JPWO2010010797A1 - Pharmaceutical composition containing a compound that inhibits DYRK - Google Patents

Pharmaceutical composition containing a compound that inhibits DYRK Download PDF

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JPWO2010010797A1
JPWO2010010797A1 JP2010521663A JP2010521663A JPWO2010010797A1 JP WO2010010797 A1 JPWO2010010797 A1 JP WO2010010797A1 JP 2010521663 A JP2010521663 A JP 2010521663A JP 2010521663 A JP2010521663 A JP 2010521663A JP WO2010010797 A1 JPWO2010010797 A1 JP WO2010010797A1
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dyrk1b
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JP5747142B2 (en
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萩原正敏
浩司 澁谷
浩司 澁谷
英理子 大西
英理子 大西
靖 小川
靖 小川
孝充 細谷
孝充 細谷
俊行 平松
俊行 平松
吉田　実代
実代 吉田
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
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    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴う疾病の治療又は予防のための医薬組成物の提供。ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの蛋白質リン酸化活性を阻害する化合物を有効成分とする、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴う疾病の治療又は予防のための医薬組成物。Provided is a pharmaceutical composition for treating or preventing a disease associated with at least one increased activity of DYRK1A, DYRK1B, and DYRK2. Pharmaceutical composition for the treatment or prevention of a disease associated with at least one increased activity of DYRK1A, DYRK1B, and DYRK2, comprising a compound that inhibits at least one protein phosphorylation activity of DYRK1A, DYRK1B, and DYRK2 as an active ingredient object.

Description

本発明は、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つを阻害するベンゾチアゾール誘導体、それを含有する医薬組成物、及び、それを用いた疾病の治療・予防方法に関する。   The present invention relates to a benzothiazole derivative that inhibits at least one of DYRK1A, DYRK1B, and DYRK2, a pharmaceutical composition containing the same, and a method for treating or preventing a disease using the same.

蛋白質リン酸化酵素（キナーゼ）は、細胞内情報伝達に必須であり、それらの発現異常あるいは活性異常は、様々な疾患を惹起することが知られている。そのため、様々なリン酸化酵素が創薬標的因子として着目され、標的のリン酸化酵素に特異的なインヒビターの探索が世界中で行われている。   Protein kinases (kinases) are essential for intracellular signal transduction, and their abnormal expression or abnormal activity is known to cause various diseases. For this reason, various phosphorylases have attracted attention as drug discovery target factors, and search for inhibitors specific to the target phosphorylase has been conducted all over the world.

リン酸化酵素ファミリーの１つに、ＤＹＲＫファミリーがある。ＤＹＲＫの名称の由来は、“dual-specificity tyrosine phosphorylation regulated kinase”である。すなわち、ＤＹＲＫファミリーは、チロシン（Ｔｙｒ）及びセリン／トレオニン（Ｓｅｒ／Ｔｈｒ）の両方を基質とするリン酸化酵素のファミリーであって、自己リン酸化の場合にのみＴｙｒリン酸化酵素として機能し、細胞内では“ｐｒｏｌｉｎｅ−ｄｉｒｅｃｔｅｄ”なＳｅｒ／Ｔｈｒリン酸化酵素として機能する。ＤＹＲＫファミリーのメンバーとして、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、ＤＹＲＫ２、ＤＹＲＫ３、ＤＹＲＫ４の５つが知られている。   One of the phosphorylating enzyme families is the DYRK family. The name of DYRK is derived from “dual-specificity tyrosine phosphorylation regulated kinase”. That is, the DYRK family is a family of phosphorylases using both tyrosine (Tyr) and serine / threonine (Ser / Thr) as substrates, and functions as Tyr kinase only in the case of autophosphorylation. It functions as a “proline-directed” Ser / Thr kinase. There are five known members of the DYRK family: DYRK1A, DYRK1B, DYRK2, DYRK3, and DYRK4.

ＤＹＲＫ１Ａは、精神神経疾患との関連性が深い。ＤＹＲＫ１Ａ遺伝子は、ダウン症候群の原因である２１番染色体トリソミーのダウン症クリティカル領域（ＤＳＣＲ）に位置する。そして、これまでに、マウスではＤＹＲＫ１Ａの単独の過剰発現によりダウン症様の精神神経症状をきたすことが報告され（非特許文献１、２）、ダウン症患者及びダウン症モデルマウスの脳内ではＤＹＲＫ１Ａ発現が上昇することが報告されている（非特許文献３）。これらのことは、ＤＹＲＫ１Ａが、ダウン症候群の発症において重要な役割を担っていることを示している。また、アルツハイマー病の原因として、Ａβの蓄積とタウ蛋白質（Ｔａｕ）の異常リン酸化が知られている。アルツハイマー病の患者では、Ａβの発現亢進とＤＹＲＫ１Ａの発現とが有意に一致することから、Ａβの発現亢進からＴａｕの異常リン酸化への橋渡しをＤＹＲＫ１Ａが担うという作業仮説も提案されている（非特許文献４）。   DYRK1A is closely related to neuropsychiatric disorders. The DYRK1A gene is located in the Down syndrome critical region (DSCR) of chromosome 21 trisomy responsible for Down syndrome. So far, it has been reported that DYRK1A overexpression in mice causes Down syndrome-like neuropsychiatric symptoms (Non-patent Documents 1 and 2), and DYRK1A expression is increased in the brains of Down syndrome patients and Down syndrome model mice. It has been reported (Non-Patent Document 3). These facts indicate that DYRK1A plays an important role in the development of Down's syndrome. Further, as a cause of Alzheimer's disease, accumulation of Aβ and abnormal phosphorylation of tau protein (Tau) are known. In patients with Alzheimer's disease, since the increased expression of Aβ and the expression of DYRK1A are in agreement, a working hypothesis that DYRK1A is responsible for bridging the increased expression of Aβ to abnormal phosphorylation of Tau has been proposed (non- Patent Document 4).

