180 ° C., preferably at a temperature between 50 ° C. and 120 ° C., and is usually completed in 1 hour to 72 hours.
  Of the compounds (I), X is —C (═O) R.⁵And R²-NR³R⁴(Wherein R³And R⁴Can be obtained by, for example, production method 2 shown below.
Production method 2:

(Where A, R¹, R³, R⁴, R⁵, Y and m are as defined above. )
[Step 7]
  Compound (L) can be obtained by reacting compound (K) with 1 equivalent to a large excess, preferably 1 equivalent to 3 equivalents of compound (E) in a solvent-free or inert solvent. it can.
  The solvent inert to the reaction may be any solvent as long as it is inert to the reaction and is not particularly limited. For example, tetrahydrofuran, dioxane, 1,2-dimethoxyethane, diisopropyl ether, benzene, toluene, xylene, ethyl acetate, Acetonitrile, dichloromethane, chloroform, 1,2-dichloroethane, N, N-dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide and the like can be used alone or in combination, among which tetrahydrofuran, chloroform, 1 , 2-dimethoxyethane or a mixed solvent thereof is preferred.
  The reaction is carried out at a temperature between 0 ° C. and 150 ° C., preferably at a temperature between 50 ° C. and 120 ° C., and is usually completed in 1 hour to 96 hours.
  Further, this reaction may be carried out by adding 1 equivalent to a large excess amount, preferably 1 equivalent to 10 equivalents of a base, if necessary.
  Examples of the base include organic bases such as triethylamine, N, N-diisopropylethylamine, DBU, N, N-dimethylaniline, pyridine, quinoline, potassium carbonate, sodium carbonate, lithium carbonate, sodium hydrogen carbonate, potassium hydroxide, hydroxide Inorganic bases such as sodium, lithium hydroxide and potassium tert-butoxide, basic anion exchange resins such as Amberlyst A-21 (Rohm and Haas), AG1-X8 (BioRad), polyvinylpyridine, morpholinomethyl A base supported on a solid phase such as polystyrene can be used, among which triethylamine, N, N-diisopropylethylamine or DBU is preferable.
  Compound (E) can be obtained commercially or by Comprehensive Organic Transformations, 2nd edition (by Comprehensive Organic Transformations, the second edition), R. C. Larock, John Wiley and Sons. Incorporated (John Wiley & Sons Inc.) (1999) etc. can obtain.
  Compound (K) can be obtained as a commercially available product or according to the method described in, for example, WO 00/76980, WO 00/27826, WO 01/83460, WO 97/09315, JP 2001-89452 A, WO 99/31073, and the like. .
[Step 8]
  Compound (M) can be obtained by treating compound (L) obtained in step 7 with an oxidizing agent in an amount of 1 equivalent to a large excess, preferably 1 equivalent to 5 equivalents, in a solvent inert to the reaction. it can.
  The solvent inert to the reaction may be any solvent as long as it is inert to the reaction, and is not particularly limited. For example, dichloromethane, chloroform, 1,2-dichloroethane, tetrahydrofuran, dioxane, diethyl ether, diisopropyl ether, methanol, ethanol , Isopropyl alcohol, benzene, toluene, xylene, ethyl acetate, acetonitrile, water and the like can be used alone or in admixture thereof, with dichloromethane being preferred.
  As the oxidizing agent, for example, methacrofiltered benzoic acid, benzoyl peroxide, peracetic acid, hydrogen peroxide, sodium periodate, etc. can be used.
  The reaction is carried out at a temperature between 0 ° C. and 100 ° C., preferably at a temperature between 0 ° C. and 50 ° C., and is usually completed in 10 minutes to 24 hours.
  In addition, in the compound (M), the compound in which m is 1 and the compound in which m is 2 are obtained, for example, by adjusting conditions such as the number of equivalents of the oxidizing agent and the temperature of the reaction. Sometimes. When mixed, the ratio is not particularly limited, and in any case, the ratio can be used as it is in the next step.
[Step 9]
  Compound (I-ii) can be obtained by reacting compound (M) obtained in step 8 with 1 to 5 equivalents of compound (H) in a solvent inert to the reaction.
  The solvent inert to the reaction is not particularly limited as long as it is inert to the reaction. For example, dichloromethane, chloroform, 1,2-dichloroethane, N, N-dimethylformamide, dimethylacetamide, N-methyl Pyrrolidone, dimethyl sulfoxide, tetrahydrofuran, dioxane, 1,2-dimethoxyethane, diisopropyl ether, benzene, toluene, xylene, ethyl acetate, acetonitrile, and the like can be used alone or in combination, among which tetrahydrofuran or 1,2 -Dimethoxyethane is preferred.
  The reaction is carried out at a temperature between 20 ° C. and 150 ° C., preferably between 50 ° C. and 120 ° C., and is usually completed in 1 hour to 7 days.
  Compound (H) can be obtained commercially or by Comprehensive Organic Transformations, 2nd edition (Comprehensive Organic Transformations, the second edition), by John Lay, RC Wyland and Sons. Incorporated (John Wiley & Sons Inc.) (1999) etc. can obtain.
  Of the compounds (I-ii), R⁵Compound (I-iib), in which is hydroxy, is R among compounds (I-ii)⁵Is R^5a(Wherein R^5aIs R⁵Can be obtained also from the compound (I-ia) which is a substituted or unsubstituted lower alkoxy in the definition of
[Step 10]

(Wherein R¹, A, R³, R⁴, R^5aAnd Y are as defined above)
  Compound (I-iib) can be obtained by treating compound (I-ia) obtained in step 9 with a base in an amount of 1 equivalent to 10 equivalents, preferably 1 equivalent to 5 equivalents, in a solvent.
  The solvent is not particularly limited, and examples thereof include a mixed solvent of a protic solvent such as methanol, ethanol, propanol, and butanol and water, or a mixed solvent of an aprotic solvent such as tetrahydrofuran and dioxane and water. A mixed solvent of ethanol and water is preferable.
  Examples of the base include inorganic bases such as sodium hydroxide, lithium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate and lithium carbonate, Amberlyst A-21 (manufactured by Rohm and Haas), AG1-X8 (Bio-Rad). Etc.) and the like can be used.
  The reaction is carried out at a temperature between 0 ° C. and 150 ° C., preferably at a temperature between 20 ° C. and 100 ° C., and is usually completed in 1 hour to 48 hours.
  Of the compounds (I-ii), R⁵-NR⁶R⁷The compound (I-iic) can be obtained, for example, by Production Method 3 shown below.
Production method 3:

(Wherein R¹, A, R³, R⁴, R⁶, R⁷And Y are as defined above)
[Step 11]
  In the presence of 1 to 10 equivalents of a condensing agent, 1 equivalent to 5 equivalents, preferably 1 equivalent to 3 equivalents, of compound (I-iib) obtained in Step 10 of Production Method 2 in a solvent inert to the reaction The compound (N) can be obtained by reacting with 1-hydroxybenzotriazole.
  The solvent inert to the reaction is not particularly limited as long as it is inert to the reaction. For example, dichloromethane, chloroform, 1,2-dichloroethane, N, N-dimethylformamide, dimethylacetamide, N-methyl Pyrrolidone, dimethyl sulfoxide, tetrahydrofuran, dioxane, diethyl ether, diisopropyl ether, benzene, toluene, xylene, ethyl acetate, acetonitrile, water, etc. can be used alone or as a mixture thereof, especially chloroform, tetrahydrofuran or a mixture thereof A solvent is preferred.
  Examples of the condensing agent include dicyclohexylcarbodiimide, diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminopropyl) carbodiimide or its hydrochloride, and N-ethyl-N ′-(3-dimethylaminopropyl supported on polystyrene. ) Carbodiimide, N-benzyl-N′-cyclohexylcarbodiimide supported on polystyrene, benzotriazol-1-yl-tris (dimethylamino) phosphonium hexafluorophosphide salt, diphenylphosphoryl azide, etc. Ethyl-N ′-(3-dimethylaminopropyl) carbodiimide or a hydrochloride thereof or N-ethyl-N ′-(3-dimethylaminopropyl) carbodiimide supported on polystyrene is preferred.
  The reaction is carried out at a temperature between 0 ° C. and 100 ° C., preferably at a temperature between 0 ° C. and 60 ° C., and is usually completed in 1 hour to 120 hours.
[Step 12]
  The compound (N) obtained in step 11 is used in a solvent-free or reaction-inert solvent in an amount of 1 equivalent to a large excess, preferably 1 equivalent to 5 equivalents of HNR.⁶R⁷(Wherein R⁶And R⁷Are the same as defined above, to give compound (I-ic).
  The solvent inert to the reaction may be any solvent as long as it is inert to the reaction and is not particularly limited. For example, tetrahydrofuran, dioxane, 1,2-dimethoxyethane, diisopropyl ether, benzene, toluene, xylene, ethyl acetate, Acetonitrile, dichloromethane, chloroform, 1,2-dichloroethane, N, N-dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, water and the like can be used alone or in combination, among which tetrahydrofuran and water The mixed solvent is preferable.
  The reaction is carried out at a temperature between 0 ° C. and 100 ° C., preferably at a temperature between 20 ° C. and 60 ° C., and is usually completed in 1 hour to 96 hours.
  In addition, this reaction may be carried out by adding 1 equivalent to 10 equivalents, preferably 1 equivalent to 5 equivalents of a base, if necessary.
  Examples of the base include organic bases such as triethylamine, N, N-diisopropylethylamine, DBU, N, N-dimethylaniline, pyridine, quinoline, potassium carbonate, sodium carbonate, lithium carbonate, sodium hydrogen carbonate, potassium hydroxide, hydroxide Inorganic bases such as sodium, lithium hydroxide and potassium tert-butoxide, basic anion exchange resins such as Amberlyst A-21 (Rohm and Haas), AG1-X8 (BioRad), polyvinylpyridine, morpholinomethyl A base supported on a solid phase such as polystyrene can be used, and among them, potassium carbonate or DBU is preferable.
[Step 13]
  Of the compounds (I-ic) obtained by Production Method 3, R⁷In the same manner as in Step 10 of Production Method 2, compound (I-ica) having carboxy lower alkyl (wherein the carboxy lower alkyl has the same meaning as above) in R⁷In addition, the compound (I-iib) having lower alkoxycarbonyl lower alkyl (wherein lower alkoxycarbonyl lower alkyl is as defined above) can also be obtained by hydrolysis treatment.
  In addition, by treating the compound (I-icic) in which the lower alkoxy moiety of the lower alkoxycarbonyl lower alkyl is tert-butoxy in the compound (I-iicb) with 1 equivalent to a large excess of acid in a solvent. A compound (I-icica) can also be obtained.
  The solvent is not particularly limited as long as it is inert to the reaction. For example, dichloromethane, chloroform, 1,2-dichloroethane, N, N-dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide , Tetrahydrofuran, dioxane, diethyl ether, diisopropyl ether, benzene, toluene, xylene, ethyl acetate, acetonitrile, water and the like can be used singly or in combination, with dichloromethane being preferred.
  As the acid, for example, a carboxylic acid such as trifluoroacetic acid, a mineral acid such as hydrochloric acid, a sulfonic acid such as trifluoromethanesulfonic acid, and benzenesulfonic acid can be used, and among them, trifluoroacetic acid is preferable.
  The reaction is carried out at a temperature between 0 ° C. and 150 ° C., preferably at a temperature between 0 ° C. and 50 ° C., and is usually completed in 1 hour to 48 hours.
  Of the compounds (I), X is hydroxymethyl, R²-NR³R⁴(Wherein R³And R⁴Can be obtained by, for example, Production Method 4 shown below.
Production method 4:

(Where A, m, R¹, R³, R⁴, R^5aAnd Y are as defined above)
[Step 14]
  Of the compounds (L) obtained in Step 7 of Production Method 2, R⁵Is R^5a(Wherein R^5aIs the same as defined above) by treating the compound (La) with a reducing agent in an inert solvent for reaction with 2 to 10 equivalents, preferably 2 to 6 equivalents of a reducing agent. Obtainable.
  The solvent inert to the reaction may be any solvent as long as it is inert to the reaction, and is not particularly limited. For example, diethyl ether, tetrahydrofuran, dimethoxyethane, dioxane, benzene, toluene, xylene and the like can be used alone or in combination. It can be used by mixing.
  As the reducing agent, for example, lithium aluminum hydride, diisobutylaluminum hydride and the like can be used, and among them, lithium aluminum hydride is preferable.
  The reaction is carried out at a temperature between −78 ° C. and the boiling point of the solvent, preferably between 0 ° C. and 50 ° C., and is usually completed in 1 minute to 10 hours.
[Step 15]
  In the same manner as in Step 4 of Production Method 1, compound (O) obtained in Step 14 is added in an amount of 1 equivalent to a large excess, preferably 1 equivalent to 5 equivalents, in a solvent inert to the reaction. Compound (P) can be obtained by treatment with an oxidizing agent.
  Various conditions such as a solvent, an oxidizing agent, a reaction temperature, and a reaction time are the same as those described in Step 4.
[Step 16]
  The compound (P) obtained in step 15 is used in the absence or presence of a solvent inert to the reaction in the presence or absence of 1 equivalent to a large excess, preferably 1 equivalent to 10 equivalents of a base. Compound (I-iii) can be obtained by reacting with an equivalent amount of compound (H).
  The solvent inert to the reaction is not particularly limited as long as it is inert to the reaction. For example, dichloromethane, chloroform, 1,2-dichloroethane, N, N-dimethylformamide, dimethylacetamide, N-methyl Pyrrolidone, dimethyl sulfoxide, tetrahydrofuran, dioxane, 1,2-dimethoxyethane, diisopropyl ether, benzene, toluene, xylene, ethyl acetate, acetonitrile, and the like can be used alone or in combination. -Dimethoxyethane is preferred.
  As the base, for example, a base supported on a solid phase such as triethylamine, N, N-diisopropylethylamine, N-methylmorpholine, morpholinomethylpolystyrene or the like can be used, and among them, N, N-diisopropylethylamine is preferable.
  The reaction is carried out at a temperature between 20 ° C. and 150 ° C., preferably at a temperature between 80 ° C. and 120 ° C., and is usually completed in 1 hour to 96 hours.
  In compound (I), X is substituted hydroxymethyl, R²-NR³R⁴(Wherein R³And R⁴Can be obtained by, for example, Production Method 5 shown below.
Production method 5:

(Where A, R¹, R³, R⁴And Y are as defined above, and R⁸Represents lower alkyl, and the lower alkyl has the same meaning as the lower alkyl (i).
[Step 17]
  Compound (Q) can be obtained by treating compound (O) obtained in step 14 of production method 4 with an oxidizing agent in a solvent.
  As the oxidizing agent, various reagents used in the oxidation reaction of alcohol can be arbitrarily used. For example, manganese dioxide, pyridinium chlorochromate (PCC), pyridinium dichromate (PDC), chromium oxide (IV) -pyridicy complex , Potassium dichromate, sodium dichromate, cerium (IV) ammonium nitrate, etc., among which manganese dioxide is preferred.
  The solvent is not particularly limited as long as it is inert to the reaction. For example, dichloromethane, chloroform, 1,2-dichloroethane, N, N-dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide, tetrahydrofuran , Dioxane, 1,2-dimethoxyethane, diisopropyl ether, benzene, toluene, xylene, ethyl acetate, acetonitrile, pyridine, water and the like can be used singly or as a mixture thereof, among which chloroform is preferable.
  The reaction is carried out at a temperature between −78 ° C. and the boiling point of the solvent, preferably between 0 ° C. and 50 ° C., and is usually completed in 1 hour to 96 hours.
[Step 18]
  Compound (R) is obtained by reacting compound (Q) obtained in step 17 with a corresponding alkylating agent in an amount of 1 to 5 equivalents, preferably 2 to 3 equivalents, in a solvent inert to the reaction. be able to.
  Corresponding alkylating agents include, for example, R⁸MgBr (wherein R⁸Is as defined above), R⁸Li (wherein R⁸Are as defined above. More specifically, R⁸When is methyl, for example, methylmagnesium bromide, methyllithium, or the like can be used, and among these, methylmagnesium bromide is preferable.
  Any solvent inert to the reaction may be used as long as it is inert to the reaction. For example, tetrahydrofuran, dioxane, 1,2-dimethoxyethane, diisopropyl ether and the like can be used alone or in combination. Tetrahydrofuran is preferred.
  The reaction is carried out at a temperature between −78 ° C. and the boiling point of the solvent, preferably at a temperature between −40 ° C. and 20 ° C., and is usually completed in 1 hour to 96 hours.
[Step 19]
  From compound (R) obtained in step 18, in the same manner as in steps 15 and 16 of production method 4, among compounds (I-iiib), X^ACompound (I-iiiba) in which is mono-substituted hydroxymethyl can be obtained.
[Step 20]
  In addition, among compounds (I-iiib), X^ACompound (I-iiibb) in which is di-substituted hydroxymethyl can be obtained by treating the compound obtained in Step 18 in the same manner as in Step 17, Step 18 and then Step 19.
Production method 6:
  In addition to the method described in Production Method 4, compound (I-iiia) can also be obtained according to the following production method.

(Where A, R¹, R³, R⁴, R^5aAnd Y are as defined above)
[Step 21]
  Compound (I-iii) obtained by treating Compound (I-ia) obtained in Step 9 of Production Method 2 with a reducing agent in an amount of 1 equivalent to 10 equivalents, preferably 1.5 equivalents to 3 equivalents, in a solvent. Can be obtained.
  Any solvent may be used as long as it is inert to the reaction, and is not particularly limited. For example, pentane, hexane, benzene, toluene, xylene, diethyl ether, tetrahydrofuran, dimethoxyethane, dioxane and the like can be used alone or in combination. Can be used.
  As the reducing agent, for example, lithium aluminum hydride, diisobutylaluminum hydride, bis (methoxyethoxy) aluminum hydride and the like can be used, and among them, bis (methoxyethoxy) aluminum hydride is preferable.
  The reaction is carried out at a temperature between −78 ° C. and the boiling point of the solvent, preferably 0 ° C. to 30 ° C., and is usually completed in 10 minutes to 24 hours.
Production method 7:
  Compound (I) can be obtained, for example, by the method described in WO92 / 01675, WO97 / 09315, WO99 / 65897, JP-A-2001-89452, or the like, in addition to the production methods shown above. Also, for example, WO92 / 01675, WO97 / 09315, WO99 / 65897, JP 2001-89452, edited by E. Klingsberg, The Chemistry of Heterocyclic Compounds (The Chemistry of Heterocyclic Compounds). ), Vol. 14, Pyridine and Its Derivatives, Part 1, New York, Interscience (1960), Part 2 (1961), Part 3 (1962), Part 4 (1964), edited by GR Newkom, The Chemistry of Heterocyclic Compounds, vol. 14. A compound obtained by the method described in Pyridine and Its Derivatives, Part 5 (1984), or the like or an intermediate thereof, for example, Synlett, 2000 , P. 625-626, Journal of Chemical Society, 1952, p. 800-803, Tetrahedron, 1987, 43, p. 2557-2564, Journal of Heterocyclic Chemistry, 1987, Vol. 24, p. 85-89, Journal of Organic Chemistry, 1995, Vol. 60, p. The target compound can be obtained by performing functional group conversion according to the method described in 1875-1877 or the like.
  The conversion of each functional group in the compound (I), the raw material compound and the intermediate and the conversion of the functional group contained in the substituent can be performed by a known method [for example, Comprehensive Organic Transformations 2nd Edition (Comprehensive Organic Transformations]. , The second edition), the method described by John Wiley & Sons Inc. (1999)] by R. C. Larock, etc. .
  A compound (I) having a desired functional group at a desired position can be obtained by appropriately combining the above methods and the like.
  Isolation and purification of the intermediate and the target compound in the above production method can be performed by appropriately combining methods used in ordinary organic synthesis, for example, filtration, extraction, washing, drying, concentration, crystallization, various chromatography, and the like. it can. Furthermore, it can be performed by a purification method commonly used in a general parallel synthesis method, for example, a purification method using a scavenger resin, an ion exchange resin or the like. In addition, the intermediate can be subjected to the next reaction without any particular purification.
  Compound (I) and compound (IA) may have isomers such as positional isomers, geometric isomers, optical isomers or tautomers, but all possible isomers including these isomers And mixtures in any ratio of the isomers are used in the therapeutic agents of the invention or are encompassed by the invention.
  When it is desired to obtain a salt of compound (I) or compound (IA), it can be purified as it is when a salt of compound (I) or compound (IA) is obtained. When it is obtained in a free form, compound (I) or compound (IA) may be dissolved or suspended in a suitable solvent, and an acid or base may be added for isolation and purification.
  In addition, Compound (I), Compound (IA) or a pharmacologically acceptable salt thereof may exist in the form of an adduct with water or various solvents, and these adducts are also present in the present invention. Used in therapeutic agents or included in the present invention.
  Specific examples of the compound (I) obtained by the present invention are shown in Tables 1 to 6.

  Next, the pharmacological action of typical compound (I) will be specifically described with reference to test examples.
Test Example 1: GPR88 function inhibitory action (GPR88-specific constitutive activity inhibitory action)
  GPR88 assay cells 1 constructed in Reference Example 5 were placed on a white plate at 10 per well.⁵In order to induce and express GPR88 individually, 17β-estradiol (17β-estradiol, hereinafter abbreviated as E2; manufactured by Sigma-Aldrich) was diluted with a medium so as to be 10 nmol / L in the reaction solution. Compound and add 5% CO₂The reaction was performed at 37 ° C. for 6 hours in an incubator. Thereafter, Steady Glo Luciferase Assay System (Promega) solution was added to stop the reaction, and the amount of luminescence for 1 second was measured with a top count (Packard).
  The activity (antagonism) of the test compound was expressed as an inhibition rate calculated based on the number of counts per second when E2 was added and when E2 was not added, as shown in the following formula.
          Inhibition rate (%) = [1-{(A−B) / (C−B)}] × 100
  In the formula, A, B, and C each represent the following meanings.
A: Counts per second when E2 and test compound were added
B: Counts per second when E2 and test compound are not added
C: Count per second when only E2 is added
  IC₅₀The value was calculated from the inhibition rate by the linear approximation analysis method of the Logit-Log conversion method. The results are shown in Table 7.

  In addition, Table 8 shows the inhibition rate (%) when cells were treated under the condition that the final concentration of the test compound was 3 μmol / L.

  From the above results, it was shown that Compound (I) has a GPR88 function inhibitory action (GPR88 antagonistic action).
  In addition, according to the method described in WO03 / 087366, when a similar assay is performed using an assay cell that induces and expresses another constitutively active GPCR (for example, GPR3, GPR4, GPR6, or GPR12), compound (I ) Showed no inhibitory activity. From this, it was shown that Compound (I) specifically inhibits the constitutive activity of GPR88.
  In addition, GPR88 assay cells 2 and 3 constructed in Reference Examples 6 and 7, respectively, should be used for searching for a GPR88-specific constitutive activity inhibitor (a substance having a GPR88 function suppressing action), as in GPR88 assay cell 1. Can do.
Test Example 2: Motor function improvement by intracerebroventricular administration of antisense oligonucleotide against GPR88 gene (evaluation in model animals with dopamine receptor antagonist)
  Phosphorothioate-type antisense oligonucleotide (Strg-AS1: requested to synthesize by Genset) (10 μg / 20 μL) for GPR88 gene having the base sequence shown in SEQ ID NO: 7 was dissolved in physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.). Then, this solution was repeatedly administered into the cerebral ventricles of mice 6 times every 12 hours using a two-stage needle from 3 days before administration of the dopamine receptor antagonist SCH23390 described later. Coomassie solution was administered intraventricularly to another mouse in the same manner, the brain was removed, and it was confirmed that the administration solution was diffused in the brain. Three hours after the final intracerebroventricular administration of Strg-AS1 (10 μg / 20 μL), subcutaneous injection of dopamine receptor antagonist SCH23390 (0.5 mg / kg; diluted by Sigma to 0.05 mg / mL with physiological saline) Thus, an insufficiency state was created. 30 minutes after the subcutaneous administration of SCH23390, the time until the animal begins to move up to 1 minute, with both hind limbs hung on an acrylic stand that is 4.5 cm high and 1.0 cm wide, one by one (Fixed time) was measured. The degree of motor dysfunction was expressed using this immobility time as an index. In addition, the group which did not administer Strg-AS1 and administered only SCH23390 subcutaneously was made into the negative control group.
  As a result, the average immobility time in the negative control group to which only SCH23390 was administered subcutaneously was 58 seconds. On the other hand, the immobility time in the group in which Strg-AS1 was administered intraventricularly decreased to 34.4 seconds, and an effect of improving motor function was observed.
  That is, the repeated administration of Strg-AS1 improved the dyskinetic state induced by the dopamine receptor antagonist, and thus it became clear that the GPR88 gene is related to the motor regulation of the dopamine nervous system. Drugs targeting the drug are effective in the treatment and / or prevention of diseases based on abnormalities in the brain dopamine nervous system (eg Parkinson's disease, involuntary movements, extrapyramidal disease, depression, schizophrenia, etc.) Was suggested.
  Strg-AS1 is a phosphorothioate oligo DNA comprising a sequence complementary to the 401st to 418th sequences of the base sequence shown in SEQ ID NO: 5, including the translation start point of mouse GPR88 mRNA. In addition, since the base sequence of Strg-AS1 is complementary to not only mouse GPR88 mRNA but also the 425th to 442nd sequences of the base sequence shown in SEQ ID NO: 4, including the translation start point of human GPR88 mRNA, Even when administered, it is considered that the expression of the GPR88 gene is suppressed, and the same action as that shown in the following and Test Example 2 is exhibited.
Test Example 3: Central function improvement by intraventricular administration of antisense oligonucleotide against GPR88 gene (evaluation in reserpine-treated animals)
  From 3 days before the administration of reserpine described later, Strg-AS1 (10 μg / 20 μL) dissolved in physiological saline was repeatedly administered into the ventricle of the mouse 6 times every 12 hours using a two-stage needle. Coomassie solution was administered intraventricularly to another mouse in the same manner, the brain was removed, and it was confirmed that the administration solution was diffused in the brain. 20 hours prior to the last intracerebroventricular administration of Strg-AS1 (10 μg / 20 μL), reserpine (2.5 mg / kg: Apopron injection; first pharmaceutical diluted to 0.25 mg / mL with distilled water) was injected subcutaneously. A central dysfunction state was created. Twenty-four hours after reserpine administration, the measurement was carried out one by one in the same manner as in Test Example 2, and the time until the mouse started to move up to 1 minute (immobility time) was measured. The degree of central dysfunction was expressed using this immobility time as an index. In addition, the group which did not administer Strg-AS1 but administered only reserpine subcutaneously was made into the negative control group.
  As a result, the average immobility time in the negative control group to which only reserpine was administered subcutaneously was 60 seconds. On the other hand, the immobility time in the group in which Strg-AS1 was administered intracerebroventricularly decreased to 39.6 seconds, and the effect of improving the central function was recognized.
  That is, the central dysfunction induced by reserpine administration was improved by repeated administration of Strg-AS1, which is a phosphorothioate-type antisense oligonucleotide for the GPR88 gene. That is, drugs targeting the GPR88 gene are those that are based on abnormalities in the catecholamine nervous system in the brain (based on catecholamine depletion) (eg Parkinson's disease, involuntary movement, extrapyramidal disease, depression, schizophrenia, etc.) It was suggested to be effective for treatment and / or prevention.
Test Example 4: Effect on dopamine receptor antagonist SCH23390-induced catalepsy
  Experiments were performed using 10 male ddY mice (body weight 22 to 25 g, Nippon SLC) 5 weeks old. SCH23390 (manufactured by Sigma) was dissolved in physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) and injected subcutaneously into mice at 0.5 mg / kg. The test compound (Compound 37) was suspended in distilled water (manufactured by Otsuka Pharmaceutical Co., Ltd.) and used. 30 minutes before subcutaneous injection of SCH23390, a suspension containing Compound 37 or distilled water alone (control) was orally administered (0.1 mL per 10 g of mouse body weight). One hour after administration of Compound 37, each mouse was placed on an acrylic stand vertically 4.5 cm in height and 1.0 cm in width, and only both forelimbs and only both hindlimbs of the mouse were sequentially suspended to measure catalepsy. Table 9 shows the criteria for determining catalepsy score.

  The intensity of action was expressed as the average value of the catalepsy score of 10 animals per group (5 points). The result is shown in FIG.
  From FIG. 1, improvement of catalepsy symptoms (improved motor function) induced by administration of a dopamine receptor antagonist is observed by administration of Compound 37, and Compound (I) is a disease based on an abnormality of the dopamine nervous system (for example, Parkinson Disease, involuntary movement, extrapyramidal disease, depression, schizophrenia, etc.) was suggested to be effective for treatment and / or prevention.
Test Example 5: Effect on reserpine-induced catalepsy
  Catalepsy induced by administration of antipsychotic drugs such as reserpine is regarded as a symptom model of Parkinson's disease and depression [Journal of Neural Transmission, Vol. 8, pp. 39-71. (1994)].
  Experiments were performed using 10 male ddY mice (body weight 22 to 25 g, Nippon SLC) 5 weeks old. During the preliminary breeding period, the animals were raised in an animal room with a room temperature of 22 ± 3 ° C. and a humidity of 50 ± 20%, and food and water were freely taken. Reserpine (5 mg / kg: Apopron Note: Daiichi Pharmaceutical Co., Ltd. diluted with distilled water) was subcutaneously administered and the catalepsy-inducing action was observed 18 hours later. The catalepsy score was 5 (Criteria for Table 4 in Test Example 4). The mouse | mouth which shows was selected for the experiment. The test compound (compound 37) was used as a suspension in distilled water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.), and the suspension containing compound 37 or distilled water for injection alone (control) was orally administered (per mouse body weight of 10 g). 0.1 mL), and 1 hour after administration of Compound 37, each mouse was placed on a platform of 4.5 cm in height and 1.0 cm in width, and only the forelimbs and only the hindlimbs of the mouse were sequentially suspended, and catalepsy was measured.
  The intensity of action was expressed as the average value of the catalepsy score of 10 animals per group (5 points). The result is shown in FIG.
  From FIG. 2, the catalepsy improvement (improvement of central function) induced by reserpine administration is observed by administration of compound 37, and compound (I) is a disease based on abnormalities of catecholamine nervous system in the brain (based on catecholamine depletion) It was suggested that it is effective for the treatment and / or prevention of (for example, Parkinson's disease, involuntary movement, extrapyramidal disease, depression, schizophrenia, etc.).
Test Example 6: Increased spontaneous exercise amount
  Experiments were performed using 10 male ddY mice (body weight 22 to 25 g, Nippon SLC) 5 weeks old. One hour before administration of Compound 263, the mice were placed in an acrylic box having a width of 40 cm, a depth of 40 cm, and a height of 40 cm to acclimatize to the environment. Compound 263 was used after being suspended in distilled water containing 0.5% carboxymethylcellulose (CMC; manufactured by Otsuka Pharmaceutical Co., Ltd.). Only a suspension or a solvent containing Compound 263 (2.5 mg / mL) was intraperitoneally administered (0.1 mL per 10 g of mouse body weight). Using a spontaneous exercise amount measuring device (SCANET MV-10MT, Toyo Sangyo Co., Ltd., Pharmaceutical Equipment Division), three items of grooming behavior, walking behavior and hindlimbing behavior were measured for 4 hours immediately after administration. The exercise in the light period was measured from 10:00 am and the exercise in the dark period was measured from 7 pm.
  As a result, in the light period, the grooming behaviors in the negative control group administered with the solvent and the group administered with compound 263 were 11013 counts and 17830 counts, respectively, the walking behaviors were 6745 counts and 13613 counts, respectively, Both 19 count and 52 count, and the compound 263 administration group increased.
  Since the amount of locomotor activity increased by administration of Compound 263, GPR88 function inhibitors are diseases involving motor dysfunction, such as Parkinson's disease, involuntary movement, extrapyramidal syndrome, Huntington's chorea, cerebrovascular disease, spinocerebellar degeneration. It was suggested that it is effective for treatment and / or prevention.
  On the other hand, in the dark period, the grooming behaviors in the negative control group administered with the solvent and the group administered with compound 263 were 201564 count and 108411 count, respectively, the walking behavior was 98254 count and 86667 count, respectively, and the back-standing behavior was 1439 respectively. Counts and 541 counts were both decreased in the compound 263 administration group.
  As described above, in mice that are nocturnal animals, administration of Compound 263 increased the amount of light during exercise and decreased the amount of activity during dark. This suggests that GPR88 modifies the optical tuning of the biological clock to match the period of the bright and dark environment of the outside world. Therefore, it was suggested that GPR88 function control agents are effective for the treatment and / or prevention of rhythm disorders such as jet lag and seasonal emotional disorder.
  Compound (I) or a pharmacologically acceptable salt thereof can be administered alone as it is, but it is usually desirable to provide it as various pharmaceutical preparations. These pharmaceutical preparations are used for animals and humans.
  The pharmaceutical preparation according to the present invention contains an antibody against GPR88 or compound (I) or a pharmacologically acceptable salt thereof alone or as a mixture with any other active ingredient for treatment as an active ingredient. be able to. In addition, these pharmaceutical preparations are produced by any method well known in the technical field of pharmaceutics by mixing the active ingredient with one or more pharmacologically acceptable carriers.
  As the administration route, it is desirable to use one that is most effective in the treatment, and examples thereof include oral administration and parenteral administration such as intravenous administration.
  Examples of dosage forms include tablets and injections.
  In preparation of tablets, for example, excipients such as lactose and mannitol, disintegrants such as starch, lubricants such as magnesium stearate, binders such as hydroxypropylcellulose, surfactants such as fatty acid esters, glycerin and the like Any plasticizer may be used according to a conventional method.
  In preparing the injection, water, physiological saline, vegetable oil, solubilizer, preservative, antioxidant and the like may be used according to a conventional method.
  The effective amount and frequency of administration of Compound (I) or a pharmacologically acceptable salt thereof vary depending on the administration form, patient age, body weight, nature or severity of the condition to be treated, etc. It is preferable to administer 0.1 to 100 mg / kg for adults in 3 to 4 divided doses. However, these doses and the number of doses vary depending on the various conditions described above.

FIG. 1 shows the intensity of the action of the monosodium salt of compound 37 on the dopamine receptor antagonist SCH23390-induced catalepsy as the catalepsy score. The vertical axis represents the catalepsy score (full score of 5 points), and the horizontal axis represents the dose (mg / kg) of the monosodium salt of Compound 37. In the bar graph, the left side shows the case of the control group, and the other cases show the case of the compound 37 monosodium salt administration group. * Indicates a significant difference of p <0.05 (Steel test), and ** indicates a significant difference of p <0.01 (Steel test).
FIG. 2 shows the intensity of the action of the monosodium salt of Compound 37 on reserpine-induced catalepsy as the catalepsy score. The vertical axis represents the catalepsy score (full score of 5 points), and the horizontal axis represents the dose (mg / kg) of the monosodium salt of Compound 37. In the bar graph, the left side shows the case of the control group, and the right side shows the case of the compound 37 monosodium salt administration group. * Indicates a significant difference of p <0.05 (Steel test).