ＤＹＲＫ１Ｂは、悪性腫瘍との関連性が深い。例えば、膵癌組織においてＤＹＲＫ１Ｂの発現が上昇すること（非特許文献５）、横紋筋肉腫においてＤＹＲＫ１Ｂが多く発現していること（非特許文献６）が開示されている。そして、ＤＹＲＫ１Ｂは、ストレス応答経路により活性化され、チェックポイントキナーゼとして機能し、損傷した腫瘍細胞を休止状態に停止させて腫瘍細胞の修復をさせる、という作業仮説も提案されている（非特許文献７）。   DYRK1B is closely related to malignant tumors. For example, it is disclosed that the expression of DYRK1B is increased in pancreatic cancer tissues (Non-Patent Document 5), and that DYRK1B is highly expressed in rhabdomyosarcoma (Non-Patent Document 6). A working hypothesis has also been proposed that DYRK1B is activated by a stress response pathway, functions as a checkpoint kinase, and stops damaged tumor cells in a dormant state to repair the tumor cells (Non-Patent Document). 7).

ＤＹＲＫ２については、ＤＮＡ損傷に応答してｐ５３を制御し、アポトーシスを誘導することが示唆されている（非特許文献８）。   It has been suggested that DYRK2 regulates p53 in response to DNA damage and induces apoptosis (Non-patent Document 8).

一方、ベンゾチアゾール誘導体とリン酸化酵素との関係においては、リン酸化酵素であるＣｌｋ１及びＣｌｋ４のリン酸化活性を阻害できるベンゾチアゾール誘導体が開示されている（特許文献１、非特許文献９）。   On the other hand, regarding the relationship between a benzothiazole derivative and a phosphorylating enzyme, a benzothiazole derivative capable of inhibiting the phosphorylating activity of Clk1 and Clk4 which are phosphorylating enzymes has been disclosed (Patent Document 1, Non-Patent Document 9).

US 2005/0171026 A1US 2005/0171026 A1

Branchi I et al. J Neuropathol Exp Neurol. 2004 May;63(5):429-40.Branchi I et al. J Neuropathol Exp Neurol. 2004 May; 63 (5): 429-40. Altafaj X et al. Hum Mol Genet. 2001 Sep 1;10(18):1915-23.Altafaj X et al. Hum Mol Genet. 2001 Sep 1; 10 (18): 1915-23. Dowjat WK et al. Neurosci Lett. 2007 Feb 8;413(1):77-81. Epub 2006 Dec 4.Dowjat WK et al. Neurosci Lett. 2007 Feb 8; 413 (1): 77-81. Epub 2006 Dec 4. Kimura R et al. Hum Mol Genet. 2007 Jan 1;16(1):15-23. Epub 2006 Nov 29.Kimura R et al. Hum Mol Genet. 2007 Jan 1; 16 (1): 15-23. Epub 2006 Nov 29. Deng X et al. Cancer Res. 2006 Apr 15;66(8):4149-58.Deng X et al. Cancer Res. 2006 Apr 15; 66 (8): 4149-58. Mercer SE et al. Cancer Res. 2006 May 15;66(10):5143-50.Mercer SE et al. Cancer Res. 2006 May 15; 66 (10): 5143-50. Friedman E. J Cell Biochem. 2007 Oct 1;102(2):274-9.Friedman E. J Cell Biochem. 2007 Oct 1; 102 (2): 274-9. Taira N et al. Mol Cell. 2007 Mar 23;25(6):794-6.Taira N et al. Mol Cell. 2007 Mar 23; 25 (6): 794-6. Muraki M et al.J Biol Chem. 2004 Jun 4;279(23):24246-54. Epub 2004 Mar 8.Muraki M et al. J Biol Chem. 2004 Jun 4; 279 (23): 24246-54. Epub 2004 Mar 8.

ＤＹＲＫは疾患と密接な関係を持つリン酸化酵素であり、ＤＹＲＫのリン酸化活性を阻害できれば疾患の治療や予防に有用であると考えられる。しかしながら、ＤＹＲＫのリン酸化活性を効果的に阻害できる医薬組成物は未だ報告されていない。   DYRK is a phosphorylating enzyme closely related to a disease, and if DYRK phosphorylation activity can be inhibited, it is considered useful for the treatment and prevention of the disease. However, a pharmaceutical composition that can effectively inhibit the phosphorylation activity of DYRK has not been reported yet.

そこで、本発明は、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴う疾病の治療又は予防のための医薬組成物、及びそれを用いた治療方法を提供する。   Therefore, the present invention provides a pharmaceutical composition for treating or preventing a disease accompanied by at least one increased activity of DYRK1A, DYRK1B, and DYRK2, and a treatment method using the same.

本発明の医薬組成物は、一つの態様として、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの蛋白質リン酸化活性を阻害する化合物を有効成分とする医薬組成物であって、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴う疾病の治療又は予防のための医薬組成物である。   The pharmaceutical composition of the present invention, as one embodiment, is a pharmaceutical composition comprising as an active ingredient a compound that inhibits at least one protein phosphorylation activity of DYRK1A, DYRK1B, and DYRK2, and includes DYRK1A, DYRK1B, and A pharmaceutical composition for treating or preventing a disease associated with at least one increased activity of DYRK2.

また、本発明の医薬組成物は、その他の態様として、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴う疾病の治療又は予防のための医薬組成物であって、有効成分として下記式（Ｉ）の化合物、又は、その製薬上許容される塩若しくは溶媒和物を含有する医薬組成物である。