（式中、Ｒ^１およびＡはそれぞれ前記と同義である）
参考例８で得られた４−クロロ−５−シアノ−２−メチルチオピリミジン（０．２５ｍｏｌ／Ｌクロロホルム溶液，０．２０ｍＬ，０．０５０ｍｍｏｌ）とＲ^１ＡｒＮＨ（式中、Ｒ^１およびＡｒはそれぞれ前記と同義である）で表される化合物Ｅ（１．０ｍｏｌ／Ｌクロロホルム溶液，０．０６０ｍＬ，０．０６０ｍｍｏｌ）の混合液に、モルホリノメチルポリスチレン（３５ｍｇ，０．１２ｍｍｏｌ）を加え、５０℃にて１日間攪拌した。反応混合物にベンゾイルクロリドポリマーバウンド（２３ｍｇ，０．０５８ｍｍｏｌ）およびクロロホルム（０．５０ｍＬ）を加え、室温にてさらに１日間攪拌した。反応混合物中の樹脂を濾別し、樹脂をクロロホルムとメタノールの混合溶媒（クロロホルム／メタノール＝３／１，１．２ｍＬ）で洗浄した。得られた溶液を全て合わせ、溶媒を留去して化合物Ｆを得た。
工程２：化合物Ｇの合成

（式中、Ｒ^１、Ａおよびｍはそれぞれ前記と同義である）
工程１で得られた化合物Ｆをジクロロメタン（０．２０ｍＬ）に溶解し、その溶液にメタクロ濾過安息香酸（０．５０ｍｏｌ／Ｌジクロロメタン溶液，０．１２５ｍＬ，０．０６３ｍｍｏｌ）を加え、室温にて０．５〜１時間攪拌した。反応混合物に飽和重曹水（０．２０ｍＬ）を加えて分液した。有機層を飽和重曹水（０．２０ｍＬ）で洗浄し、硫酸マグネシウムと珪藻土の混合物（硫酸マグネシウム／珪藻土＝１／１，１４０ｍｇ）を加えて攪拌した。硫酸マグネシウムと珪藻土の混合物を濾別し、硫酸マグネシウムと珪藻土の混合物をジクロロメタン（０．８０ｍＬ）で洗浄した。得られた溶液を全て合わせ、溶媒を留去して化合物Ｇを得た。
工程３：化合物Ｊの合成

（式中、Ｒ^１、Ａ、Ｒ^３およびＲ^４はそれぞれ前記と同義である）
工程２で得られた化合物Ｇをテトラヒドロフラン（０．２０ｍＬ）に溶解し、その溶液にＲ^３Ｒ^４ＮＨ（式中、Ｒ^３およびＲ^４はそれぞれ前記と同義である）で表される化合物Ｈ（１．０ｍｏｌ／Ｌクロロホルム溶液，０．０６０ｍＬ，０．０６０ｍｍｏｌ）を加え、室温にて１日間攪拌した。反応混合物にクロロホルム（０．４０ｍＬ）、ベンゾイルクロリドポリマーバウンド（２３ｍｇ，０．０５８ｍｍｏｌ）、ポリ４−ビニルピリジン（２３ｍｇ）を加え、室温にて１日間攪拌した。反応混合物中の樹脂を濾別し、樹脂をクロロホルムとメタノールの混合溶媒（クロロホルム／メタノール＝３／１，１．４ｍＬ）で洗浄した。得られた溶液を全て合わせ、溶媒を留去して化合物Ｊを得た。
工程４：化合物２〜化合物３６の合成
工程３で得られた化合物ＪをＮ，Ｎ−ジメチルホルムアミド（０．１０ｍＬ）に溶解し、その溶液にアジ化アンモニウムのＮ，Ｎ−ジメチルホルムアミド溶液（１．０ｍｏｌ／Ｌ，０．０８０ｍＬ，０．０８０ｍｍｏｌ）を加え、１００℃にて１日間撹拌した。反応液から溶媒を留去し、残渣をクロロホルムと１，１，１，３，３，３−ヘキサフルオロイソプロピルアルコールの混合溶媒（クロロホルム／１，１，１，３，３，３−ヘキサフルオロイソプロピルアルコール＝３／１，０．４０ｍＬ）に溶解し、５％クエン酸水溶液で洗浄した。有機層を濃縮した後、残渣をエタノール（０．３００ｍＬ）に溶解し、その溶液にイオン交換樹脂ＡＧ１−Ｘ８（バイオラッド社製）を加え、室温にて２時間撹拌した。反応混合物中の樹脂を濾取し、樹脂をメタノール（０．６０ｍＬ）で洗浄した後、樹脂にクロロホルムとメタノールの混合溶媒（クロロホルム／メタノール＝１／１，０．４０ｍＬ）次いで塩化水素の酢酸エチル溶液（４ｍｏｌ／Ｌ，０．０５０ｍＬ）を加え、目的物を溶出した。さらに樹脂をクロロホルムとメタノールの混合溶媒（クロロホルム／メタノール＝１／１，１．２ｍＬ）で洗浄し、得られた溶出液と洗浄液を合わせて濃縮し、目的とする化合物２〜化合物３６をそれぞれ通算収率約３０％で得た。
得られた化合物２〜化合物３６はそれぞれ質量分析により同定した。各化合物の分析結果は第１表に機器データとして記載する。
実施例３：化合物３７〔４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボン酸〕の合成
工程１：４−（２−ベンジルフェニルアミノ）−２−メチルチオピリミジン−５−カルボン酸エチルの合成
市販の４−クロロ−２−メチルチオピリミジン−５−イルカルボン酸エチル（１５．０ｇ，６４．５ｍｍｏｌ）を１，２−ジメトキシエタン（１５０ｍＬ）に溶解し、その溶液にＮ，Ｎ−ジイソプロピルエチルアミン（２７．６ｍＬ，１６１ｍｍｏｌ）および２−ベンジルアニリン（１３．０ｇ，７０．９ｍｍｏｌ）を加え、加熱還流下、１８時間攪拌した。反応液を減圧濃縮し、得られた残渣を酢酸エチルで希釈した後、水を加えて分液した。有機層を５％クエン酸水溶液および飽和食塩水で順次洗浄した。有機層を無水硫酸マグネシウムで乾燥した後、溶媒を減圧留去した。得られた残渣をエタノールから再結晶することにより、白色結晶として４−（２−ベンジルフェニルアミノ）−２−メチルチオピリミジン−５−カルボン酸エチル（１９．６ｇ，５１．６ｍｍｏｌ，収率８０％）を得た。
^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：１．３８（ｔ，Ｊ＝６．９Ｈｚ，３Ｈ），２．３６（ｓ，３Ｈ），４．０２（ｓ，２Ｈ），４．３４（ｑ，Ｊ＝６．９Ｈｚ，２Ｈ），７．１０−７．２７（ｍ，８Ｈ），７．８２（ｄ，Ｊ＝７．９Ｈｚ，１Ｈ），８．７０（ｓ，１Ｈ），１０．００（ｂｒ ｓ，１Ｈ）
ＡＰＣＩ−ＭＳ：ｍ／ｚ３８０（Ｍ＋Ｈ）^＋
工程２：４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボン酸エチルの合成
工程１で得られた化合物（５．０ｇ，１３．２ｍｍｏｌ）をジクロロメタン（５０ｍＬ）に溶解し、氷冷下、その溶液にメタクロ濾過安息香酸（２．７０ｇ，１５．６ｍｍｏｌ）を１０分間かけて加え、室温にて３０分間攪拌した。さらに氷冷下、反応液にメタクロ濾過安息香酸（１．１３ｇ，７．５３ｍｍｏｌ）を５分間かけて加え、室温にて２０分間攪拌した。反応液をジクロロメタンで希釈し、飽和重曹水を加えて分液した。有機層を無水硫酸マグネシウムで乾燥した後、溶媒を減圧留去した。得られた残渣を１，２−ジメトキシエタン（５０ｍＬ）に溶解し、その溶液にシクロヘキシルメチルアミン（２．５７ｍＬ，１９．８ｍｍｏｌ）を加え、加熱還流下、２．５時間攪拌した。反応液を減圧濃縮し、得られた残渣をエタノールから再結晶することにより、白色結晶として４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボン酸エチル（４．８７ｇ，１１．０ｍｍｏｌ，収率８３％）を得た。
^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ_６，８０℃）δ（ｐｐｍ）：０．７４−０．９８（ｍ，２Ｈ），１．０１−１．２４（ｍ，３Ｈ），１．２９（ｔ，Ｊ＝７．１Ｈｚ，３Ｈ），１．４０−１．７３（ｍ，６Ｈ），３．０６（ｂｒ ｓ，２Ｈ），３．９８（ｓ，２Ｈ），４．２５（ｑ，Ｊ＝７．１Ｈｚ，２Ｈ），７．０６−７．２４（ｍ，８Ｈ），７．４０（ｂｒ ｓ，１Ｈ），７．９５（ｄ，Ｊ＝８．２Ｈｚ，１Ｈ），８．５２（ｂｒ ｓ，１Ｈ），９．８５（ｂｒ ｓ，１Ｈ）
ＡＰＣＩ−ＭＳ：ｍ／ｚ４４５（Ｍ＋Ｈ）^＋
ＩＲ（ＫＢｒ）：２４６８，２４５８，１６６６，１６２９，１６０８，１５７７，１５４６，１５２１，１５０２，１４９４，１４６３，１４４６，１４３０，１３９４，１３９０，１３６９ｃｍ^−１
工程３：４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボン酸の合成
工程２で得られた化合物（１．００ｇ，２．２５ｍｍｏｌ）をエタノール（７ｍＬ）に溶解し、その溶液に水酸化ナトリウム水溶液（１．０ｍｏｌ／Ｌ，７．０ｍＬ，６．５ｍｍｏｌ）を加え、６０℃にて４時間攪拌した。反応液にエタノール（７ｍＬ）および水酸化ナトリウム水溶液（４．０ｍｏｌ／Ｌ，１．０ｍＬ，４．０ｍｍｏｌ）を加え、加熱還流下、３０分間攪拌した。
さらに反応液に水酸化ナトリウム水溶液（４．０ｍｏｌ／Ｌ，１．０ｍＬ，４．０ｍｍｏｌ）を加え、加熱還流下、２時間攪拌した。反応液を半分の体積まで減圧濃縮し、塩酸（１．０ｍｏｌ／Ｌ）で反応液のｐＨ値を４．５に調整した。析出した結晶を濾取し、白色結晶として化合物３７（０．９９０ｇ，２．２５ｍｍｏｌ，定量的収率）を得た。
^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ_６）δ（ｐｐｍ）：０．７８−０．９９（ｍ，２Ｈ），１．０３−１．２９（ｍ，３Ｈ），１．４７−１．８１（ｍ，６Ｈ），３．０７（ｂｒ ｓ，２Ｈ），４．０３（ｓ，２Ｈ），６．８９−７．１０（ｍ，１Ｈ），７．１２−７．３５（ｍ，８Ｈ），７．５０（ｂｒ ｓ，１Ｈ），８．４０（ｄ，Ｊ＝７．９Ｈｚ，１Ｈ），８．４８（ｓ，１Ｈ），１１．９７（ｂｒ ｓ，１Ｈ）
ＡＰＣＩ−ＭＳ：ｍ／ｚ４１７（Ｍ＋Ｈ）^＋
また、対応するナトリウム塩は以下の方法に従って調製した。
上記で得られた化合物３７（１．７ｇ，４．０８ｍｍｏｌ）をテトラヒドロフラン（５ｍＬ）に溶解し、その溶液に水酸化ナトリウム水溶液（１．０ｍｏｌ／Ｌ，４．１ｍＬ，４．１ｍｍｏｌ）を加え、室温にて１時間攪拌した後、反応液を減圧濃縮した。得られた残渣を水で再結晶化することにより、白色結晶として化合物３７の１ナトリウム塩（１．６９ｇ，３．８５ｍｍｏｌ，収率９４％）を得た。
^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ_６，）δ（ｐｐｍ）：０．７６−１．００（ｍ，２Ｈ），１．０２−１．３２（ｍ，３Ｈ），１．４４−１．９０（ｍ，６Ｈ），３．０７（ｂｒ ｓ，２Ｈ），４．０３（ｓ，２Ｈ），６．７９−７．４９（ｍ，９Ｈ），８．４７（ｓ，１Ｈ），８．５７（ｄ，Ｊ＝７．２Ｈｚ，１Ｈ），１３．０８（ｂｒ ｓ，１Ｈ）
ＡＰＣＩ−ＭＳ：ｍ／ｚ４１７（Ｍ＋Ｈ）^＋
ＩＲ（ＫＢｒ）：３０４５，２９９１，２９６６，２９５４，２９３３，２９１４，２８４６，１６６４，１６３１，１６１０，１５９２，１５７７，１５５２，１５３９，１５３５，１５１０ｃｍ^−１
実施例４：化合物３８〔４−［（２−ベンジルフェニル）（メチル）アミノ］−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボン酸〕の合成
工程１：４−［（２−ベンジルフェニル）（メチル）アミノ］−２−メチルチオピリミジン−５−カルボン酸エチルの合成
市販の４−クロロ−２−メチルチオピリミジン−５−カルボン酸エチル（３．９０ｇ，１６．８ｍｍｏｌ）を１，２−ジメトキシエタン（３０ｍＬ）に溶解し、その溶液にＮ，Ｎ−ジイソプロピルエチルアミン（７．８ｍＬ，４６ｍｍｏｌ）および参考例２で得られた２−ベンジル−Ｎ−メチルアニリン（３．０ｇ，１５ｍｍｏｌ）を加え、加熱還流下、２４時間攪拌した。反応液を減圧濃縮した。得られた残渣に酢酸エチルおよび水を加えて分液した後、有機層を５％クエン酸水溶液および飽和食塩水で順次洗浄した。有機層を無水硫酸マグネシウムで乾燥した後、溶媒を減圧留去した。得られた残渣を２−プロパノールから再結晶することにより、白色結晶として４−［（２−ベンジルフェニル）（メチル）アミノ］−２−メチルチオピリミジン−５−カルボン酸エチル（５．０６ｇ，１２．９ｍｍｏｌ，収率７７％）を得た。
^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ_６）δ（ｐｐｍ）：１．０４（ｔ，Ｊ＝７．１Ｈｚ，３Ｈ），２．４１（ｓ，３Ｈ），３．１８（ｓ，３Ｈ），３．７０（ｑ，Ｊ＝７．１Ｈｚ，２Ｈ），３．８０（ｓ，２Ｈ），７．０７−７．２９（ｍ，９Ｈ），８．１９（ｓ，１Ｈ）
工程２：４−［（２−ベンジルフェニル）（メチル）アミノ］−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボン酸エチルの合成
工程１で得られた４−［（２−ベンジルフェニル）（メチル）アミノ］−２−メチルチオピリミジン−５−カルボン酸エチル（３．００ｇ，７．６２ｍｍｏｌ）をジクロロメタン（３０ｍＬ）に溶解し、氷冷下、その溶液にメタクロ濾過安息香酸（１．５７ｇ，９．１５ｍｍｏｌ）を５分間かけて加え、室温にて３０分間攪拌した。さらに氷冷下、反応液にメタクロ濾過安息香酸（０．６５ｇ，３．８ｍｍｏｌ）を加え、室温にて３０分間攪拌した。反応液をジクロロメタンで希釈し、飽和重曹水を加えて分液した。有機層を無水硫酸マグネシウムで乾燥した後、溶媒を減圧留去した。得られた残渣を１，２−ジメトキシエタン（３０ｍＬ）に溶解し、その溶液にシクロヘキシルメチルアミン（１．４８ｍＬ，１１．４ｍｍｏｌ）を加え、加熱還流下、１．５時間攪拌した。反応液を放冷した後、生じた結晶を濾取した。得られた粗結晶をエタノールから再結晶することにより、白色結晶として４−［（２−ベンジルフェニル）（メチル）アミノ］−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボン酸エチル（２．８７ｇ，６．２７ｍｍｏｌ，収率８２％）を得た。
^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ_６，８０℃）δ（ｐｐｍ）：０．８５−０．９３（ｍ，２Ｈ），１．０３（ｔ，Ｊ＝７．１Ｈｚ，３Ｈ），１．０９−１．２４（ｍ，３Ｈ），１．５１−１．６５（ｍ，６Ｈ），３．０５（ｂｒ ｓ，２Ｈ），３．０７（ｓ，３Ｈ），３．７３（ｑ，Ｊ＝７．１Ｈｚ，２Ｈ），３．８２（ｓ，２Ｈ），６．９７（ｂｒ ｓ，１Ｈ），７．０３−７．２８（ｍ，９Ｈ），８．１２（ｓ，１Ｈ）
工程３：４−［（２−ベンジルフェニル）（メチル）アミノ］−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボン酸の合成
工程２で得られた４−［（２−ベンジルフェニル）（メチル）アミノ］−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボン酸エチル（０．５ｇ，１．０９ｍｍｏｌ）をエタノール（５ｍＬ）に溶解し、その溶液に水酸化ナトリウム水溶液（１．０ｍｏｌ／Ｌ，１０ｍＬ，１０ｍｍｏｌ）を加え、加熱還流下、２４時間攪拌した。反応液を半分の体積まで減圧濃縮し、塩酸（１．０ｍｏｌ／Ｌ）で反応液のｐＨ値を４．５に調整した。析出した結晶を濾取し、白色結晶として化合物３８（４７４ｍｇ，１．０９ｍｍｏｌ，定量的収率）を得た。
^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ_６，８０℃）δ（ｐｐｍ）：主なピークとして０．８３−０．９１（ｍ，２Ｈ），１．１２−１．２５（ｍ，３Ｈ），１．４８−１．６３（ｍ，６Ｈ），３．０４（ｂｒ ｓ，２Ｈ），３．１８（ｓ，３Ｈ），３．８７（ｓ，２Ｈ），７．００−７．４１（ｍ，１０Ｈ），８．２２（ｓ，１Ｈ）
ＡＰＣＩ−ＭＳ：ｍ／ｚ４３１（Ｍ＋Ｈ）^＋
ＩＲ（ＫＢｒ）：２９３０，２８４０，２３８０，２３５０，１６５５，１５１０，１４９０，１３８５，１３７５ｃｍ^−１
実施例５：化合物３９〔４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボヒドロキサム酸〕の合成
工程１：４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルアミノ）ピリミジン−５−カルボン酸＝ベンゾトリアゾール−１−イルの合成
実施例３で得られる化合物３７（５．００ｇ，１２．０ｍｍｏｌ）をクロロホルム（５０ｍＬ）に溶解し、氷冷下、その溶液にＮ−エチル−Ｎ’−（３−ジメチルアミノプロピル）カルボジイミド塩酸塩（４．６０ｇ，２４．０ｍｍｏｌ）および１−ヒドロキシベンゾトリアゾール（３．３０ｇ，２４．４ｍｍｏｌ）を加え、室温にて５時間攪拌した。さらに反応液にＮ−エチル−Ｎ’−（３−ジメチルアミノプロピル）カルボジイミド塩酸塩（２．３０ｇ，１２．０ｍｍｏｌ）および１−ヒドロキシベンゾトリアゾール（１．６０ｇ，１２．０ｍｍｏｌ）を加え、室温にて２時間攪拌した。反応液を酢酸エチルで希釈し、水を加えて分液した後、有機層を５％クエン酸水溶液および飽和重曹水で順次洗浄した。有機層を無水硫酸マグネシウムで乾燥した後、溶媒を減圧留去することにより、淡黄色結晶として４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルアミノ）ピリミジン−５−カルボン酸＝ベンゾトリアゾール−１−イル（５．６７ｇ，１０．６ｍｍｏｌ，収率８９％）を得た。
^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：０．７６−１．０８（ｍ，２Ｈ），１．０９−１．３３（ｍ，３Ｈ），１．４３−１．８２（ｍ，６Ｈ），３．１４（ｔ，Ｊ＝６．５Ｈｚ，１．５Ｈ），３．３５（ｔ，Ｊ＝６．３Ｈｚ，０．５Ｈ），３．９２（ｓ，０．５Ｈ），３．９３（ｓ，１．５Ｈ），５．４７（ｔ，Ｊ＝５．９Ｈｚ，０．２５Ｈ），５．８９（ｔ，Ｊ＝５．９Ｈｚ，０．７５Ｈ），６．９９−７．３０（ｍ，９Ｈ），７．４２−７．６０（ｍ，２Ｈ），７．６８（ｄ，Ｊ＝７．９Ｈｚ，０．２５Ｈ），７．８０（ｄ，Ｊ＝７．９Ｈｚ，０．７５Ｈ），８．１０（ｄ，Ｊ＝８．２Ｈｚ，１Ｈ），８．９５（ｓ，０．７５Ｈ），９．０３（ｂｒ ｓ，０．２５Ｈ），９．０８（ｓ，０．２５Ｈ），９．１９（ｂｒ ｓ，０．７５Ｈ）
工程２：〔４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボヒドロキサム酸〕の合成
工程１で得られた４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルアミノ）ピリミジン−５−カルボン酸＝ベンゾトリアゾール−１−イル（１．４０ｇ，２．６２ｍｍｏｌ）をテトラヒドロフラン（２０ｍＬ）と水（２０ｍＬ）の混合溶媒に溶解し、氷冷下、その溶液にヒドロキシルアミン塩酸塩（５４６ｍｇ，７．８８ｍｍｏｌ）および炭酸カリウム（１．２７ｇ，９．１８ｍｍｏｌ）を加え、室温にて１８時間攪拌した。反応液を酢酸エチルで希釈し、水を加えて分液した後、有機層を飽和重曹水にて洗浄した。有機層を無水硫酸マグネシウムで乾燥した後、溶媒を減圧留去した。得られた残渣をシリカゲルカラムクロマトグラフィーで精製した。得られた油状物質を酢酸エチルから再結晶することにより、淡黄色結晶として化合物３９（４９０ｍｇ，１．１４ｍｍｏｌ，収率４３％）を得た。
^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ_６，８０℃）δ（ｐｐｍ）：０．７６−０．９９（ｍ，２Ｈ），１．０１−１．２６（ｍ，３Ｈ），１．４５−１．７４（ｍ，６Ｈ），３．０６（ｂｒ ｓ，２Ｈ），４．００（ｓ，２Ｈ），６．９９−７．２６（ｍ，９Ｈ），８．１１（ｄｄ，Ｊ＝１．２，８．１Ｈｚ，１Ｈ），８．３３（ｓ，１Ｈ），８．６５（ｂｒ ｓ，１Ｈ），１０．７７（ｂｒ ｓ，１Ｈ）
ＡＰＣＩ−ＭＳ：ｍ／ｚ４３２（Ｍ＋Ｈ）^＋
実施例６：化合物４０〔４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボキサミド〕の合成
実施例５の工程１で得られた４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルアミノ）ピリミジン−５−カルボン酸＝ベンゾトリアゾール−１−イル（２００ｍｇ，０．３７ｍｍｏｌ）をＮ，Ｎ−ジメチルホルムアミド（５ｍＬ）に溶解し、氷冷下、その溶液にアンモニア水（２８％，５ｍＬ）を加え、室温にて２時間攪拌した。反応液を酢酸エチルで希釈し、水を加えて分液した後、有機層を飽和重曹水および飽和食塩水で順次洗浄した。有機層を無水硫酸マグネシウムで乾燥した後、溶媒を減圧留去した。得られた残渣を２−プロパノールから再結晶することにより、白色結晶として化合物４０（７７ｍｇ，０．１８ｍｍｏｌ，収率５０％）を得た。
^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ_６，７０℃）δ（ｐｐｍ）：０．８６−０．９４（ｍ，２Ｈ），１．１２−１．２５（ｍ，３Ｈ），１．４９−１．６８（ｍ，６Ｈ），３．１２（ｂｒ ｔ，Ｊ＝６．１Ｈｚ，２Ｈ），３．９９（ｓ，２Ｈ），６．９８−７．２４（ｍ，９Ｈ），７．２８（ｂｒ ｓ，２Ｈ），８．１７（ｄ，Ｊ＝８．１Ｈｚ，１Ｈ），８．５０（ｓ，１Ｈ），１１．３２（ｂｒ ｓ，１Ｈ）
ＡＰＣＩ−ＭＳ：ｍ／ｚ４１６（Ｍ＋Ｈ）^＋
実施例７：化合物４１〔４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−イル−Ｎ−（２−ヒドロキシエチル）カルボキサミド〕の合成
実施例５の工程１で得られた４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルアミノ）ピリミジン−５−カルボン酸＝ベンゾトリアゾール−１−イル（２５０ｍｇ，０．５８ｍｍｏｌ）をアセトニトリル（５ｍＬ）とＮ，Ｎ−ジメチルホルムアミド（２．５ｍＬ）の混合溶媒に溶解し、氷冷下、その溶液に２−アミノエタノール（７１μＬ，１．４６ｍｍｏｌ）および１，８−ジアザビシクロ［５．４．０］−ウンデク−７−エン（ＤＢＵ）（２１７μＬ，１．４６ｍｍｏｌ）を加え、室温にて１８時間攪拌した。反応液を酢酸エチルで希釈し、５％クエン酸水溶液を加えて分液した後、有機層を飽和重曹水および飽和食塩水で順次洗浄した。有機層を無水硫酸マグネシウムで乾燥した後、溶媒を減圧留去した。得られた残渣を２−プロパノールから再結晶することにより、白色結晶として化合物４１（８６ｍｇ，０．１９ｍｍｏｌ，収率３２％）を得た。
^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：主なピークとして０．６９−０．９０（ｍ，２Ｈ），０．９６−１．２６（ｍ，３Ｈ），１．３１−１．８０（ｍ，６Ｈ），３．００（ｂｒ ｓ，２Ｈ），３．５４（ｂｒ ｓ，２Ｈ），３．８１（ｂｒ ｓ，２Ｈ），４．００（ｓ，２Ｈ），６．９４−７．２８（ｍ，９Ｈ），７．７７（ｂｒ ｓ，１Ｈ），８．１６（ｂｒ ｓ，１Ｈ），８．６５（ｂｒ ｓ，１Ｈ），１１．４８（ｂｒ ｓ，１Ｈ）
ＡＰＣＩ−ＭＳ：ｍ／ｚ４６０（Ｍ＋Ｈ）^＋
ＩＲ（ＫＢｒ）：３１２５，２８９０，１７３４，１７１６，１６９５，１６６８，１６６２，１６５５，１６０１，１５８１，１５５８，１５５１，１５２７，１４９７，１４７３，１４５４，１４３１，１４１７ｃｍ^−１
実施例８：化合物４２〔２−［４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−イルカルボニルアミノ］酢酸〕の合成
工程１：２−［４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−イルカルボニルアミノ］酢酸＝ｔｅｒｔ−ブチルの合成
実施例５の工程１で得られた４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルアミノ）ピリミジン−５−カルボン酸＝ベンゾトリアゾール−１−イル（２５０ｍｇ，０．４７ｍｍｏｌ）をアセトニトリル（２ｍＬ）とＮ，Ｎ−ジメチルホルムアミド（２ｍＬ）の混合溶媒に溶解し、氷冷下、その溶液にグリシンｔｅｒｔ−ブチル塩酸塩（１９６ｍｇ，１．１７ｍｍｏｌ）およびＤＢＵ（３５０μＬ，２．３４ｍｍｏｌ）を加え、室温にて６時間攪拌した。反応液を酢酸エチルで希釈し、５％クエン酸水溶液を加えて分液した後、有機層を飽和重曹水および飽和食塩水で順次洗浄した。有機層を無水硫酸マグネシウムで乾燥した後、溶媒を減圧留去し、淡黄色油状物質として２−［４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−イルカルボニルアミノ］酢酸＝ｔｅｒｔ−ブチル（２４６ｍｇ，０．４６ｍｍｏｌ，収率９９％）を得た。
^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ_６，８０℃）δ（ｐｐｍ）：０．７４−０．９９（ｍ，２Ｈ），１．０２−１．２８（ｍ，３Ｈ），１．４２（ｓ，９Ｈ），１．４３−１．７７（ｍ，６Ｈ），３．０５（ｂｒ ｓ，２Ｈ），３．８５（ｄ，Ｊ＝５．８Ｈｚ，２Ｈ），３．９７（ｓ，２Ｈ），６．９７−７．４３（ｍ，９Ｈ），８．０９（ｄｄ，Ｊ＝１．２，８．１Ｈｚ，１Ｈ），８．４３（ｔ，Ｊ＝５．８Ｈｚ，１Ｈ），８．５１（ｓ，１Ｈ），１０．９３（ｂｒ ｓ，１Ｈ）
工程２：２−［４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−イルカルボニルアミノ］酢酸の合成
工程１で得られた２−［４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−イルカルボニルアミノ］酢酸＝ｔｅｒｔ−ブチル（２４６ｍｇ，０．４６ｍｍｏｌ）をジクロロメタン（３ｍＬ）に溶解し、氷冷下、その溶液にトリフルオロ酢酸（５ｍＬ）を加え、室温にて２時間攪拌した。反応液を減圧濃縮し、得られた残渣をエタノール（３ｍＬ）に溶解し、その溶液に水酸化ナトリウム水溶液（１．０ｍｏｌ／Ｌ，１．８４ｍＬ，１．８４ｍｍｏｌ）を加え、室温にて１５分間攪拌した。塩酸（１．０ｍｏｌ／Ｌ）で反応液のｐＨ値を４．５に調整し、水（５ｍＬ）を加え、析出した結晶を濾取した。得られた粗結晶を２−プロパノールから再結晶することにより、白色結晶として化合物４２（１７６ｍｇ，０．３７ｍｍｏｌ，収率８１％）を得た。
^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：０．７４−０．９９（ｍ，２Ｈ），１．００−１．３２（ｍ，３Ｈ），１．４０−１．５５（ｍ，１Ｈ），１．５６−１．９０（ｍ，５Ｈ），３．０５（ｄ，Ｊ＝６．３Ｈｚ，２Ｈ），３．９９（ｓ，２Ｈ），４．０５（ｄ，Ｊ＝５．６Ｈｚ，２Ｈ），７．０２−７．２３（ｍ，９Ｈ），７．５９（ｄ，Ｊ＝６．３Ｈｚ，１Ｈ），８．３３（ｂｒ ｓ，１Ｈ），８．７８（ｓ，１Ｈ），８．８４（ｂｒ ｓ，１Ｈ），１１．５３（ｓ，１Ｈ）
ＡＰＣＩ−ＭＳ：ｍ／ｚ４７４（Ｍ＋Ｈ）^＋
ＩＲ（ＫＢｒ）：３３８０，３２４０，３０６５，２９３０，２８６８，１７０１，１６９７，１６７０，１６６２，１６５２，１６０２，１５８５，１５６０，１５４３，１５２４，１４９８，１４７３，１４５２，１４３７，１４１７，１４１２ｃｍ^−１
実施例９：化合物４３〔２−［４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−イルカルボニル−Ｎ−メチルアミノ］酢酸〕の合成
工程１：２−［４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−イルカルボニル−Ｎ−メチルアミノ］酢酸エチルの合成
実施例５の工程１で得られた４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルアミノ）ピリミジン−５−カルボン酸＝ベンゾトリアゾール−１−イル（１．０ｇ，１．８７ｍｍｏｌ）をアセトニトリル（１０ｍＬ）とＮ，Ｎ−ジメチルホルムアミド（１０ｍＬ）の混合溶媒に溶解し、氷冷下、その溶液にＮ−メチルアミノ酢酸エチル塩酸塩（４３０ｍｇ，２．８１ｍｍｏｌ）およびＤＢＵ（８４０μＬ，５．６２ｍｍｏｌ）を加え、室温にて１時間攪拌した。反応液を酢酸エチルで希釈し、５％クエン酸水溶液を加えて分液した後、有機層を飽和重曹水および飽和食塩水で洗浄した。有機層を無水硫酸マグネシウムで乾燥した後、溶媒を減圧留去し、淡黄色油状物質として２−［４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−イルカルボニル−Ｎ−メチルアミノ］酢酸エチル（９９０ｍｇ，１．８７ｍｍｏｌ，定量的収率）を得た。
^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ_６，８０℃）δ（ｐｐｍ）：０．７６−０．９８（ｍ，２Ｈ），１．０１−１．２７（ｍ，３Ｈ），１．１８（ｔ，Ｊ＝７．１Ｈｚ，３Ｈ），１．４２−１．７７（ｍ，６Ｈ），３．０４（ｂｒ ｓ，２Ｈ），３．０７（ｓ，３Ｈ），３．９３（ｓ，２Ｈ），４．１３（ｑ，Ｊ＝７．１Ｈｚ，２Ｈ），４．１６（ｓ，２Ｈ），６．９７（ｂｒ ｓ，１Ｈ），７．０１−７．２４（ｍ，８Ｈ），７．９０（ｄ，Ｊ＝８．１Ｈｚ，１Ｈ），８．２１（ｓ，１Ｈ），８．９０（ｂｒ ｓ，１Ｈ）
工程２：２−［４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−イルカルボニル−Ｎ−メチルアミノ］酢酸の合成
工程１で得られた２−［４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−イルカルボニル−Ｎ−メチルアミノ］酢酸エチル（９９０ｍｇ，１．８７ｍｍｏｌ）をエタノール（２０ｍＬ）に溶解し、その溶液に水酸化ナトリウム水溶液（１．０ｍｏｌ／Ｌ，４．０ｍＬ，４．０ｍｍｏｌ）を加え、室温にて１８時間攪拌した。反応液を半分の体積まで濃縮し、水（２０ｍＬ）で希釈した。塩酸水溶液（１．０ｍｏｌ／Ｌ）で反応液のｐＨ値を４．５に調整し、析出した結晶を濾取した。得られた粗結晶をエタノールから再結晶することにより、白色結晶として化合物４３（３０５ｍｇ，０．６３ｍｍｏｌ，収率３４％）を得た。
^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ_６，８０℃）δ（ｐｐｍ）：主なピークとして０．７７−１．００（ｍ，２Ｈ），１．０４−１．３２（ｍ，３Ｈ），１．４４−１．７４（ｍ，６Ｈ），３．０７（ｂｒ ｓ，２Ｈ），３．０８（ｓ，３Ｈ），３．９６（ｓ，２Ｈ），４．１２（ｓ，２Ｈ），６．９７（ｂｒ ｓ，１Ｈ），７．０４−７．２５（ｍ，８Ｈ），７．９２（ｄ，Ｊ＝８．１Ｈｚ，１Ｈ），８．０２（ｓ，１Ｈ），８．９３（ｂｒ，１Ｈ）
ＡＰＣＩ−ＭＳ：ｍ／ｚ４８８（Ｍ＋Ｈ）^＋
ＩＲ（ＫＢｒ）：２７８０，１６５３，１６４７，１６３７，１６１８，１６１０，１６０１，１５８３，１５６０，１５４５，１５３５，１５２９，１５０８，１４９５，１４５０，１４２３，１４００ｃｍ^−１
実施例１０：化合物４４〔４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）−５−ヒドロキシメチルピリミジン〕の合成
実施例５の工程１で得られた４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルアミノ）ピリミジン−５−カルボン酸＝ベンゾトリアゾール−１−イル（２．００ｇ，３．７５ｍｍｏｌ）をテトラヒドロフラン（２０ｍＬ）に溶解し、アルゴン雰囲気中、氷冷下、その溶液に水素化リチウムアルミニウム（０．２８ｇ，７．５ｍｍｏｌ）を加え、室温にて１時間攪拌した。氷冷下、反応液に水（０．３０ｍＬ）、水酸化ナトリウム水溶液（４．０ｍｏｌ／Ｌ，０．３０ｍＬ）および水（１．０ｍＬ）を順次加え、室温にて１５分間攪拌した。析出した固体を珪藻土を通して濾過し、クロロホルムで洗浄した。濾液および洗浄液を合わせ、水で希釈したのち、クロロホルムで抽出した。有機層を無水硫酸マグネシウムで乾燥した後、溶媒を減圧留去した。得られた残渣をシリカゲルカラムクロマトグラフィーで精製することにより、淡黄色結晶として化合物４４（１．６９ｇ，０．７６ｍｍｏｌ，収率２０％）を得た。
^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：主なピークとして０．７４−１．０１（ｍ，２Ｈ），１．０２−１．３１（ｍ，３Ｈ），１．４５−１．８１（ｍ，６Ｈ），３．１５（ｔ，Ｊ＝６．３Ｈｚ，２Ｈ），４．０３（ｓ，２Ｈ），４．２７（ｓ，２Ｈ），７．０５−７．２６（ｍ，９Ｈ），７．４１（ｓ，１Ｈ），７．６０（ｓ，１Ｈ），８．１０（ｂｒ ｓ，１Ｈ）
ＡＰＣＩ−ＭＳ：ｍ／ｚ４０３（Ｍ＋Ｈ）^＋
ＩＲ（ＫＢｒ）：３５００，２９００，２８５８，１９１５，１８９４，１８８８，１８７５，１８６７，１７１３，１７０９，１７０５，１６９３，１６８７，１６７８，１６７２，１６５７，１６５１，１６２２，１６１２ｃｍ^−１
実施例１１：化合物４５〔４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）−５−（１−ヒドロキシエチル）ピリミジン〕の合成
工程１：４−（２−ベンジルフェニルアミノ）−５−ヒドロキシメチル−２−メチルチオピリミジンの合成
実施例３の工程１で得られた４−（２−ベンジルフェニルアミノ）−２−メチルチオピリミジン−５−カルボン酸エチル（５．０ｇ，１３．２ｍｍｏｌ）をテトラヒドロフラン（５０ｍＬ）に溶解し、アルゴン雰囲気中、氷冷下、その溶液に水素化リチウムアルミニウム（０．６ｇ，１５．８ｍｍｏｌ）を加え、室温にて３．５時間攪拌した。さらに、アルゴン雰囲気中、氷冷下、反応液に水素化リチウムアルミニウム（０．１５ｇ，４．０ｍｍｏｌ）を加え、室温にて１．５時間攪拌した。氷冷下、反応液に水（０．８ｍＬ）、水酸化ナトリウム水溶液（４ｍｏｌ／Ｌ，０．８ｍＬ）および水（２．４ｍＬ）を順次加え、室温にて１５分間攪拌した。析出した固体を珪藻土を通して濾過し、クロロホルムで洗浄した。濾液および洗浄液を合わせ、水で希釈した後、クロロホルムで抽出した。有機層を無水硫酸マグネシウムで乾燥した後、溶媒を減圧留去し、淡黄色油状物質として４−（２−ベンジルフェニルアミノ）−５−ヒドロキシメチル−２−メチルチオピリミジン（３．７０ｇ，１１．０ｍｍｏｌ，収率８３％）を得た。
^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＣＤＣｌ_３，）δ（ｐｐｍ）：主なピークとして２．４０（ｓ，３Ｈ），４．０３（ｓ，２Ｈ），４．３４（ｓ，２Ｈ），７．１０−７．３２（ｍ，８Ｈ），７．６６（ｓ，１Ｈ），７．６８（ｓ，１Ｈ），８．００（ｄ，Ｊ＝８．２Ｈｚ，１Ｈ）
工程２：４−（２−ベンジルフェニルアミノ）−５−ホルミル−２−メチルチオピリミジンの合成
工程１で得られた４−（２−ベンジルフェニルアミノ）−５−ヒドロキシメチル−２−メチルチオピリミジン（３．７０ｍｇ，１１．０ｍｍｏｌ）をクロロホルム（１００ｍＬ）に溶解し、その溶液に二酸化マンガン（８．０ｇ，１１０ｍｍｏｌ）を加え、室温にて１８時間攪拌した。反応液を珪藻土を通して濾過し、濾別した固体をクロロホルムで洗浄した。濾液および洗浄液を合わせて減圧濃縮し、得られた残渣をエタノールから再結晶することにより、淡黄色結晶として４−（２−ベンジルフェニルアミノ）−５−ホルミル−２−メチルチオピリミジン（１．９４ｇ，５．７８ｍｍｏｌ，収率５３％）を得た。
^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＣＤＣｌ_３，）δ（ｐｐｍ）：２．４１（ｓ，３Ｈ），４．０５（ｓ，２Ｈ），７．０８−７．２８（ｍ，８Ｈ），７．８８（ｄ，Ｊ＝８．１Ｈｚ，１Ｈ），８．３７（ｓ，１Ｈ），９．７２（ｓ，１Ｈ），１０．２８（ｂｒ ｓ，１Ｈ）
工程３：４−（２−ベンジルフェニルアミノ）−５−（１−ヒドロキシエチル）−２−メチルチオピリミジンの合成
アルゴン雰囲気下、工程２で得られた４−（２−ベンジルフェニルアミノ）−５−ホルミル−２−メチルチオピリミジン（５００ｍｇ，１．４９ｍｍｏｌ）をテトラヒドロフラン（１０ｍＬ）に溶解し、その溶液に２０℃にて臭化メチルマグネシウム（０．９３ｍｏｌ／Ｌテトラヒドロフラン溶液，４．０ｍＬ，３．７ｍｍｏｌ）を加え、−２０℃にて１．５時間攪拌した。反応液に−２０℃にて飽和塩化アンモニウム水溶液を徐々に加えた。反応液を酢酸エチルで抽出し、有機層を水および飽和食塩水で順次洗浄した。有機層を無水硫酸マグネシウムで乾燥した後、溶媒を減圧留去し、淡黄色油状物質として４−（２−ベンジルフェニルアミノ）−５−（１−ヒドロキシエチル）−２−メチルチオピリミジン（５６０ｍｇ，１．４９ｍｍｏｌ，定量的収率）を得た。
^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：主なピークとして１．３３（ｄ，Ｊ＝６．８Ｈｚ，３Ｈ），２．３７（ｓ，３Ｈ），４．０２（ｓ，２Ｈ），４．６０（ｑ，Ｊ＝６．８Ｈｚ，１Ｈ），７．０７−７．２９（ｍ，８Ｈ），７．６０（ｓ，１Ｈ），８．０１（ｄ，Ｊ＝７．２Ｈｚ，１Ｈ），８．２５（ｓ，１Ｈ）
工程４：４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）−５−（１−ヒドロキシエチル）ピリミジンの合成
工程３で得られた４−（２−ベンジルフェニルアミノ）−５−（１−ヒドロキシエチル）−２−メチルチオピリミジン（７３．４ｍｇ，０．２４ｍｍｏｌ）をジクロロメタン（２ｍＬ）に溶解し、氷冷下、その溶液にメタクロ濾過安息香酸（５０ｍｇ，０．２９ｍｍｏｌ）を加え、室温にて３０分間攪拌した。反応液をジクロロメタンで希釈し、飽和重曹水を加えて分液した。有機層を無水硫酸マグネシウムで乾燥した後、溶媒を減圧留去した。得られた残渣をジオキサン（３ｍＬ）に溶解し、その溶液にＮ，Ｎ−ジイソプロピルエチルアミン（０．０８０ｍＬ，０．４８ｍｍｏｌ）およびシクロヘキシルメチルアミン（０．０６０ｍＬ，０．４８ｍｍｏｌ）を加え、加熱還流下、４日間攪拌した。反応液を酢酸エチルで希釈し、水を加えて分液した後、有機層を飽和食塩水で洗浄した。有機層を無水硫酸マグネシウムで乾燥した後、溶媒を減圧留去し、残渣をシリカゲルカラムクロマトグラフィーで精製することにより、淡黄色油状物質として化合物４５（８６．１ｍｇ，０．２０７ｍｍｏｌ，収率８６％）を得た。
^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：主なピークとして０．６８−１．０３（ｍ，２Ｈ），１．０４−１．３７（ｍ，３Ｈ），１．４５−１．８８（ｍ，６Ｈ），２．２５（ｄ，Ｊ＝６．６Ｈｚ，３Ｈ），３．１８（ｄｄ，Ｊ＝４．５，６．４Ｈｚ，２Ｈ），３．５８（ｑ，Ｊ＝６．６Ｈｚ，１Ｈ），４．０６（ｓ，２Ｈ），６．９７−７．２８（ｍ，９Ｈ），７．６１（ｓ，１Ｈ），８．２３（ｄ，Ｊ＝７．２Ｈｚ，１Ｈ），９．９３（ｂｒ ｓ，１Ｈ）
ＡＰＣＩ−ＭＳ：ｍ／ｚ３９９（Ｍ−ＯＨ）^＋
実施例１２：化合物４６〔４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）−５−（１−ヒドロキシ−１−メチルエチル）ピリミジン〕の合成
工程１：５−アセチル−４−（２−ベンジルフェニルアミノ）−２−メチルチオピリミジンの合成
実施例１１の工程３で得られた４−（２−ベンジルフェニルアミノ）−５−（１−ヒドロキシエチル）−２−メチルチオピリミジン（５２４ｍｇ，１．４９ｍｍｏｌ）をクロロホルム（１０ｍＬ）に溶解し、その溶液に二酸化マンガン（１．３ｇ，１５ｍｍｏｌ）を加え、室温にて１２時間攪拌した。さらにその溶液にクロロホルム（１０ｍＬ）および二酸化マンガン（１．３ｇ，１５ｍｍｏｌ）を加え、加熱還流下、２時間攪拌した。反応液を珪藻土を通して濾過し、濾別した固体をクロロホルムで洗浄した。濾液および洗浄液を合わせて減圧濃縮することにより淡黄色結晶として５−アセチル−４−（２−ベンジルフェニルアミノ）−２−メチルチオピリミジン（３８１ｍｇ，１．０９ｍｍｏｌ，収率７３％）を得た。
^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＣＤＣｌ_３，）δ（ｐｐｍ）：２．３７（ｓ，３Ｈ），２．５４（ｓ，３Ｈ），４．０３（ｓ，２Ｈ），７．０８−７．２９（ｍ，８Ｈ），７．８０（ｄ，Ｊ＝７．９Ｈｚ，１Ｈ），８．６３（ｓ，１Ｈ），１０．９４（ｂｒ ｓ，１Ｈ）
工程２：４−（２−ベンジルフェニルアミノ）−５−（１−ヒドロキシ−１−メチルエチル）−２−メチルチオピリミジンの合成
アルゴン雰囲気下、工程１で得られた５−アセチル−４−（２−ベンジルフェニルアミノ）−２−メチルチオピリミジン（２７２ｍｇ，０．７８ｍｍｏｌ）をテトラヒドロフラン（５ｍＬ）に溶解し、その溶液に−２０℃にて臭化メチルマグネシウム（０．９３ｍｏｌ／Ｌテトラヒドロフラン溶液，２．５ｍＬ，２．３ｍｍｏｌ）を加え、−２０℃にて１時間、０℃にて１時間攪拌した。さらに、反応液に０℃にて臭化メチルマグネシウム（０．９３ｍｏｌ／Ｌテトラヒドロフラン溶液，２．５ｍＬ，２．３ｍｍｏｌ）を加え、室温にて２．５時間攪拌した。反応液に０℃にて飽和塩化アンモニウム水溶液を徐々に加え、酢酸エチルで抽出した後、有機層を水および飽和食塩水で順次洗浄した。有機層を無水硫酸マグネシウムで乾燥した後、溶媒を減圧留去し、淡黄色油状物質として４−（２−ベンジルフェニルアミノ）−５−（１−ヒドロキシ−１−メチルエチル）−２−メチルチオピリミジン（２６４ｍｇ，０．７２ｍｍｏｌ，収率９３％）を得た。
^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：主なピークとして１．４６（ｓ，６Ｈ），２．３７（ｓ，３Ｈ），４．０１（ｓ，２Ｈ），７．０１−７．２９（ｍ，８Ｈ），７．７８（ｓ，１Ｈ），７．９４（ｄ，Ｊ＝７．７Ｈｚ，１Ｈ），８．８４（ｂｒ ｓ，１Ｈ）
工程３：４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）−５−（１−ヒドロキシ−１−メチルエチル）ピリミジンの合成
工程２で得られる４−（２−ベンジルフェニルアミノ）−５−（１−ヒドロキシ−１−メチルエチル）−２−メチルチオピリミジン（２８４ｍｇ，０．７８ｍｍｏｌ）をジクロロメタン（５ｍＬ）に溶解し、氷冷下、その溶液にメタクロ濾過安息香酸（２００ｍｇ，１．１７ｍｍｏｌ）を加え、室温にて１時間攪拌した。さらに氷冷下、反応液にメタクロ濾過安息香酸（１３３ｍｇ，０．７８ｍｍｏｌ）を加え、室温にて３０分間攪拌した。反応液をジクロロメタンで希釈し、飽和重曹水を加えて分液した。有機層を無水硫酸マグネシウムで乾燥した後、溶媒を減圧留去した。得られた残渣をジオキサン（３ｍＬ）に溶解し、その溶液にＮ，Ｎ−ジイソプロピルエチルアミン（０．３０ｍＬ，２．３ｍｍｏｌ）およびシクロヘキシルメチルアミン（０．２２ｍＬ，２．３ｍｍｏｌ）を加え、加熱還流下、４日間攪拌した。反応液を酢酸エチルで希釈し、水を加えて分液した後、有機層を飽和食塩水で洗浄した。有機層を無水硫酸マグネシウムで乾燥した後、溶媒を減圧留去し、得られた残渣をシリカゲルカラムクロマトグラフィーで精製することにより、油状物質を得た。得られた油状物質をジエチルエーテルから再結晶することにより、淡黄色結晶として化合物４６（８０．５ｍｇ，０．１８ｍｍｏｌ，収率２４％）を得た。
^１Ｈ−ＮＭＲ（２７０ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：０．７６−１．０２（ｍ，２Ｈ），１．１０−１．３５（ｍ，３Ｈ），１．４３−１．８０（ｍ，６Ｈ），２．４７（ｓ，６Ｈ），３．１５（ｔ，Ｊ＝６．３Ｈｚ，２Ｈ），４．０３（ｓ，１Ｈ），４．０７（ｓ，２Ｈ），５．５５（ｂｒ ｓ，１Ｈ），７．１１−７．２５（ｍ，９Ｈ），８．０３（ｄ，Ｊ＝８．２Ｈｚ，１Ｈ），８．５８（ｓ，１Ｈ）
ＡＰＣＩ−ＭＳ：ｍ／ｚ４１５（Ｍ−ＣＨ_３）^＋
ＩＲ（ＫＢｒ）：３２６０，２８００，２６００，１９９４，１９６９，１９５１，１９４４，１９３２，１９２５，１６２４，１５７８，１５４５，１５２４，１４９８，１４７３，１４６６，１４５４，１４３９，１４２３ｃｍ^−１
実施例１３：化合物４７〔Ｎ−［４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボニル］−４−トルエンスルホンアミド〕の合成
参考例１０で得られたベンゾトリアゾール−１−イル−４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボキシラート（０．２０ｇ，０．３７ｍｍｏｌ）をアセトニトリル（４ｍＬ）およびＮ，Ｎ−ジメチルホルムアミド（２ｍＬ）の混合溶媒に溶解し、氷冷下、４−トルエンスルホンアミド（０．１０ｇ，０．５６ｍｍｏｌ）およびＤＢＵ（８０μＬ，０．５６ｍｍｏｌ）を加え、室温で３時間攪拌した。さらに４−トルエンスルホンアミド（６０ｍｇ，０．３７ｍｍｏｌ）およびＤＢＵ（５０μＬ，０．３７ｍｍｏｌ）を加え、室温で１８時間攪拌した。反応液を酢酸エチルで希釈し、５％クエン酸水溶液、飽和重曹水および飽和食塩水で順次洗浄した。有機層を無水硫酸マグネシウムで乾燥した後、溶媒を減圧留去し、得られた残渣をシリカゲルカラムクロマトグラフィーで精製した。得られた粗結晶を２−プロパノールから再結晶することにより白色結晶として標記化合物（９７ｍｇ，０．１７ｍｍｏｌ，収率４６％）を得た。
^１Ｈ−ＮＭＲ（２７０ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：０．７０−０．９０（ｍ，２Ｈ），０．９５−１．２４（ｍ，３Ｈ），１．３０−１．７５（ｍ，６Ｈ），２．３４（ｓ，３Ｈ），３．０７（ｔ，Ｊ＝６．３Ｈｚ，２Ｈ），３．９４（ｓ，２Ｈ），７．０５−７．２２（ｍ，１１Ｈ），７．６６（ｄ，Ｊ＝７．７Ｈｚ，１Ｈ），７．７５（ｄ，Ｊ＝８．２Ｈｚ，２Ｈ），８．０８（ｂｒ ｓ，１Ｈ），８．５１（ｓ，１Ｈ），１２．４５（ｓ，１Ｈ）
ＡＰＣＩ−ＭＳ：ｍ／ｚ５６８（Ｍ−Ｈ）⁻
ＩＲ（ＫＢｒ）：２８７５，２８５０，１６５６，１６４８，１６４１，１６０１，１５８３，１５５８，１５４１，１５１６，１５１２，１５０８，１４８８，１４５２，１４０２，１３７１ｃｍ^−１
実施例１４：化合物４８〔Ｎ−［４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボニル］メタンスルホンアミド〕の合成
参考例１０で得られたベンゾトリアゾール−１−イル ４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボキシラート（０．２０ｇ，０．３７ｍｍｏｌ）をアセトニトリル（４ｍＬ）およびＮ，Ｎ−ジメチルホルムアミド（２ｍＬ）の混合溶媒に溶解し、氷冷下、メタンスルホンアミド（５０ｍｇ，０．５６ｍｍｏｌ）およびＤＢＵ（８０μＬ，０．５６ｍｍｏｌ）を加え、室温で３時間攪拌した。さらにメタンスルホンアミド（３０ｍｇ，０．３７ｍｍｏｌ）およびＤＢＵ（５０μＬ，０．３７ｍｍｏｌ）を加え、室温で１８時間攪拌した。反応液を酢酸エチルで希釈し、５％クエン酸水溶液、飽和重曹水および飽和食塩水で順次洗浄した。有機層を無水硫酸マグネシウムで乾燥した後、溶媒を減圧留去し、得られた残渣をシリカゲルカラムクロマトグラフィーで精製した。得られた粗結晶を２−プロパノールから再結晶することにより白色結晶として標記化合物（３９ｍｇ，０．０８０ｍｍｏｌ，収率２２％）を得た。
^１Ｈ−ＮＭＲ（２７０ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：０．７４−０．９５（ｍ，２Ｈ），０．９９−１．３０（ｍ，３Ｈ），１．４０−１．８０（ｍ，６Ｈ），２．９４（ｓ，３Ｈ），３．０７（ｔ，Ｊ＝６．１Ｈｚ，２Ｈ），４．１３（ｓ，２Ｈ），７．１３（ｍ，９Ｈ），７．６３（ｄ，Ｊ＝７．１Ｈｚ，１Ｈ），８．００（ｂｒ ｓ，１Ｈ），８．５９（ｓ，１Ｈ），１２．３３（ｓ，１Ｈ）
ＡＰＣＩ−ＭＳ：ｍ／ｚ４９２（Ｍ−Ｈ）⁻
ＩＲ（ＫＢｒ）：２９２０，２８５０，１６５５，１６３７，１６０８，１５８７，１５７８，１５６０，１５４５，１５４１，１５３５，１５２２，１４５４，１４０２，１３２３，１２７７ｃｍ^−１
実施例１５：化合物４９〔Ｎ−［４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボニル］トリフルオロメタンスルホンアミド〕の合成
参考例１０で得られたベンゾトリアゾール−１−イル ４−（２−ベンジルフェニルアミノ）−２−（シクロヘキシルメチルアミノ）ピリミジン−５−カルボキシラート（０．３０ｇ，０．７０ｍｍｏｌ）をアセトニトリル（６ｍＬ）およびＮ，Ｎ−ジメチルホルムアミド（６ｍＬ）の混合溶媒に溶解し、氷冷下、トリフルオロメタンスルホンアミド（２５９ｍｇ，１．７４ｍｍｏｌ）およびＤＢＵ（２６０μＬ，１．７４ｍｍｏｌ）を加え、室温で１８時間攪拌した。反応液を酢酸エチルで希釈し、５％クエン酸水溶液、飽和重曹水および飽和食塩水で順次洗浄した。有機層を無水硫酸マグネシウムで乾燥した後、溶媒を減圧留去した。得られた残渣を２−プロパノールから再結晶することにより白色結晶として標記化合物（０．２５ｇ，０．４６ｍｍｏｌ，収率６５％）を得た。
^１Ｈ−ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ_６）δ（ｐｐｍ）：０．６４−０．９３（ｍ，２Ｈ），０．９９−１．２３（ｍ，３Ｈ），１．２９−１．７８（ｍ，６Ｈ），２．９７（ｔ，Ｊ＝６．３Ｈｚ，２Ｈ），３．９９（ｓ，２Ｈ），７．０５−７．３４（ｍ，９Ｈ），７．７３（ｄ，Ｊ＝６．９Ｈｚ，１Ｈ），８．２４（ｂｒ，１Ｈ），８．２６（ｓ，１Ｈ），１１．８８（ｓ，１Ｈ）
ＡＰＣＩ−ＭＳ：ｍ／ｚ５４８（Ｍ＋Ｈ）^＋
ＩＲ（ＫＢｒ）：１７５７，１７４９，１７３２，１７１６，１６５３，１６４１，１５８３，１５５６，１５１０，１４９３，１３３１，１３１５，１１９４ｃｍ^−１
実施例１６：化合物５０〜２３４の合成
工程１
市販のエチル ４−クロロ−２−メチルチオ−５−ピリミジンカルボキシラートの１．７５ｇ／３０ｍＬテトラヒドロフラン（ＴＨＦ）溶液３００μＬ（１７．５ｍｇ，７５．２μｍｏｌ）に、各種アミン試薬（１００μｍｏｌ）およびモリホリノメチルポリスチレン（４２．０ｍｇ，１４７μｍｏｌ）を加え、６０℃で一晩攪拌した。ＴＨＦ（０．３ｍＬ）とベンゾイルクロリドポリマーバウンド（４５．０ｍｇ，９０μｍｏｌ）を加えてさらに一晩攪拌した後、不溶物を濾過、洗浄し、得られた濾液と洗浄液を合わせて濃縮し、残渣を得た。
工程２
工程１で得られた残渣をジクロロメタン（０．３ｍＬ）に溶解し、ｍ−クロ濾過安息香酸（含量６５％，２６．５ｍｇ，１００μｍｏｌ）を加えて１時間攪拌した。反応液をジクロロメタン（０．１ｍＬ）で希釈し、飽和重曹水で洗浄し、有機層を無水硫酸マグネシウムで乾燥した後、溶媒を留去した。得られた残渣をＴＨＦ（０．２ｍＬ）に溶解し、各種アミン（１００μｍｏｌ）を加え、６０℃で一晩攪拌した。クロロホルム（０．４ｍＬ）、モリホリノメチルポリスチレン（４２．０ｍｇ，１４７μｍｏｌ）およびベンゾイルクロリドポリマーバウンド（９０．０ｍｇ，１８０μｍｏｌ）を加えてさらに一晩攪拌した後、不溶物を濾過、洗浄し、得られた濾液と洗浄液を合わせて濃縮し、残渣を得た。
工程３
工程２で得られた残渣をエタノール（０．３ｍＬ）およびＴＨＦ（０．０５ｍＬ）の混合溶媒に溶解し、１ｍｏｌ／Ｌ ＮａＯＨ水溶液（１４５μＬ）を加えて６５℃で一晩攪拌した。メタノール（０．４ｍＬ）および１ｍｏｌ／Ｌ ＨＣｌ水溶液（１４５μＬ）を加えて攪拌した後、溶媒を留去し、得られた残渣にクロロホルム（０．３ｍＬ）、メタノール（０．１ｍＬ）およびヘキサフルオロイソプロピルアルコール（０．２ｍＬ）を加えてよく攪拌した。不溶物を濾別し、得られた濾液を濃縮することにより目的化合物５０〜２３４を得た。
以下に、実施例１６で得られた代表的化合物の収率、^１Ｈ ＮＭＲスペクトルデータおよびマススペクトルデータを示す。
２−（３−クロロベンジルアミノ）−４−（２−フェニルプロピルアミノ）ピリミジン−５−カルボン酸（化合物６４）
収率：５６％（４工程）
^１Ｈ−ＮＭＲ（３００ＭＨｚ，ＣＤＣｌ_３，８０℃）δ（ｐｐｍ）：１．１８（ｄ，Ｊ＝７．０Ｈｚ，３Ｈ），２．９９（ｍ，１Ｈ），３．５５（ｍ，２Ｈ），４．５９（ｄ，Ｊ＝６．２Ｈｚ，２Ｈ），７．１４−７．３５（ｍ，９Ｈ），７．７２（ｂｒ ｓ，１Ｈ），８．１９（ｂｒ ｔ，１Ｈ），８．３７（ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ３９７（Ｍ＋Ｈ）^＋
４−（２−フェニルプロピルアミノ）−２−（３−トリフルオロメチルベンジルアミノ）ピリミジン−５−カルボン酸（化合物７１）
収率：５６％（４工程）
^１Ｈ−ＮＭＲ（３００ＭＨｚ，ＤＭＳＯ−ｄ_６，８０℃）δ（ｐｐｍ）：１．１６（ｄ，Ｊ＝６．８Ｈｚ，３Ｈ），２．９８（ｍ，１Ｈ），３．５４（ｍ，２Ｈ），４．５９（ｄ，Ｊ＝６．４Ｈｚ，０．４Ｈ），４．６６（ｄ，Ｊ＝６．２Ｈｚ，１．６Ｈ），６．９５−７．７９（ｍ，９Ｈ），８．００（ｂｒ ｓ，０．２Ｈ），８．１７（ｂｒ ｓ，０．８Ｈ），８．３８（ｓ，０．２Ｈ），８．５９（ｓ，０．８Ｈ），１０．９０（ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ４３１（Ｍ＋Ｈ）^＋
２−（シクロヘキシルメチルアミノ）−４−（１−メチル−２−フェノキシエチルアミノ）ピリミジン−５−カルボン酸（化合物１０６）
収率：４６％（４工程）
^１Ｈ−ＮＭＲ（３００ＭＨｚ，ＣＤＣｌ_３，８０℃）δ（ｐｐｍ）：０．９２−１．０９（ｍ，２Ｈ），１．１３−１．２７（ｍ，３Ｈ），１．４３（ｄ，Ｊ＝６．８Ｈｚ，３Ｈ），１．５８−１．８０（ｍ，６Ｈ），３．２６（ｔ，Ｊ＝５．７Ｈｚ，２Ｈ），３．９６（ｄｄ，Ｊ＝６．０，９．１Ｈｚ，１Ｈ），４．１４（ｄｄ，Ｊ＝４．６，９．１Ｈｚ，１Ｈ），４．６１（ｍ，１Ｈ），６．９３（ｍ，３Ｈ），７．２３（ｍ，２Ｈ），８．３７（ｓ，１Ｈ），９．７７（ｂｒ ｓ，１Ｈ），１０．３（ｂｒ ｄ，Ｊ＝５．９Ｈｚ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ３８５（Ｍ＋Ｈ）^＋
４−（１−メチル−２−フェノキシエチルアミノ）−２−（３−トリフルオロメチルベンジルアミノ）ピリミジン−５−カルボン酸（化合物１１２）
収率：６５％（４工程）
^１Ｈ−ＮＭＲ（３００ＭＨｚ，ＣＤＣｌ_３，８０℃）δ（ｐｐｍ）：１．２９（ｄ，Ｊ＝６．６Ｈｚ，３Ｈ），３．８６（ｄｄ，Ｊ＝５．３，９．０Ｈｚ，１Ｈ），３．９６（ｄｄ，Ｊ＝４．４，９．０Ｈｚ，１Ｈ），４．４６（ｍ，１Ｈ），４．５５（ｄ，Ｊ＝５．１Ｈｚ，２Ｈ），６．８０（ｄ，Ｊ＝８．３Ｈｚ，２Ｈ），６．８８（ｔ，Ｊ＝７．５Ｈｚ，１Ｈ），７．１６（ｔ，Ｊ＝７．５Ｈｚ，２Ｈ），７．３０（ｔ，Ｊ＝７．７Ｈｚ，１Ｈ），７．４３（ｄ，Ｊ＝７．９Ｈｚ，１Ｈ），７．４８（ｄ，Ｊ＝７．５Ｈｚ，１Ｈ），７．５６（ｓ，１Ｈ），８．４１（ｓ，１Ｈ），１０．１６（ｂｒ ｓ，１Ｈ），１０．４７（ｂｒ ｔ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ４４７（Ｍ＋Ｈ）^＋
２−（ベンジルブチルアミノ）−４−（２−フェノキシエチルアミノ）ピリミジン−５−カルボン酸（化合物１１７）
収率：７４％（４工程）
^１Ｈ−ＮＭＲ（３００ＭＨｚ，ＣＤＣｌ_３，８０℃）δ（ｐｐｍ）：０．８９（ｔ，Ｊ＝７．３Ｈｚ，３Ｈ），１．３１（ｓｅｘｔｅｔ，Ｊ＝７．３Ｈｚ，２Ｈ），１．６０（ｑｕｉｎｔｅｔ，Ｊ＝７．３Ｈｚ，２Ｈ），３．６１（ｂｒ ｓ，２Ｈ），３．８２（ｂｒ ｓ，２Ｈ），４．０４（ｂｒ ｓ，２Ｈ），４．８９（ｓ，２Ｈ），６．８３−６．９３（ｍ，３Ｈ），７．１９−７．２９（ｍ，７Ｈ），８．１６（ｂｒ ｓ，１Ｈ），８．６９（ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ４２１（Ｍ＋Ｈ）^＋
２−（オクタヒドロイソキノリン−２−イル）−４−（２−フェノキシエチルアミノ）ピリミジン−５−カルボン酸（化合物１２１）
収率：３９％（４工程）
^１Ｈ−ＮＭＲ（３００ＭＨｚ，ＣＤＣｌ_３，８０℃）δ（ｐｐｍ）：１．２７−１．９４（ｍ，１２Ｈ），３．２８−３．４２（ｍ，２Ｈ），３．８７（ｑ，Ｊ＝５．８Ｈｚ，２Ｈ），４．１６（ｔ，Ｊ＝５．８Ｈｚ，２Ｈ），４．２６−４．４２（ｍ，２Ｈ），６．９０−６．９５（ｍ，３Ｈ），７．２４（ｔ，Ｊ＝７．７Ｈｚ，２Ｈ），８．１４（ｂｒ ｓ，１Ｈ），８．６３（ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ３９７（Ｍ＋Ｈ）^＋
４−（２−フェノキシエチルアミノ）−２−（３−トリフルオロメチルベンジルアミノ）ピリミジン−５−カルボン酸（化合物１２３）
収率：６０％（４工程）
^１Ｈ−ＮＭＲ（３００ＭＨｚ，ＤＭＳＯ−ｄ_６，８０℃）δ（ｐｐｍ）：３．７５（ｑ，Ｊ＝５．７Ｈｚ，２Ｈ），４．０４（ｔ，Ｊ＝５．７Ｈｚ，２Ｈ），４．５７（ｄ，Ｊ＝６．２Ｈｚ，２Ｈ），６．９０（ｄ，Ｊ＝７．３Ｈｚ，２Ｈ），６．９２（ｔ，Ｊ＝７．３Ｈｚ，１Ｈ），７．２５（ｔ，Ｊ＝７．３Ｈｚ，２Ｈ），７．４７−７．６３（ｍ，４Ｈ），７．７８（ｂｒ ｓ，１Ｈ），８．３９（ｂｒ ｓ，１Ｈ），８．４０（ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ４３３（Ｍ＋Ｈ）^＋
４−（２−フェノキシフェニルアミノ）−２−［（ピリジン−２−イルメチル）アミノ］ピリミジン−５−カルボン酸（化合物１３８）
収率：５０％（４工程）
^１Ｈ−ＮＭＲ（３００ＭＨｚ，ＤＭＳＯ−ｄ_６，８０℃）δ（ｐｐｍ）：４．６６（ｄ，Ｊ＝６．０Ｈｚ，２Ｈ），６．９４−７．１０（ｍ，６Ｈ），７．２０−７．３５（ｍ，４Ｈ），７．７１（ｄｔ，Ｊ＝１．８，７．５Ｈｚ，１Ｈ），８．０２（ｂｒ ｓ，１Ｈ），８．５４（ｔ，Ｊ＝４．２Ｈｚ，１Ｈ），８．５６（ｓ，１Ｈ），１０．８８（ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ４１４（Ｍ＋Ｈ）^＋
２−（シクロヘキシルメチルアミノ）−４−（２−フェノキシフェニルアミノ）ピリミジン−５−カルボン酸（化合物１３９）
収率：７０％（４工程）
^１Ｈ−ＮＭＲ（３００ＭＨｚ，ＤＭＳＯ−ｄ_６，８０℃）δ（ｐｐｍ）：０．９２−１．０９（ｍ，２Ｈ），１．１３−１．２７（ｍ，３Ｈ），１．６２−１．７７（ｍ，６Ｈ），３．２１（ｔ，Ｊ＝６．４Ｈｚ，２Ｈ），６．９５−７．１８（ｍ，６Ｈ），７．２９−７．３６（ｍ，２Ｈ），７．５３（ｂｒ ｓ，１Ｈ），８．５１（ｓ，１Ｈ），８．７３（ｂｒ ｓ，１Ｈ），１０．８７（ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ４１９（Ｍ＋Ｈ）^＋
２−（２−メチルベンジルアミノ）−４−（２−フェノキシフェニルアミノ）ピリミジン−５−カルボン酸（化合物１４２）
収率：７０％（４工程）
^１Ｈ−ＮＭＲ（３００ＭＨｚ，ＤＭＳＯ−ｄ_６，８０℃）δ（ｐｐｍ）：２．３２（ｓ，３Ｈ），４．５５（ｄ，Ｊ＝５．９Ｈｚ，２Ｈ），６．９４−７．３４（ｍ，１３Ｈ），７．９２（ｂｒ ｓ，１Ｈ），８．５６（ｓ，１Ｈ），１０．８４（ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ４２７（Ｍ＋Ｈ）^＋
２−（ベンジルブチルアミノ）−４−（２−フェノキシフェニルアミノ）ピリミジン−５−カルボン酸（化合物１４３）
収率：２０％（４工程）
^１Ｈ−ＮＭＲ（３００ＭＨｚ，ＣＤＣｌ_３，８０℃）δ（ｐｐｍ）：０．９２（ｔ，Ｊ＝７．３Ｈｚ，３Ｈ），１．３５（ｓｅｘｔｅｔ，Ｊ＝７．３Ｈｚ，２Ｈ），１．６５（ｑｕｉｎｔｅｔ，Ｊ＝７．３Ｈｚ，２Ｈ），３．６５（ｂｒ ｓ，２Ｈ），４．９４（ｓ，２Ｈ），６．９６−７．０４（ｍ，６Ｈ）７．２０−７．３２（ｍ，８Ｈ），８．７７（ｓ，１Ｈ），１０．４（ｂｒ ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ４６９（Ｍ＋Ｈ）^＋
４−（２−フェノキシフェニルアミノ）−２−（３−トリフルオロメチルベンジルアミノ）ピリミジン−５−カルボン酸（化合物１４８）
収率：６３％（４工程）
^１Ｈ−ＮＭＲ（３００ＭＨｚ，ＤＭＳＯ−ｄ_６，８０℃）δ（ｐｐｍ）：４．６４（ｄ，Ｊ＝６．２Ｈｚ，２Ｈ），６．９３−７．０９（ｍ，７Ｈ），７．２９（ｄｄ，Ｊ＝７．５，８．４Ｈｚ，２Ｈ），７．５１−７．６７（ｍ，４Ｈ），８．１３（ｂｒ ｓ，１Ｈ），８．５６（ｓ，１Ｈ），１０．８３（ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ４８１（Ｍ＋Ｈ）^＋
４−（４−フェニルピペリジン−１−イル）−２−（３−トリフルオロメチルベンジルアミノ）ピリミジン−５−カルボン酸（化合物１７７）
収率：７２％（４工程）
^１Ｈ−ＮＭＲ（３００ＭＨｚ，ＤＭＤＯ−ｄ_６，８０℃）δ（ｐｐｍ）：１．５６（ｍ，２Ｈ），１．７６（ｍ，２Ｈ），２．７７（ｔｔ，Ｊ＝３．８，１１．９Ｈｚ，１Ｈ），３．００（ｄｔ，Ｊ＝２．３，１４．４Ｈｚ，２Ｈ），４．０９（ｄ，Ｊ＝１３．２Ｈｚ，２Ｈ），４．５６（ｄ，Ｊ＝６．２Ｈｚ，２Ｈ），７．１４−７．３０（ｍ，５Ｈ），７．４８−７．６６（ｍ，５Ｈ），８．３７（ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ４５７（Ｍ＋Ｈ）^＋
実施例１７：化合物２３５〜３９３の合成
工程１
市販のエチル ４−クロロ−２−メチルチオ−５−ピリミジンカルボキシラートの１．５４ｇ／２２ｍＬ ＴＨＦ溶液２００μＬ（１４．０ｍｇ，６０．２μｍｏｌ）に、各種アミン試薬（９０μｍｏｌ）およびモリホリノメチルポリスチレン（４２．０ｍｇ，１４７μｍｏｌ）を加え、６０℃で一晩攪拌した。ＴＨＦ（０．３ｍＬ）とベンゾイルクロリドポリマーバウンド（４５．０ｍｇ，９０μｍｏｌ）を加え、さらに一晩攪拌した後、不溶物を濾過、洗浄し、得られた濾液と洗浄液を合わせて濃縮し、残渣を得た。
工程２
工程１で得られた残渣をジクロロメタン（０．２ｍＬ）に溶解し、ｍ−クロ濾過安息香酸（含量６５％，２３．９ｍｇ，９０．０μｍｏｌ）を加えて室温で１時間攪拌した。反応液をジクロロメタン（０．１ｍＬ）で希釈し、飽和重曹水で洗浄し、有機層を無水硫酸マグネシウムで乾燥した後、溶媒を留去した。得られた残渣をＴＨＦ（０．２ｍＬ）に溶解し、各種アミン（９０μｍｏｌ）を加え、６０℃で一晩攪拌した。クロロホルム（０．４ｍＬ）、モルホリノメチルポリスチレン（４２．０ｍｇ，１４７μｍｏｌ）およびベンゾイルクロリドポリマーバウンド（９０．０ｍｇ，１８０μｍｏｌ）を加えてさらに一晩攪拌した後、不溶物を濾過、洗浄し、得られた濾液と洗浄液を合わせて濃縮し、残渣を得た。
工程３
工程２で得られた残渣をトルエン（０．５ｍＬ）に溶解し、６５重量％水素化ビス（２−メトキシエトキシ）アルミニウム トルエン溶液（６２．４μＬ，２０８μｍｏｌ）を加え、０℃で４時間攪拌した。３ｍｏｌ／Ｌ水酸化ナトリウム水溶液（３００μＬ）を２回に分けて加えた後、有機層を飽和食塩水で洗浄した。有機層を無水硫酸マグネシウムで乾燥した後、溶媒を留去することにより目的化合物２３５〜３９３を得た。
以下に、実施例１７で得られた代表的化合物の収率、^１Ｈ ＮＭＲスペクトルデータおよびマススペクトルデータを示す。
２−（シクロヘキシルメチルアミノ）−５−ヒドロキシメチル−４−（１−メチル−２−フェノキシエチルアミノ）ピリミジン（化合物２５６）
収率：３８％（４工程）
^１Ｈ−ＮＭＲ（３００ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：０．８９−１．１０（ｍ，２Ｈ），１．１５−１．２４（ｍ，３Ｈ），１．３９（ｄ，Ｊ＝６．８Ｈｚ，３Ｈ），１．４７−１．７８（ｍ，６Ｈ），３．２１（ｄｔ，Ｊ＝２．６，６．６Ｈｚ，２Ｈ），３．９６（ｄｄ，Ｊ＝５．６，９．１Ｈｚ，１Ｈ），４．１０（ｄｄ，Ｊ＝３．８，９．１Ｈｚ，１Ｈ），４．４２（ｓ，２Ｈ），４．４６−４．６３（ｍ，１Ｈ），４．８６（ｂｒ ｔ，１Ｈ），５．６８（ｄ，Ｊ＝７．１Ｈｚ，１Ｈ），６．９４（ｔ，Ｊ＝８．６Ｈｚ，３Ｈ），７．２３−７．３０（ｍ，２Ｈ），７．５９（ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ３７１（Ｍ＋Ｈ）^＋
２−（ベンジルブチルアミノ）−５−ヒドロキシメチル−４−（２−フェノキシエチルアミノ）ピリミジン（化合物２６４）
収率：５５％（４工程）
^１Ｈ−ＮＭＲ（３００ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：０．８９（ｔ，Ｊ＝７．３Ｈｚ，３Ｈ），１．３１（ｓｅｘｔｅｔ，Ｊ＝７．１Ｈｚ，２Ｈ），１．５９（ｍ，２Ｈ），３．５５（ｔ，Ｊ＝７．５Ｈｚ，２Ｈ），３．７８（ｂｒ ｓ，２Ｈ），３．９８（ｂｒ ｓ，２Ｈ），４．４５（ｓ，２Ｈ），４．８４（ｓ，２Ｈ），５．８６（ｂｒ ｓ，１Ｈ），６．８１（ｄ，Ｊ＝８．２Ｈｚ，２Ｈ），６．９３（ｔ，Ｊ＝８．４Ｈｚ，１Ｈ），７．１８−７．３０（ｍ，７Ｈ），７．７１（ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ４０７（Ｍ＋Ｈ）^＋
５−ヒドロキシメチル−２−（オクタヒドロイソキノリン−２−イル）−４−（２−フェノキシエチルアミノ）ピリミジン（化合物２６７）
収率：４４％（４工程）
^１Ｈ−ＮＭＲ（３００ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：１．２５−１．９１（ｍ，１２Ｈ），３．２７（ｍ，２Ｈ），３．８９（ｍ，２Ｈ），４．１６（ｔ，Ｊ＝５．５Ｈｚ，２Ｈ），４．２６（ｍ，２Ｈ），４．４２（ｓ，２Ｈ），５．８５（ｂｒ ｔ，Ｊ＝５．１Ｈｚ，１Ｈ），６．８９−６．９７（ｍ，３Ｈ），７．２３−７．３０（ｍ，２Ｈ），７．６５（ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ３８３（Ｍ＋Ｈ）^＋
５−ヒドロキシメチル−４−（４−フェニルブチルアミノ）−２−（３−トリフルオロメチルベンジルアミノ）ピリミジン（化合物２７８）
収率：６０％（４工程）
^１Ｈ−ＮＭＲ（２７０ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：１．５７−１．６８（ｍ，４Ｈ），２．６０（ｔ，Ｊ＝７．３Ｈｚ，２Ｈ），３．３８（ｑ，Ｊ＝６．３Ｈｚ，２Ｈ），４．４０（ｓ，２Ｈ），４．６０（ｄ，Ｊ＝６．１Ｈｚ，２Ｈ），５．３６（ｂｒ ｓ，１Ｈ），５．６５（ｂｒ ｔ，１Ｈ），７．１２−７．２９（ｍ，５Ｈ），７．３９（ｔ，Ｊ＝７．４Ｈｚ，１Ｈ），７．４８（ｄ，Ｊ＝５．８Ｈｚ，１Ｈ），７．５１（ｄ，Ｊ＝６．５Ｈｚ，１Ｈ），７．５２（ｓ，１Ｈ），７．６０（ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ４３１（Ｍ＋Ｈ）^＋
２−（３−フルオロベンジルアミノ）−５−ヒドロキシメチル−４−（インダン−２−イルアミノ）ピリミジン（化合物２８７）
収率：６９％（４工程）
^１Ｈ−ＮＭＲ（２７０ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：２．２０（ｂｒ ｓ，１Ｈ），２．７９（ｄｄ，Ｊ＝６．３，１６．０Ｈｚ，２Ｈ），３．２７（ｄｄ，Ｊ＝７．４，１６．０Ｈｚ，２Ｈ），４．３８（ｓ，２Ｈ），４．５７（ｄ，Ｊ＝６．０Ｈｚ，２Ｈ），４．７９（ｍ，１Ｈ），５．３５（ｂｒ ｓ，１Ｈ），５．８５（ｄ，Ｊ＝６．６Ｈｚ，１Ｈ），６．９２（ｄｔ，Ｊ＝２．１，８．４Ｈｚ，１Ｈ），７．０７（ｔ，Ｊ＝７．８Ｈｚ，２Ｈ），７．１３−７．２９（ｍ，５Ｈ），７．５１（ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ３６５（Ｍ＋Ｈ）^＋
４−（２，３−ジヒドロベンゾ［１，４］ジオキシン−６−イルアミノ）−５−ヒドロキシメチル−２−（３−トリフルオロメチルベンジルアミノ）ピリミジン（化合物２９９）
収率：５９％（４工程）
^１Ｈ−ＮＭＲ（２７０ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：４．２３（ｓ，４Ｈ），４．５０（ｓ，２Ｈ），４．６１（ｄ，，Ｊ＝６．３Ｈｚ，２Ｈ），５．４６（ｂｒ ｓ，１Ｈ），６．７２（ｄ，Ｊ＝８．８Ｈｚ，１Ｈ），６．８１（ｄｄ，Ｊ＝２．５，８．７Ｈｚ，１Ｈ），７．２６（ｓ，１Ｈ），７．３９（ｔ，Ｊ＝７．６Ｈｚ，１Ｈ），７．４８（ｂｒ ｄ，Ｊ＝７．２Ｈｚ，２Ｈ），７．５９（ｓ，１Ｈ），７．６２（ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ４３３（Ｍ＋Ｈ）^＋
２−（シクロヘキシルメチルアミノ）−５−ヒドロキシメチル−４−（２−フェノキシフェニルアミノ）ピリミジン（化合物３２０）
収率：２７％（４工程）
^１Ｈ−ＮＭＲ（３００ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：０．９１−１．０３（ｍ，２Ｈ），１．１３−１．３１（ｍ，３Ｈ），１．６５−１．８３（ｍ，６Ｈ），３．２５（ｔ，Ｊ＝６．２Ｈｚ，２Ｈ），４．４０（ｓ，１Ｈ），５．０６（ｂｒ ｓ，１Ｈ），６．９６−７．２２（ｍ，６Ｈ），７．２７−７．３２（ｍ，３Ｈ），７．７１（ｓ，１Ｈ），８．４１（ｓ，１Ｈ），８．６２（ｄ，Ｊ＝６．２，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ４０５（Ｍ＋Ｈ）^＋
５−ヒドロキシメチル−２−（２−メチルベンジルアミノ）−４−（２−フェノキシフェニルアミノ）ピリミジン（化合物３２１）
収率：３３％（４工程）
^１Ｈ−ＮＭＲ（２７０ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：２．３６（ｓ，３Ｈ），４．４２（ｓ，２Ｈ），４．５８（ｄ，Ｊ＝５．４Ｈｚ，２Ｈ），５．２３（ｂｒ ｓ，１Ｈ），６．９３−７．０８（ｍ，６Ｈ），７．１２−７．３２（ｍ，７Ｈ），７．７０（ｓ，１Ｈ），８．３５（ｓ，１Ｈ），８．４０（ｄ，Ｊ＝７．６Ｈｚ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ４１３（Ｍ＋Ｈ）^＋
５−ヒドロキシメチル−４−（２−フェノキシエチルアミノ）−２−（３−トリフルオロメチルベンジルアミノ）ピリミジン（化合物３８６）
収率：４８％（４工程）
^１Ｈ−ＮＭＲ（３００ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：３．７８（ｑ，Ｊ＝５．３Ｈｚ，２Ｈ），４．００（ｔ，Ｊ＝５．３Ｈｚ，２Ｈ），４．４３（ｓ，２Ｈ），４．６２（ｄ，Ｊ＝６．０Ｈｚ，２Ｈ），５．３２（ｂｒ ｔ，１Ｈ），６．０５（ｂｒ ｔ，１Ｈ），６．８３（ｄ，Ｊ＝７．９Ｈｚ，２Ｈ），６．９３（ｔ，Ｊ＝７．３Ｈｚ，１Ｈ），７．２４−７．２９（ｍ，２Ｈ），７．３９（ｔ，Ｊ＝７．１Ｈｚ，１Ｈ），７．４９（ｄ，Ｊ＝６．４Ｈｚ，１Ｈ），７．５１（ｄ，Ｊ＝７．０Ｈｚ，１Ｈ），７．５９（ｓ，２Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ４１９（Ｍ＋Ｈ）^＋
５−ヒドロキシメチル−４−（２−フェノキシフェニルアミノ）−２−（３−トリフルオロメチルベンジルアミノ）ピリミジン（化合物３８７）
収率：２７％（４工程）
^１Ｈ−ＮＭＲ（２７０ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：４．４１（ｓ，２Ｈ），４．６５（ｄ，Ｊ＝６．１Ｈｚ，２Ｈ），５．５０（ｂｒ ｓ，１Ｈ），６．９２−７．０７（ｍ，６Ｈ），７．２３−７．６６（ｍ，２Ｈ），７．３９−７．６６（ｍ，５Ｈ），８．３１（ｂｒ ｄ，Ｊ＝６．４Ｈｚ，１Ｈ），８．４０（ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ４６７（Ｍ＋Ｈ）^＋
５−ヒドロキシメチル−４−（２−フェノキシフェニルアミノ）−２−［（ピリジン−２−イルメチル）アミノ］ピリミジン（化合物３８８）
収率：２５％（４工程）
^１Ｈ−ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ_６）δ（ｐｐｍ）：４．２６（ｄ，Ｊ＝４．４Ｈｚ，２Ｈ），４．５６（ｄ，Ｊ＝５．９Ｈｚ，２Ｈ），５．３２（ｔ，Ｊ＝４．８Ｈｚ，１Ｈ），６．９３−７．４７（ｍ，１２Ｈ），７．７１（ｄｔ，Ｊ＝１．８，７．７Ｈｚ，１Ｈ），７．７６（ｓ，１Ｈ），８．５２（ｄ，Ｊ＝４．０Ｈｚ，１Ｈ），８．７１（ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ４００（Ｍ＋Ｈ）^＋
２−（ベンジルブチルアミノ）−５−ヒドロキシメチル−４−（２−フェノキシフェニルアミノ）ピリミジン（化合物３８９）
収率：３１％（４工程）
^１Ｈ−ＮＭＲ（２７０ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：０．９２（ｔ，Ｊ＝７．２Ｈｚ，３Ｈ），１．３５（ｓｅｘｔｅｔ，Ｊ＝７．２Ｈｚ，２Ｈ），１．６０−１．６９（ｍ，２Ｈ），３．５８（ｔ，Ｊ＝７．７Ｈｚ，２Ｈ），４．３９（ｓ，２Ｈ），４．８７（ｓ，２Ｈ），６．９３−７．０７（ｍ，６Ｈ），７．１８−７．３２（ｍ，８Ｈ），７．７７（ｓ，１Ｈ），８．２８（ｂｒ ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ４５５（Ｍ＋Ｈ）^＋
５−ヒドロキシメチル−４−（１−メチル−２−フェノキシエチルアミノ）−２−（３−トリフルオロメチルベンジルアミノ）ピリミジン（化合物３９０）
収率：４６％（４工程）
^１Ｈ−ＮＭＲ（３００ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：１．３２（ｄ，Ｊ＝６．８Ｈｚ，３Ｈ），３．８６（ｄｄ，Ｊ＝５．１，９．１Ｈｚ，１Ｈ），３．９５（ｄｄ，Ｊ＝３．８，９．１Ｈｚ，１Ｈ），４．４２（ｓ，２Ｈ），４．４６−４．５３（ｍ，１Ｈ），４．６０（ｄ，Ｊ＝５．７Ｈｚ，２Ｈ），５．３０（ｂｒ ｓ，１Ｈ），５．７７（ｄ，Ｊ＝７．９Ｈｚ，１Ｈ），６．８７（ｄｄ，Ｊ＝１．１，７．７Ｈｚ，２Ｈ），６．９３（ｔｔ，Ｊ＝１．１，７．３Ｈｚ，１Ｈ），７．２１−７．２８（ｍ，２Ｈ），７．３８（ｔ，Ｊ＝７．７Ｈｚ，１Ｈ），７．４７（ｄ，Ｊ＝９．３Ｈｚ，１Ｈ），７．５１（ｄ，Ｊ＝７．８Ｈｚ，１Ｈ），７．５９（ｂｒ ｓ，１Ｈ），７．６１（ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ４３３（Ｍ＋Ｈ）^＋
５−ヒドロキシメチル−４−（２−フェニルプロピルアミノ）−２−（３−トリラルオロメチルベンジルアミノ）ピリミジン（化合物３９１）
収率：４８％（４工程）
^１Ｈ−ＮＭＲ（３００ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：１．２４（ｄ，Ｊ＝７．０Ｈｚ，３Ｈ），２．９８（ｓｅｘｔｅｔ，Ｊ＝７．０Ｈｚ，１Ｈ），３．４２（ｍ，１Ｈ），３．６２（ｍ，１Ｈ），４．３０（ｓ，２Ｈ），４．６５（ｄ，Ｊ＝５．９Ｈｚ，２Ｈ），５．２８（ｂｒ ｓ，１Ｈ），５．５５（ｂｒ ｓ，１Ｈ），７．１１−７．３０（ｍ，５Ｈ），７．４１（ｔ，Ｊ＝７．３Ｈｚ，１Ｈ），７．４９（ｄ，Ｊ＝８．４Ｈｚ，１Ｈ），７．５２（ｄ，Ｊ＝６．３Ｈｚ，１Ｈ），７．５３（ｓ，１Ｈ），７．６１（ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ４１７（Ｍ＋Ｈ）^＋
２−（３−クロロベンジルアミノ）−５−ヒドロキシメチル−４−（２−フェニルプロピルアミノ）ピリミジン（化合物３９２）
収率：４１％（４工程）
^１Ｈ−ＮＭＲ（３００ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：１．２６（ｄ，Ｊ＝７．０Ｈｚ，３Ｈ），２．９８（ｓｅｘｔｅｔ，Ｊ＝７．３Ｈｚ，１Ｈ），３．４３（ｍ，１Ｈ），３．６５（ｍ，１Ｈ），４．３１（ｓ，２Ｈ），４．５８（ｄ，Ｊ＝５．９Ｈｚ，２Ｈ），５．２３（ｂｒ ｓ，１Ｈ），５．５４（ｂｒ ｓ，１Ｈ），７．１２−７．３５（ｍ，９Ｈ），７．５５（ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ３８３（Ｍ＋Ｈ）^＋
５−ヒドロキシメチル−４−（４−フェニルピペリジン−１−イル）−２−（３−トリフルオロメチルベンジルアミノ）ピリミジン（化合物３９３）
収率：６４％（４工程）
^１Ｈ−ＮＭＲ（３００ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：１．７３（ｍ，２Ｈ），１．８８（ｍ，２Ｈ），２．７５（ｔｔ，Ｊ＝４．０，１２．１Ｈｚ，１Ｈ），２．９６（ｄｔ，Ｊ＝２．６，１３．２Ｈｚ，２Ｈ），４．３９（ｂｒ ｄ，Ｊ＝１３．２Ｈｚ，２Ｈ），４．５１（ｓ，２Ｈ），４．６４（ｄ，Ｊ＝６．２Ｈｚ，２Ｈ），５．３４（ｂｒ ｓ，１Ｈ），７．１８−７．３３（ｍ，５Ｈ），７．４１（ｔ，Ｊ＝７．７Ｈｚ，１Ｈ），７．４８（ｄ，Ｊ＝７．７Ｈｚ，１Ｈ），７．５３（ｄ，Ｊ＝７．７Ｈｚ，１Ｈ），７．６１（ｓ，１Ｈ），７．８８（ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ４４３（Ｍ＋Ｈ）^＋