［上記式（Ｉ）において、
Ａは、Ｓ（硫黄原子）又はＳｅ（セレニウム原子）であり、Ｘは、ハロゲン置換されてもよいＣ１−Ｃ１０アルキル基であり、
Ｙは、Ｈ（水素原子）、ハロゲン置換されてもよいＣ１−Ｃ１０アルキル基、Ｃ２−Ｃ６アルケニル基、−Ｒ１ＯＨ、−ＣＯＯＲ２、及び、−Ｒ１ＯＣＯＲ３から選択され、
Ｚは、ハロゲン置換されてもよいＣ１−Ｃ１０アルキル基、ハロゲン、−ＮＨＣＯＲ２、−ＯＲ２、及び、−ＯＣＯＲ３から選択され、
Ｒ１は、Ｃ１−Ｃ１０アルキレン基であり、
Ｒ２は、Ｈ、又は、Ｃ１−Ｃ１０アルキル基であり、
Ｒ３は、Ｃ１−Ｃ１０アルキル基、Ｃ１−Ｃ３アルコキシ基若しくはＣ１−Ｃ６アルキル基で置換されてもよいアリール基、及び、Ｃ１−Ｃ６アルキル基で置換されてもよいへテロアリール基から選択される。］In another aspect, the pharmaceutical composition of the present invention is a pharmaceutical composition for the treatment or prevention of a disease associated with at least one increased activity of DYRK1A, DYRK1B, and DYRK2, which has the following formula as an active ingredient: A pharmaceutical composition comprising the compound (I) or a pharmaceutically acceptable salt or solvate thereof.

[In the above formula (I),
A is S (sulfur atom), or Se (selenium atom), X is a halogen-substituted C 1 may be -C 10 alkyl group,
Y is selected from H (hydrogen atom), C 1 -C 10 alkyl group optionally substituted by halogen, C 2 -C 6 alkenyl group, —R 1 OH, —COOR 2 , and —R 1 OCOR 3. ,
Z is a halogen substituted may be C 1 -C 10 alkyl group, halogen, -NHCOR 2, -OR 2, and is selected from -OCOR 3,
R 1 is a C 1 -C 10 alkylene group,
R 2 is H or a C 1 -C 10 alkyl group,
R 3 may be substituted with a C 1 -C 10 alkyl group, an aryl group that may be substituted with a C 1 -C 3 alkoxy group or a C 1 -C 6 alkyl group, and a C 1 -C 6 alkyl group. Selected from good heteroaryl groups. ]

本発明の治療又は予防方法は、一つの態様において、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴う疾病の治療又は予防の方法であって、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの蛋白質リン酸化活性を阻害する化合物を有効成分とする医薬組成物を対象に有効量投与することを含む治療又は予防方法である。   In one embodiment, the treatment or prevention method of the present invention is a method for treating or preventing a disease associated with at least one increased activity of DYRK1A, DYRK1B, and DYRK2, and includes at least one of DYRK1A, DYRK1B, and DYRK2. A therapeutic or prophylactic method comprising administering to a subject an effective amount of a pharmaceutical composition comprising a compound that inhibits one protein phosphorylation activity as an active ingredient.

また、本発明の治療又は予防方法は、その他の態様として、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴う疾病の治療又は予防の方法であって、本発明の医薬組成物を対象に有効量投与することを含む治療又は予防方法である。   In another aspect, the method for treating or preventing the present invention is a method for treating or preventing a disease associated with at least one increased activity of DYRK1A, DYRK1B, and DYRK2, and is intended for the pharmaceutical composition of the present invention. Is a therapeutic or prophylactic method comprising administering an effective amount to the patient.

本発明の医薬組成物及び治療又は予防方法によれば、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つのリン酸化活性を阻害できることから、好ましくは、これらのリン酸化酵素の活性亢進を伴う疾病の治療又は予防ができる。   According to the pharmaceutical composition and the treatment or prevention method of the present invention, since at least one phosphorylation activity of DYRK1A, DYRK1B, and DYRK2 can be inhibited, it is preferable to treat a disease associated with an increased activity of these phosphorylases. Or it can be prevented.

図１は、培養細胞内におけるベンゾチアゾール誘導体によるタウ蛋白質リン酸化阻害を示す一例のグラフである。FIG. 1 is a graph showing an example of inhibition of tau protein phosphorylation by a benzothiazole derivative in cultured cells. 図２は、培養細胞内におけるベンゾチアゾール誘導体によるタウ蛋白質リン酸化阻害を示すその他の例のグラフである。FIG. 2 is a graph of another example showing inhibition of tau protein phosphorylation by a benzothiazole derivative in cultured cells. 図３は、ＤＹＲＫ１Ａ過剰発現モデルにおける発生異常とベンゾチアゾール誘導体投与による抑制の一例を示す写真である。ｍＲＮＡ非投与群（ａ、ｄ、ｇ）、２５０ｐｇ ＤＹＲＫ１Ａ ｍＲＮＡ投与群（ｂ、ｅ、ｈ）、５００ｐｇ ＤＹＲＫ１Ａ ｍＲＮＡ投与群（ｃ、ｆ、ｉ）。ベンゾチアゾール誘導体非投与群（ａ、ｂ、ｃ）、製造例００１のベンゾチアゾール誘導体投与群（ｄ、ｅ、ｆ）、製造例０１９のベンゾチアゾール誘導体投与群（ｇ、ｈ、ｉ）。FIG. 3 is a photograph showing an example of abnormal development and suppression by administration of a benzothiazole derivative in a DYRK1A overexpression model. mRNA non-administration group (a, d, g), 250 pg DYRK1A mRNA administration group (b, e, h), 500 pg DYRK1A mRNA administration group (c, f, i). The benzothiazole derivative non-administered group (a, b, c), the benzothiazole derivative administered group (d, e, f) of Production Example 001, and the benzothiazole derivative administered group (g, h, i) of Production Example 019. 図４は、ＤＹＲＫ１Ａ過剰発現モデルにおける発生異常とベンゾチアゾール誘導体投与による抑制の一例を示すグラフである。実験結果の個体を、正常、眼形成異常（軽度）、及び眼形成異常（重度）の３つに分類し、各実験群におけるそれぞれの割合を百分率で示した。図４ａはｍＲＮＡ非投与群のグラフであり、図４ｂは２５０ｐｇ ｍＲＮＡ投与群のグラフであり、図４ｃは５００ｐｇ ｍＲＮＡ投与群のグラフである。FIG. 4 is a graph showing an example of abnormal development and suppression by administration of a benzothiazole derivative in a DYRK1A overexpression model. Individuals of the experimental results were classified into normal, ocular dysplasia (mild), and ocular dysplasia (severe), and the respective ratios in each experimental group were shown as percentages. 4a is a graph of the group not administered with mRNA, FIG. 4b is a graph of the group administered with 250 pg mRNA, and FIG. 4c is a graph of the group administered with 500 pg mRNA. 図５は、ＤＹＲＫ１Ａ過剰発現モデルにおける発生異常（頭部縮小）とベンゾチアゾール誘導体投与による抑制について、各実験群における頭部縮小個体の割合を百分率で示したグラフの一例である。図５ａはｍＲＮＡ非投与群及び２５０ｐｇ ｍＲＮＡ投与群のグラフであり、図５ｂはｍＲＮＡ非投与群及び５００ｐｇ ｍＲＮＡ投与群のグラフである。FIG. 5 is an example of a graph showing the percentage of individuals with reduced head in each experimental group in terms of developmental abnormality (head reduction) and suppression by administration of a benzothiazole derivative in the DYRK1A overexpression model. FIG. 5a is a graph of the mRNA non-administered group and the 250 pg mRNA administered group, and FIG. 5b is a graph of the mRNA non-administered group and the 500 pg mRNA administered group. 図６は、ＤＹＲＫ１Ａ過剰発現モデル（ｍＲＮＡ（＋）レーン）における神経発生マーカー発現異常とベンゾチアゾールによる回復を示す図である。FIG. 6 is a graph showing abnormal expression of neurogenesis marker and recovery by benzothiazole in a DYRK1A overexpression model (mRNA (+) lane). 図７は、横紋筋肉腫細胞株ＲＭＳ−ＹＭに対するベンゾチアゾール誘導体の細胞増殖抑制効果及び抗がん剤作用増強効果を調べた結果の一例を示すグラフである。図７ａは、ベンゾチアゾール誘導体のみを用いた場合の結果を示し、図７ｂは、抗がん剤としてビンクリスチンを用いた場合の結果を示し、図７ｃは、抗がん剤としてアクチノマイシンＤを用いた場合の結果を示す。FIG. 7 is a graph showing an example of the results of examining the cell growth inhibitory effect and anticancer agent enhancing effect of a benzothiazole derivative on rhabdomyosarcoma cell line RMS-YM. FIG. 7a shows the results when only the benzothiazole derivative is used, FIG. 7b shows the results when vincristine is used as the anticancer agent, and FIG. 7c uses actinomycin D as the anticancer agent. The result is shown. 図８は、ＤＹＲＫ１Ｂ高発現ヒト膵管癌細胞株Ｐａｎｃ−１に対するベンゾチアゾール誘導体の細胞増殖抑制効果及び抗がん剤作用増強効果を調べた結果の一例を示すグラフである。FIG. 8 is a graph showing an example of the results of examining the cell growth inhibitory effect and the anticancer agent enhancing effect of a benzothiazole derivative against DYRK1B highly expressing human pancreatic duct cancer cell line Panc-1.

本発明は、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴い発現される個体の異常が、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの蛋白質リン酸化活性を阻害する化合物により抑制可能であるという知見に基づく。   In the present invention, an abnormality of an individual expressed with at least one increased activity of DYRK1A, DYRK1B, and DYRK2 can be suppressed by a compound that inhibits at least one protein phosphorylation activity of DYRK1A, DYRK1B, and DYRK2. Based on the knowledge that there is.

本発明は、また、下記式（Ｉ）のベンゾチアゾール誘導体が蛋白質リン酸化酵素であるＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２のリン酸化活性に対して阻害能を有するという知見に基づく。なお、下記式（Ｉ）のベンゾチアゾール誘導体は、上記特許文献１及び非特許文献９に開示されたベンゾチアゾール誘導体と一部重複する。しかしながら、これらの文献は、上記式（Ｉ）に含まれる一部のベンゾチアゾール誘導体がＣｌｋファミリー（Ｃｌｋ１及び４）のリン酸化酵素の阻害剤となりうることのみを開示し、ＤＹＲＫファミリーのリン酸化酵素については言及がない。   The present invention is also based on the finding that the benzothiazole derivative of the following formula (I) has the ability to inhibit the phosphorylating activity of DYRK1A, DYRK1B, and DYRK2 which are protein kinases. In addition, the benzothiazole derivative of the following formula (I) partially overlaps with the benzothiazole derivative disclosed in Patent Document 1 and Non-Patent Document 9. However, these documents only disclose that some benzothiazole derivatives included in the above formula (I) can be inhibitors of the Clk family (Clk1 and 4) phosphorylase, and the DYRK family phosphorylase There is no mention about.

すなわち、本発明は下記を含む；
［１］ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの蛋白質リン酸化活性を阻害する化合物を有効成分とする、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴う疾病の治療又は予防のための医薬組成物。
［２］前記疾病が、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性阻害が治療又は予防に有効な疾病である［１］の医薬組成物。
［３］前記疾病が、精神神経疾患及び悪性腫瘍を含む［１］又は［２］の医薬組成物。
［４］前記精神神経疾患が、ダウン症候群及びアルツハイマー病を含む［３］の医薬組成物。
［５］前記悪性腫瘍が、膵管がん及び横紋筋肉腫を含む［３］の医薬組成物。
［６］前記化合物が、ベンゾチアゾール誘導体である、［１］から［５］のいずれかの医薬組成物。
［７］ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴う疾病の治療又は予防のための医薬組成物であって、有効成分として下記式（Ｉ）の化合物、又は、その製薬上許容される塩若しくは溶媒和物を含む、医薬組成物。