実施例１８：２−（シクロヘキシルメチルアミノ）−４−（２−フェノキシエチルアミノ）ピリミジン−５−カルボン酸（化合物１１６）の合成
工程１：エチル ２−メチルチオ−４−（２−フェノキシエチルアミノ）ピリミジン−５−カルボキシラートの合成
市販のエチル ４−クロロ−２−メチルチオ−５−ピリミジンカルボキシラート（１１．３ｇ，４８．６ｍｍｏｌ）をテトラヒドロフラン（３００ｍＬ）に溶解し、Ｎ，Ｎ−ジイソプロピルエチルアミン（１６．９ｍＬ，９７．０ｍｍｏｌ）および２−フェノキシエチルアミン（９．５４ｍＬ，７２．９ｍｍｏｌ）を加え、室温で５時間攪拌した。反応液を減圧留去し、酢酸エチルで希釈した。有機層を水および飽和食塩水で順次洗浄し、無水硫酸マグネシウムで乾燥した後、溶媒を減圧留去した。得られた残渣をエタノールから再結晶することにより白色結晶として標記化合物（１６．１ｇ，４８．３ｍｍｏｌ，定量的収率）を得た。
^１Ｈ−ＮＭＲ（２７０ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：１．３５（ｔ，Ｊ＝７．１Ｈｚ，３Ｈ）２．５２（ｓ，３Ｈ），３．９６（ｑ，Ｊ＝５．５Ｈｚ，２Ｈ），４．１６（ｔ，Ｊ＝５．５Ｈｚ，２Ｈ），４．３３（ｑ，Ｊ＝７．１Ｈｚ，２Ｈ），６．９１−６．９８（ｍ，３Ｈ），７．２６（ｔ，Ｊ＝８．６Ｈｚ，２Ｈ），８．５４（ｂｒ ｓ，１Ｈ），８．６３（ｓ，１Ｈ）
工程２：エチル ２−（シクロヘキシルメチルアミノ）−４−（２−フェノキシエチルアミノ）ピリミジン−５−カルボキシラートの合成
工程１で得られたエチル ２−メチルチオ−４−（２−フェノキシエチルアミノ）ピリミジン−５−カルボキシラート（１０．０ｇ，３０．２ｍｍｏｌ）をジクロロメタン（２００ｍＬ）に溶解し、氷冷下ｍ−クロ濾過安息香酸（含量６５％，１２．０ｇ，４５．２ｍｍｏｌ）を１０分間かけて加え、室温で１時間攪拌した。反応液をジクロロメタンで希釈し、飽和重曹水で洗浄した。有機層を無水硫酸マグネシウムで乾燥した後、溶媒を減圧留去した。得られた残渣をテトラヒドロフラン（２００ｍＬ）に溶解し、シクロヘキシルメチルアミン（５．８９ｍＬ，４５．２ｍｍｏｌ）を加え、加熱還流下１０時間攪拌した。反応液を減圧留去し、得られた粗結晶をエタノールから再結晶することにより白色結晶として標記化合物（９．７４ｇ，２４．４ｍｍｏｌ，収率８１％）を得た。
^１Ｈ−ＮＭＲ（２７０ＭＨｚ，ＣＤＣｌ_３，８０℃）δ（ｐｐｍ）：０．９３−１．０６（ｍ，２Ｈ），１．１１−１．２７（ｍ，３Ｈ），１．３２（ｔ，Ｊ＝７．１Ｈｚ，３Ｈ），１．５１−１．７８（ｍ，６Ｈ），３．２８（ｔ，Ｊ＝６．６Ｈｚ，２Ｈ），３．８７（ｑ，Ｊ＝５．７Ｈｚ，２Ｈ），４．１５（ｔ，Ｊ＝５．７Ｈｚ，２Ｈ），４．２９（ｑ，Ｊ＝７．１Ｈｚ，２Ｈ），５．２２（ｂｒ ｓ，１Ｈ），６．８９−６．９５（ｍ，３Ｈ），７．２１−７．２８（ｍ，２Ｈ），８．３４（ｂｒ ｓ，１Ｈ），８．５５（ｓ，１Ｈ）
工程３：２−（シクロヘキシルメチルアミノ）−４−（２−フェノキシエチルアミノ）ピリミジン−５−カルボン酸（化合物１１６）の合成
工程２で得られたエチル ２−（シクロヘキシルメチルアミノ）−４−（２−フェノキシエチルアミノ）ピリミジン−５−カルボキシラート（１．００ｇ，２．５１ｍｍｏｌ）をエタノール（１０ｍＬ）に溶解し、３ｍｏｌ／Ｌ水酸化ナトリウム水溶液（１０．０ｍＬ，３０．０ｍｍｏｌ）を加え、５０℃で５時間攪拌した。反応液を半分の体積まで減圧濃縮し、１ｍｏｌ／Ｌ塩酸水溶液で反応液のｐＨを４．５に調整した。析出した結晶を濾取することにより白色結晶として標記化合物（８８０ｍｇ，２．２５ｍｍｏｌ，収率９５％）を得た。
^１Ｈ−ＮＭＲ（３００ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：０．９０−１．０１（ｍ，２Ｈ），１．０８−１．２５（ｍ，３Ｈ），１．５９−１．８０（ｍ，６Ｈ），３．２６（ｔ，Ｊ＝５．９Ｈｚ，２Ｈ），３．９３（ｑ，Ｊ＝５．５Ｈｚ，２Ｈ），４．１８（ｔ，Ｊ＝５．５Ｈｚ，２Ｈ），６．９１−６．９８（ｍ，３Ｈ），７．２５−７．３１（ｍ，２Ｈ），８．３６（ｓ，１Ｈ），９．８９（ｂｒ ｓ，１Ｈ），１０．３７（ｂｒ ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ３７１（Ｍ＋Ｈ）^＋
実施例１９：２−（シクロヘキシルメチルアミノ）−５−ヒドロキシメチル−４−（２−フェノキシエチルアミノ）ピリミジン（化合物２６３）の合成
実施例１８の工程２で得られたエチル ２−（シクロヘキシルメチルアミノ）−４−（２−フェノキシエチルアミノ）ピリミジン−５−カルボキシラート（１．００ｇ，２．５１ｍｍｏｌ）をトルエン（２０ｍＬ）に溶解し、フラスコ内をアルゴンガスで置換後、０℃で６５重量％水素化ビス（２−メトキシエトキシ）アルミニウム トルエン溶液（３．００ｍＬ，９．９９ｍｍｏｌ）を加え、０℃で２時間攪拌した。３ｍｏｌ／Ｌ水酸化ナトリウム水溶液を加えて反応を停止させ、酢酸エチルで希釈した。有機層を水、飽和食塩水で洗浄後、無水硫酸マグネシウムで乾燥し、溶媒を減圧留去した。得られた残渣をエタノールから再結晶することにより白色結晶として標記化合物（５００ｍｇ，１．４０ｍｍｏｌ，収率５６％）を得た。
^１Ｈ−ＮＭＲ（３００ＭＨｚ，ＣＤＣｌ_３）δ（ｐｐｍ）：０．９３−１．００（ｍ，２Ｈ），１．１６−１．２４（ｍ，３Ｈ），１．５１−１．７９（ｍ，６Ｈ），３．２０（ｔ，Ｊ＝６．４Ｈｚ，２Ｈ），３．８４（ｑ，Ｊ＝５．５Ｈｚ，２Ｈ），４．１４（ｔ，Ｊ＝５．５Ｈｚ，２Ｈ），４．３９（ｓ，２Ｈ），４．８７（ｂｒ ｔ，Ｊ＝５．５Ｈｚ，１Ｈ），６．０１（ｂｒ ｔ，Ｊ＝５．７Ｈｚ，１Ｈ），６．８９−６．９７（ｍ，３Ｈ），７．２４−７．３０（ｍ，２Ｈ），７．４９（ｓ，１Ｈ）
ＥＳＩ−ＭＳ：ｍ／ｚ３５７（Ｍ＋Ｈ）^＋
製剤例１：錠剤
常法により、次の組成からなる錠剤を調製する。
処方 化合物１ ２０ｍｇ
乳糖 １４３．４ｍｇ
馬鈴薯デンプン ３０ｍｇ
ヒドロキシプロピルセルロース ６ｍｇ
ステアリン酸マグネシウム ０．６ｍｇ
２００ｍｇ
製剤例２：注射剤
常法により、次の組成からなる注射剤を調製する。
処方 化合物３７ ２ｍｇ
精製ダイズ油 ２００ｍｇ
精製卵黄レシチン ２４ｍｇ
注射用グリセリン ５０ｍｇ
注射用蒸留水 １．７２ｍＬ
２．００ｍＬ  EXAMPLES The present invention will be described more specifically with reference to examples and reference examples. However, the scope of the present invention is not limited by these examples.
  The physicochemical data of each compound in the following examples and reference examples were measured by the following instruments.
¹H NMR: JEOL EX-270 type (270 MHz) or JEOL GX-270 type (270 MHz)
IR: HORIBA FT-200
MS: Micromass Quattro (APCI method) or MicroMass ZQ2000 (ESI method)
Reference Example 1 Construction of host cell KJMGER8 expressing Gal4-ER and inducible expression vectors pAGal9-d and pAGal9-nd
(1) Construction of Gal4-ER expression plasmid pGERbsrR2
  pSV2bsr (Kaken Pharmaceutical Co., Ltd.)PvuII andEcoAfter cutting with RI, Klenow processing and 2.6 kbPvuII (blunt end)-EcoRI (blunt end) fragments were obtained.
  yeast(Saccharomyces cerevisiae) Derived transcription factor Gal4p DNA binding region and estrogen receptor ligand binding region chimeric protein (Gal4-ER) gene [Cell,54, 199 (1988), Proc. Natl. Acad. Sci. USA,90, 1657 (1993)] containing ERαAF2 in pM (distributed by Prof. Shigeaki Kato of the University of Tokyo)AatII andNdeAfter cutting with I, Klenow processing,AatII (blunt end)-NdeI (blunt end) fragment was obtained.
  Derived from the above pSV2bsrPvuII (blunt end)-EcoFrom RI (blunt end) fragment and ERαAF2 in pMAatII (blunt end)-NdeA Gal4-ER expression plasmid pGERbsrR2 was constructed by ligating the I (blunt end) fragment.
(2) Construction of an inducible expression plasmid for firefly luciferase
  pcDNA3 (Invitrogen)XhoAfter cutting with I, Klenow processing,XhoI (blunt end) fragment was obtained. By joining the fragments,XhoPcDNA3 in which the I cleavage site was eliminated was constructed.XhoPcDNA3 from which the I-cleavage site has been eliminatedKpnAfter cutting with I, Klenow processing,KpnI (blunt end) fragment was obtained. By joining the fragments,XhoI andKpnPcDNA3 in which the I cleavage site was eliminated was constructed. The plasmidBglAfter cutting with II, Klenow processing,BglII (blunt end) fragment was obtained.
  pAMoERC3Sc (Japanese Patent Laid-Open No. 05-336963)XhoI andNsiAfter cutting with I, Klenow processing and 2.2 kb including the oriP sequenceXhoI (blunt end)-NsiI (blunt end) fragment was obtained.
  aboveXhoI cleavage site andKpnDerived from pcDNA3 in which the I cleavage site has been deletedBglFrom the II (blunt end) fragment and pAMoERC3ScXhoI (blunt end)-NsiPlasmid pcDNA3-oriP was constructed by ligating I (blunt end) fragments. pcDNA3-oriPXhoI andHincut with dIII,XhoI-HinA dIII fragment was obtained.
  pSE0luc2 (WO98 / 14474)XhoI andNcoAfter cutting with I, Klenow treatment and ampicillin resistance gene is includedXhoI (blunt end)-NcoI (blunt end) fragment was obtained. Plasmid pASd1-luc1 was constructed by ligating the fragments. pASd1-luc1XhoI andHinAfter cutting with dIII, 0.11 kbXhoI-HinA dIII fragment was obtained.
  Derived from pcDNA3-oriPXhoI-Hinderived from dIII fragment and pASd1-luc1XhoI-HinThe dIII fragment was ligated to construct plasmid pcDNA3-oriP-Sd1. pcDNA3-oriP-Sd1XhoI andKpnCut with I,XhoI-KpnI fragment was obtained.
  Four types of DNAs having the base sequences represented by SEQ ID NOs: 8, 9, 10 and 11 were synthesized. The synthetic DNA is mixed and annealed to form a double-stranded DNA having a polyadenylation signal. Each of the synthetic DNAs was phosphorylated using T4 polynucleotide kinase, mixed and annealed to obtain double-stranded DNA.
  Derived from the double-stranded DNA and pcDNA3-oriP-Sd1XhoI-AspThe plasmid pcDNA3-oriP-Sd1-pA was constructed by ligating the 718 fragment. pcDNA3-oriP-Sd1-pAXhoAfter cutting with I, Klenow processing,XhoI (blunt end) fragment was obtained.
  pFR-luc (manufactured by Stratagene)HindIII andBamCut with HI, Klenow processing, 0.14 kbHindIII (blunt end)-BamHI (blunt end) fragments were obtained.
  Derived from pcDNA3-oriP-Sd1-pAXhoDerived from I (blunt end) fragment and pFR-lucHindIII (blunt end)-BamThe HI (blunt end) fragment was ligated to produce plasmid pAGalSd1. pAGalSd1 contains a promoter having a sequence in which a Gal4p response element (UASG) is repeated five times. pAGalSd1EcoAfter cutting with RI, Klenow processing,EcoRI (blunt end) fragments were obtained.
  pSE0luc2 (WO98 / 14474)HindIII andSacAfter cutting with I, it is 1.7 kb containing the firefly luciferase gene by Klenow treatment.HindIII (blunt end)-SacI (blunt end) fragment was obtained.
  Derived from pSE0luc2HindIII (blunt end)-ScaFrom the I (blunt end) fragment and pAGalSd1EcoPlasmid pAGalSd1-luc was constructed by ligating RI (blunt end) fragments.
  The two existing in pAGalSd1-luc aboveHinOne of the dIII sites located away from the firefly luciferase geneHinpAGalSd4-luc was constructed by eliminating only the dIII site by Klenow treatment.
  PAGalSd4-luc aboveAspAfter cutting at 718,Stu9.5 kb derived from pAGalSd4-lucAsp718-StuI fragment was obtained. The DNA fragment was treated with Klenow and self-ligated to construct plasmid pAGal9-luc.
(3) Construction of inducible expression vectors pAGal9-d and pAGal9-nd
  Plasmid pAGal9-luc constructed in (2) above isHindIII andSacCut with I and 6.9 kb including oriPHindIII-SacI fragment was obtained.
  pAMo-d (Japanese Patent Laid-Open No. 2001-211885)HindIII andSacCleaved with I, tetracycline resistance gene (Tc^R)includingHindIII-SacI fragment was obtained.
  Derived from the above pAGal9-lucHindIII-SacFrom the I fragment and pAMo-dHindIII-SacBy ligating the I fragment, a plasmid pAGal9-d was constructed in which the firefly luciferase gene portion in pAGal9-luc was replaced with the stuffer sequence of pAMo-d.
  pAMo-nd (JP 2001-211885)HindIII-SacI cut and contains a tetracycline resistance geneHindIII-SacI fragment was obtained.
  Derived from the above pAGal9-lucHindIII-SacI fragment, and from pAMo-ndHindIII-SacBy ligating the I fragment, a plasmid pAGal9-nd was constructed in which the firefly luciferase gene portion in pAGal9-luc was replaced with the Staff sequence of pAMo-nd.
(4) Construction of a cell line KJMGER8 in which the Gal4-ER expression plasmid pGERbsrR2 is incorporated into the chromosomal DNA of Namalwa KJM-1 cells
  PGERbsrR2 prepared in (1) was dissolved in TE buffer [10 mmol / LTris-HCl (PH8.0), 1 mmol / L ethylenediaminetetraacetic acid] so as to be 1 μg / μL, and then electroporation method [Cytotechnology,3133 (1990)], the plasmid was transformed into Namalwa KJM-1 cells [Cytotechnology,1, 151 (1988)], 1.6 × 10⁶4 μg per cell was introduced to obtain transformed cells. Namalwa KJM-1 cells are a serum-free conditioned B cell line that expresses the EBNA-1 gene.
  The transformant was added to 8 mL of RPMI1640 · ITPSG medium [RPMI1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) at a 1/40 amount of 7.5% NaHCO 3.₃3% 200 mmol / L L-glutamine solution (Invitrogen), 0.5% penicillin / streptomycin solution (Invitrogen, 5,000 units / mL penicillin, 5,000 μg / mL streptomycin), 10 mmol / L N-2 -Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (N-2-hydroxyethylperazine-N'-2-ethanesulfonic acid; HEPES), 3 μg / mL insulin, 5 μg / mL transferrin, 5 mmol / L sodium pyruvate, 125 nmol / L-sodium selenite, a medium supplemented with 1 mg / mL galactose]₂The cells were cultured for 24 hours at 37 ° C. in an incubator.
  After culturing, Blastcidin S (KK-400: manufactured by Kaken Pharmaceutical Co., Ltd.) was added to 2.0 μg / mL, and dispensed into a 96-well plate (500 to 2000 cells / hole). A number of stable transformants (single clones) in which pGERbsrR2 was incorporated into the chromosomal DNA were obtained. Each transformant was subcultured in RPMI1640 • ITPSG medium containing 2.0 μg / mL of blasticidin S.
  An excellent stable transformant KJMGER8 cell having a high induction ratio and a low background during non-induction was selected from the stable transformants by the method described below.
  Firefly luciferase-inducible expression plasmid pAGalSd1-luc was introduced into each transformant by electroporation and cultured for 2 days.
  After culturing, E2 (E8875: Sigma-Aldrich) (final concentration 10 nmol / L) was added, and after further culturing for 24 hours, firefly luciferase activity was measured. The activity was measured using a luminometer LB953 (manufactured by Berthold) and a cell lysis buffer [1% Triton X-100, 100 mmol / L KH.₂PO₄(PH 7.8) 1 mmol / L dithiothreitol] 100 μL was automatically injected into the culture solution, and then the substrate solution [25 mmol / L glycylglycine (pH 7.8), 15 mmol / L MgSO 4₄5 mmol / L ATP, 0.33 mmol / L luciferin] 300 μL was automatically injected, and the amount of luminescence for 10 seconds was measured to obtain luciferase activity. For comparison, luciferase activity was also measured under the condition where E2 was not added.
  By comparing the luciferase activity under the condition of E2 addition and the luciferase activity under the condition of no addition of E2, the induction ratio of gene expression was calculated, and the clone with high induction ratio and low luciferase activity under the condition of no addition of E2 KJMGER8 cells were selected.
(5) Induced expression of firefly luciferase gene using KJMGER8 as a host
  The firefly luciferase-inducible expression plasmid pAGal9-luc constructed in (2) or the control plasmid (pAGal9-nd) constructed in (3) was added to the KJMGER8 cells selected in (4) by the electroporation method. × 10⁶4 μg was introduced per cell, and stable transformants (KJMGER8 / pAGal9-luc and KJMGER8 / pAGal9-nd) were obtained.
  The stable transformant was suspended in 8 mL of RPMI1640 • ITPSG medium, and CO₂The cells were cultured for 24 hours at 37 ° C. in an incubator.
  After the culture, blasticidin S (0.2 μg / mL) and geneticin (Geneticin; manufactured by Invitrogen) (0.5 mg / mL) were added, and further cultured for 14 days to obtain a stable transformant. The transformed strain was subcultured in RPMI1640 • ITPSG medium containing Blasticidin S (0.2 μg / mL) and Geneticin (Invitrogen) (0.5 mg / mL).
  E2 (E8875: manufactured by Sigma) (final concentration: 10 nmol / L) was added to the transformant and cultured for 24 hours, and then luciferase activity was measured in the same manner as described above. For comparison, luciferase activity was also measured under the condition where E2 was not added.
  The induction ratio of gene expression was calculated by comparing the luciferase activity under the condition of E2 addition and the luciferase activity under the condition of no addition of E2. As a result, the induction rate of gene expression in KJMGER8 / pAGal9-luc was about 10,000 times. The luciferase activity in KJMGER8 / pAGal9-luc under the condition of no addition of E2 was very low (about 60 RLU / 10 seconds). This result shows that the luciferase gene is hardly expressed under the condition of no addition of E2.
  As described above, in the system using pAGal9-luc as an inducible expression plasmid and KJMGER8 as a host, the luciferase activity during non-induction was very low, and the luciferase activity during induction was very high. The structure of pAGal9-luc is a structure in which a firefly luciferase gene is inserted as a reporter gene that is induced to be expressed by pAGal9-nd or pAGal9-d. Therefore, the system using KJMGER8 as a host and pAGal9-nd or pAGal-d as an inducible expression vector is an extremely excellent inducible expression system in which the gene expression level when not induced is very low and the induction rate of gene expression is high. It turned out to be.
Reference Example 2 Production of host cells GBC7 and GBCR2 expressing Gal4-ER and introduced with a reporter plasmid
(1) Construction of reporter plasmids pACREluc and pACREluc using firefly luciferase as a reporter
  pACREluc and pACREluc, which are reporter plasmids capable of expressing the firefly luciferase gene under the control of the cAMP response element (CRE), were constructed by the following method. pACREluc and pACREluc have a hygromycin resistance gene and Epstein-Barr virus oriP.
  pAMo [J. Biol. Chem. ,268, 22782 (1993), also known as pAMoPRC3Sc (Japanese Patent Laid-Open No. 05-336963)]ClaPartially digested with I to obtain a DNA fragment cleaved at one place. The DNA fragmentMluPartial digestion with I, 9.5 kbClaI-MluI fragment was obtained. pAGE248 [J. Biol. Chem. ,269, 14730 (1994)]ClaI andMluCut with I and 1.5 kb containing the hygromycin resistance geneClaI-MluI fragment was obtained. derived from pAMoClaI-MluI fragment, and from pAGE248ClaI-MluThe I fragment was ligated to construct plasmid pAMoh.
  Created pAMohXhoI andHinContains hygromycin resistance gene after digestion with dIIIXhoI-HinA dIII fragment was obtained. pAGal9-lucSalI andHincut with dIII, including oriP, UASGSalI-HinA dIII fragment was obtained. derived from pAGal9-lucSalI-HindIII fragment, and from pAMoh aboveXhoI-HinPlasmid pAGal9h was constructed by ligating the dIII fragment.
  pBluescript II KS + (Stratagene)SalI andXhoAfter digestion with I, dephosphorylation using alkaline phosphatase (derived from E. coli C75, manufactured by Takara Shuzo Co., Ltd.), containing ampicillin resistance geneSalI-XhoI fragment was obtained. Double-stranded DNA containing two CRE sequences was prepared by annealing synthetic oligonucleotides having the base sequences represented by SEQ ID NOs: 12 and 13. The double-stranded DNA and the above derived from pBluescript II KS +SalI-XhoThe I fragment was ligated to construct a plasmid pBS-CREI containing two CRE sequences. pBS-CREI has the double-stranded DNASalI cleavage site andXhoA plasmid in which I cleavage sites are incorporated in the direction of regeneration, and each has one of the cleavage sites.
  pBS-CREIScaI andXhoCleaved with I and contains ori of phage f1ScaI-XhoI fragment was obtained. pBS-CREIScaI andSalCut with I and include ColE1 oriScaI-SalI fragment was obtained. derived from pBS-CREIScaI-XhoI fragment andScaI-SalThe I fragment was ligated to create pBS-CREII containing 4 CRE sequences.
  PBS-CREII aboveScaI andXhoCleaved with I and contains ori of phage f1ScaI-XhoI fragment was obtained. pBS-CREIIScaI andSalCut with I and include ColE1 oriScaI-SalI fragment was obtained. derived from pBS-CREIIScaI-XhoI fragment andScaI-SalThe I fragment was ligated to create pBS-CREIV containing 8 CRE sequences.
  PBS-CREIV aboveScaI andXhoCleaved with I and contains ori of phage f1ScaI-XhoI fragment was obtained. pBS-CREIVScaI andSalCut with I and include ColE1 oriScaI-SalI fragment was obtained. derived from pBS-CREIVScaI-XhoI fragment andScaI-SalThe I fragment was ligated to create pBS-CREVIII containing 16 CRE sequences.
  PBS-CREVIII aboveXhoAfter cutting with I, Klenow processing,HinContains 16 CREs by cutting with dIIIHindIII-XhoI (blunt end) fragment was obtained. pAGalSd1MluI andHincut with dIII, 1.4 kbMluI-HinA dIII fragment was obtained. pAGal9hXbaAfter cutting with I, Klenow processing,MluBy cutting with IXbaI (blunt end)-MluI fragment was obtained. derived from pBS-CREVIIIHindIII-XhoI (blunt end) fragment, derived from pAGalSd1MluI-HindIII fragment, and from pAGal9hXbaI (blunt end)-MluThe I fragment was ligated to produce plasmid pACREh.
  PAGal9-luc prepared in (2) of Reference Example 2XhoI andNotCut with I and contains the firefly luciferase geneXhoI-NotI fragment was obtained. pACREhXhoI andNotCleaved with I and contains CRE sequenceXhoI-NotI fragment was obtained. derived from pAGal9-lucXhoI-NotI fragment, and from pACREhXhoI-NotPlasmid pACREluc was constructed by ligating the I fragment.
  PACCREluc aboveHinKlenow treatment after cutting with dIII,XhoIncluding CRE by cutting with IHindIII (blunt end)-XhoIncludes I fragment and firefly luciferase geneHindIII (blunt end)-XhoEach I fragment was obtained. The above two types derived from pACRElucHindIII (blunt end)-XhoBy linking the I fragment upstream of the CRE sequence in pACREluc.HinPlasmid pACRElucH in which the dIII site disappeared was prepared.
  pGL3-Enhancer vector (manufactured by Promega)HindIII andHpaCleaved with I and contains luc + gene (modified firefly luciferase gene)HindIII-HpaI fragment was obtained. PACRElucH prepared aboveNotAfter cutting with I, Klenow processing,HinIncluding CRE by cutting with dIIIHinA dIII-NotI (blunt end) fragment was obtained. derived from pGL3-Enhancer vectorHindIII-HpaI fragment, and from pACRElucHHindIII-NotThe reporter plasmid pACREplus was made by ligating the I (blunt end) fragment.
(2) Construction of Namalwa KJM-1 cells introduced with reporter plasmid pACREplus
  After the reporter plasmid pACREplus prepared in (1) above was dissolved in TE buffer so as to be 1 μg / μL, the plasmid was transferred to Namalwa KJM-1 cells by electroporation method at 1.6 × 10 6.⁶4 μg per cell was introduced to obtain transformed cells.
  The transformed cells are suspended in 8 mL of RPMI1640 • ITPSG medium, and CO₂The cells were cultured for 24 hours at 37 ° C. in an incubator. After the culture, hygromycin B (300 μg / mL) was added, and further cultured for 14 days to obtain a stable transformant. The transformed strain was subcultured in RPMI1640-ITPSG medium containing hygromycin B (300 μg / mL).
  In order to confirm whether gene expression via CRE occurs, the transformed strain was treated with 5′-N-ethylcarboxamide adenosine (NECA: Sigma-Aldrich), which is an agonist of type 2a adenosine receptor (A2a). ), Forskolin (manufactured by Sigma-Aldrich) as an activator of adenylate cyclase, or A23187 (manufactured by Research Biochemicals International) as a calcium ionophore under the following conditions I was stimulated.
  That is, the stable transformant is 1 × 10 4 per well of 48-well plate.⁵After cell aliquots and adding NECA (final concentration 100 nmol / L), forskolin (final concentration 100 μmol / L), or A23187 (final concentration 10 μmol / L), CO₂The cells were cultured at 37 ° C. for 5 hours in an incubator and stimulated.
  After stimulation, firefly luciferase activity was measured using the method described in (4) of Reference Example 1.
  As a result, the firefly luciferase activity was increased about 7 times by NECA stimulation and about 10 times by forskolin stimulation. On the other hand, A23187 stimulation hardly changed the firefly luciferase activity.
  From the above results, it has been clarified that in Namalwa KJM-1 cells into which pACREplus has been introduced, the luciferase gene is expressed by stimulation that promotes transcription from CRE.
  That is, by using Namalwa KJM-1 cells into which pACREplus has been introduced, it is possible to search for substances having an activity to promote transcription from CRE.
  Namalwa KJM-1 cells into which pACREplus had been introduced were stimulated with NECA, and firefly luciferase activity was measured after 6, 24, 48, 72, and 96 hours. As a result, the activity reached its maximum at 6 hours and was almost halved at 24 hours. . Therefore, when stimulating the cells with an arbitrary substance, it was considered preferable to measure the activity 6 hours after the stimulation.
(3) Construction of a stable transformant in which the reporter plasmid pACREplus is incorporated into the chromosomal DNA of KJMGER8
  It is a restriction enzyme existing in one place in oriP in pACREplus produced in (1)HpaI,SpeI, orBalPACREplus cleaved with I was dissolved in TE buffer so as to have a concentration of 1 μg / μL, and then was electroporated to 1.6 × 10 6 in KJMGER8 prepared in (4) of Reference Example 1.⁶4 μg per cell was introduced to obtain transformed cells.
  The transformed cells are suspended in 8 mL of RPMI1640 • ITPSG medium, and CO₂The cells were cultured for 24 hours at 37 ° C. in an incubator. After the culture, blasticidin S (2.0 μg / mL) and hygromycin B (300 μg / mL) were added, and further cultured for 7 days. After confirming the number of viable cells, blasticidin S (2.0 μg / mL), hygromycin B (300 μg / mL) and KJMGER8 were added at 1 × 10⁵In RPMI1640 / ITPSG medium containing cells / mL, dilute to 150 cells / mL, dispense into 96-well plates (average 30 cells / well), and culture, actually form 1 colony per well Thus, 420 stable transformants (single clones) were obtained. Each transformant was passaged in RPMI 1640 • ITPSG medium containing blasticidin S (2.0 μg / mL) and hygromycin B (300 μg / mL).
(4) Selection of stable transformants (single clones) with excellent properties
  Each clone obtained in (3) above is seeded in a 96-well plate (1-2 × 10⁴Cells / well), NECA (final concentration 100 nmol / L) and CO₂The cells were cultured at 37 ° C. for 6 hours in an incubator.
  After the culture, firefly luciferase activity in each well was measured using the method described in (4) of Reference Example 1. As a measuring instrument, Micro Lumat LB96P (manufactured by Bertrand) was used. 17 clones with high activity were selected.
  Using the 17 clones, NECa stimulation was carried out in the same manner as in (2) above, and 8 clones in which the luciferase activity was increased by 60 times or more were selected as compared with the case where NECA stimulation was not performed. The 8 clones were named GBC1, GBC2, GBC3, GBC4, GBC5, GBC6, GBC7 and GBC8, respectively.
  PAGal9-GPR12 [plasmid for inducing and expressing GPR12, a constitutively active G protein-coupled receptor: see (3) in Reference Example 4 below] was added to the 8 clones according to the above electroporation method. 6x10⁶4 μg was introduced per cell to obtain a transformed strain.
  The transformed strain was suspended in 8 mL of RPMI1640 · ITPSG medium, and CO₂The cells were cultured for 24 hours at 37 ° C. in an incubator.
  After culturing, blasticidin S (2.0 μg / mL), hygromycin B (300 μg / mL) and geneticin (500 μg / mL) were added, and further cultured for 14 days to obtain a stable transformant. The transformed strain was subcultured in RPMI1640 · ITPSG medium containing blasticidin S (2.0 μg / mL), hygromycin B (300 μg / mL) and geneticin (500 μg / mL).
  E2 (final concentration 10 nmol / L) was added to each transformant, and after culturing for 24 hours, luciferase activity was measured by the same method as in Reference Example 1 (4). For comparison, luciferase activity was also measured under the condition where E2 was not added.
  Three clones (GBC5, GBC6, and GBC7) that had a high rate of increase in luciferase activity when E2 was added were selected. The rates of increase in GBC5, GBC6 and GBC7 were 56, 193 and 364 times, respectively.
  A type 2 vasopressin receptor (V2) induced expression plasmid pAGal9-V2 (see (1) of Reference Example 4 below) was introduced into the 3 clones in the same manner as described above to obtain a stable transformant, and E2 stimulation was performed. After inducing expression of the transgene (V2 gene), the luciferase activity when stimulated with vasopressin was examined. At the same time, the activity without E2 stimulation and the activity without vasopressin stimulation were examined.
  As a result, GBC7, which showed high luciferase activity (599 times) in response to vasopressin only when stimulated with E2, was selected as a superior strain.
(5) Construction of a reporter plasmid pACLERluc using Renilla luciferase as a reporter
  pRL-SV40 vector (manufactured by Promega)XbaAfter cutting with I and Klenow processing,HinCleaved with dIII and contains the Renilla luciferase geneHindIII-XbaI (blunt end) fragment was obtained. PACRElucH constructed in (1) aboveNotAfter cutting with I, Klenow processing,HinIncluding CRE by cutting with dIIIHindIII-NotI (blunt end) fragment was obtained. derived from pRL-SV40 vectorHindIII-XbaI (blunt end) fragment, and from pACRElucHHindIII-NotPlasmid pACLERluc was constructed by ligating the I (blunt end) fragment.
(6) Construction of a stable transformant in which the reporter plasmid pACLERluc is incorporated into the chromosomal DNA of KJMGER8
  According to the method described in (3) above, pACLERluc was introduced into KJMGER8 to obtain 96 stable transformants (single clones). Each transformant was passaged in RPMI 1640 • ITPSG medium containing blasticidin S (2.0 μg / mL) and hygromycin B (300 μg / mL).
(7) Selection of stable transformants (single clones) having excellent properties
  According to the method described in (4) above, except that the activity of Renilla luciferase is measured instead of measuring the activity of firefly luciferase, clones having excellent properties among the clones obtained in (6) above are used. Made a selection.
  Each clone obtained in (6) above is plated on a 96-well plate (1-2 × 10⁴Cells / well), NECA (final concentration 100 nmol / L) and CO₂The cells were cultured at 37 ° C. for 6 hours in an incubator. Thereafter, coelenterazine h (Molecular Probes) (final concentration 250 nmol / L) was added to measure Renilla luciferase activity. For the activity measurement, a Wallac 1420 ARVOsx multi-label counter (manufactured by Wallac Bertoled Japan) was used. Nine clones with high activity were selected. The nine clones were named GBCR1, GBCR2, GBCR3, GBCR4, GBCR5, GBCR6, GBCR7, GBCR8 and GBCR9, respectively.
  Of these 9 clones, 2 clones in which Renilla luciferase activity was increased 18-fold or more were selected as compared with the case of NECA stimulation and no NECA stimulation. The two clones were named GBCR1 and GBCR2, respectively.
  PAGal9-GPR12 was introduced into the two clones to obtain stable transformants. The transformed strain was stimulated with E2 for 24 hours, and then Renilla luciferase activity was measured. For comparison, Renilla luciferase activity was also measured under the condition where E2 was not added. One clone (GBCR2) having a high increase rate in Renilla luciferase activity when E2 was added was selected as a superior strain. The rate of increase was 364 times.
Reference Example 3 Chimeric Gα_sOf host cells GBCC13 and GBCRC6 that expresses Gal4-ER and introduces a reporter plasmid
(1) Total RNA was obtained from Nomalwa KJM-1 cells using RNeasy Mini Kit (manufactured by QIAGEN). Single-stranded cDNA was synthesized by SUPERSCRIPT First-Strand Synthesis System for RT-PCR (manufactured by Invitrogen) using 5 μg of the total RNA as a template. The single-stranded cDNA was diluted 50-fold with water and used as a PCR template.
  To the above single-stranded cDNA (10 μL), Gα_s4Gene-specific primer (20 pmol each), 2.5 μmol / L dNTP mixed solution, 4 μL, dimethyl sulfoxide 2.5 μL, 5 unit / μLPyrobest DNA polymerase (Takara Shuzo) 0.25 μL, 10 × reaction buffer 5 μL of Takara Shuzo was added, and sterilized water was added to make a total volume of 50 μL. Gα_s4As gene-specific primers, synthetic DNAs having the sequences described in SEQ ID NOs: 14 and 15 were used. Each of these primersHindIII site andAsp718 sites have been introduced. Using a thermal cycler DNA engine (manufactured by MJ Research) for 5 minutes at 95 ° C, PCR was performed under the conditions of 30 cycles at 94 ° C for 30 seconds, 65 ° C for 1 minute, and 72 ° C for 2 minutes. went. The DNA fragment amplified by PCR was recovered by agarose gel electrophoresis. The amplified fragmentHindIII andAspCut at 718,HindIII-Asp718 fragments were obtained.
  Plasmid pAMohHindIII andAspAfter cutting at 718,HindIII-Asp718 fragments were obtained.
  Derived from PCR fragmentHindIII-Asp718 fragment, and from pAMohHindIII-AspBy binding the 718 fragment, Gα_s4The expression plasmid pAMoh-Gs4 was constructed.
(2) Construction of plasmid pAMoh-Gs-q
  Gα_sOf the C-terminal 5 amino acids of Gα_qChimeric Gα substituted with 5 amino acids of C-terminal_s(Hereafter, Gα_sqThe expression plasmid pAMoh-Gs-q was constructed as follows.
  PAMoh-Gs4 constructed in the above (1)XbaI andAspInclude oriP by cutting at 718XbaI-Asp718 fragments were obtained. In addition, pAMoh-Gs4XbaAfter cutting with I,SphGα lacking the C-terminus by partial digestion with I_s4CodeXbaI-SphI fragment was obtained.
  By synthesizing a synthetic DNA having the nucleotide sequences represented by SEQ ID NOs: 16 and 17 with T4 polynucleotide kinase and annealing, Gα_qA double-stranded DNA containing a region encoding the C-terminal 5 amino acids was obtained.
  This double-stranded DNA, derived from pAMoh-Gs4XbaI-Asp718 fragments andXbaI-SphPAMoh-Gs-q was constructed by binding the I fragment.
(3) Construction of plasmid pAMoh-Gs-i
  Gα_sOf the C-terminal 5 amino acids of Gα_iChimeric Gα substituted with 5 amino acids of C-terminal_s(Hereafter, Gα_siThe expression plasmid pAMoh-Gs-i was constructed as follows.
  By synthesizing a synthetic DNA having the nucleotide sequences represented by SEQ ID NOs: 18 and 19 with T4 polynucleotide kinase and annealing, Gα_iA double-stranded DNA containing a region encoding the C-terminal 5 amino acids was obtained. This double-stranded DNA, derived from pAMoh-Gs4 obtained in (2) aboveXbaI-AspFrom 718 fragment and pAMoh-Gs4XbaI-SphPAMoh-Gs-i was constructed by binding the I fragment.
(4) Construction of plasmids pACRElucMoGs-qMoGs-i and pAMopGs-qMoGs-i
  Gα_sqAnd Gα_siExpression plasmids pAMopGs-qMoGs-i, and Gα_sqAnd Gα_siPACRElucMoGs-qMoGs-i, which is an expression plasmid and a reporter plasmid using firefly luciferase as a reporter, was constructed as follows.
  PACREluc prepared in (1) of Reference Example 2ClaAfter cutting with I, Klenow processing,CpoCleave with I to include the oriP and CRE-firefly luciferase gene partsCpoI-ClaI (blunt end) fragment was obtained. PAMoh-Gs-i prepared in (3) aboveBssHII andCpoCut with I and Gα_siCodeBssHII-CpoI fragment was obtained. pAGE248 [J. Biol. Chem.269, 14730 (1994)]XhoAfter cutting with I, Klenow processing,BssCleaved with HII and contains a portion of the Moloney murine leukemia virus LTR promoter (hereinafter abbreviated as Mo promoter) sequenceXhoI (blunt end)-BssThe HII fragment was obtained. derived from pACRElucCpoI-ClaI (blunt end) fragment, derived from pAMoh-Gs-iBssHII-CpoI fragment, derived from pAGE248XhoI (blunt end)-BssThe HII fragment was ligated to construct plasmid pACRElucMoGs-i.
  PAMoh-Gs-q prepared in (2) aboveBssHII andCpoCut with I and Gα_sqCodeBssHII-CpoI fragment was obtained. derived from pAMoh-Gs-qBssHII-CpoI fragment, as well as from pACREluc obtained aboveCpoI-ClaFrom the I (blunt end) fragment and pAGE248XhoI (blunt end)-BssThe HII fragment was ligated to construct plasmid pACRElucMoGs-q.
  The pACRElucMoGs-i created above isNaeI andBssCleaved with HII and contains Mo promoterNaeI-BssHII fragmentCpoI andBssGα cut with HII_siCodeCpoI-BssEach HII fragment was obtained. Furthermore, pACRElucMoGs-q created above isNheI andCpoCleaved with I and contains CRE-firefly luciferase geneNheI-CpoI fragmentNheI andNaeCut with I and Gα_sqCodeNheI-NaeEach I fragment was obtained.
  Derived from these pACRElucMoGs-iNaeI-BssHII fragment andCpoI-BssHII fragment, as well as from pACRElucMoGs-qNheI-CpoI fragment andNheI-NaeFour fragments of the I fragment were ligated to construct plasmid pACRElucMoGs-qMoGs-i.
  The constructed pACRElucMoGs-qMoGs-i plasmidSalI andNotDigested with I, treated with Klenow,SalI (blunt end)-NotI (blunt end) fragment was obtained. The fragment was self-ligated to construct a plasmid pAMohGs-qMoGs-i in which the CRE reporter unit was removed from pACRElucMoGs-qMoGs-i.
  PAMohGs-qMoGs-i aboveCpoAfter cutting with I, Klenow processing,AseNo hygromycin resistance gene by cutting with ICpoI (blunted) -AseI fragment was obtained. pPUR (manufactured by Clontech; GenBank Accession No. U07648)BamAfter cutting with HI, Klenow processing,AseContains puromycin resistance gene by cutting with IBamHI (blunt end)-AseI fragment was obtained. Derived from this pPURBamHI (blunt end)-AseI fragment, and from pAMohGs-qMoGs-i aboveCpoPAMopGs-qMoGs-i was constructed by ligating the I (blunted) -AseI fragment and replacing the hygromycin resistance gene in pAMohGs-qMoGs-i with a puromycin resistance gene.
(5) Construction of a stable transformant in which the plasmid pAMopGs-qMoGs-i is incorporated into the chromosomal DNA of GBC7
  It is a restriction enzyme present in one place in oriP in pAMopGs-qMoGs-i constructed in (4) aboveSpeAfter dissolving pAMopGs-qMoGs-i cleaved with I in TE buffer so as to be 1 μg / μL, the cell line GBC7 constructed in (4) of Reference Example 2 described above was added 1.6 × 10⁶4 μg per cell was introduced to obtain transformed cells.
  The transformed strain was suspended in 8 mL of RPMI1640 • ITPSG medium, and CO₂The cells were cultured for 24 hours at 37 ° C. in an incubator.
  After the culture, blasticidin S (2.0 μg / mL), hygromycin B (300 μg / mL), and puromycin (manufactured by Sigma) (2.0 μg / mL) were added, and further cultured for 7 days. .
  After culturing, the number of viable cells of the transformant was confirmed, and blasticidin S (2.0 μg / mL), hygromycin B (300 μg / mL), puromycin (2.0 μg / mL), and KJMGER8 (1 × 10⁶In a RPMI1640 / ITPSG medium containing cells / mL), the transformant is diluted to 150 cells / mL, dispensed into a 96-well plate (30 cells / hole), and cultured. A transformant (single clone) was obtained. Each transformant was passaged in RPMI 1640 • ITPSG medium containing blasticidin S (2.0 μg / mL), hygromycin B (300 μg / mL), and puromycin (2.0 μg / mL).
(6) Selection of stable transformants (single clones) having excellent properties
  93 clones obtained in (5) above are seeded in a 96-well plate (1-2 × 10⁴Cell / well), Lipofectamine 2000 (manufactured by Invitrogen), a type 1 angiotensin II receptor-induced expression plasmid pAGal9-AT1 (see (2) of Reference Example 4 below) was introduced into each clone. A transformant was obtained. The specific method of gene transfer was performed according to the instructions for Lipofectamine 2000, and 0.3 μg of plasmid and 0.8 μL of Lipofectamine 2000 were used per well.
  After culturing the transformed strain for one day, hygromycin B (300 μg / mL), blasticidin S (2.0 μg / mL), puromycin (2.0 μg / mL), and geneticin (500 μg / mL) were added. The mixture was added and cultured at 37 ° C. for 7 days. E2 (final concentration 10 nmol / L) was added to each well, and further cultured for 1 day. Angiotensin II (manufactured by Peptide Institute, Inc.) (final concentration 100 nmol / L) was added to each well, and after culturing for 6 hours, firefly luciferase activity was measured using the method of Reference Example 2 (4). At the same time, the activity when not stimulated with angiotensin II was examined. As a result, 10 clones that showed high firefly luciferase activity in response to angiotensin II were selected.
  PAGal9-AT1 was again added to the 10 clones by electroporation method at 1.6 × 10 6.⁶4 μg was introduced per cell to obtain a transformed strain.
  The transformed strain was suspended in 8 mL of RPMI1640 · ITPSG medium, and CO₂The cells were cultured for 24 hours at 37 ° C. in an incubator.
  After culturing, blasticidin S (2.0 μg / mL), hygromycin B (300 μg / mL), puromycin (2.0 μg / mL), and geneticin (500 μg / mL) are added, and further cultured for 14 days. A stable transformant was obtained. The transformant is RPMI1640 • ITPSG medium containing blasticidin S (2.0 μg / mL), hygromycin B (300 μg / mL), puromycin (2.0 μg / mL), and geneticin (500 μg / mL). Passed on.
  E2 (final concentration 10 nmol / L) was added to each transformant and cultured for 24 hours, then angiotensin II (final concentration 100 nmol / L) was added, and further cultured for 6 hours, then described in (4) of Reference Example 2 The firefly luciferase activity was measured using the method described above. At the same time, the activity without stimulation with E2 and the activity without stimulation with angiotensin II were measured. GBCC13, which showed high firefly luciferase activity (85 times) in response to angiotensin II only when stimulated with E2, was selected as a superior strain.
(7) Construction of stable transformant in which plasmid pAMopGs-qMoGs-i is incorporated into the chromosomal DNA of GBCR2.
  In accordance with the method described in (5) above, pAMopGs-qMoGs-i constructed in (4) was introduced into the cell line GBCR2 constructed in (6) of Reference Example 2 above, whereby 94 stable transformations were performed. A strain (single clone) was obtained. Each transformant was passaged in RPMI 1640 • ITPSG medium containing blasticidin S (2.0 μg / mL), hygromycin B (300 μg / mL), and puromycin (2.0 μg / mL).
(8) Selection of stable transformants (single clones) having excellent properties
  A clone having excellent properties was selected from the clones obtained in (7) above. The method described in (6) above was followed except that the activity of Renilla luciferase was measured instead of measuring the activity of firefly luciferase. The activity of Renilla luciferase was measured according to the method described in (7) of Reference Example 2.
  PAGal9-AT1 was introduced into each clone obtained in (7) above to obtain transformed cells. Each transformed cell was treated with E2 and then stimulated with angiotensin II to measure Renilla luciferase activity. At the same time, the activity without stimulation with angiotensin II was measured. Thirteen clones that showed high Renilla luciferase activity in response to angiotensin II were selected.
  PAGal9-AT1 was again introduced into the 13 clones by electroporation to obtain transformed cells. Each transformed cell was treated with E2 and then stimulated with angiotensin II to measure Renilla luciferase activity. At the same time, the activity without stimulation with E2 and the activity without stimulation with angiotensin II were measured. GBCRC6, which showed high Renilla luciferase activity (132 times) in response to angiotensin II only when stimulated with E2, was selected as a superior strain.
Reference Example 4 Construction of various GPCR-induced expression plasmids and GPR88-induced expression plasmids
(1) Construction of type 2 vasopressin receptor (V2) inducible expression plasmid pAGal9-V2
  As a template, single-stranded cDNA prepared from human kidney-derived mRNA (1 μg; manufactured by Clontech) was used as a V2 gene-specific primer and a synthetic DNA having the sequences described in SEQ ID NOs: 20 and 21. Acquired. Each V2 gene specific primer containsHindIII site andAsp718 sites have been introduced.
  Obtained V2 gene amplified fragmentHindIII andAspCut at 718,HindIII-Asp718 fragments were obtained.
  Plasmid pAGal9-dHindIII andAspCut at 718,HindIII-Asp718 fragments were obtained.
  Derived from the above V2 gene amplified fragmentHindIII-AspFrom 718 fragment and pAGal9-dHindIII-AspBy joining the 718 fragment, an inducible expression plasmid pAGal9-V2 of V2 was constructed.
(2) Construction of type 1 angiotensin II receptor (AT1) inducible expression plasmid pAGal9-AT1
  Plasmid pAR1.8 [Nature,351, 230 (1991)]HindIII andNotCleaved with I and contains the bovine AT1 geneHindIII-NotI fragment was obtained.
  PAGal9-luc prepared in (2) of Reference Example 1HindIII andNotCleaved with I and does not contain firefly luciferase geneHindIII-NotI fragment was obtained.
  derived from pAR1.8HindIII-NotI fragment and from pAGal9-lucHindIII-NotThe I fragment was ligated to construct pAGal9-AT1.
(3) Construction of GPR12 inducible expression plasmid
  As a template, a single-stranded cDNA prepared from mRNA derived from human corpus callosum (1 μg; manufactured by Clontech) was used as a GPR12 gene [Genomics,28347 (1995), GenBank accession number: U18548] GPR12 gene was obtained by PCR using synthetic DNA having the sequence described in SEQ ID NO: 22 or 23 as a specific primer. As the DNA polymerase, KOD DNA polymerase (Toyobo Co., Ltd.) was used. As a buffer for PCR, a 10-fold concentration buffer added to the enzyme used was used. PCR is performed using a thermal cycler DNA Engine (manufactured by MJ Research) at 95 ° C for 5 minutes, followed by a reaction consisting of 94 ° C for 1 minute, 60 ° C for 1 minute, and 72 ° C for 1 minute. 30 cycles were performed.
  Each GPR12 gene-specific primer containsHindIII site andNotI site has been introduced. Amplified fragmentHindIII andNotAfter cutting with I, a fragment containing the GPR12 gene was recovered by agarose gel electrophoresis. The cleaved fragment was ligated to the corresponding restriction enzyme site of plasmid pAGal9-nd (HindIII-NotInducible expression plasmid pAGal9-GPR12 was constructed by integrating between I).
  The sequence of the DNA fragment incorporated into the plasmid was determined and confirmed to encode GPR12. That is, using the primer specific to the sequence in pAGal9-nd (synthetic DNA having the sequences shown in SEQ ID NOs: 24 and 25), the sequences on the 5 'side and 3' side of the cDNA were determined. Synthetic DNA specific to the determined sequence was prepared and used as a primer, and further the base sequence was determined. By repeating this operation, the entire nucleotide sequence of the cDNA was determined. For the determination of the base sequence, DNA sequencer 377 of Perkin Elmer and reaction kit (ABI Prism)^TM  BigDye^TM  Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) was used.
(4) Construction of inducible expression plasmid for GPR88
  The GPR88 gene was obtained by PCR using human chromosomal DNA (100 ng; manufactured by Clontech) as a template and synthetic DNA having the sequences described in SEQ ID NOs: 26 and 27 as human GPR88 gene (SEQ ID NO: 1) specific primers. As the DNA polymerase, PLATINUM Pfx DNA polymerase (Invitrogen) was used. As a buffer for PCR, a 10-fold concentration buffer added to the enzyme used was used. PCR was performed using Thermal Cycler DNA Engine (manufactured by MJ Research) at 98 ° C. for 2 minutes, followed by 30 cycles of 98 ° C. for 15 seconds, 62 ° C. for 30 seconds, and 68 ° C. for 1 minute. . Each GPCR88 gene specific primer containsHindIII site andXhoI site has been introduced. Amplified fragmentXhoAfter cutting with I and Klenow processingHincleaved with dIII and contains the GPCR88 geneHindIII-XhoI (blunt end) fragment was recovered by agarose gel electrophoresis. After cutting the plasmid pAGal9-nd with NotI and treating with KlenowHincut with dIII,HindIII-NotI (blunt end) fragment was recovered by agarose gel electrophoresis. Contains GPR88 gene obtained by PCRHindIII-XhoFrom I (blunt end) fragment and pAGal9-ndHindIII-NotA GPR88-induced expression plasmid pAGal9-GPR88 was constructed by ligating with an I (blunt end) fragment.
  The sequence of the DNA fragment incorporated into the plasmid pAGal9-nd was determined according to the method described in (3) above, and it was confirmed that it encoded GPCR88.
Reference Example 5 Construction and utilization of GPR88 assay cells using KJMGER8 as a host
  GPR88-induced expression plasmid pAGal9-GPR88 (2 μg) constructed in (4) of Reference Example 4 and Gα constructed in (4) of Reference Example 3_sqAnd Gα_siExpression plasmid and reporter plasmid pACRElucMoGs-qMoGs-i (2 μg) was prepared by electroporation method at 1.6 × 10 6.⁶The cells were co-introduced into the cell line KJMGER8 selected in (4) of Reference Example 1 of cells. The transformed strain was suspended in 8 mL of RPMI1640 · ITPSG medium, and CO₂The cells were cultured for 24 hours at 37 ° C. in an incubator. After culturing, blasticidin S (2.0 μg / mL), hygromycin B (300 μg / mL) and geneticin (500 μg / mL) were added, and further cultured for 14 days to obtain a stable transformant (GPR88 assay cell 1). Obtained). The transformed strain was subcultured in RPMI1640 · ITPSG medium containing blasticidin S (2.0 μg / mL), hygromycin B (300 μg / mL) and geneticin (500 μg / mL).
  Similarly, control plasmid pAGal9-nd (2 μg) constructed in (3) of Reference Example 1 and Gα constructed in (4) of Reference Example 3_siAnd Gα_sqThe expression plasmid / reporter plasmid pACRElucMoGs-qMoGs-i (2 μg) was co-introduced into KJMGER8 to obtain a stable transformant (hereinafter referred to as control cell 1).
  Dispense GPR88 assay cells 1 and control cells 1 into 96-well plates, respectively (about 1 × 10⁴Cells / well) and cultured at 37 ° C. for 1 day. After adding E2 (final concentration 10 nmol / L) to each well and further culturing for 1 day, Steady Glo Luciferase Assay System (manufactured by Promega) is added, and firefly luciferase activity is measured using a top count (manufactured by Packard). It was measured. Moreover, the same experiment was conducted without adding E2, and the firefly luciferase activity was measured.
  The induction ratio was obtained by dividing the activity when E2 was added by the activity when no activity was added. The induction factor in control cell 1 was about 1. On the other hand, the induction ratio of GPR88 assay cell 1 was about 4 to 6 times. GPR88 was found to be a constitutively active GPCR because a signal was passed only by inducing expression.
Reference Example 6 Construction and utilization of GPR88 assay cells using GBCC13 as a host
  The GPR88 inducible expression plasmid pAGal9-GPR88 (4 μg) constructed in (4) of Reference Example 4 was obtained by electroporation at 1.6 × 10 6.⁶The cells were introduced into the cell line GBCC13 selected in Reference Example 3 (6). The transformed strain was suspended in 8 mL of RPMI1640 · ITPSG medium, and CO₂The cells were cultured for 24 hours at 37 ° C. in an incubator. After culturing, blasticidin S (2.0 μg / mL), hygromycin B (300 μg / mL), puromycin (2.0 μg / mL) and geneticin (500 μg / mL) were added, and further cultured for 14 days. A stable transformant (referred to as GPR88 assay cell 2) was obtained. The transformed strain was treated with RPMI1640 • ITPSG medium containing blasticidin S (2.0 μg / mL), hygromycin B (300 μg / mL), puromycin (2.0 μg / mL) and geneticin (500 μg / mL). Passed.
  Similarly, the control plasmid pAGal9-nd (4 μg) constructed in (3) of Reference Example 1 was introduced into GBCC13 to obtain a stable transformant (referred to as control cell 2).
  Dispense GPR88 assay cell 2 or control cell 2 into a 96-well plate (approximately 1 × 10⁴Cells / well) and cultured at 37 ° C. for 1 day. After adding E2 (final concentration 10 nmol / L) to each well and further culturing for 1 day, Steady Glo Luciferase Assay System (Promega) was added, and firefly luciferase activity was measured using a top count (Packard) did. Moreover, the same experiment was conducted without adding E2, and the firefly luciferase activity was measured.
  The induction ratio was obtained by dividing the activity when E2 was added by the activity when no activity was added. The induction factor in control cell 2 was about 1. On the other hand, the induction ratio of GPR88 assay cell 2 was about 4-18 times. GPR88 was found to be a constitutively active GPCR because a signal was passed only by inducing expression.
Reference Example 7 Construction of GPR88 assay cells using GBCRC6 as a host
  An arbitrary GPCR assay cell can be constructed by introducing an inducible expression plasmid of GPCR into GBCRC6 constructed in Reference Example 3.
  The GPR88 inducible expression plasmid pAGal9-GPR88 (4 μg) constructed in (4) of Reference Example 4 was obtained by electroporation.⁶The cells were introduced into GBCRC6. The transformed strain was suspended in 8 mL of RPMI1640 · ITPSG medium, and CO₂The cells were cultured for 24 hours at 37 ° C. in an incubator. After culturing, blasticidin S (2.0 μg / mL), hygromycin B (300 μg / mL), puromycin (2.0 μg / mL) and geneticin (500 μg / mL) were added, and further cultured for 14 days. A stable transformant (referred to as GPR88 assay cell 3) was obtained. The transformed strain was treated with RPMI1640 • ITPSG medium containing blasticidin S (2.0 μg / mL), hygromycin B (300 μg / mL), puromycin (2.0 μg / mL) and geneticin (500 μg / mL). Passed.
  Similarly, the control plasmid pAGal9-nd (4 μg) constructed in (3) of Reference Example 1 was introduced into GBCRC6 to obtain a stable transformant (referred to as control cell 3).
  Dispense GPR88 assay cells 3 or control cells 3 into 96-well plates (approximately 1 × 10⁴Incubate at 37 ° C for 1 day. E2 (final concentration: 10 nmol / L) was added to each well and further cultured for 1 day, then coelenterazine h (Molecular Probes) (final concentration: 250 nmol / L) was added to measure Renilla luciferase activity. For the activity measurement, a Wallac 1420 ARVOsx multi-label counter (manufactured by Wallac Bertoled Japan) or FDSS6000 (manufactured by Hamamatsu Photonics) was used. Moreover, the same experiment was conducted without adding E2, and Renilla luciferase activity was measured. The induction ratio was obtained by dividing the activity when E2 was added by the activity when no activity was added.
  The induction factor in control cell 3 was about 1. On the other hand, the induction ratio of GPR88 assay cell 3 was about 15 to 40 times. GPR88 was found to be a constitutively active GPCR because a signal was passed only by inducing expression.
Reference Example 8: Synthesis of 4-chloro-5-cyano-2-methylthiopyrimidine
Step 1: Synthesis of 5-cyano-3,4-dihydro-2-methylthiopyrimidin-4-one sodium salt
  Methyl isothiourea sulfate (90.5 g, 0.325 mmol) was dissolved in an aqueous sodium hydroxide solution (2 mol / L, 325 mL), and the resulting aqueous solution was cooled to ice with ethyl 2-ethoxymethylene-2-cyanoacetate (100 g,. 591 mmol) in ethanol (350 mL) was added dropwise little by little while taking care not to exceed an internal temperature of 15 ° C. After all the ethanol solution was added, aqueous sodium hydroxide solution (2 mol / L, 300 mL) was added little by little, ethanol (150 mL) was further added, and the mixture was stirred overnight at room temperature. Crystals precipitated in the reaction mixture were collected by filtration and washed with ethanol (500 mL). The obtained white crystals were dried under reduced pressure to obtain 5-cyano-3,4-dihydro-2-methylthiopyrimidin-4-one sodium salt (113.3 g, yield 100%).
Step 2: Synthesis of 4-chloro-5-cyano-2-methylthiopyrimidine
  Phosphorus oxychloride (150 mL) was added to 5-cyano-3,4-dihydro-2-methylthiopyrimidin-4-one (30.0 g, 0.159 mmol) obtained above, and the mixture was heated to reflux for 7 hours. After the reaction mixture was allowed to cool, phosphorus oxychloride was distilled off under reduced pressure. The obtained residue was poured into ice (about 1000 mL), and the precipitated pale yellow solid was collected by filtration and washed with water. The solid was dried under reduced pressure to obtain 4-chloro-5-cyano-2-methylthiopyrimidine (11.9 g, yield 40%).
Reference Example 9: Synthesis of 2-benzyl-N-methylaniline
Step 1: Synthesis of 2-benzyltrifluoroacetanilide
  2-Benzylaniline (5.00 g, 27.3 mmol) was dissolved in dichloromethane (100 mL) and triethylamine (7.6 mL, 55 mmol) and trifluoroacetic anhydride (4.17 mL, 30.30) were added to the solution at −20 ° C. 0 mmol) was added and stirred for 30 minutes. After stirring at room temperature for 1 hour, the reaction solution was diluted with dichloromethane, water was added to separate the solution, and the organic layer was washed with saturated aqueous sodium hydrogen carbonate and saturated brine. After the organic layer was dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure to obtain 2-benzyltrifluoroacetanilide (7.51 g, 26.9 mmol, yield 99%) as pale yellow crystals.
¹1 H NMR (270 MHz, CDCl₃) Δ (ppm): 4.00 (s, 2H), 7.14 (d, J = 6.6 Hz, 2H), 7.23-7.40 (m, 6H), 7.79 (br s, 1H), 7.81 (d, J = 7.7 Hz, 1H)
Step 2: Synthesis of 2-benzyl-N-methyltrifluoroacetanilide
  2-Benzyltrifluoroacetanilide (7.51 g, 26.9 mmol) obtained in Step 1 was dissolved in N, N-dimethylformamide (50 mL), and potassium carbonate (7.54 g, 54) was added to the solution under ice cooling. .6 mmol) and methyl iodide (3.40 mL, 54.6 mmol) were added, and the mixture was stirred at room temperature for 18 hours. The reaction solution was diluted with ethyl acetate, water was added to separate the solution, and the organic layer was washed with saturated brine. After the organic layer was dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure to obtain 2-benzyl-N-methyltrifluoroacetanilide (7.49 g, 25.6 mmol, yield 95%).
¹1 H NMR (270 MHz, CDCl₃) Δ (ppm): 3.13 (s, 3H), 3.94 (s, 2H), 7.11-7.35 (m, 9H)
Step 3: Synthesis of 2-benzyl-N-methylaniline
  2-Benzyl-N-methyltrifluoroacetanilide (7.49 g, 25.6 mmol) obtained in step 2 is dissolved in methanol (50 mL), and potassium carbonate (7.54 g, 54.6 mmol) is added to the solution. The mixture was stirred for 5 hours under reflux. The reaction solution was diluted with ethyl acetate, water was added to separate the solution, and the organic layer was washed with saturated brine. After the organic layer was dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure to obtain 2-benzyl-N-methylaniline (4.70 g, 23.8 mmol, yield 92%).
¹1 H NMR (270 MHz, CDCl₃) Δ (ppm): 2.74 (s, 3H), 3.50 (br s, 1H), 3.85 (s, 2H), 6.63 (d, J = 8.1 Hz, 1H), 6 .71 (t, J = 7.3 Hz, 1H), 7.01 (d, J = 7.3 Hz, 1H), 7.13-7.29 (m, 6H)
Reference Example 10: Synthesis of benzotriazol-1-yl 4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carboxylate
Step 1: Synthesis of ethyl 4- (benzylphenylamino) -2-methylthio-5-pyrimidinecarboxylate
  Commercially available ethyl 4-chloro-2-methylthio-5-pyrimidinecarboxylate (15.0 g, 64.5 mmol) was dissolved in 1,2-dimethoxyethane (150 mL) and N, N-diisopropylethylamine (27.6 mL, 161 mmol) and 2-benzylaniline (13.0 g, 70.9 mmol) were added, and the mixture was stirred for 18 hours with heating under reflux. The reaction solution was distilled off under reduced pressure and diluted with ethyl acetate. The organic layer was washed successively with water, 5% aqueous citric acid solution and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was recrystallized from ethanol to obtain the title compound (19.6 g, 51.6 mmol, yield 80%) as white crystals.
¹H-NMR (270 MHz, CDCl₃) Δ (ppm): 1.38 (t, J = 6.9 Hz, 3H), 2.36 (s, 3H), 4.02 (s, 2H), 4.34 (q, J = 6.9 Hz) , 2H), 7.10-7.27 (m, 8H), 7.82 (d, J = 7.9 Hz, 1H), 8.70 (s, 1H), 10.00 (brs, 1H)
APCI-MS: m / z 380 (M + H)⁺
Step 2: Synthesis of ethyl 4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carboxylate
  Ethyl 4- (benzylphenylamino) -2-methylthio-5-pyrimidinecarboxylate (5.00 g, 13.2 mmol) obtained in step 1 was dissolved in dichloromethane (50 mL), and m-chlorofiltered benzoate was cooled with ice. Acid (65% content, 2.70 g, 15.6 mmol) was added over 10 minutes and stirred at room temperature for 30 minutes. Further, m-chlorofiltered benzoic acid (content 65%, 1.13 g, 7.53 mmol) was added over 5 minutes under ice cooling, and the mixture was stirred at room temperature for 20 minutes. The reaction mixture was diluted with dichloromethane and washed with saturated aqueous sodium hydrogen carbonate. The organic layer was dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was dissolved in 1,2-dimethoxyethane (50 mL), cyclohexylmethylamine (2.57 mL, 19.8 mmol) was added, and the mixture was stirred for 2.5 hours with heating under reflux. The reaction solution was distilled off under reduced pressure, and the obtained residue was recrystallized from ethanol to obtain the title compound (4.87 g, 11.0 mmol, yield 83%) as white crystals.
¹H-NMR (270 MHz, DMSO-d₆, 80 ° C.) δ (ppm): 0.74-0.98 (m, 2H), 1.01-1.24 (m, 3H), 1.29 (t, J = 7.1 Hz, 3H), 1.40-1.73 (m, 6H), 3.06 (br s, 2H), 3.98 (s, 2H), 4.25 (q, J = 7.1 Hz, 2H), 7.06 −7.24 (m, 8H), 7.40 (br, 1H), 7.95 (d, J = 8.2 Hz, 1H), 8.52 (brs, 1H), 9.85 (brs) , 1H)
APCI-MS: 445 (M + H)⁺
IR (KBr): 2468, 2458, 1666, 1629, 1608, 1577, 1546, 1521, 1502, 1494, 1463, 1446, 1430, 1394, 1390, 1369 cm^-1
Step 3: Synthesis of 4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carboxylic acid
  Ethyl 4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carboxylate (1.00 g, 2.25 mmol) obtained in step 2 was dissolved in ethanol (7 mL), and 1 mol / L Aqueous sodium hydroxide solution (7.00 mL, 7.00 mmol) was added, and the mixture was stirred at 60 ° C. for 4 hr. Subsequently, ethanol (7 mL) and a 4 mol / L sodium hydroxide aqueous solution (1.00 mL, 4.00 mmol) were added, and the mixture was stirred for 30 minutes while heating under reflux. Furthermore, 4 mol / L sodium hydroxide aqueous solution (1.00 mL, 4.00 mmol) was added, and it stirred under heating-refluxing for 2 hours. The reaction solution was concentrated under reduced pressure to half the volume, and the pH of the reaction solution was adjusted to 4.5 with 1 mol / L hydrochloric acid aqueous solution. The precipitated crystals were collected by filtration to give the title compound (0.99 g, 2.2 mmol, quantitative yield) as white crystals.
¹H-NMR (270 MHz, DMSO-d₆) Δ (ppm): 0.78-0.99 (m, 2H), 1.03-1.29 (m, 3H), 1.47-1.81 (m, 6H), 3.07 (br) s, 2H), 4.03 (s, 2H), 6.89-7.10 (m, 1H), 7.12-7.35 (m, 8H), 7.50 (brs, 1H), 8.40 (d, J = 7.9 Hz, 1H), 8.48 (s, 1H), 11.97 (brs, 1H)
APCI-MS: m / z 417 (M + H)⁺
IR (KBr): 2918, 2841, 1664, 1655, 1649, 1637, 1632, 1610, 1589, 1578, 1570, 1552, 1541, 1535, 1510, 1454, 1417, 1381, 1362 cm^-1
Step 4: Synthesis of benzotriazol-1-yl 4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carboxylate
  4- (2-Benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carboxylic acid (5.00 g, 12.0 mmol) obtained in Step 3 was dissolved in chloroform (50 mL), and the mixture was cooled with ice. , N-ethyl-N ′-(3-dimethylaminopropyl) carbodiimide hydrochloride (4.60 g, 24.0 mmol) and 1-hydroxybenzotriazole (3.30 g, 24.4 mmol) were added, and the mixture was stirred at room temperature for 5 hours. did. Further, N-ethyl-N ′-(3-dimethylaminopropyl) carbodiimide hydrochloride (2.30 g, 12.0 mmol) and 1-hydroxybenzotriazole (1.60 g, 12.0 mmol) were added, and the mixture was stirred at room temperature for 2 hours. did. The reaction mixture was diluted with ethyl acetate and washed successively with water, 5% aqueous citric acid solution and saturated aqueous sodium hydrogen carbonate. The organic layer was dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure to obtain the title compound (5.67 g, 10.6 mmol, 89% yield) as pale yellow crystals. This compound was used in the next step without further purification.
¹H-NMR (270 MHz, CDCl₃) Δ (ppm): 0.76-1.08 (m, 2H), 1.09-1.33 (m, 3H), 1.43-1.82 (m, 6H), 3.14 (t , J = 6.5 Hz, 1.5H), 3.35 (t, J = 6.3 Hz, 0.5H), 3.92 (s, 0.5H), 3.93 (s, 1.5H) 5.47 (t, J = 5.9 Hz, 0.25H), 5.89 (t, J = 5.9 Hz, 0.75H), 6.99-7.30 (m, 9H), 7. 42-7.60 (m, 2H), 7.68 (d, J = 7.9 Hz, 0.25H), 7.80 (d, J = 7.9 Hz, 0.75H), 8.10 (d , J = 8.2 Hz, 1H), 8.95 (s, 0.75H), 9.03 (brs, 0.25H), 9.08 (s, 0.25H), 9.19 (brs). , 0.75H)
Example 1 Synthesis of Compound 1 [4- (2-Benzylphenylamino) -2- (cyclohexylmethylamino) -5- (2H-tetrazol-5-yl) pyrimidine]
Step 1: Synthesis of 4- (2-benzylphenylamino) -5-cyano-2-methylthiopyrimidine
  4-Chloro-5-cyano-2-methylthiopyrimidine (800 mg, 4.31 mmol) obtained in Reference Example 8 was dissolved in tetrahydrofuran (8 mL), and 2-benzylaniline (869 mg, 4.74 mmol) and Triethylamine (0.90 mL, 6.46 mmol) was added, and the mixture was stirred at room temperature for 24 hours. The reaction solution was diluted with water and extracted with chloroform. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography, and 4- (2-benzylphenylamino) -5-cyano-2-methylthiopyrimidine (1.15 g, 3.46 mmol, yield 80%) was obtained as pale yellow crystals. Got.
¹1 H NMR (270 MHz, CDCl₃) Δ (ppm): 2.34 (s, 3H), 4.00 (s, 2H), 6.85 (brs, 1H), 7.12-7.32 (m, 8H), 7.61 (D, J = 6.3 Hz, 1H), 8.27 (s, 1H)
Step 2: Synthesis of 4- (2-benzylphenylamino) -5-cyano-2- (cyclohexylmethylamino) pyrimidine
  4- (2-Benzylphenylamino) -5-cyano-2-methylthiopyrimidine (1.15 g, 3.46 mmol) obtained in Step 1 was dissolved in dichloromethane (20 mL), and the solution was mixed with Filtered benzoic acid (65%, 1.38 g, 5.2 mmol) was added over 5 minutes and stirred at room temperature for 5 hours. The reaction mixture was diluted with saturated aqueous sodium hydrogen carbonate and extracted with dichloromethane. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was dissolved in tetrahydrofuran (20 mL), cyclohexylmethylamine (0.54 mL, 4.15 mmol) was added to the solution, and the mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with water, and the precipitated crystals were collected by filtration to give pale yellow crystals of 4- (2-benzylphenylamino) -5-cyano-2- (cyclohexylmethylamino) pyrimidine (1.21 g, 3 0.04 mmol, 88% yield).
¹1 H NMR (270 MHz, CDCl₃) Δ (ppm): 0.76-1.02 (m, 2H), 1.05-1.34 (m, 3H), 1.36-1.91 (m, 6H), 3.07 (t , J = 6.4 Hz, 0.63H), 3.24 (t, J = 6.4 Hz, 0.37H), 3.98 (s, 0.74H), 4.01 (s, 1.26H) , 5.24 (br s, 0.37H), 5.59 (br s, 0.63H), 6.56 (br s, 0.37H), 6.70 (br s, 0.63H), 7 .06-7.42 (m, 8H), 7.60 (d, J = 7.6 Hz, 0.37H), 7.76 (d, J = 7.9 Hz, 0.63H), 8.12 ( s, 0.63H), 8.23 (s, 0.37H)
APCI-MS: m / z 396 (M-H)⁻
Step 3: Synthesis of 4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) -5- (2H-tetrazol-5-yl) pyrimidine
  4- (2-Benzylphenylamino) -5-cyano-2- (cyclohexylmethylamino) pyrimidine (1.00 g, 2.52 mmol) obtained in Step 2 was dissolved in N, N-dimethylformamide (8 mL). To the solution were added ammonium chloride (404 mg, 7.55 mmol) and sodium azide (491 mg, 7.55 mmol), and the mixture was stirred at 100 ° C. for 5 hours. The reaction solution was diluted with a saturated aqueous ammonium chloride solution, and the precipitated crystals were collected by filtration. The obtained crude crystals were recrystallized from a mixed solvent of ethyl acetate and hexane to obtain Compound 1 (693 mg, 1.57 mmol, yield 62%) as pale yellow crystals.
¹1 H NMR (270 MHz, DMSO-d₆80 ° C.) δ (ppm): 0.74-0.99 (m, 2H), 1.01-1.25 (m, 3H), 1.40-1.73 (m, 6H), 05 (brs, 2H), 4.04 (s, 2H), 7.08-7.30 (m, 9H), 7.60 (brs, 1H), 7.88 (d, J = 7. 8 Hz, 1H), 8.61 (s, 1H), 10.36 (brs, 1H).
APCI-MS: m / z 439 (M-H)⁻
IR (KBr): 2922, 2891, 1655, 1647, 1637, 1593, 1570, 1560, 1541, 1527, 1508, 1483, 1454 cm^-1
  The corresponding hydrochloride salt was prepared according to the following method.
  The above compound 1 (1.56 g, 3.54 mmol) was suspended in ethanol (30 mL), and a hydrogen chloride ethyl acetate solution (4 mol / L, 9.00 mL, 36.0 mmol) was added to the suspension. And stirred for 2 hours. The precipitated crystals were collected by filtration and recrystallized from ethanol to obtain Compound 1 monohydrochloride (1.59 g, 3.33 mmol, yield 94%).
¹1 H NMR (270 MHz, DMSO-d₆) Δ (ppm): 0.65-1.00 (m, 2H), 1.01-1.30 (m, 3H), 1.31-1.81 (m, 6H), 2 as main peaks .95 (t, J = 5.9 Hz, 0.8H), 3.19 (t, J = 5.9 Hz, 0.2H), 4.05 (s, 2H), 6.99-7.20 ( m, 5H), 7.22-7.42 (m, 3H), 7.63 (d, J = 5.9 Hz, 0.8H), 7.72 (d, J = 5.9 Hz, 0.2H) ), 8.62 (br s, 0.8H), 8.82 (s, 1H), 8.88 (br s, 0.2H), 10.62 (s, 0.2H), 10.68 ( s, 0.8H)
APCI-MS: m / z 441 (M + H)⁺
IR (KBr): 1684, 1670, 1662, 1653, 1649, 1635, 1630, 1593, 1572, 1560, 1541, 1524, 1497, 1473, 1458, 1412, 1367 cm^-1
Example 2: Synthesis of compounds 2 to 36
Step 1: Synthesis of Compound F