［上記式（Ｉ）において、Ａは、Ｓ（硫黄原子）又はＳｅ（セレニウム原子）であり、Ｘは、ハロゲン置換されてもよいＣ１−Ｃ１０アルキル基であり、Ｙは、Ｈ（水素原子）、ハロゲン置換されてもよいＣ１−Ｃ１０アルキル基、Ｃ２−Ｃ６アルケニル基、−Ｒ１ＯＨ、−ＣＯＯＲ２、及び、−Ｒ１ＯＣＯＲ３から選択され、Ｚは、ハロゲン置換されてもよいＣ１−Ｃ１０アルキル基、ハロゲン、−ＮＨＣＯＲ２、−ＯＲ２、及び、−ＯＣＯＲ３から選択され、
Ｒ１は、Ｃ１−Ｃ１０アルキレン基であり、Ｒ２は、Ｈ、又は、Ｃ１−Ｃ１０アルキル基であり、Ｒ３は、Ｃ１−Ｃ１０アルキル基、Ｃ１−Ｃ３アルコキシ基若しくはＣ１−Ｃ６アルキル基で置換されてもよいアリール基、及び、Ｃ１−Ｃ６アルキル基で置換されてもよいへテロアリール基から選択される。］
［８］前記疾病が、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性阻害が治療又は予防に有効な疾病である、［７］の医薬組成物。
［９］前記疾病が、精神神経疾患及び悪性腫瘍を含む、［７］又は［８］の医薬組成物。
［１０］前記精神神経疾患が、ダウン症候群及びアルツハイマー病を含む、［９］の医薬組成物。
［１１］前記悪性腫瘍が、膵管がん及び横紋筋肉腫を含む、［９］の医薬組成物。
［１２］ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴う疾病の治療又は予防の方法であって、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの蛋白質リン酸化活性を阻害する化合物を有効成分とする医薬組成物を対象に有効量投与することを含む、治療又は予防方法。
［１３］前記対象又はその一部においてＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つが活性亢進していることを確認することを含む、［１２］の治療又は予防方法。
［１４］前記疾病が、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性阻害が治療又は予防に有効な疾病である、［１２］又は［１３］の治療又は予防方法。
［１５］前記疾病が、精神神経疾患及び悪性腫瘍を含む、［１２］から［１４］のいずれかの治療又は予防方法。
［１６］前記精神神経疾患が、ダウン症候群及びアルツハイマー病を含む、［１５］の治療又は予防方法。
［１７］前記悪性腫瘍が、膵管がん及び横紋筋肉腫を含む、［１５］の治療又は予防方法。
［１８］前記化合物が、ベンゾチアゾール誘導体である、［１２］から［１７］のいずれかの治療又は予防方法。
［１９］ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴う疾病の治療又は予防の方法であって、［７］から［１１］のいずれかに記載の医薬組成物を対象に有効量投与することを含む、治療又は予防方法。
［２０］前記対象又はその一部においてＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つが活性亢進していることを確認することを含む、［１９］の治療又は予防方法。
［２１］前記疾病が、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性阻害が治療又は予防に有効な疾病である、［１９］又は［２０］の治療又は予防方法。
［２２］前記疾病が、精神神経疾患及び悪性腫瘍を含む、［１９］から［２１］のいずれかの治療又は予防方法。
［２３］前記精神神経疾患が、ダウン症候群及びアルツハイマー病を含む、［２２］の治療又は予防方法。
［２４］前記悪性腫瘍が、膵管がん及び横紋筋肉腫を含む、［２２］の治療又は予防方法。
［２５］下記式（Ｉ）化合物、又は、その製薬上許容される塩若しくは溶媒和物。

［上記式（Ｉ）において、Ａは、Ｓ（硫黄原子）又はＳｅ（セレニウム原子）であり、Ｘは、ハロゲン置換されてもよいＣ１−Ｃ１０アルキル基であり、Ｙは、Ｈ（水素原子）、ハロゲン置換されてもよいＣ１−Ｃ１０アルキル基、Ｃ２−Ｃ６アルケニル基、−Ｒ１ＯＨ、−ＣＯＯＲ２、及び、−Ｒ１ＯＣＯＲ３から選択され、Ｚは、ハロゲン置換されてもよいＣ１−Ｃ１０アルキル基、ハロゲン、−ＮＨＣＯＲ２、−ＯＲ２、及び、−ＯＣＯＲ３から選択され、
Ｒ１は、Ｃ１−Ｃ１０アルキレン基であり、Ｒ２は、Ｈ、又は、Ｃ１−Ｃ１０アルキル基であり、Ｒ３は、Ｃ１−Ｃ１０アルキル基、Ｃ１−Ｃ３アルコキシ基若しくはＣ１−Ｃ６アルキル基で置換されてもよいアリール基、及び、Ｃ１−Ｃ６アルキル基で置換されてもよいへテロアリール基から選択され、但し、ＡがＳかつＸ及びＹがメチル基の場合、Ｚは、フッ素原子（Ｆ）、ヒドロキシ基、メトキシ基、エトキシ基、及び、アセトキシ基を除く上記範囲から選択され、ＡがＳかつＸがエチル基かつＹがメチル基の場合、Ｚは、メトキシ基を除く上記範囲から選択される。］
［２６］モデル動物の初期胚にＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つのｍＲＮＡを誘導して発生異常を起こしうる状態とすること、前記初期胚を候補化合物と接触させること、及び、候補化合物を接触させなかった場合と比較して候補化合物による発生異常の抑制効果を評価することを含む、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴う疾病の治療又は予防のための医薬組成物の有効成分のスクリーニング方法。That is, the present invention includes:
[1] For the treatment or prevention of a disease accompanied by at least one DYRK1A, DYRK1B, and DYRK2 activity enhancement, comprising a compound that inhibits at least one protein phosphorylation activity of DYRK1A, DYRK1B, and DYRK2 as an active ingredient Pharmaceutical composition.
[2] The pharmaceutical composition according to [1], wherein the disease is a disease in which at least one activity inhibition of DYRK1A, DYRK1B, and DYRK2 is effective for treatment or prevention.
[3] The pharmaceutical composition according to [1] or [2], wherein the disease includes a neuropsychiatric disorder and a malignant tumor.
[4] The pharmaceutical composition of [3], wherein the neuropsychiatric disorder includes Down's syndrome and Alzheimer's disease.
[5] The pharmaceutical composition according to [3], wherein the malignant tumor includes pancreatic duct cancer and rhabdomyosarcoma.
[6] The pharmaceutical composition according to any one of [1] to [5], wherein the compound is a benzothiazole derivative.
[7] A pharmaceutical composition for the treatment or prevention of a disease accompanied by at least one increased activity of DYRK1A, DYRK1B, and DYRK2, comprising a compound of the following formula (I) as an active ingredient, or a pharmaceutically acceptable salt thereof A pharmaceutical composition comprising a salt or solvate obtained.