(Wherein R¹And A are as defined above)
  4-chloro-5-cyano-2-methylthiopyrimidine (0.25 mol / L chloroform solution, 0.20 mL, 0.050 mmol) obtained in Reference Example 8 and R¹ArNH (wherein R¹And Ar are as defined above), morpholinomethylpolystyrene (35 mg, 0.12 mmol) is added to a mixture of compound E (1.0 mol / L chloroform solution, 0.060 mL, 0.060 mmol) represented by And stirred at 50 ° C. for 1 day. Benzoyl chloride polymer bound (23 mg, 0.058 mmol) and chloroform (0.50 mL) were added to the reaction mixture, and the mixture was further stirred at room temperature for 1 day. The resin in the reaction mixture was filtered off, and the resin was washed with a mixed solvent of chloroform and methanol (chloroform / methanol = 3/1, 1.2 mL). All of the obtained solutions were combined, and the solvent was distilled off to obtain Compound F.
Step 2: Synthesis of Compound G

(Wherein R¹, A and m are as defined above.
  Compound F obtained in Step 1 was dissolved in dichloromethane (0.20 mL), and methacrofiltered benzoic acid (0.50 mol / L dichloromethane solution, 0.125 mL, 0.063 mmol) was added to the solution. Stir for 5 to 1 hour. Saturated aqueous sodium hydrogen carbonate (0.20 mL) was added to the reaction mixture to separate the layers. The organic layer was washed with saturated aqueous sodium hydrogen carbonate (0.20 mL), and a mixture of magnesium sulfate and diatomaceous earth (magnesium sulfate / diatomaceous earth = 1, 1,140 mg) was added and stirred. The mixture of magnesium sulfate and diatomaceous earth was filtered off, and the mixture of magnesium sulfate and diatomaceous earth was washed with dichloromethane (0.80 mL). All of the obtained solutions were combined, and the solvent was distilled off to obtain Compound G.
Step 3: Synthesis of Compound J

(Wherein R¹, A, R³And R⁴Are as defined above)
  Compound G obtained in step 2 is dissolved in tetrahydrofuran (0.20 mL), and R is added to the solution.³R⁴NH (wherein R³And R⁴Were each synonymous with the above, and compound H (1.0 mol / L chloroform solution, 0.060 mL, 0.060 mmol) was added, and the mixture was stirred at room temperature for 1 day. Chloroform (0.40 mL), benzoyl chloride polymer bound (23 mg, 0.058 mmol), and poly-4-vinylpyridine (23 mg) were added to the reaction mixture, and the mixture was stirred at room temperature for 1 day. The resin in the reaction mixture was filtered off, and the resin was washed with a mixed solvent of chloroform and methanol (chloroform / methanol = 3/1, 1.4 mL). All of the obtained solutions were combined, and the solvent was distilled off to obtain Compound J.
Step 4: Synthesis of Compound 2 to Compound 36
  Compound J obtained in Step 3 was dissolved in N, N-dimethylformamide (0.10 mL), and an N, N-dimethylformamide solution of ammonium azide (1.0 mol / L, 0.080 mL, 0) was added to the solution. 0.080 mmol) was added, and the mixture was stirred at 100 ° C. for 1 day. The solvent was distilled off from the reaction solution, and the residue was mixed with chloroform and 1,1,1,3,3,3-hexafluoroisopropyl alcohol (chloroform / 1,1,1,3,3,3-hexafluoroisopropyl alcohol). (Alcohol = 3/1, 0.40 mL) and washed with a 5% aqueous citric acid solution. After the organic layer was concentrated, the residue was dissolved in ethanol (0.300 mL), and ion exchange resin AG1-X8 (manufactured by Bio-Rad) was added to the solution, followed by stirring at room temperature for 2 hours. The resin in the reaction mixture was collected by filtration, and the resin was washed with methanol (0.60 mL). Then, the resin was mixed with chloroform and methanol (chloroform / methanol = 1/1, 0.40 mL), and then hydrogen chloride in ethyl acetate. A solution (4 mol / L, 0.050 mL) was added to elute the target product. Further, the resin was washed with a mixed solvent of chloroform and methanol (chloroform / methanol = 1/1, 1.2 mL), and the obtained eluate and the washing solution were combined and concentrated, and the target compounds 2 to 36 were respectively added. Yield about 30%.
  The obtained compounds 2 to 36 were each identified by mass spectrometry. The analysis results for each compound are listed in Table 1 as instrument data.
Example 3: Synthesis of compound 37 [4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carboxylic acid]
Step 1: Synthesis of ethyl 4- (2-benzylphenylamino) -2-methylthiopyrimidine-5-carboxylate
  Commercially available ethyl 4-chloro-2-methylthiopyrimidin-5-ylcarboxylate (15.0 g, 64.5 mmol) was dissolved in 1,2-dimethoxyethane (150 mL), and N, N-diisopropylethylamine (27 6 mL, 161 mmol) and 2-benzylaniline (13.0 g, 70.9 mmol) were added, and the mixture was stirred for 18 hours under heating to reflux. The reaction mixture was concentrated under reduced pressure, and the resulting residue was diluted with ethyl acetate, and then water was added to separate the layers. The organic layer was washed successively with 5% aqueous citric acid solution and saturated brine. The organic layer was dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was recrystallized from ethanol to give ethyl 4- (2-benzylphenylamino) -2-methylthiopyrimidine-5-carboxylate (19.6 g, 51.6 mmol, yield 80%) as white crystals. Got.
¹1 H NMR (270 MHz, CDCl₃) Δ (ppm): 1.38 (t, J = 6.9 Hz, 3H), 2.36 (s, 3H), 4.02 (s, 2H), 4.34 (q, J = 6.9 Hz) , 2H), 7.10-7.27 (m, 8H), 7.82 (d, J = 7.9 Hz, 1H), 8.70 (s, 1H), 10.00 (brs, 1H)
APCI-MS: m / z 380 (M + H)⁺
Step 2: Synthesis of ethyl 4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carboxylate
  The compound obtained in Step 1 (5.0 g, 13.2 mmol) was dissolved in dichloromethane (50 mL), and methacrofiltered benzoic acid (2.70 g, 15.6 mmol) was added to the solution over 10 minutes under ice cooling. In addition, the mixture was stirred at room temperature for 30 minutes. Further, methacrofiltered benzoic acid (1.13 g, 7.53 mmol) was added to the reaction solution over 5 minutes under ice cooling, and the mixture was stirred at room temperature for 20 minutes. The reaction mixture was diluted with dichloromethane, and saturated aqueous sodium hydrogen carbonate was added to separate the layers. The organic layer was dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was dissolved in 1,2-dimethoxyethane (50 mL), cyclohexylmethylamine (2.57 mL, 19.8 mmol) was added to the solution, and the mixture was stirred for 2.5 hours under heating to reflux. The reaction solution was concentrated under reduced pressure, and the obtained residue was recrystallized from ethanol to give ethyl 4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carboxylate (4. 87 g, 11.0 mmol, yield 83%).
¹1 H NMR (270 MHz, DMSO-d₆, 80 ° C.) δ (ppm): 0.74-0.98 (m, 2H), 1.01-1.24 (m, 3H), 1.29 (t, J = 7.1 Hz, 3H), 1.40-1.73 (m, 6H), 3.06 (br s, 2H), 3.98 (s, 2H), 4.25 (q, J = 7.1 Hz, 2H), 7.06 −7.24 (m, 8H), 7.40 (br s, 1H), 7.95 (d, J = 8.2 Hz, 1H), 8.52 (br s, 1H), 9.85 (br s, 1H)
APCI-MS: m / z 445 (M + H)⁺
IR (KBr): 2468, 2458, 1666, 1629, 1608, 1577, 1546, 1521, 1502, 1494, 1463, 1446, 1430, 1394, 1390, 1369 cm^-1
Step 3: Synthesis of 4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carboxylic acid
  The compound obtained in Step 2 (1.00 g, 2.25 mmol) was dissolved in ethanol (7 mL), and an aqueous sodium hydroxide solution (1.0 mol / L, 7.0 mL, 6.5 mmol) was added to the solution. Stir at 60 ° C. for 4 hours. Ethanol (7 mL) and aqueous sodium hydroxide solution (4.0 mol / L, 1.0 mL, 4.0 mmol) were added to the reaction solution, and the mixture was stirred for 30 minutes under heating to reflux.
  Further, a sodium hydroxide aqueous solution (4.0 mol / L, 1.0 mL, 4.0 mmol) was added to the reaction solution, and the mixture was stirred for 2 hours while heating under reflux. The reaction solution was concentrated under reduced pressure to half the volume, and the pH value of the reaction solution was adjusted to 4.5 with hydrochloric acid (1.0 mol / L). The precipitated crystals were collected by filtration to obtain Compound 37 (0.990 g, 2.25 mmol, quantitative yield) as white crystals.
¹1 H NMR (270 MHz, DMSO-d₆) Δ (ppm): 0.78-0.99 (m, 2H), 1.03-1.29 (m, 3H), 1.47-1.81 (m, 6H), 3.07 (br) s, 2H), 4.03 (s, 2H), 6.89-7.10 (m, 1H), 7.12-7.35 (m, 8H), 7.50 (brs, 1H), 8.40 (d, J = 7.9 Hz, 1H), 8.48 (s, 1H), 11.97 (brs, 1H)
APCI-MS: m / z 417 (M + H)⁺
  The corresponding sodium salt was prepared according to the following method.
  Compound 37 (1.7 g, 4.08 mmol) obtained above was dissolved in tetrahydrofuran (5 mL), and an aqueous sodium hydroxide solution (1.0 mol / L, 4.1 mL, 4.1 mmol) was added to the solution. After stirring at room temperature for 1 hour, the reaction solution was concentrated under reduced pressure. The obtained residue was recrystallized from water to obtain a monosodium salt of Compound 37 (1.69 g, 3.85 mmol, 94% yield) as white crystals.
¹1 H NMR (270 MHz, DMSO-d₆)) Δ (ppm): 0.76-1.00 (m, 2H), 1.02-1.32 (m, 3H), 1.4-1.90 (m, 6H), 3.07 ( br s, 2H), 4.03 (s, 2H), 6.79-7.49 (m, 9H), 8.47 (s, 1H), 8.57 (d, J = 7.2 Hz, 1H) ), 13.08 (br s, 1H)
APCI-MS: m / z 417 (M + H)⁺
IR (KBr): 3045, 2991, 2966, 2954, 2933, 2914, 2846, 1664, 1631, 1610, 1592, 1577, 1552, 1539, 1535, 1510 cm^-1
Example 4: Synthesis of compound 38 [4-[(2-benzylphenyl) (methyl) amino] -2- (cyclohexylmethylamino) pyrimidine-5-carboxylic acid]
Step 1: Synthesis of ethyl 4-[(2-benzylphenyl) (methyl) amino] -2-methylthiopyrimidine-5-carboxylate
  Commercially available ethyl 4-chloro-2-methylthiopyrimidine-5-carboxylate (3.90 g, 16.8 mmol) was dissolved in 1,2-dimethoxyethane (30 mL), and N, N-diisopropylethylamine (7 8 mL, 46 mmol) and 2-benzyl-N-methylaniline (3.0 g, 15 mmol) obtained in Reference Example 2 were added, and the mixture was stirred for 24 hours while heating under reflux. The reaction solution was concentrated under reduced pressure. Ethyl acetate and water were added to the resulting residue for liquid separation, and the organic layer was washed successively with 5% aqueous citric acid solution and saturated brine. The organic layer was dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was recrystallized from 2-propanol to give ethyl 4-[(2-benzylphenyl) (methyl) amino] -2-methylthiopyrimidine-5-carboxylate (5.06 g, 12. 9 mmol, yield 77%).
¹1 H NMR (270 MHz, DMSO-d₆) Δ (ppm): 1.04 (t, J = 7.1 Hz, 3H), 2.41 (s, 3H), 3.18 (s, 3H), 3.70 (q, J = 7.1 Hz) , 2H), 3.80 (s, 2H), 7.07-7.29 (m, 9H), 8.19 (s, 1H)
Step 2: Synthesis of ethyl 4-[(2-benzylphenyl) (methyl) amino] -2- (cyclohexylmethylamino) pyrimidine-5-carboxylate
  Ethyl 4-[(2-benzylphenyl) (methyl) amino] -2-methylthiopyrimidine-5-carboxylate (3.00 g, 7.62 mmol) obtained in step 1 was dissolved in dichloromethane (30 mL) and iced. Under cooling, methacrofiltered benzoic acid (1.57 g, 9.15 mmol) was added over 5 minutes and stirred at room temperature for 30 minutes. Further, methacrofiltered benzoic acid (0.65 g, 3.8 mmol) was added to the reaction solution under ice cooling, and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was diluted with dichloromethane, and saturated aqueous sodium hydrogen carbonate was added to separate the layers. The organic layer was dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was dissolved in 1,2-dimethoxyethane (30 mL), cyclohexylmethylamine (1.48 mL, 11.4 mmol) was added to the solution, and the mixture was stirred for 1.5 hours with heating under reflux. After allowing the reaction solution to cool, the resulting crystals were collected by filtration. The obtained crude crystals were recrystallized from ethanol to give ethyl 4-[(2-benzylphenyl) (methyl) amino] -2- (cyclohexylmethylamino) pyrimidine-5-carboxylate (2.87 g) as white crystals. 6.27 mmol, yield 82%).
¹1 H NMR (270 MHz, DMSO-d₆, 80 ° C.) δ (ppm): 0.85-0.93 (m, 2H), 1.03 (t, J = 7.1 Hz, 3H), 1.09-1.24 (m, 3H), 1.51-1.65 (m, 6H), 3.05 (br s, 2H), 3.07 (s, 3H), 3.73 (q, J = 7.1 Hz, 2H), 3.82 (S, 2H), 6.97 (br s, 1H), 7.03-7.28 (m, 9H), 8.12 (s, 1H)
Step 3: Synthesis of 4-[(2-benzylphenyl) (methyl) amino] -2- (cyclohexylmethylamino) pyrimidine-5-carboxylic acid
  Ethyl 4-[(2-benzylphenyl) (methyl) amino] -2- (cyclohexylmethylamino) pyrimidine-5-carboxylate (0.5 g, 1.09 mmol) obtained in step 2 was added to ethanol (5 mL). It melt | dissolved, the sodium hydroxide aqueous solution (1.0 mol / L, 10 mL, 10 mmol) was added to the solution, and it stirred under heating-refluxing for 24 hours. The reaction solution was concentrated under reduced pressure to half the volume, and the pH value of the reaction solution was adjusted to 4.5 with hydrochloric acid (1.0 mol / L). The precipitated crystals were collected by filtration to obtain Compound 38 (474 mg, 1.09 mmol, quantitative yield) as white crystals.
¹1 H NMR (270 MHz, DMSO-d₆, 80 ° C.) δ (ppm): 0.83-0.91 (m, 2H), 1.12-1.25 (m, 3H), 1.48-1.63 (m, 6H) as main peaks ), 3.04 (br s, 2H), 3.18 (s, 3H), 3.87 (s, 2H), 7.00-7.41 (m, 10H), 8.22 (s, 1H) )
APCI-MS: m / z 431 (M + H)⁺
IR (KBr): 2930, 2840, 2380, 2350, 1655, 1510, 1490, 1385, 1375 cm^-1
Example 5: Synthesis of compound 39 [4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carbohydroxamic acid]
Step 1: Synthesis of 4- (2-benzylphenylamino) -2- (cyclohexylamino) pyrimidine-5-carboxylic acid = benzotriazol-1-yl
  Compound 37 (5.00 g, 12.0 mmol) obtained in Example 3 was dissolved in chloroform (50 mL), and N-ethyl-N ′-(3-dimethylaminopropyl) carbodiimide hydrochloride was added to the solution under ice cooling. (4.60 g, 24.0 mmol) and 1-hydroxybenzotriazole (3.30 g, 24.4 mmol) were added, and the mixture was stirred at room temperature for 5 hours. Further, N-ethyl-N ′-(3-dimethylaminopropyl) carbodiimide hydrochloride (2.30 g, 12.0 mmol) and 1-hydroxybenzotriazole (1.60 g, 12.0 mmol) were added to the reaction solution, and the mixture was brought to room temperature. And stirred for 2 hours. The reaction solution was diluted with ethyl acetate, and water was added for liquid separation, and then the organic layer was washed successively with 5% aqueous citric acid solution and saturated aqueous sodium hydrogen carbonate. After drying the organic layer with anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure to give 4- (2-benzylphenylamino) -2- (cyclohexylamino) pyrimidine-5-carboxylic acid = benzotriazole- as pale yellow crystals. 1-yl (5.67 g, 10.6 mmol, 89% yield) was obtained.
¹1 H NMR (270 MHz, CDCl₃) Δ (ppm): 0.76-1.08 (m, 2H), 1.09-1.33 (m, 3H), 1.43-1.82 (m, 6H), 3.14 (t , J = 6.5 Hz, 1.5H), 3.35 (t, J = 6.3 Hz, 0.5H), 3.92 (s, 0.5H), 3.93 (s, 1.5H) 5.47 (t, J = 5.9 Hz, 0.25H), 5.89 (t, J = 5.9 Hz, 0.75H), 6.99-7.30 (m, 9H), 7. 42-7.60 (m, 2H), 7.68 (d, J = 7.9 Hz, 0.25H), 7.80 (d, J = 7.9 Hz, 0.75H), 8.10 (d , J = 8.2 Hz, 1H), 8.95 (s, 0.75H), 9.03 (brs, 0.25H), 9.08 (s, 0.25H), 9.19 (brs). , 0.75H)
Step 2: Synthesis of [4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carbohydroxamic acid]
  4- (2-Benzylphenylamino) -2- (cyclohexylamino) pyrimidine-5-carboxylic acid = benzotriazol-1-yl (1.40 g, 2.62 mmol) obtained in Step 1 was added to tetrahydrofuran (20 mL). Dissolve in a mixed solvent of water (20 mL), add hydroxylamine hydrochloride (546 mg, 7.88 mmol) and potassium carbonate (1.27 g, 9.18 mmol) to the solution under ice cooling, and stir at room temperature for 18 hours. did. The reaction solution was diluted with ethyl acetate, and water was added for liquid separation, and then the organic layer was washed with saturated aqueous sodium hydrogen carbonate. The organic layer was dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography. The obtained oily substance was recrystallized from ethyl acetate to obtain Compound 39 (490 mg, 1.14 mmol, yield 43%) as pale yellow crystals.
¹1 H NMR (270 MHz, DMSO-d₆, 80 ° C.) δ (ppm): 0.76-0.99 (m, 2H), 1.01-1.26 (m, 3H), 1.45-1.74 (m, 6H), 06 (br s, 2H), 4.00 (s, 2H), 6.99-7.26 (m, 9H), 8.11 (dd, J = 1.2, 8.1 Hz, 1H), 8 .33 (s, 1H), 8.65 (br s, 1H), 10.77 (br s, 1H)
APCI-MS: m / z 432 (M + H)⁺
Example 6: Synthesis of compound 40 [4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carboxamide]
  4- (2-Benzylphenylamino) -2- (cyclohexylamino) pyrimidine-5-carboxylic acid = benzotriazol-1-yl (200 mg, 0.37 mmol) obtained in Step 1 of Example 5 was replaced with N, N. -Dissolved in dimethylformamide (5 mL), added with aqueous ammonia (28%, 5 mL) to the solution under ice cooling, and stirred at room temperature for 2 hours. The reaction solution was diluted with ethyl acetate, and water was added for liquid separation, and then the organic layer was washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine. The organic layer was dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was recrystallized from 2-propanol to obtain Compound 40 (77 mg, 0.18 mmol, yield 50%) as white crystals.
¹1 H NMR (270 MHz, DMSO-d₆, 70 ° C.) δ (ppm): 0.86-0.94 (m, 2H), 1.12-1.25 (m, 3H), 1.49-1.68 (m, 6H), 12 (brt, J = 6.1 Hz, 2H), 3.99 (s, 2H), 6.98-7.24 (m, 9H), 7.28 (brs, 2H), 8.17 ( d, J = 8.1 Hz, 1H), 8.50 (s, 1H), 11.32 (brs, 1H)
APCI-MS: m / z 416 (M + H)⁺
Example 7: Synthesis of compound 41 [4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidin-5-yl-N- (2-hydroxyethyl) carboxamide]
  4- (2-Benzylphenylamino) -2- (cyclohexylamino) pyrimidine-5-carboxylic acid = benzotriazol-1-yl (250 mg, 0.58 mmol) obtained in Step 1 of Example 5 was added to acetonitrile (5 mL). ) And N, N-dimethylformamide (2.5 mL), and under ice cooling, 2-aminoethanol (71 μL, 1.46 mmol) and 1,8-diazabicyclo [5.4. ] -Undec-7-ene (DBU) (217 μL, 1.46 mmol) was added, and the mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with ethyl acetate, 5% aqueous citric acid solution was added to separate the layers, and the organic layer was washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine. The organic layer was dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was recrystallized from 2-propanol to obtain Compound 41 (86 mg, 0.19 mmol, yield 32%) as white crystals.
¹1 H NMR (270 MHz, CDCl₃) Δ (ppm): 0.69-0.90 (m, 2H), 0.96-1.26 (m, 3H), 1.31-1.80 (m, 6H), 3 as main peaks 0.00 (brs, 2H), 3.54 (brs, 2H), 3.81 (brs, 2H), 4.00 (s, 2H), 6.94-7.28 (m, 9H) 7.77 (brs, 1H), 8.16 (brs, 1H), 8.65 (brs, 1H), 11.48 (brs, 1H)
APCI-MS: m / z 460 (M + H)⁺
IR (KBr): 3125, 2890, 1734, 1716, 1695, 1668, 1662, 1655, 1601, 1581, 1558, 1551, 1527, 1497, 1473, 1454, 1431, 1417 cm^-1
Example 8: Synthesis of compound 42 [2- [4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidin-5-ylcarbonylamino] acetic acid]
Step 1: Synthesis of 2- [4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidin-5-ylcarbonylamino] acetic acid = tert-butyl
  4- (2-Benzylphenylamino) -2- (cyclohexylamino) pyrimidine-5-carboxylic acid = benzotriazol-1-yl (250 mg, 0.47 mmol) obtained in Step 1 of Example 5 was added to acetonitrile (2 mL). ) And N, N-dimethylformamide (2 mL), and glycine tert-butyl hydrochloride (196 mg, 1.17 mmol) and DBU (350 μL, 2.34 mmol) were added to the solution under ice cooling, Stir at room temperature for 6 hours. The reaction mixture was diluted with ethyl acetate, 5% aqueous citric acid solution was added to separate the layers, and the organic layer was washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine. After the organic layer was dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure to give 2- [4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidin-5-ylcarbonylamino as a pale yellow oily substance. Acetic acid = tert-butyl (246 mg, 0.46 mmol, yield 99%) was obtained.
¹1 H NMR (270 MHz, DMSO-d₆, 80 ° C.) δ (ppm): 0.74-0.99 (m, 2H), 1.02-1.28 (m, 3H), 1.42 (s, 9H), 1.43-1. 77 (m, 6H), 3.05 (br s, 2H), 3.85 (d, J = 5.8 Hz, 2H), 3.97 (s, 2H), 6.97-7.43 (m , 9H), 8.09 (dd, J = 1.2, 8.1 Hz, 1H), 8.43 (t, J = 5.8 Hz, 1H), 8.51 (s, 1H), 10.93 (Br s, 1H)
Step 2: Synthesis of 2- [4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidin-5-ylcarbonylamino] acetic acid
  2- [4- (2-Benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidin-5-ylcarbonylamino] acetic acid = tert-butyl (246 mg, 0.46 mmol) obtained in step 1 was added to dichloromethane (3 mL). ), And trifluoroacetic acid (5 mL) was added to the solution under ice cooling, followed by stirring at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was dissolved in ethanol (3 mL). To the solution was added aqueous sodium hydroxide (1.0 mol / L, 1.84 mL, 1.84 mmol), and the mixture was stirred at room temperature for 15 minutes. Stir. The pH value of the reaction solution was adjusted to 4.5 with hydrochloric acid (1.0 mol / L), water (5 mL) was added, and the precipitated crystals were collected by filtration. The obtained crude crystals were recrystallized from 2-propanol to obtain Compound 42 (176 mg, 0.37 mmol, yield 81%) as white crystals.
¹1 H NMR (270 MHz, CDCl₃) Δ (ppm): 0.74-0.99 (m, 2H), 1.00-1.32 (m, 3H), 1.40-1.55 (m, 1H), 1.56-1 .90 (m, 5H), 3.05 (d, J = 6.3 Hz, 2H), 3.99 (s, 2H), 4.05 (d, J = 5.6 Hz, 2H), 7.02 −7.23 (m, 9H), 7.59 (d, J = 6.3 Hz, 1H), 8.33 (brs, 1H), 8.78 (s, 1H), 8.84 (brs) , 1H), 11.53 (s, 1H)
APCI-MS: m / z 474 (M + H)⁺
IR (KBr): 3380, 3240, 3065, 2930, 2868, 1701, 1697, 1670, 1662, 1652, 1602, 1585, 1560, 1543, 1524, 1498, 1473, 1452, 1437, 1417, 1412 cm^-1
Example 9: Synthesis of compound 43 [2- [4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidin-5-ylcarbonyl-N-methylamino] acetic acid]
Step 1: Synthesis of ethyl 2- [4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidin-5-ylcarbonyl-N-methylamino] acetate
  4- (2-Benzylphenylamino) -2- (cyclohexylamino) pyrimidine-5-carboxylic acid = benzotriazol-1-yl (1.0 g, 1.87 mmol) obtained in Step 1 of Example 5 was added to acetonitrile. (10 mL) and N, N-dimethylformamide (10 mL) are dissolved in a mixed solvent. Under ice cooling, N-methylaminoethyl acetate hydrochloride (430 mg, 2.81 mmol) and DBU (840 μL, 5.62 mmol) are added to the solution. ) And stirred at room temperature for 1 hour. The reaction solution was diluted with ethyl acetate, 5% aqueous citric acid solution was added to separate the solution, and the organic layer was washed with saturated aqueous sodium hydrogen carbonate and saturated brine. After drying the organic layer over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure to give 2- [4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidin-5-ylcarbonyl- as a pale yellow oily substance. N-methylamino] ethyl acetate (990 mg, 1.87 mmol, quantitative yield) was obtained.
¹1 H NMR (270 MHz, DMSO-d₆, 80 ° C.) δ (ppm): 0.76-0.98 (m, 2H), 1.01-1.27 (m, 3H), 1.18 (t, J = 7.1 Hz, 3H), 1.42-1.77 (m, 6H), 3.04 (br s, 2H), 3.07 (s, 3H), 3.93 (s, 2H), 4.13 (q, J = 7) .1 Hz, 2H), 4.16 (s, 2H), 6.97 (brs, 1H), 7.01-7.24 (m, 8H), 7.90 (d, J = 8.1 Hz, 1H), 8.21 (s, 1H), 8.90 (brs, 1H)
Step 2: Synthesis of 2- [4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidin-5-ylcarbonyl-N-methylamino] acetic acid
  Ethyl 2- [4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidin-5-ylcarbonyl-N-methylamino] acetate (990 mg, 1.87 mmol) obtained in Step 1 was added to ethanol (990 mg, 1.87 mmol). 20 mL), an aqueous sodium hydroxide solution (1.0 mol / L, 4.0 mL, 4.0 mmol) was added to the solution, and the mixture was stirred at room temperature for 18 hours. The reaction was concentrated to half volume and diluted with water (20 mL). The pH value of the reaction solution was adjusted to 4.5 with an aqueous hydrochloric acid solution (1.0 mol / L), and the precipitated crystals were collected by filtration. The obtained crude crystals were recrystallized from ethanol to obtain Compound 43 (305 mg, 0.63 mmol, yield 34%) as white crystals.
¹1 H NMR (270 MHz, DMSO-d₆, 80 ° C.) δ (ppm): 0.77-1.00 (m, 2H), 1.04-1.32 (m, 3H), 1.4-1.74 (m, 6H) as main peaks ), 3.07 (brs, 2H), 3.08 (s, 3H), 3.96 (s, 2H), 4.12 (s, 2H), 6.97 (brs, 1H), 7 .04-7.25 (m, 8H), 7.92 (d, J = 8.1 Hz, 1H), 8.02 (s, 1H), 8.93 (br, 1H)
APCI-MS: m / z 488 (M + H)⁺
IR (KBr): 2780, 1653, 1647, 1637, 1618, 1610, 1601, 1583, 1560, 1545, 1535, 1529, 1508, 1495, 1450, 1423, 1400 cm^-1
Example 10: Synthesis of compound 44 [4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) -5-hydroxymethylpyrimidine]
  4- (2-Benzylphenylamino) -2- (cyclohexylamino) pyrimidine-5-carboxylic acid = benzotriazol-1-yl (2.00 g, 3.75 mmol) obtained in Step 1 of Example 5 was added to tetrahydrofuran. (20 mL), lithium aluminum hydride (0.28 g, 7.5 mmol) was added to the solution in an argon atmosphere under ice cooling, and the mixture was stirred at room temperature for 1 hour. Under ice cooling, water (0.30 mL), aqueous sodium hydroxide (4.0 mol / L, 0.30 mL) and water (1.0 mL) were sequentially added to the reaction solution, and the mixture was stirred at room temperature for 15 minutes. The precipitated solid was filtered through diatomaceous earth and washed with chloroform. The filtrate and washings were combined, diluted with water and extracted with chloroform. The organic layer was dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography to obtain compound 44 (1.69 g, 0.76 mmol, yield 20%) as pale yellow crystals.
¹1 H NMR (270 MHz, CDCl₃) Δ (ppm): 0.74-1.01 (m, 2H), 1.02-1.31 (m, 3H), 1.45-1.81 (m, 6H), 3 as main peaks .15 (t, J = 6.3 Hz, 2H), 4.03 (s, 2H), 4.27 (s, 2H), 7.05-7.26 (m, 9H), 7.41 (s) , 1H), 7.60 (s, 1H), 8.10 (brs, 1H)
APCI-MS: m / z 403 (M + H)⁺
IR (KBr): 3500, 2900, 2858, 1915, 1894, 1888, 1875, 1867, 1713, 1709, 1705, 1693, 1687, 1678, 1672, 1657, 1651, 1622, 1612 cm^-1
Example 11: Synthesis of compound 45 [4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) -5- (1-hydroxyethyl) pyrimidine]
Step 1: Synthesis of 4- (2-benzylphenylamino) -5-hydroxymethyl-2-methylthiopyrimidine
  Ethyl 4- (2-benzylphenylamino) -2-methylthiopyrimidine-5-carboxylate (5.0 g, 13.2 mmol) obtained in Step 1 of Example 3 was dissolved in tetrahydrofuran (50 mL), and the atmosphere was argon. Under ice cooling, lithium aluminum hydride (0.6 g, 15.8 mmol) was added to the solution, and the mixture was stirred at room temperature for 3.5 hours. Furthermore, lithium aluminum hydride (0.15 g, 4.0 mmol) was added to the reaction mixture under ice cooling in an argon atmosphere, and the mixture was stirred at room temperature for 1.5 hours. Under ice cooling, water (0.8 mL), aqueous sodium hydroxide (4 mol / L, 0.8 mL) and water (2.4 mL) were sequentially added to the reaction solution, and the mixture was stirred at room temperature for 15 minutes. The precipitated solid was filtered through diatomaceous earth and washed with chloroform. The filtrate and the washing solution were combined, diluted with water, and extracted with chloroform. The organic layer was dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure to give 4- (2-benzylphenylamino) -5-hydroxymethyl-2-methylthiopyrimidine (3.70 g, 11.0 mmol) as a pale yellow oily substance. Yield 83%).
¹1 H NMR (270 MHz, CDCl₃)) Δ (ppm): 2.40 (s, 3H), 4.03 (s, 2H), 4.34 (s, 2H), 7.10-7.32 (m, 8H) as main peaks , 7.66 (s, 1H), 7.68 (s, 1H), 8.00 (d, J = 8.2 Hz, 1H)
Step 2: Synthesis of 4- (2-benzylphenylamino) -5-formyl-2-methylthiopyrimidine
  4- (2-Benzylphenylamino) -5-hydroxymethyl-2-methylthiopyrimidine (3.70 mg, 11.0 mmol) obtained in Step 1 was dissolved in chloroform (100 mL), and manganese dioxide (8 0.0 g, 110 mmol) was added, and the mixture was stirred at room temperature for 18 hours. The reaction solution was filtered through diatomaceous earth, and the filtered solid was washed with chloroform. The filtrate and washings were combined and concentrated under reduced pressure, and the resulting residue was recrystallized from ethanol to give 4- (2-benzylphenylamino) -5-formyl-2-methylthiopyrimidine (1.94 g, 5.78 mmol, 53% yield).
¹1 H NMR (270 MHz, CDCl₃)) Δ (ppm): 2.41 (s, 3H), 4.05 (s, 2H), 7.08-7.28 (m, 8H), 7.88 (d, J = 8.1 Hz, 1H), 8.37 (s, 1H), 9.72 (s, 1H), 10.28 (brs, 1H)
Step 3: Synthesis of 4- (2-benzylphenylamino) -5- (1-hydroxyethyl) -2-methylthiopyrimidine
  Under an argon atmosphere, 4- (2-benzylphenylamino) -5-formyl-2-methylthiopyrimidine (500 mg, 1.49 mmol) obtained in Step 2 was dissolved in tetrahydrofuran (10 mL), and the solution was heated to 20 ° C. Then, methylmagnesium bromide (0.93 mol / L tetrahydrofuran solution, 4.0 mL, 3.7 mmol) was added, and the mixture was stirred at −20 ° C. for 1.5 hours. Saturated aqueous ammonium chloride solution was gradually added to the reaction solution at −20 ° C. The reaction mixture was extracted with ethyl acetate, and the organic layer was washed successively with water and saturated brine. After drying the organic layer over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and 4- (2-benzylphenylamino) -5- (1-hydroxyethyl) -2-methylthiopyrimidine (560 mg, 1) was obtained as a pale yellow oily substance. .49 mmol, quantitative yield).
¹1 H NMR (270 MHz, CDCl₃) Δ (ppm): 1.33 (d, J = 6.8 Hz, 3H), 2.37 (s, 3H), 4.02 (s, 2H), 4.60 (q, J) as main peaks = 6.8 Hz, 1 H), 7.07-7.29 (m, 8 H), 7.60 (s, 1 H), 8.01 (d, J = 7.2 Hz, 1 H), 8.25 (s) , 1H)
Step 4: Synthesis of 4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) -5- (1-hydroxyethyl) pyrimidine
  4- (2-Benzylphenylamino) -5- (1-hydroxyethyl) -2-methylthiopyrimidine (73.4 mg, 0.24 mmol) obtained in step 3 was dissolved in dichloromethane (2 mL), and the mixture was ice-cooled. To the solution, methacrofiltered benzoic acid (50 mg, 0.29 mmol) was added and stirred at room temperature for 30 minutes. The reaction mixture was diluted with dichloromethane, and saturated aqueous sodium hydrogen carbonate was added to separate the layers. The organic layer was dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was dissolved in dioxane (3 mL), and N, N-diisopropylethylamine (0.080 mL, 0.48 mmol) and cyclohexylmethylamine (0.060 mL, 0.48 mmol) were added to the solution. Stir for 4 days. The reaction solution was diluted with ethyl acetate, water was added to separate the solution, and the organic layer was washed with saturated brine. The organic layer was dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography to give Compound 45 (86.1 mg, 0.207 mmol, yield 86%) as a pale yellow oily substance. )
¹1 H NMR (270 MHz, CDCl₃) Δ (ppm): 0.68-1.03 (m, 2H), 1.04-1.37 (m, 3H), 1.45-1.88 (m, 6H), 2 as main peaks .25 (d, J = 6.6 Hz, 3H), 3.18 (dd, J = 4.5, 6.4 Hz, 2H), 3.58 (q, J = 6.6 Hz, 1H), 4. 06 (s, 2H), 6.97-7.28 (m, 9H), 7.61 (s, 1H), 8.23 (d, J = 7.2 Hz, 1H), 9.93 (br s , 1H)
APCI-MS: m / z 399 (M-OH)⁺
Example 12: Synthesis of compound 46 [4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) -5- (1-hydroxy-1-methylethyl) pyrimidine]
Step 1: Synthesis of 5-acetyl-4- (2-benzylphenylamino) -2-methylthiopyrimidine
  4- (2-Benzylphenylamino) -5- (1-hydroxyethyl) -2-methylthiopyrimidine (524 mg, 1.49 mmol) obtained in Step 3 of Example 11 was dissolved in chloroform (10 mL). Manganese dioxide (1.3 g, 15 mmol) was added to the solution and stirred at room temperature for 12 hours. Further, chloroform (10 mL) and manganese dioxide (1.3 g, 15 mmol) were added to the solution, and the mixture was stirred for 2 hours while heating under reflux. The reaction solution was filtered through diatomaceous earth, and the filtered solid was washed with chloroform. The filtrate and washings were combined and concentrated under reduced pressure to give 5-acetyl-4- (2-benzylphenylamino) -2-methylthiopyrimidine (381 mg, 1.09 mmol, 73% yield) as pale yellow crystals.
¹1 H NMR (270 MHz, CDCl₃)) Δ (ppm): 2.37 (s, 3H), 2.54 (s, 3H), 4.03 (s, 2H), 7.08-7.29 (m, 8H), 7.80 (D, J = 7.9 Hz, 1H), 8.63 (s, 1H), 10.94 (br s, 1H)
Step 2: Synthesis of 4- (2-benzylphenylamino) -5- (1-hydroxy-1-methylethyl) -2-methylthiopyrimidine
  Under an argon atmosphere, 5-acetyl-4- (2-benzylphenylamino) -2-methylthiopyrimidine (272 mg, 0.78 mmol) obtained in Step 1 was dissolved in tetrahydrofuran (5 mL), and −20 ° C. was added to the solution. Was added methylmagnesium bromide (0.93 mol / L tetrahydrofuran solution, 2.5 mL, 2.3 mmol), and the mixture was stirred at −20 ° C. for 1 hour and at 0 ° C. for 1 hour. Further, methylmagnesium bromide (0.93 mol / L tetrahydrofuran solution, 2.5 mL, 2.3 mmol) was added to the reaction solution at 0 ° C., and the mixture was stirred at room temperature for 2.5 hours. A saturated aqueous ammonium chloride solution was gradually added to the reaction solution at 0 ° C., and the mixture was extracted with ethyl acetate. The organic layer was washed successively with water and saturated brine. After drying the organic layer over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure to give 4- (2-benzylphenylamino) -5- (1-hydroxy-1-methylethyl) -2-methylthiopyrimidine as a pale yellow oily substance. (264 mg, 0.72 mmol, 93% yield) was obtained.
¹1 H NMR (270 MHz, CDCl₃) Δ (ppm): 1.46 (s, 6H), 2.37 (s, 3H), 4.01 (s, 2H), 7.01-7.29 (m, 8H) as main peaks, 7.78 (s, 1H), 7.94 (d, J = 7.7 Hz, 1H), 8.84 (brs, 1H)
Step 3: Synthesis of 4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) -5- (1-hydroxy-1-methylethyl) pyrimidine
  4- (2-Benzylphenylamino) -5- (1-hydroxy-1-methylethyl) -2-methylthiopyrimidine (284 mg, 0.78 mmol) obtained in step 2 was dissolved in dichloromethane (5 mL) and ice-cooled. Under this condition, methacrofiltered benzoic acid (200 mg, 1.17 mmol) was added to the solution, and the mixture was stirred at room temperature for 1 hour. Further, methacrofiltered benzoic acid (133 mg, 0.78 mmol) was added to the reaction solution under ice cooling, and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was diluted with dichloromethane, and saturated aqueous sodium hydrogen carbonate was added to separate the layers. The organic layer was dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was dissolved in dioxane (3 mL), and N, N-diisopropylethylamine (0.30 mL, 2.3 mmol) and cyclohexylmethylamine (0.22 mL, 2.3 mmol) were added to the solution. Stir for 4 days. The reaction solution was diluted with ethyl acetate, water was added to separate the solution, and the organic layer was washed with saturated brine. The organic layer was dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and the resulting residue was purified by silica gel column chromatography to obtain an oily substance. The obtained oily substance was recrystallized from diethyl ether to obtain Compound 46 (80.5 mg, 0.18 mmol, yield 24%) as pale yellow crystals.
¹H-NMR (270 MHz, CDCl₃) Δ (ppm): 0.76-1.02 (m, 2H), 1.10-1.35 (m, 3H), 1.43-1.80 (m, 6H), 2.47 (s) , 6H), 3.15 (t, J = 6.3 Hz, 2H), 4.03 (s, 1H), 4.07 (s, 2H), 5.55 (brs, 1H), 7.11 −7.25 (m, 9H), 8.03 (d, J = 8.2 Hz, 1H), 8.58 (s, 1H)
APCI-MS: m / z 415 (M-CH₃)⁺
IR (KBr): 3260, 2800, 2600, 1994, 1969, 1951, 1944, 1932, 1925, 1624, 1578, 1545, 1524, 1498, 1473, 1466, 1454, 1439, 1423 cm^-1
Example 13: Synthesis of Compound 47 [N- [4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carbonyl] -4-toluenesulfonamide]
  Benzotriazol-1-yl-4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carboxylate (0.20 g, 0.37 mmol) obtained in Reference Example 10 was added to acetonitrile (4 mL). ) And N, N-dimethylformamide (2 mL), and 4-toluenesulfonamide (0.10 g, 0.56 mmol) and DBU (80 μL, 0.56 mmol) were added under ice-cooling at room temperature. Stir for 3 hours. 4-Toluenesulfonamide (60 mg, 0.37 mmol) and DBU (50 μL, 0.37 mmol) were further added, and the mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with ethyl acetate and washed successively with 5% aqueous citric acid solution, saturated aqueous sodium hydrogen carbonate and saturated brine. The organic layer was dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and the resulting residue was purified by silica gel column chromatography. The obtained crude crystals were recrystallized from 2-propanol to obtain the title compound (97 mg, 0.17 mmol, yield 46%) as white crystals.
¹H-NMR (270 MHz, CDCl₃) Δ (ppm): 0.70-0.90 (m, 2H), 0.95-1.24 (m, 3H), 1.30-1.75 (m, 6H), 2.34 (s) , 3H), 3.07 (t, J = 6.3 Hz, 2H), 3.94 (s, 2H), 7.05-7.22 (m, 11H), 7.66 (d, J = 7 .7 Hz, 1 H), 7.75 (d, J = 8.2 Hz, 2 H), 8.08 (br s, 1 H), 8.51 (s, 1 H), 12.45 (s, 1 H)
APCI-MS: m / z 568 (M-H)⁻
IR (KBr): 2875, 2850, 1656, 1648, 1641, 1601, 1583, 1558, 1541, 1516, 1512, 1508, 1488, 1452, 1402, 1371 cm^-1
Example 14: Synthesis of compound 48 [N- [4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carbonyl] methanesulfonamide]
  Benzotriazol-1-yl 4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carboxylate (0.20 g, 0.37 mmol) obtained in Reference Example 10 was added to acetonitrile (4 mL). And methanesulfonamide (50 mg, 0.56 mmol) and DBU (80 μL, 0.56 mmol) were added under ice cooling, and the mixture was stirred at room temperature for 3 hours. Further, methanesulfonamide (30 mg, 0.37 mmol) and DBU (50 μL, 0.37 mmol) were added, and the mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with ethyl acetate and washed successively with 5% aqueous citric acid solution, saturated aqueous sodium hydrogen carbonate and saturated brine. The organic layer was dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and the resulting residue was purified by silica gel column chromatography. The obtained crude crystals were recrystallized from 2-propanol to obtain the title compound (39 mg, 0.080 mmol, yield 22%) as white crystals.
¹H-NMR (270 MHz, CDCl₃) Δ (ppm): 0.74-0.95 (m, 2H), 0.99-1.30 (m, 3H), 1.40-1.80 (m, 6H), 2.94 (s) , 3H), 3.07 (t, J = 6.1 Hz, 2H), 4.13 (s, 2H), 7.13 (m, 9H), 7.63 (d, J = 7.1 Hz, 1H) ), 8.00 (br s, 1H), 8.59 (s, 1H), 12.33 (s, 1H)
APCI-MS: m / z 492 (M-H)⁻
IR (KBr): 2920, 2850, 1655, 1637, 1608, 1587, 1578, 1560, 1545, 1541, 1535, 1522, 1454, 1402, 1323, 1277 cm^-1
Example 15: Synthesis of compound 49 [N- [4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carbonyl] trifluoromethanesulfonamide]
  Benzotriazol-1-yl 4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carboxylate (0.30 g, 0.70 mmol) obtained in Reference Example 10 was added to acetonitrile (6 mL). And N, N-dimethylformamide (6 mL) were dissolved, and trifluoromethanesulfonamide (259 mg, 1.74 mmol) and DBU (260 μL, 1.74 mmol) were added under ice cooling, followed by stirring at room temperature for 18 hours. . The reaction mixture was diluted with ethyl acetate and washed successively with 5% aqueous citric acid solution, saturated aqueous sodium hydrogen carbonate and saturated brine. The organic layer was dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was recrystallized from 2-propanol to give the title compound (0.25 g, 0.46 mmol, yield 65%) as white crystals.
¹H-NMR (270 MHz, DMSO-d₆) Δ (ppm): 0.64-0.93 (m, 2H), 0.99-1.23 (m, 3H), 1.29-1.78 (m, 6H), 2.97 (t , J = 6.3 Hz, 2H), 3.99 (s, 2H), 7.05-7.34 (m, 9H), 7.73 (d, J = 6.9 Hz, 1H), 8.24. (Br, 1H), 8.26 (s, 1H), 11.88 (s, 1H)
APCI-MS: m / z 548 (M + H)⁺
IR (KBr): 1757, 1749, 1732, 1716, 1653, 1641, 1583, 1556, 1510, 1493, 1331, 1315, 1194 cm^-1
Example 16: Synthesis of compounds 50-234
Process 1
  To 300 μL (17.5 mg, 75.2 μmol) of a commercially available ethyl 4-chloro-2-methylthio-5-pyrimidinecarboxylate 1.75 g / 30 mL tetrahydrofuran (THF) solution, various amine reagents (100 μmol) and morpholinomethyl Polystyrene (42.0 mg, 147 μmol) was added and stirred at 60 ° C. overnight. After adding THF (0.3 mL) and benzoyl chloride polymer bound (45.0 mg, 90 μmol) and further stirring overnight, the insoluble matter was filtered and washed. The obtained filtrate and washing solution were combined and concentrated, and the residue was concentrated. Obtained.
Process 2
  The residue obtained in Step 1 was dissolved in dichloromethane (0.3 mL), m-chlorofiltered benzoic acid (content 65%, 26.5 mg, 100 μmol) was added, and the mixture was stirred for 1 hour. The reaction mixture was diluted with dichloromethane (0.1 mL), washed with saturated aqueous sodium hydrogen carbonate, the organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated. The obtained residue was dissolved in THF (0.2 mL), various amines (100 μmol) were added, and the mixture was stirred at 60 ° C. overnight. Chloroform (0.4 mL), morpholinomethylpolystyrene (42.0 mg, 147 μmol) and benzoyl chloride polymer bound (90.0 mg, 180 μmol) were added, and the mixture was further stirred overnight, and then the insoluble material was filtered and washed. The obtained filtrate and washing solution were combined and concentrated to obtain a residue.
Process 3
  The residue obtained in step 2 was dissolved in a mixed solvent of ethanol (0.3 mL) and THF (0.05 mL), 1 mol / L NaOH aqueous solution (145 μL) was added, and the mixture was stirred at 65 ° C. overnight. Methanol (0.4 mL) and 1 mol / L aqueous HCl (145 μL) were added and stirred, and then the solvent was distilled off. Chloroform (0.3 mL), methanol (0.1 mL) and hexafluoroisopropyl were added to the resulting residue. Alcohol (0.2 mL) was added and stirred well. Insoluble materials were filtered off, and the obtained filtrate was concentrated to give the target compounds 50 to 234.
  Below, the yield of the representative compound obtained in Example 16,¹1 shows H NMR spectral data and mass spectral data.
2- (3-Chlorobenzylamino) -4- (2-phenylpropylamino) pyrimidine-5-carboxylic acid (Compound 64)
Yield: 56% (4 steps)
¹H-NMR (300 MHz, CDCl₃, 80 ° C.) δ (ppm): 1.18 (d, J = 7.0 Hz, 3H), 2.99 (m, 1H), 3.55 (m, 2H), 4.59 (d, J = 6.2 Hz, 2 H), 7.14-7.35 (m, 9 H), 7.72 (br s, 1 H), 8.19 (br t, 1 H), 8.37 (s, 1 H)
ESI-MS: m / z 397 (M + H)⁺
4- (2-Phenylpropylamino) -2- (3-trifluoromethylbenzylamino) pyrimidine-5-carboxylic acid (Compound 71)
Yield: 56% (4 steps)
¹H-NMR (300 MHz, DMSO-d₆, 80 ° C.) δ (ppm): 1.16 (d, J = 6.8 Hz, 3H), 2.98 (m, 1H), 3.54 (m, 2H), 4.59 (d, J = 6.4 Hz, 0.4 H), 4.66 (d, J = 6.2 Hz, 1.6 H), 6.95-7.79 (m, 9 H), 8.00 (br s, 0.2 H) , 8.17 (brs, 0.8H), 8.38 (s, 0.2H), 8.59 (s, 0.8H), 10.90 (s, 1H)
ESI-MS: m / z 431 (M + H)⁺
2- (Cyclohexylmethylamino) -4- (1-methyl-2-phenoxyethylamino) pyrimidine-5-carboxylic acid (Compound 106)
Yield: 46% (4 steps)
¹H-NMR (300 MHz, CDCl₃, 80 ° C.) δ (ppm): 0.92-1.09 (m, 2H), 1.13-1.27 (m, 3H), 1.43 (d, J = 6.8 Hz, 3H), 1.58-1.80 (m, 6H), 3.26 (t, J = 5.7 Hz, 2H), 3.96 (dd, J = 6.0, 9.1 Hz, 1H), 4.14 (Dd, J = 4.6, 9.1 Hz, 1H), 4.61 (m, 1H), 6.93 (m, 3H), 7.23 (m, 2H), 8.37 (s, 1H) ), 9.77 (br s, 1H), 10.3 (br d, J = 5.9 Hz, 1H)
ESI-MS: m / z 385 (M + H)⁺
4- (1-Methyl-2-phenoxyethylamino) -2- (3-trifluoromethylbenzylamino) pyrimidine-5-carboxylic acid (Compound 112)
Yield: 65% (4 steps)
¹H-NMR (300 MHz, CDCl₃, 80 ° C.) δ (ppm): 1.29 (d, J = 6.6 Hz, 3H), 3.86 (dd, J = 5.3, 9.0 Hz, 1H), 3.96 (dd, J = 4.4, 9.0 Hz, 1H), 4.46 (m, 1H), 4.55 (d, J = 5.1 Hz, 2H), 6.80 (d, J = 8.3 Hz, 2H) , 6.88 (t, J = 7.5 Hz, 1H), 7.16 (t, J = 7.5 Hz, 2H), 7.30 (t, J = 7.7 Hz, 1H), 7.43 ( d, J = 7.9 Hz, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.56 (s, 1H), 8.41 (s, 1H), 10.16 (brs) , 1H), 10.47 (br t, 1H)
ESI-MS: m / z 447 (M + H)⁺
2- (Benzylbutylamino) -4- (2-phenoxyethylamino) pyrimidine-5-carboxylic acid (Compound 117)
Yield: 74% (4 steps)
¹H-NMR (300 MHz, CDCl₃, 80 ° C.) δ (ppm): 0.89 (t, J = 7.3 Hz, 3H), 1.31 (sextet, J = 7.3 Hz, 2H), 1.60 (quintet, J = 7.3 Hz) , 2H), 3.61 (br s, 2H), 3.82 (br s, 2H), 4.04 (br s, 2H), 4.89 (s, 2H), 6.83-6.93. (M, 3H), 7.19-7.29 (m, 7H), 8.16 (br s, 1H), 8.69 (s, 1H)
ESI-MS: m / z 421 (M + H)⁺
2- (Octahydroisoquinolin-2-yl) -4- (2-phenoxyethylamino) pyrimidine-5-carboxylic acid (Compound 121)
Yield: 39% (4 steps)
¹H-NMR (300 MHz, CDCl₃, 80 ° C.) δ (ppm): 1.27-1.94 (m, 12H), 3.28-3.42 (m, 2H), 3.87 (q, J = 5.8 Hz, 2H), 4.16 (t, J = 5.8 Hz, 2H), 4.26-4.42 (m, 2H), 6.90-6.95 (m, 3H), 7.24 (t, J = 7 .7 Hz, 2H), 8.14 (br s, 1H), 8.63 (s, 1H)
ESI-MS: m / z 397 (M + H)⁺
4- (2-phenoxyethylamino) -2- (3-trifluoromethylbenzylamino) pyrimidine-5-carboxylic acid (Compound 123)
Yield: 60% (4 steps)
¹H-NMR (300 MHz, DMSO-d₆, 80 ° C.) δ (ppm): 3.75 (q, J = 5.7 Hz, 2H), 4.04 (t, J = 5.7 Hz, 2H), 4.57 (d, J = 6.2 Hz) , 2H), 6.90 (d, J = 7.3 Hz, 2H), 6.92 (t, J = 7.3 Hz, 1H), 7.25 (t, J = 7.3 Hz, 2H), 7 .47-7.63 (m, 4H), 7.78 (br s, 1H), 8.39 (br s, 1H), 8.40 (s, 1H)
ESI-MS: m / z 433 (M + H)⁺
4- (2-phenoxyphenylamino) -2-[(pyridin-2-ylmethyl) amino] pyrimidine-5-carboxylic acid (Compound 138)
Yield: 50% (4 steps)
¹H-NMR (300 MHz, DMSO-d₆, 80 ° C.) δ (ppm): 4.66 (d, J = 6.0 Hz, 2H), 6.94-7.10 (m, 6H), 7.20-7.35 (m, 4H), 7.71 (dt, J = 1.8, 7.5 Hz, 1H), 8.02 (br s, 1H), 8.54 (t, J = 4.2 Hz, 1H), 8.56 (s, 1H), 10.88 (s, 1H)
ESI-MS: m / z 414 (M + H)⁺
2- (Cyclohexylmethylamino) -4- (2-phenoxyphenylamino) pyrimidine-5-carboxylic acid (Compound 139)
Yield: 70% (4 steps)
¹H-NMR (300 MHz, DMSO-d₆, 80 ° C.) δ (ppm): 0.92-1.09 (m, 2H), 1.13-1.27 (m, 3H), 1.62-1.77 (m, 6H), 21 (t, J = 6.4 Hz, 2H), 6.95-7.18 (m, 6H), 7.29-7.36 (m, 2H), 7.53 (brs, 1H), 8 .51 (s, 1H), 8.73 (br s, 1H), 10.87 (s, 1H)
ESI-MS: m / z 419 (M + H)⁺
2- (2-Methylbenzylamino) -4- (2-phenoxyphenylamino) pyrimidine-5-carboxylic acid (Compound 142)
Yield: 70% (4 steps)
¹H-NMR (300 MHz, DMSO-d₆, 80 ° C.) δ (ppm): 2.32 (s, 3H), 4.55 (d, J = 5.9 Hz, 2H), 6.94-7.34 (m, 13H), 7.92 ( br s, 1H), 8.56 (s, 1H), 10.84 (s, 1H)
ESI-MS: m / z 427 (M + H)⁺
2- (Benzylbutylamino) -4- (2-phenoxyphenylamino) pyrimidine-5-carboxylic acid (Compound 143)
Yield: 20% (4 steps)
¹H-NMR (300 MHz, CDCl₃, 80 ° C.) δ (ppm): 0.92 (t, J = 7.3 Hz, 3H), 1.35 (sextet, J = 7.3 Hz, 2H), 1.65 (quintet, J = 7.3 Hz) , 2H), 3.65 (br s, 2H), 4.94 (s, 2H), 6.96-7.04 (m, 6H) 7.20-7.32 (m, 8H), 8. 77 (s, 1H), 10.4 (br s, 1H)
ESI-MS: m / z 469 (M + H)⁺
4- (2-phenoxyphenylamino) -2- (3-trifluoromethylbenzylamino) pyrimidine-5-carboxylic acid (Compound 148)
Yield: 63% (4 steps)
¹H-NMR (300 MHz, DMSO-d₆, 80 ° C.) δ (ppm): 4.64 (d, J = 6.2 Hz, 2H), 6.93-7.09 (m, 7H), 7.29 (dd, J = 7.5, 8 .4 Hz, 2H), 7.51-7.67 (m, 4H), 8.13 (br s, 1H), 8.56 (s, 1H), 10.83 (s, 1H)
ESI-MS: m / z 481 (M + H)⁺
4- (4-Phenylpiperidin-1-yl) -2- (3-trifluoromethylbenzylamino) pyrimidine-5-carboxylic acid (Compound 177)
Yield: 72% (4 steps)
¹1 H-NMR (300 MHz, DMDO-d₆, 80 ° C.) δ (ppm): 1.56 (m, 2H), 1.76 (m, 2H), 2.77 (tt, J = 3.8, 11.9 Hz, 1H), 3.00 ( dt, J = 2.3, 14.4 Hz, 2H), 4.09 (d, J = 13.2 Hz, 2H), 4.56 (d, J = 6.2 Hz, 2H), 7.14-7 .30 (m, 5H), 7.48-7.66 (m, 5H), 8.37 (s, 1H)
ESI-MS: m / z 457 (M + H)⁺
Example 17: Synthesis of compounds 235-393
Process 1
  Commercially available ethyl 4-chloro-2-methylthio-5-pyrimidinecarboxylate in 1.54 g / 22 mL THF solution (200 μL, 14.0 mg, 60.2 μmol) was mixed with various amine reagents (90 μmol) and morpholinomethyl polystyrene (42 0.0 mg, 147 μmol) was added, and the mixture was stirred at 60 ° C. overnight. After adding THF (0.3 mL) and benzoyl chloride polymer bound (45.0 mg, 90 μmol) and further stirring overnight, the insoluble matter was filtered and washed. The obtained filtrate and washing solution were combined and concentrated, and the residue was concentrated. Obtained.
Process 2
  The residue obtained in Step 1 was dissolved in dichloromethane (0.2 mL), m-chlorofiltered benzoic acid (content 65%, 23.9 mg, 90.0 μmol) was added, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with dichloromethane (0.1 mL), washed with saturated aqueous sodium hydrogen carbonate, the organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated. The obtained residue was dissolved in THF (0.2 mL), various amines (90 μmol) were added, and the mixture was stirred at 60 ° C. overnight. Chloroform (0.4 mL), morpholinomethylpolystyrene (42.0 mg, 147 μmol) and benzoyl chloride polymer bound (90.0 mg, 180 μmol) were added and the mixture was further stirred overnight. The filtrate and the washing solution were combined and concentrated to obtain a residue.
Process 3
  The residue obtained in step 2 was dissolved in toluene (0.5 mL), 65 wt% bis (2-methoxyethoxy) aluminum hydride toluene solution (62.4 μL, 208 μmol) was added, and the mixture was stirred at 0 ° C. for 4 hours. . A 3 mol / L aqueous sodium hydroxide solution (300 μL) was added in two portions, and the organic layer was washed with saturated brine. The organic layer was dried over anhydrous magnesium sulfate, and then the solvent was distilled off to obtain the target compounds 235 to 393.
  Below, the yield of the representative compound obtained in Example 17,¹1 shows H NMR spectral data and mass spectral data.
2- (Cyclohexylmethylamino) -5-hydroxymethyl-4- (1-methyl-2-phenoxyethylamino) pyrimidine (Compound 256)
Yield: 38% (4 steps)
¹H-NMR (300 MHz, CDCl₃) Δ (ppm): 0.89-1.10 (m, 2H), 1.15 to 1.24 (m, 3H), 1.39 (d, J = 6.8 Hz, 3H), 1.47 -1.78 (m, 6H), 3.21 (dt, J = 2.6, 6.6 Hz, 2H), 3.96 (dd, J = 5.6, 9.1 Hz, 1H), 4. 10 (dd, J = 3.8, 9.1 Hz, 1H), 4.42 (s, 2H), 4.46-4.63 (m, 1H), 4.86 (brt, 1H), 5 .68 (d, J = 7.1 Hz, 1H), 6.94 (t, J = 8.6 Hz, 3H), 7.23-7.30 (m, 2H), 7.59 (s, 1H)
ESI-MS: m / z 371 (M + H)⁺
2- (Benzylbutylamino) -5-hydroxymethyl-4- (2-phenoxyethylamino) pyrimidine (Compound 264)
Yield: 55% (4 steps)
¹H-NMR (300 MHz, CDCl₃) Δ (ppm): 0.89 (t, J = 7.3 Hz, 3H), 1.31 (sextet, J = 7.1 Hz, 2H), 1.59 (m, 2H), 3.55 (t , J = 7.5 Hz, 2H), 3.78 (brs, 2H), 3.98 (brs, 2H), 4.45 (s, 2H), 4.84 (s, 2H), 5. 86 (brs, 1H), 6.81 (d, J = 8.2 Hz, 2H), 6.93 (t, J = 8.4 Hz, 1H), 7.18-7.30 (m, 7H) , 7.71 (s, 1H)
ESI-MS: m / z 407 (M + H)⁺
5-Hydroxymethyl-2- (octahydroisoquinolin-2-yl) -4- (2-phenoxyethylamino) pyrimidine (Compound 267)
Yield: 44% (4 steps)
¹H-NMR (300 MHz, CDCl₃) Δ (ppm): 1.25-1.91 (m, 12H), 3.27 (m, 2H), 3.89 (m, 2H), 4.16 (t, J = 5.5 Hz, 2H) ), 4.26 (m, 2H), 4.42 (s, 2H), 5.85 (brt, J = 5.1 Hz, 1H), 6.89-6.97 (m, 3H), 7 .23-7.30 (m, 2H), 7.65 (s, 1H)
ESI-MS: m / z 383 (M + H)⁺
5-hydroxymethyl-4- (4-phenylbutylamino) -2- (3-trifluoromethylbenzylamino) pyrimidine (Compound 278)
Yield: 60% (4 steps)
¹H-NMR (270 MHz, CDCl₃) Δ (ppm): 1.57-1.68 (m, 4H), 2.60 (t, J = 7.3 Hz, 2H), 3.38 (q, J = 6.3 Hz, 2H), 4 .40 (s, 2H), 4.60 (d, J = 6.1 Hz, 2H), 5.36 (brs, 1H), 5.65 (brt, 1H), 7.12-7.29 (M, 5H), 7.39 (t, J = 7.4 Hz, 1H), 7.48 (d, J = 5.8 Hz, 1H), 7.51 (d, J = 6.5 Hz, 1H) 7.52 (s, 1H), 7.60 (s, 1H)
ESI-MS: m / z 431 (M + H)⁺
2- (3-Fluorobenzylamino) -5-hydroxymethyl-4- (indan-2-ylamino) pyrimidine (Compound 287)
Yield: 69% (4 steps)
¹H-NMR (270 MHz, CDCl₃) Δ (ppm): 2.20 (br s, 1H), 2.79 (dd, J = 6.3, 16.0 Hz, 2H), 3.27 (dd, J = 7.4, 16.0 Hz) , 2H), 4.38 (s, 2H), 4.57 (d, J = 6.0 Hz, 2H), 4.79 (m, 1H), 5.35 (brs, 1H), 5.85. (D, J = 6.6 Hz, 1H), 6.92 (dt, J = 2.1, 8.4 Hz, 1H), 7.07 (t, J = 7.8 Hz, 2H), 7.13- 7.29 (m, 5H), 7.51 (s, 1H)
ESI-MS: m / z 365 (M + H)⁺
4- (2,3-dihydrobenzo [1,4] dioxin-6-ylamino) -5-hydroxymethyl-2- (3-trifluoromethylbenzylamino) pyrimidine (Compound 299)
Yield: 59% (4 steps)
¹H-NMR (270 MHz, CDCl₃) Δ (ppm): 4.23 (s, 4H), 4.50 (s, 2H), 4.61 (d, J = 6.3 Hz, 2H), 5.46 (brs, 1H), 6.72 (d, J = 8.8 Hz, 1H), 6.81 (dd, J = 2.5, 8.7 Hz, 1H), 7.26 (s, 1H), 7.39 (t, J = 7.6 Hz, 1H), 7.48 (br d, J = 7.2 Hz, 2H), 7.59 (s, 1H), 7.62 (s, 1H)
ESI-MS: m / z 433 (M + H)⁺
2- (Cyclohexylmethylamino) -5-hydroxymethyl-4- (2-phenoxyphenylamino) pyrimidine (Compound 320)
Yield: 27% (4 steps)
¹H-NMR (300 MHz, CDCl₃) Δ (ppm): 0.91-1.03 (m, 2H), 1.13-1.31 (m, 3H), 1.65-1.83 (m, 6H), 3.25 (t , J = 6.2 Hz, 2H), 4.40 (s, 1H), 5.06 (brs, 1H), 6.96-7.22 (m, 6H), 7.27-7.32 ( m, 3H), 7.71 (s, 1H), 8.41 (s, 1H), 8.62 (d, J = 6.2, 1H)
ESI-MS: m / z 405 (M + H)⁺
5-Hydroxymethyl-2- (2-methylbenzylamino) -4- (2-phenoxyphenylamino) pyrimidine (Compound 321)
Yield: 33% (4 steps)
¹H-NMR (270 MHz, CDCl₃) Δ (ppm): 2.36 (s, 3H), 4.42 (s, 2H), 4.58 (d, J = 5.4 Hz, 2H), 5.23 (brs, 1H), 6 .93-7.08 (m, 6H), 7.12-7.32 (m, 7H), 7.70 (s, 1H), 8.35 (s, 1H), 8.40 (d, J = 7.6Hz, 1H)
ESI-MS: m / z 413 (M + H)⁺
5-Hydroxymethyl-4- (2-phenoxyethylamino) -2- (3-trifluoromethylbenzylamino) pyrimidine (Compound 386)
Yield: 48% (4 steps)
¹H-NMR (300 MHz, CDCl₃) Δ (ppm): 3.78 (q, J = 5.3 Hz, 2H), 4.00 (t, J = 5.3 Hz, 2H), 4.43 (s, 2H), 4.62 (d , J = 6.0 Hz, 2H), 5.32 (br t, 1H), 6.05 (br t, 1H), 6.83 (d, J = 7.9 Hz, 2H), 6.93 (t , J = 7.3 Hz, 1H), 7.24-7.29 (m, 2H), 7.39 (t, J = 7.1 Hz, 1H), 7.49 (d, J = 6.4 Hz, 1H), 7.51 (d, J = 7.0 Hz, 1H), 7.59 (s, 2H)
ESI-MS: m / z 419 (M + H)⁺
5-hydroxymethyl-4- (2-phenoxyphenylamino) -2- (3-trifluoromethylbenzylamino) pyrimidine (Compound 387)
Yield: 27% (4 steps)
¹H-NMR (270 MHz, CDCl₃) Δ (ppm): 4.41 (s, 2H), 4.65 (d, J = 6.1 Hz, 2H), 5.50 (brs, 1H), 6.92-7.07 (m, 6H), 7.23-7.66 (m, 2H), 7.39-7.66 (m, 5H), 8.31 (brd, J = 6.4 Hz, 1H), 8.40 (s). , 1H)
ESI-MS: m / z 467 (M + H)⁺
5-Hydroxymethyl-4- (2-phenoxyphenylamino) -2-[(pyridin-2-ylmethyl) amino] pyrimidine (Compound 388)
Yield: 25% (4 steps)
¹H-NMR (270 MHz, DMSO-d₆) Δ (ppm): 4.26 (d, J = 4.4 Hz, 2H), 4.56 (d, J = 5.9 Hz, 2H), 5.32 (t, J = 4.8 Hz, 1H) , 6.93-7.47 (m, 12H), 7.71 (dt, J = 1.8, 7.7 Hz, 1H), 7.76 (s, 1H), 8.52 (d, J = 4.0Hz, 1H), 8.71 (s, 1H)
ESI-MS: m / z 400 (M + H)⁺
2- (Benzylbutylamino) -5-hydroxymethyl-4- (2-phenoxyphenylamino) pyrimidine (Compound 389)
Yield: 31% (4 steps)
¹H-NMR (270 MHz, CDCl₃) Δ (ppm): 0.92 (t, J = 7.2 Hz, 3H), 1.35 (sextet, J = 7.2 Hz, 2H), 1.60-1.69 (m, 2H), 3 .58 (t, J = 7.7 Hz, 2H), 4.39 (s, 2H), 4.87 (s, 2H), 6.93-7.07 (m, 6H), 7.18-7 .32 (m, 8H), 7.77 (s, 1H), 8.28 (br s, 1H)
ESI-MS: m / z 455 (M + H)⁺
5-hydroxymethyl-4- (1-methyl-2-phenoxyethylamino) -2- (3-trifluoromethylbenzylamino) pyrimidine (Compound 390)
Yield: 46% (4 steps)
¹H-NMR (300 MHz, CDCl₃) Δ (ppm): 1.32 (d, J = 6.8 Hz, 3H), 3.86 (dd, J = 5.1, 9.1 Hz, 1H), 3.95 (dd, J = 3. 8, 9.1 Hz, 1 H), 4.42 (s, 2 H), 4.46-4.53 (m, 1 H), 4.60 (d, J = 5.7 Hz, 2 H), 5.30 ( br s, 1H), 5.77 (d, J = 7.9 Hz, 1H), 6.87 (dd, J = 1.1, 7.7 Hz, 2H), 6.93 (tt, J = 1. 1, 7.3 Hz, 1 H), 7.21-7.28 (m, 2 H), 7.38 (t, J = 7.7 Hz, 1 H), 7.47 (d, J = 9.3 Hz, 1 H) ), 7.51 (d, J = 7.8 Hz, 1H), 7.59 (brs, 1H), 7.61 (s, 1H)
ESI-MS: m / z 433 (M + H)⁺
5-Hydroxymethyl-4- (2-phenylpropylamino) -2- (3-triololomethylbenzylamino) pyrimidine (Compound 391)
Yield: 48% (4 steps)
¹H-NMR (300 MHz, CDCl₃) Δ (ppm): 1.24 (d, J = 7.0 Hz, 3H), 2.98 (sextet, J = 7.0 Hz, 1H), 3.42 (m, 1H), 3.62 (m) , 1H), 4.30 (s, 2H), 4.65 (d, J = 5.9 Hz, 2H), 5.28 (brs, 1H), 5.55 (brs, 1H), 7. 11-7.30 (m, 5H), 7.41 (t, J = 7.3 Hz, 1H), 7.49 (d, J = 8.4 Hz, 1H), 7.52 (d, J = 6) .3 Hz, 1 H), 7.53 (s, 1 H), 7.61 (s, 1 H)
ESI-MS: m / z 417 (M + H)⁺
2- (3-Chlorobenzylamino) -5-hydroxymethyl-4- (2-phenylpropylamino) pyrimidine (Compound 392)
Yield: 41% (4 steps)
¹H-NMR (300 MHz, CDCl₃) Δ (ppm): 1.26 (d, J = 7.0 Hz, 3H), 2.98 (sextet, J = 7.3 Hz, 1H), 3.43 (m, 1H), 3.65 (m , 1H), 4.31 (s, 2H), 4.58 (d, J = 5.9 Hz, 2H), 5.23 (brs, 1H), 5.54 (brs, 1H), 7. 12-7.35 (m, 9H), 7.55 (s, 1H)
ESI-MS: m / z 383 (M + H)⁺
5-hydroxymethyl-4- (4-phenylpiperidin-1-yl) -2- (3-trifluoromethylbenzylamino) pyrimidine (Compound 393)
Yield: 64% (4 steps)
¹H-NMR (300 MHz, CDCl₃) Δ (ppm): 1.73 (m, 2H), 1.88 (m, 2H), 2.75 (tt, J = 4.0, 12.1 Hz, 1H), 2.96 (dt, J = 2.6, 13.2 Hz, 2H), 4.39 (brd, J = 13.2 Hz, 2H), 4.51 (s, 2H), 4.64 (d, J = 6.2 Hz, 2H) ), 5.34 (brs, 1H), 7.18-7.33 (m, 5H), 7.41 (t, J = 7.7 Hz, 1H), 7.48 (d, J = 7. 7 Hz, 1 H), 7.53 (d, J = 7.7 Hz, 1 H), 7.61 (s, 1 H), 7.88 (s, 1 H)
ESI-MS: m / z 443 (M + H)⁺
Example 18: Synthesis of 2- (cyclohexylmethylamino) -4- (2-phenoxyethylamino) pyrimidine-5-carboxylic acid (Compound 116)
Step 1: Synthesis of ethyl 2-methylthio-4- (2-phenoxyethylamino) pyrimidine-5-carboxylate
  Commercially available ethyl 4-chloro-2-methylthio-5-pyrimidinecarboxylate (11.3 g, 48.6 mmol) was dissolved in tetrahydrofuran (300 mL) and N, N-diisopropylethylamine (16.9 mL, 97.0 mmol) and 2-phenoxyethylamine (9.54 mL, 72.9 mmol) was added, and the mixture was stirred at room temperature for 5 hours. The reaction solution was distilled off under reduced pressure and diluted with ethyl acetate. The organic layer was washed successively with water and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was recrystallized from ethanol to obtain the title compound (16.1 g, 48.3 mmol, quantitative yield) as white crystals.
¹H-NMR (270 MHz, CDCl₃) Δ (ppm): 1.35 (t, J = 7.1 Hz, 3H) 2.52 (s, 3H), 3.96 (q, J = 5.5 Hz, 2H), 4.16 (t, J = 5.5 Hz, 2H), 4.33 (q, J = 7.1 Hz, 2H), 6.91-6.98 (m, 3H), 7.26 (t, J = 8.6 Hz, 2H) ), 8.54 (br s, 1H), 8.63 (s, 1H)
Step 2: Synthesis of ethyl 2- (cyclohexylmethylamino) -4- (2-phenoxyethylamino) pyrimidine-5-carboxylate
  Ethyl 2-methylthio-4- (2-phenoxyethylamino) pyrimidine-5-carboxylate (10.0 g, 30.2 mmol) obtained in step 1 was dissolved in dichloromethane (200 mL), and m-chlorinated under ice-cooling. Filtered benzoic acid (content 65%, 12.0 g, 45.2 mmol) was added over 10 minutes and stirred at room temperature for 1 hour. The reaction mixture was diluted with dichloromethane and washed with saturated aqueous sodium hydrogen carbonate. The organic layer was dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was dissolved in tetrahydrofuran (200 mL), cyclohexylmethylamine (5.89 mL, 45.2 mmol) was added, and the mixture was stirred with heating under reflux for 10 hr. The reaction solution was distilled off under reduced pressure, and the resulting crude crystals were recrystallized from ethanol to obtain the title compound (9.74 g, 24.4 mmol, yield 81%) as white crystals.
¹H-NMR (270 MHz, CDCl₃, 80 ° C.) δ (ppm): 0.93-1.06 (m, 2H), 1.11-1.27 (m, 3H), 1.32 (t, J = 7.1 Hz, 3H), 1.51-1.78 (m, 6H), 3.28 (t, J = 6.6 Hz, 2H), 3.87 (q, J = 5.7 Hz, 2H), 4.15 (t, J = 5.7 Hz, 2H), 4.29 (q, J = 7.1 Hz, 2H), 5.22 (brs, 1H), 6.89-6.95 (m, 3H), 7.21- 7.28 (m, 2H), 8.34 (br s, 1H), 8.55 (s, 1H)
Step 3: Synthesis of 2- (cyclohexylmethylamino) -4- (2-phenoxyethylamino) pyrimidine-5-carboxylic acid (Compound 116)
  Ethyl 2- (cyclohexylmethylamino) -4- (2-phenoxyethylamino) pyrimidine-5-carboxylate (1.00 g, 2.51 mmol) obtained in step 2 was dissolved in ethanol (10 mL), and 3 mol / L Aqueous sodium hydroxide solution (10.0 mL, 30.0 mmol) was added, and the mixture was stirred at 50 ° C. for 5 hours. The reaction solution was concentrated under reduced pressure to half the volume, and the pH of the reaction solution was adjusted to 4.5 with 1 mol / L hydrochloric acid aqueous solution. The precipitated crystals were collected by filtration to give the title compound (880 mg, 2.25 mmol, yield 95%) as white crystals.
¹H-NMR (300 MHz, CDCl₃) Δ (ppm): 0.90 to 1.01 (m, 2H), 1.08 to 1.25 (m, 3H), 1.59-1.80 (m, 6H), 3.26 (t , J = 5.9 Hz, 2H), 3.93 (q, J = 5.5 Hz, 2H), 4.18 (t, J = 5.5 Hz, 2H), 6.91-6.98 (m, 3H), 7.25-7.31 (m, 2H), 8.36 (s, 1H), 9.89 (brs, 1H), 10.37 (brs, 1H)
ESI-MS: m / z 371 (M + H)⁺
Example 19: Synthesis of 2- (cyclohexylmethylamino) -5-hydroxymethyl-4- (2-phenoxyethylamino) pyrimidine (Compound 263)
  Dissolve ethyl 2- (cyclohexylmethylamino) -4- (2-phenoxyethylamino) pyrimidine-5-carboxylate (1.00 g, 2.51 mmol) obtained in Step 2 of Example 18 in toluene (20 mL). The flask was purged with argon gas, 65 wt% bis (2-methoxyethoxy) aluminum hydride toluene solution (3.00 mL, 9.99 mmol) was added at 0 ° C, and the mixture was stirred at 0 ° C for 2 hours. 3 mol / L aqueous sodium hydroxide solution was added to stop the reaction, and the mixture was diluted with ethyl acetate. The organic layer was washed with water and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was recrystallized from ethanol to give the title compound (500 mg, 1.40 mmol, yield 56%) as white crystals.
¹H-NMR (300 MHz, CDCl₃) Δ (ppm): 0.93-1.00 (m, 2H), 1.16-1.24 (m, 3H), 1.51-1.79 (m, 6H), 3.20 (t , J = 6.4 Hz, 2H), 3.84 (q, J = 5.5 Hz, 2H), 4.14 (t, J = 5.5 Hz, 2H), 4.39 (s, 2H), 4 .87 (br t, J = 5.5 Hz, 1H), 6.01 (br t, J = 5.7 Hz, 1H), 6.89-6.97 (m, 3H), 7.24-7. 30 (m, 2H), 7.49 (s, 1H)
ESI-MS: m / z 357 (M + H)⁺
Formulation Example 1: Tablet
  A tablet having the following composition is prepared by a conventional method.
  Formulation Compound 1 20mg
        Lactose 143.4mg
        Potato starch 30mg
        Hydroxypropylcellulose 6mg
Magnesium stearate 0.6mg
                                        200mg
Formulation Example 2: Injection
  An injection having the following composition is prepared by a conventional method.
  Formulation Compound 37 2mg
        Refined soybean oil 200mg
        Purified egg yolk lecithin 24mg
        50mg glycerin for injection
Distilled water for injection 1.72mL
                                      2.00 mL