In [the formula (I), A is a S (sulfur atom), or Se (selenium atom), X is a halogen-substituted C 1 may be -C 10 alkyl group, Y is H (hydrogen Atom), a C 1 -C 10 alkyl group optionally substituted with halogen, a C 2 -C 6 alkenyl group, —R 1 OH, —COOR 2 , and —R 1 OCOR 3 , wherein Z is halogen substituted Selected from C 1 -C 10 alkyl groups, halogen, —NHCOR 2 , —OR 2 , and —OCOR 3 , which may be
R 1 is a C 1 -C 10 alkylene group, R 2 is H or a C 1 -C 10 alkyl group, R 3 is a C 1 -C 10 alkyl group, C 1 -C 3 alkoxy group group or C 1 -C 6 alkyl aryl group which may be substituted with a group, and are selected from the heteroaryl group may be substituted with C 1 -C 6 alkyl group. ]
[8] The pharmaceutical composition according to [7], wherein the disease is a disease in which at least one activity inhibition of DYRK1A, DYRK1B, and DYRK2 is effective for treatment or prevention.
[9] The pharmaceutical composition according to [7] or [8], wherein the disease includes a neuropsychiatric disorder and a malignant tumor.
[10] The pharmaceutical composition according to [9], wherein the neuropsychiatric disorder includes Down's syndrome and Alzheimer's disease.
[11] The pharmaceutical composition according to [9], wherein the malignant tumor includes pancreatic duct cancer and rhabdomyosarcoma.
[12] A method for treating or preventing a disease associated with at least one increased activity of DYRK1A, DYRK1B, and DYRK2, wherein a compound that inhibits at least one protein phosphorylation activity of DYRK1A, DYRK1B, and DYRK2 is effective A therapeutic or prophylactic method comprising administering an effective amount of a pharmaceutical composition as a component to a subject.
[13] The method of treatment or prevention according to [12], comprising confirming that at least one of DYRK1A, DYRK1B, and DYRK2 is enhanced in the subject or a part thereof.
[14] The treatment or prevention method according to [12] or [13], wherein the disease is a disease in which at least one activity inhibition of DYRK1A, DYRK1B, and DYRK2 is effective for treatment or prevention.
[15] The treatment or prevention method according to any one of [12] to [14], wherein the disease includes a neuropsychiatric disorder and a malignant tumor.
[16] The method for treating or preventing [15], wherein the neuropsychiatric disorder includes Down's syndrome and Alzheimer's disease.
[17] The method for treating or preventing [15], wherein the malignant tumor includes pancreatic duct cancer and rhabdomyosarcoma.
[18] The treatment or prevention method according to any one of [12] to [17], wherein the compound is a benzothiazole derivative.
[19] A method for the treatment or prevention of a disease accompanied by at least one increased activity of DYRK1A, DYRK1B, and DYRK2, comprising an effective amount of the pharmaceutical composition according to any one of [7] to [11] A therapeutic or prophylactic method comprising administering.
[20] The treatment or prevention method according to [19], comprising confirming that at least one of DYRK1A, DYRK1B, and DYRK2 is enhanced in the subject or a part thereof.
[21] The method of treatment or prevention according to [19] or [20], wherein the disease is a disease in which inhibition of at least one activity of DYRK1A, DYRK1B, and DYRK2 is effective for treatment or prevention.
[22] The treatment or prevention method according to any one of [19] to [21], wherein the disease includes a neuropsychiatric disorder and a malignant tumor.
[23] The method for treating or preventing [22], wherein the neuropsychiatric disorder includes Down's syndrome and Alzheimer's disease.
[24] The method for treating or preventing [22], wherein the malignant tumor includes pancreatic duct cancer and rhabdomyosarcoma.
[25] A compound of the following formula (I), or a pharmaceutically acceptable salt or solvate thereof.

In [the formula (I), A is a S (sulfur atom), or Se (selenium atom), X is a halogen-substituted C 1 may be -C 10 alkyl group, Y is H (hydrogen Atom), a C 1 -C 10 alkyl group optionally substituted with halogen, a C 2 -C 6 alkenyl group, —R 1 OH, —COOR 2 , and —R 1 OCOR 3 , wherein Z is halogen substituted Selected from C 1 -C 10 alkyl groups, halogen, —NHCOR 2 , —OR 2 , and —OCOR 3 , which may be
R 1 is a C 1 -C 10 alkylene group, R 2 is H or a C 1 -C 10 alkyl group, R 3 is a C 1 -C 10 alkyl group, C 1 -C 3 alkoxy group group or C 1 -C 6 alkyl aryl group which may be substituted with a group, and, substituted with C 1 -C 6 alkyl group selected from heteroaryl group to be, however, A is the S and X and Y In the case of a methyl group, Z is selected from the above range excluding a fluorine atom (F), a hydroxy group, a methoxy group, an ethoxy group, and an acetoxy group, A is S, X is an ethyl group, and Y is a methyl group , Z is selected from the above range excluding the methoxy group. ]
[26] Inducing at least one mRNA of DYRK1A, DYRK1B, and DYRK2 to an early embryo of a model animal so as to cause developmental abnormality, contacting the early embryo with a candidate compound, and a candidate compound A pharmaceutical composition for treating or preventing a disease associated with at least one increased activity of DYRK1A, DYRK1B, and DYRK2, comprising evaluating the inhibitory effect on developmental abnormalities caused by a candidate compound as compared to the case where no contact is made A screening method for the active ingredient of a product.