  According to the present invention, 1) a prophylactic and / or therapeutic agent for central diseases comprising, as an active ingredient, a substance having a GPR88 function inhibitory action, an antisense oligonucleotide of GPR88, a pyrimidine derivative, or a pharmacologically acceptable salt thereof. 2) GPR88 function inhibitor containing GPR88 antisense oligonucleotide, pyrimidine derivative or a pharmacologically acceptable salt thereof as an active ingredient, and 3) a compound having a preventive and / or therapeutic action for central diseases. Screening methods, 4) pyrimidine derivatives having GPR88 function-inhibiting action, or pharmacologically acceptable salts thereof, 5) antisense oligonucleotides having GPR88 function-inhibiting action, and the like are provided.

SEQ ID NO: 7-Strg-AS1, phosphorothioate antisense oligonucleotide for GPR88 gene SEQ ID NO: 8-Synthetic DNA for construction of double-stranded DNA containing polyadenylation signal
SEQ ID NO: 9-Synthetic DNA for construction of double-stranded DNA containing polyadenylation signal
SEQ ID NO: 10-Synthetic DNA for construction of double-stranded DNA containing polyadenylation signal
SEQ ID NO: 11-Synthetic DNA for construction of double-stranded DNA groove containing polyadenylation signal
SEQ ID NO: 12-2 Synthetic DNA for construction of double-stranded DNA groove containing two CRE sequences
SEQ ID NO: 13-2 Synthetic DNA for construction of double-stranded DNA containing two CRE sequences
SEQ ID NO: 14-Gα _s4 gene-specific PCR primer SEQ ID NO: 15-Gα _s4 gene-specific PCR primer SEQ ID NO: 16-Synthetic DNA for construction of double-stranded DNA encoding the C-terminal 5 amino acid residues of Gα _q
Synthetic DNA for construction of double-stranded DNA encoding the C-terminal 5 amino acid residue of SEQ ID NO: 17-Gα _q
Synthetic DNA for construction of double-stranded DNA encoding the C-terminal 5 amino acid residues of SEQ ID NO: 18-Gα _i
SEQ ID NO: 19-Synthetic DNA for construction of double-stranded DNA encoding C-terminal 5 amino acid residues of Gα _i
SEQ ID NO: 20-V2 gene specific PCR primer SEQ ID NO: 21-V2 gene specific PCR primer SEQ ID NO: 22-GPR12 gene specific PCR primer SEQ ID NO: 23-GPR12 gene specific PCR primer SEQ ID NO: 24-pAGal9-nd specific base Sequencing primer SEQ ID NO: 25-pAGal9-nd specific nucleotide sequencing primer SEQ ID NO: 26-GPR88 gene specific PCR primer SEQ ID NO: 27-GPR88 gene specific PCR primer

[Sequence Listing]

Claims (65)

A prophylactic and / or therapeutic agent for central diseases comprising, as an active ingredient, a substance having a function-inhibiting action of G protein-coupled receptor protein 88 (GPR88; G-Protein coupled receptor 88). The preventive and / or therapeutic agent according to claim 1, wherein the central disease is a disease selected from the group consisting of Parkinson's disease, depression and jet lag. (A) contacting a test compound with a GPR88-expressing cell and measuring the cellular response; and (b) comparing the cellular response of the GPR88-expressing cell without adding the test compound with the test compound. And a method for screening a compound having a preventive and / or therapeutic action for a central disease, comprising the step of confirming that the cellular response is reduced. The method according to claim 3, wherein the central disease is a disease selected from the group consisting of Parkinson's disease, depression and jet lag. A substance having GPR88 function inhibitory activity is an antisense oligonucleotide having a base sequence complementary or substantially complementary to a sequence of 10 or more consecutive base sequences represented by SEQ ID NO: 1, 3 or 5 The preventive and / or therapeutic agent for central diseases according to claim 1. The prophylactic and / or therapeutic agent for central diseases according to claim 1, wherein the substance having a GPR88 function suppressing action is an antibody against GPR88. A prophylactic and / or therapeutic agent for central diseases comprising, as an active ingredient, a compound that reduces the cellular response of GPR88 or a pharmacologically acceptable salt thereof, which is confirmed by the screening method according to claim 3. The preventive and / or therapeutic agent according to any one of claims 5 to 7, wherein the central disease is a disease selected from the group consisting of Parkinson's disease, depression and jet lag. GPR88 function inhibitor comprising as an active ingredient an antisense oligonucleotide having a base sequence complementary or substantially complementary to a sequence of 10 or more consecutive base sequences represented by SEQ ID NO: 1, 3 or 5 . An antisense oligonucleotide having the sequence represented by SEQ ID NO: 8. The antisense oligonucleotide according to claim 10, wherein the antisense oligonucleotide is a phosphorothioate oligodeoxynucleotide. A function inhibitor of GPR88 comprising the antisense oligonucleotide according to claim 10 or 11 as an active ingredient. A prophylactic and / or therapeutic agent for central diseases comprising the antisense oligonucleotide according to claim 10 or 11 as an active ingredient. The preventive and / or therapeutic agent according to claim 13, wherein the central disease is a disease selected from the group consisting of Parkinson's disease, depression and jet lag. Formula (I)

<In the formula, A is substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted aryloxyalkyl, substituted or unsubstituted aralkyloxyalkyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted Represents a substituted heterocyclic alkyl or cycloalkylalkyl;
R ¹ represents a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl or substituted or unsubstituted aralkyl,
Or A and R ¹ together with the adjacent nitrogen atom form a substituted or unsubstituted heterocyclic group;
R ² is a hydrogen atom, hydroxy, halogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted lower alkoxy, substituted or unsubstituted lower alkylthio, Substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted heterocyclic alkyl, or —NR ³ R ⁴ (wherein R ³ and R ⁴ are the same or different) Hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted Aryl, substituted or unsubstituted aralkyl Represents a substituted or unsubstituted heterocyclic group or substituted or unsubstituted heterocyclic alkyl, or R ³ and R ⁴ together with the adjacent nitrogen atom form a substituted or unsubstituted heterocyclic group) Represents
X is —C (═O) R ⁵ {wherein R ⁵ is hydroxy, substituted or unsubstituted lower alkoxy, substituted or unsubstituted aryloxy or —NR ⁶ R ⁷ , wherein R ⁶ and R ⁷ are The same or different, hydrogen atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted lower alkoxy, substituted or unsubstituted aralkyl, substituted or Represents unsubstituted heterocyclic alkyl, hydroxy or —SO ₂ R ⁸ , wherein R ⁸ represents substituted or unsubstituted lower alkyl or substituted or unsubstituted aryl,
Or R ⁶ and R ⁷ together with the adjacent nitrogen atom form a substituted or unsubstituted heterocyclic group], nitro, substituted or unsubstituted hydroxymethyl or 1,2,3,4- Represents tetrazol-5-yl,
Y represents a hydrogen atom, a halogen, or a substituted or unsubstituted lower alkyl, a pyrimidine derivative represented by> or a pharmacologically acceptable salt thereof as a GPR88 function inhibitor. The function inhibitor of GPR88 according to claim 15, wherein Y is a hydrogen atom. R ² is ^-NR 3 ^{R 4} function inhibitors GPR88 range 15 or 16 claim of claim (wherein, ^{R 3} and ^{R 4} have the same meanings as defined above) is. The function inhibitor of GPR88 according to any one of claims 15 to 17, wherein X is 1,2,3,4-tetrazol-5-yl. X is ^{-C (= O) NR 6 R} 7 ( wherein, ^{R 6} and ^{R 7} have the same meanings as defined above) function inhibitors GPR88 according to any of claims the 15 to 17, wherein a . X is -C (= ^{O) (wherein, R 5B} represents hydroxy or lower alkoxy) ^{R 5B} functional inhibitor of GPR88 according to any of claims the 15 to 17 wherein a. The function inhibitor of GPR88 according to any one of claims 15 to 17, wherein X is substituted or unsubstituted hydroxymethyl. The function inhibitor of GPR88 according to any one of claims 15 to 21, wherein A is substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, or substituted or unsubstituted heterocyclic group. The function inhibitor of GPR88 according to any one of claims 15 to 22, wherein R ¹ is a hydrogen atom. R ³ is substituted or unsubstituted lower alkyl, cycloalkylalkyl or substituted or unsubstituted aralkyl, or R ³ and R ⁴ together with the adjacent nitrogen atom form a substituted or unsubstituted heterocyclic group The function inhibitor of GPR88 according to any one of claims 17 to 23, which is formed. 24. The GPR88 of any one of claims 17 to 23, wherein R ³ is substituted or unsubstituted lower alkyl, cycloalkylalkyl, or substituted or unsubstituted aralkyl, and R ⁴ is a hydrogen atom or lower alkyl. Function inhibitor. Formula (I)

(Wherein, A, R ¹ , R ² , X and Y have the same meanings as described above), and the prevention and prevention of central diseases containing, as an active ingredient, a pyrimidine derivative represented by the formula or a pharmacologically acceptable salt thereof / Or therapeutic agent. 27. The preventive and / or therapeutic agent for central diseases according to claim 26, wherein Y is a hydrogen atom. The preventive and / or therapeutic agent for central diseases according to claim 26 or 27, wherein R ² is -NR ³ R ⁴ (wherein R ³ and R ⁴ are as defined above, respectively). The preventive and / or therapeutic agent for central diseases according to any one of claims 26 to 28, wherein X is 1,2,3,4-tetrazol-5-yl. X is ^{-C (= O) NR 6 R} 7 ( wherein, ^{R 6} and ^{R 7} have the same meanings as defined above) prevention of central diseases according to any one of claims first 26-28 wherein a and / Or therapeutic agent. X is -C (= ^{O) (wherein, R 5B} represents hydroxy or lower alkoxy) ^{R 5B} prevention and / or therapeutic agent for central diseases according to any of claims the 26 to 28 wherein a. 29. The preventive and / or therapeutic agent for central diseases according to any one of claims 26 to 28, wherein X is substituted or unsubstituted hydroxymethyl. 33. The preventive and / or therapeutic agent for central diseases according to any one of claims 26 to 32, wherein A is substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, or substituted or unsubstituted heterocyclic group. The preventive and / or therapeutic agent for central diseases according to any one of claims 26 to 33, wherein R ¹ is a hydrogen atom. R ³ is substituted or unsubstituted lower alkyl, cycloalkylalkyl or substituted or unsubstituted aralkyl, or R ³ and R ⁴ together with the adjacent nitrogen atom form a substituted or unsubstituted heterocyclic group The preventive and / or therapeutic agent for central diseases according to any one of claims 28 to 34. The central disease according to any one of claims 28 to 34, wherein R ³ is substituted or unsubstituted lower alkyl, cycloalkylalkyl, or substituted or unsubstituted aralkyl, and R ⁴ is a hydrogen atom or lower alkyl. Preventive and / or therapeutic agent. The preventive and / or therapeutic agent according to any one of claims 26 to 36, wherein the central disease is a disease selected from the group consisting of Parkinson's disease, depression and jet lag. Formula (IA)

{In the formula, A and R ¹ are as defined above, and X ^A is 1,2,3,4-tetrazol-5-yl, —C (═O) R ^5A [wherein R ^5A is hydroxy or —NR ^6A R ^7A (wherein R ^6A represents a hydrogen atom or lower alkyl, and R ^7A represents a hydrogen atom, hydroxy, hydroxy lower alkyl, carboxy lower alkyl, p-toluenesulfonyl, methanesulfonyl or trifluoromethanesulfonyl) Or substituted or unsubstituted hydroxymethyl, and (i) when X ^A is 1,2,3,4-tetrazol-5-yl, R ^3A and R ^4A are the same or different and represent hydrogen Atom, substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylalkyl or Form a substituted or unsubstituted aralkyl (provided that at the same time not a hydrogen atom) or R ^3A and R ^4A is substituted or unsubstituted together with the adjacent nitrogen atom a heterocyclic group, ( ii) When X ^A is —C (═O) R ^5A or substituted or unsubstituted hydroxymethyl, R ^3A and R ^4A are the same or different and each represents a hydrogen atom, substituted or unsubstituted lower alkyl, substituted or Represents unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted heterocyclic alkyl or substituted or unsubstituted aralkyl (but not simultaneously hydrogen atoms), or R ^3A and R ^4A Together with the adjacent nitrogen atom, substituted or unsubstituted piperazinyl, 4-methylhomopiperazinyl, 4-methyl A piperidino, 4-piperidinopiperidino, 4-benzylpiperidino, 4,4-ethylenedioxypiperidino or decahydroisoquinolin-1-yl} or a pharmacology thereof Acceptable salt. Pyrimidine derivative or a pharmacologically acceptable salt of X ^A is 1,2,3,4-tetrazol-5-yl in claims 38 wherein wherein. The pyrimidine derivative or a pharmaceutically acceptable salt thereof according to claim 38, wherein X ^A is -C (= O) R ^5A (wherein R ^5A has the same meaning as described above). The pyrimidine derivative or a pharmaceutically acceptable salt thereof according to claim 38, wherein X ^A is -C (= O) OH. The pyrimidine derivative or a pharmaceutically acceptable salt thereof according to claim 38, wherein X ^A is hydroxymethyl, 1-hydroxyethyl or 1-hydroxy-1-methylethyl. A is substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted aryloxyalkyl, substituted or unsubstituted heterocyclic group or cycloalkylalkyl, or nitrogen in which A and R ¹ are adjacent The pyrimidine derivative or a pharmaceutically acceptable salt thereof according to any one of claims 38 to 42, which forms a substituted or unsubstituted heterocyclic group together with an atom. 43. The pyrimidine derivative or a pharmaceutically acceptable salt thereof according to any one of claims 38 to 42, wherein A is substituted or unsubstituted aryl, aralkyl, heterocyclic group or cycloalkylalkyl. A is benzyl, 4-phenyl-n-butyl, 3-phenylpropyl, 3-phenyl-1-methylpropyl, 2-phenylpropyl, 2-phenoxyethyl, 2-phenoxy-1-methylethyl, 1,4-benzo A group selected from the group consisting of dioxane-6-yl, 2-benzylphenyl, 2-phenoxyphenyl, 3-phenoxyphenyl, 4-phenoxyphenyl, 4-biphenyl, biphenyl-4-ylmethyl, 2-indanyl and cyclopropylmethyl The pyrimidine derivative according to any one of claims 38 to 42 or a pharmaceutically acceptable salt thereof. The pyrimidine derivative or a pharmaceutically acceptable salt thereof according to any one of claims 38 to 45, wherein R ¹ is a hydrogen atom, lower alkyl or aralkyl. The pyrimidine derivative or a pharmaceutically acceptable salt thereof according to any one of claims 38 to 45, wherein R ¹ is a hydrogen atom. R ^3A and R ^4A are the same or different and each represents a hydrogen atom, a substituted or unsubstituted lower alkyl, cycloalkylalkyl, or substituted or unsubstituted aralkyl or a substituted or unsubstituted heterocyclic alkyl, or R ^3A and R pyrimidine derivative or a pharmacologically acceptable salt thereof according to any one of claims first 38-47 wherein ^{the 4A} together with the adjacent nitrogen atom to form a substituted or unsubstituted heterocyclic group. The pyrimidine derivative according to any one of claims 38 to 47, wherein R ^3A and R ^4A are the same or different and each is a hydrogen atom, lower alkyl, cycloalkylmethyl, substituted or unsubstituted aralkyl or heterocyclic alkyl. Its pharmacologically acceptable salt. The pyrimidine derivative according to any one of claims 38 to 47 or a pharmaceutically acceptable salt thereof, wherein R ^3A is lower alkyl, cycloalkylmethyl, or substituted or unsubstituted aralkyl, and R ^4A is a hydrogen atom. Salt. R ^3A is 3-methoxybenzyl, 3-chlorobenzyl, 3-fluorobenzyl, 2,6-difluorobenzyl, 2-methylbenzyl, 3-trifluorobenzyl, 2-pyridylmethyl, 3-pyridylmethyl, cyclohexylmethyl, cyclo A group selected from the group consisting of heptyl, 1,5-dimethyl-n-hexyl and 2,3-dihydrobenzo [1,4] dioxin-2-ylmethyl, wherein R ^4A is a hydrogen atom or R ^3A is Benzyl and R ^4A is n-butyl, R ^3A is n-hexyl and R ^4A is methyl, or R ^3A and R ^4A together with the adjacent nitrogen atom together with 4-benzylpiperidi Claims 38 and 4 forming 4,4-ethylenedioxypiperidino or decahydroisoquinolin-1-yl Pyrimidine derivative or a pharmacologically acceptable salt thereof according to any one of to 47 wherein. R ^3A is 3-methoxybenzyl, 3-chlorobenzyl, 3-fluorobenzyl, 2,6-difluorobenzyl, 2-methylbenzyl, 3-trifluorobenzyl, cyclohexylmethyl, cycloheptyl and 1,5-dimethyl-n- A group selected from the group consisting of hexyl and R ^4A is a hydrogen atom, R ^3A is benzyl and R ^4A is n-butyl, or R ^3A is n-hexyl and R ^4A is methyl Or R ^3A and R ^4A together with the adjacent nitrogen atom form 4-benzylpiperidino, 4,4-ethylenedioxypiperidino or decahydroisoquinolin-1-yl. 40. The pyrimidine derivative according to 39 or a pharmacologically acceptable salt thereof. The compound represented by the formula (IA) is 4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carboxylic acid, 4- (2-benzylphenylamino) -2- (cyclohexylmethyl) Amino) pyrimidine-5-carbohydroxamic acid, [4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carbonyl] aminoacetic acid, {[4- (2-benzylphenylamino) -2 -(Cyclohexylmethylamino) pyrimidine-5-carbonyl] methylamino} acetic acid, 4- (2-benzylphenylamino) -5-hydroxymethyl-2- (cyclohexylmethylamino) pyrimidine, N- [4- (2-benzyl) Phenylamino) -2- (cyclohexylmethylamino) pyrimidine-5 Rubonyl] -4-methylbenzenesulfonamide, N- [4- (2-benzylphenylamino) -2- (cyclohexylmethylamino) pyrimidine-5-carbonyl] methanesulfonamide, [4- (2-benzylphenylamino) 2- (cyclohexylmethylamino) pyrimidine-5-carbonyl] trifluoromethanesulfonamide, 2- (cyclohexylmethylamino) -4- (1-methyl-2-phenoxyethylamino) pyrimidine-5-carboxylic acid, 2- ( (Cyclohexylmethylamino) -4- (2-phenoxyethylamino) pyrimidine-5-carboxylic acid, 2- (cyclohexylmethylamino) -4- (2-phenoxyphenylamino) pyrimidine-5-carboxylic acid, 4- (4- Phenylpiperidin-1-yl) -2- (3 Trifluoromethylbenzylamino) pyrimidine-5-carboxylic acid, 2- (cyclohexylmethylamino) -5-hydroxymethyl-4- (1-methyl-2-phenoxyethylamino) pyrimidine, 2- (cyclohexylmethylamino) -5 -Hydroxymethyl-4- (2-phenoxyethylamino) pyrimidine, 2- (benzylbutylamino) -5-hydroxymethyl-4- (2-phenoxyethylamino) pyrimidine, 5-hydroxymethyl-2- (octahydroisoquinoline) -2-yl) -4- (2-phenoxyethylamino) pyrimidine, 2- (cyclohexylmethylamino) -5-hydroxymethyl-4- (2-phenoxyphenylamino) pyrimidine, 5-hydroxymethyl-4- (2 -Phenylpropylamino) -2- (3- Trifluoromethylbenzylamino) pyrimidine, 5-hydroxymethyl-4- (4-phenylpiperidin-1-yl) -2- (3-trifluoromethylbenzylamino) pyrimidine, 4- (2-benzylphenylamino) -2 -(3-Chlorobenzylamino) pyrimidine-5-carboxylic acid, 4- (2-benzylphenylamino) -2- (3-fluorobenzylamino) pyrimidine-5-carboxylic acid, 2- (benzylbutylamino) -4 -(2,3-dihydrobenzo [1,4] dioxin-6-ylamino) pyrimidine-5-carboxylic acid, 2- (3-methoxybenzylamino) -4- (2-phenoxyphenylamino) pyrimidine-5-carvone Acid, 2- (2,6-difluorobenzylamino) -4- (2-phenoxyphenylamino) pyric Gin-5-carboxylic acid, 2- (2-methylbenzylamino) -4- (2-phenoxyphenylamino) pyrimidine-5-carboxylic acid, 4- (2-phenoxyphenylamino) -2- (3-trifluoro Benzylamino) pyrimidine-5-carboxylic acid, 2- (3-fluorobenzylamino) -4- (4-phenylbutylamino) pyrimidine-5-carboxylic acid, 2- (benzylbutylamino) -4- (4-biphenyl) Methylamino) pyrimidine-5-carboxylic acid, 4- (2,3-dihydrobenzo [1,4] dioxin-6-ylamino) -5-hydroxymethyl-2- (3-trifluoromethylbenzylamino) pyrimidine, 4 -(2,3-dihydrobenzo [1,4] dioxin-6-ylamino) -5-hydroxymethyl-2- (2-pyridy Methylamino) pyrimidine and 2- [bis (2-methoxyethyl) amino] -4- (2,3-dihydrobenzo [1,4] dioxin-6-ylamino) -5-hydroxymethylpyrimidine, 2- (3- A pyrimidine derivative which is a compound selected from the group consisting of (fluorobenzylamino) -5-hydroxymethyl-4- (2-phenoxyethylamino) pyrimidine or a pharmaceutically acceptable salt thereof. A pharmaceutical comprising the pyrimidine derivative according to any one of claims 38 to 53 or a pharmacologically acceptable salt thereof as an active ingredient. A function inhibitor of GPR88 comprising the pyrimidine derivative according to any one of claims 38 to 53 or a pharmacologically acceptable salt thereof as an active ingredient. 54. A preventive and / or therapeutic agent for central diseases comprising the pyrimidine derivative according to any one of claims 38 to 53 or a pharmacologically acceptable salt thereof as an active ingredient. The preventive and / or therapeutic agent according to claim 56, wherein the central disease is a disease selected from the group consisting of Parkinson's disease, depression and jet lag. Use of a substance having a function-inhibiting action of GPR88 for the manufacture of a preventive and / or therapeutic agent for central diseases. An antisense oligonucleotide having a base sequence complementary or substantially complementary to a sequence of 10 or more consecutive base sequences represented by SEQ ID NO: 1, 3 or 5 for the production of a GPR88 function inhibitor Use of. Use of the pyrimidine derivative according to claim 15 or a pharmaceutically acceptable salt thereof for the manufacture of a GPR88 function inhibitor. Use of the pyrimidine derivative or a pharmaceutically acceptable salt thereof according to claim 15 for the manufacture of a preventive and / or therapeutic agent for central diseases. A method for preventing and / or treating a central disease, which comprises administering an effective amount of a substance having a function-inhibiting action of GPR88. An effective amount of an antisense oligonucleotide having a base sequence complementary or substantially complementary to a sequence of 10 or more consecutive base sequences represented by SEQ ID NO: 1, 3 or 5 is administered. A method for suppressing the function of GPR88. A method for inhibiting the function of GPR88, comprising administering an effective amount of the pyrimidine derivative or pharmacologically acceptable salt thereof according to claim 15. A method for preventing and / or treating a central disease, which comprises administering an effective amount of the pyrimidine derivative or a pharmacologically acceptable salt thereof according to claim 15.

Images