［ＤＹＲＫ１Ａ，ＤＹＲＫ１Ｂ，ＤＹＲＫ２］
本発明において、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ及びＤＹＲＫ２は、ＤＹＲＫファミリーのメンバーに属する動物のリン酸化酵素であって、好ましくは、ヒトのリン酸化酵素である。当業者であれば、例えば、下記の文献及び配列情報を参照するなどして、それぞれを容易に認識できる。また、本発明におけるＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ及びＤＹＲＫ２は、好ましくは、下記文献に記載されたｍＲＮＡ配列及びアミノ酸配列、並びに、下記ＧｅｎＢａｎｋデータベース登録番号のｍＲＮＡの塩基配列にコードされるアミノ酸配列で表される蛋白質、及び、前記蛋白質と実質同一のアミノ酸配列で表される蛋白質を含む。
［文献］
ラットＤｙｒｋ１ａ： J Biol Chem 1996 271(7):3488-95
ヒトＤＹＲＫ１Ａ： Biochem Biophys Res Commun 1996 225(1):92-9
ヒトＤＹＲＫ１Ｂ： Biochem Biophys Res Commun. 1999 254(2):474-9
ヒトＤＹＲＫ２： J Biol Chem 1998 273(40):25893-25902
［ＧｅｎＢａｎｋデータベース登録番号］
ラットＤｙｒｋ１ａ
NM_012791.1: Rattus norvegicus dual-specificity tyrosine-(Y)- phosphorylation regulated kinase 1A (Dyrk1a), mRNA
ヒトＤＹＲＫ１Ａ
NM_001396.3: Homo sapiens dual-specificity tyrosine-(Y)- phosphorylation regulated kinase 1A (DYRK1A), transcript variant 1, mRNA
NM_130438.2: Homo sapiens dual-specificity tyrosine-(Y)- phosphorylation regulated kinase 1A (DYRK1A), transcript variant 5, mRNA
NM_130437.2: Homo sapiens dual-specificity tyrosine-(Y)- phosphorylation regulated kinase 1A (DYRK1A), transcript variant 4, mRNA
NM_130436.2: Homo sapiens dual-specificity tyrosine-(Y)- phosphorylation regulated kinase 1A (DYRK1A), transcript variant 2, mRNA
NM_101395.2: Homo sapiens dual-specificity tyrosine-(Y)- phosphorylation regulated kinase 1A (DYRK1A), transcript variant 3, mRNA
ヒトＤＹＲＫ１Ｂ
NM_004714.1: Homo sapiens dual-specificity tyrosine-(Y)- phosphorylation regulated kinase 1B (DYRK1B), transcript variant a, mRNA
NM_006483.1: Homo sapiens dual-specificity tyrosine-(Y)- phosphorylation regulated kinase 1B (DYRK1B), transcript variant b, mRNA
NM_006484.1: Homo sapiens dual-specificity tyrosine-(Y)- phosphorylation regulated kinase 1B (DYRK1B), transcript variant c, mRNA
ヒトＤＹＲＫ２
NM_003583.3: Homo sapiens dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2), transcript variant 1, mRNA
NM_006482.2: Homo sapiens dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2), transcript variant 2, mRNA
なお、前記実質同一のアミノ酸配列としては、例えば、配列の相同性が約５０％以上、好ましくは約６０％以上、より好ましくは約７０％以上、さらに好ましくは約８０％以上、特に好ましくは約９０％以上、最も好ましくは約９５％以上のアミノ配列が挙げられる。[DYRK1A, DYRK1B, DYRK2]
In the present invention, DYRK1A, DYRK1B, and DYRK2 are animal kinases belonging to the DYRK family members, and preferably human kinases. Those skilled in the art can easily recognize each of them by referring to the following literature and sequence information, for example. In addition, DYRK1A, DYRK1B and DYRK2 in the present invention are preferably a protein represented by the mRNA sequence and amino acid sequence described in the following literature, and the amino acid sequence encoded by the base sequence of the mRNA of the following GenBank database registration number And a protein represented by an amino acid sequence substantially identical to the protein.
[Reference]
Rat Dyrk1a: J Biol Chem 1996 271 (7): 3488-95
Human DYRK1A: Biochem Biophys Res Commun 1996 225 (1): 92-9
Human DYRK1B: Biochem Biophys Res Commun. 1999 254 (2): 474-9
Human DYRK2: J Biol Chem 1998 273 (40): 25893-25902
[GenBank database registration number]
Rat Dyrk1a
NM_012791.1: Rattus norvegicus dual-specificity tyrosine- (Y)-phosphorylation regulated kinase 1A (Dyrk1a), mRNA
Human DYRK1A
NM_001396.3: Homo sapiens dual-specificity tyrosine- (Y)-phosphorylation regulated kinase 1A (DYRK1A), transcript variant 1, mRNA
NM_130438.2: Homo sapiens dual-specificity tyrosine- (Y)-phosphorylation regulated kinase 1A (DYRK1A), transcript variant 5, mRNA
NM_130437.2: Homo sapiens dual-specificity tyrosine- (Y)-phosphorylation regulated kinase 1A (DYRK1A), transcript variant 4, mRNA
NM_130436.2: Homo sapiens dual-specificity tyrosine- (Y)-phosphorylation regulated kinase 1A (DYRK1A), transcript variant 2, mRNA
NM_101395.2: Homo sapiens dual-specificity tyrosine- (Y)-phosphorylation regulated kinase 1A (DYRK1A), transcript variant 3, mRNA
Human DYRK1B
NM_004714.1: Homo sapiens dual-specificity tyrosine- (Y)-phosphorylation regulated kinase 1B (DYRK1B), transcript variant a, mRNA
NM_006483.1: Homo sapiens dual-specificity tyrosine- (Y)-phosphorylation regulated kinase 1B (DYRK1B), transcript variant b, mRNA
NM_006484.1: Homo sapiens dual-specificity tyrosine- (Y)-phosphorylation regulated kinase 1B (DYRK1B), transcript variant c, mRNA
Human DYRK2
NM_003583.3: Homo sapiens dual-specificity tyrosine- (Y) -phosphorylation regulated kinase 2 (DYRK2), transcript variant 1, mRNA
NM_006482.2: Homo sapiens dual-specificity tyrosine- (Y) -phosphorylation regulated kinase 2 (DYRK2), transcript variant 2, mRNA
The substantially identical amino acid sequence includes, for example, a sequence homology of about 50% or more, preferably about 60% or more, more preferably about 70% or more, still more preferably about 80% or more, and particularly preferably about 90% or more, most preferably about 95% or more amino sequences.

［用途］
本発明の医薬組成物の用途は、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴う疾病の治療又は予防である。ここで、「活性亢進」とは、正常細胞又は正常個体と比較してＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つのｍＲＮＡの転写量、蛋白質の量、活性型の量が高いこと、あるいは、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つによりリン酸化された特定のリン酸化蛋白質及びそれに由来する産物の量又は割合が高いことをいう。なお、前記特定のリン酸化蛋白質及びそれに由来する産物の具体例としては、リン酸化されたアミロイド前駆体（ｐｈｏｓｐｈｏ−ＡＰＰ）及びベータアミロイド（Ａβ）の組み合わせが挙げられる。また、活性亢進の状態であることは、従来公知の方法、例えば、ＲＴ−ＰＣＲ法や抗体を用いた検出若しくはイメージング方法などにより、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つを測定し、あるいは、前記特定のリン酸化蛋白質又はそれに由来する産物の量又は割合を測定することで確認できる。[Usage]
The use of the pharmaceutical composition of the present invention is the treatment or prevention of a disease accompanied by at least one enhanced activity of DYRK1A, DYRK1B, and DYRK2. Here, “enhanced activity” means that the amount of transcription of DYRK1A, DYRK1B, and DYRK2 at least one mRNA, the amount of protein, the amount of active form is higher than that of normal cells or normal individuals, or DYRK1A , DYRK1B, and a specific phosphorylated protein phosphorylated by at least one of DYRK2 and the amount or ratio of a product derived therefrom is high. Specific examples of the specific phosphorylated protein and products derived therefrom include a combination of phosphorylated amyloid precursor (phospho-APP) and beta amyloid (Aβ). Further, the state of increased activity is determined by measuring at least one of DYRK1A, DYRK1B, and DYRK2 by a conventionally known method, for example, a detection or imaging method using an RT-PCR method or an antibody, or It can be confirmed by measuring the amount or ratio of the specific phosphorylated protein or a product derived therefrom.

本発明の医薬組成物は、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性阻害が治療又は予防に有効な疾病に対する治療又は予防に用いることが好ましい。本発明の医薬組成物が用いられる疾病としては、精神神経疾患や、悪性腫瘍が挙げられる。前記精神神経疾患は、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴うもの、好ましくは、ＤＹＲＫ１Ａの活性亢進を伴うものであり、より好ましくは、ＤＹＲＫ１Ａの過剰発現を伴う精神神経疾患及びＴａｕリン酸化亢進を伴う精神神経疾患であり、代表的には、ダウン症候群、及びアルツハイマー病を含む。なお、前記精神神経疾患は、ダウン症候群以外、又は、アルツハイマー病以外の精神神経疾患であってもよい。前記悪性腫瘍は、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴う悪性新生物、好ましくはＤＹＲＫ１Ａ及びＤＹＲＫ１Ｂの活性亢進を伴う悪性新生物であり、より好ましくはＤＹＲＫ１Ｂの活性亢進を伴う悪性新生物である。代表的には、膵管がん及び横紋筋肉腫並びにＨＰＶによるがんを含むがこれらには限定されない。   The pharmaceutical composition of the present invention is preferably used for treatment or prevention of a disease in which inhibition of at least one activity of DYRK1A, DYRK1B, and DYRK2 is effective for treatment or prevention. Examples of diseases for which the pharmaceutical composition of the present invention is used include neuropsychiatric diseases and malignant tumors. Said neuropsychiatric disorder is accompanied by at least one increased activity of DYRK1A, DYRK1B and DYRK2, preferably accompanied by increased activity of DYRK1A, more preferably, a neuropsychiatric disorder associated with overexpression of DYRK1A and It is a neuropsychiatric disorder associated with increased Tau phosphorylation, and typically includes Down's syndrome and Alzheimer's disease. The neuropsychiatric disorder may be other than Down's syndrome or a neuropsychiatric disorder other than Alzheimer's disease. The malignant tumor is a malignant neoplasm with increased activity of at least one of DYRK1A, DYRK1B, and DYRK2, preferably a malignant neoplasm with increased activity of DYRK1A and DYRK1B, and more preferably a malignant with increased activity of DYRK1B. It is a new organism. Typically, including but not limited to pancreatic duct cancer and rhabdomyosarcoma and cancer due to HPV.

ＤＹＲＫ１Ａの活性亢進を伴う疾病、及びＤＹＲＫ１Ａの活性阻害が治療又は予防に有効な疾病としては、精神神経疾患が挙げられる。前記精神神経疾患の第一の例が、ダウン症候群である。上述したとおり、ＤＹＲＫ１Ａの遺伝子は、ダウン症候群の原因である２１番染色体トリソミーのダウン症クリティカル領域（ＤＳＣＲ）に位置し、マウスではＤＹＲＫ１Ａの単独の過剰発現によりダウン症様の精神神経症状をきたすことが報告され、ダウン症患者及びダウン症モデルマウスの脳内ではＤＹＲＫ１Ａ発現が上昇することが報告されている。さらに、後述の実施例に記載するとおり、神経系に発生異常を呈するＤＹＲＫ１Ａ過剰発現アフリカツメガエルモデルに、本発明の医薬組成物の有効成分である上記式（Ｉ）の化合物を投与すると正常な発生を導くことができる。したがって、本発明の医薬組成物であれば、ダウン症候群を含む精神神経症状を治療又は予防できるといえる。   Psychiatric and neurological diseases are examples of diseases associated with increased activity of DYRK1A and diseases in which inhibition of DYRK1A activity is effective for treatment or prevention. The first example of the neuropsychiatric disorder is Down's syndrome. As described above, the DYRK1A gene is located in the Down syndrome critical region (DSCR) of trisomy 21 that causes Down's syndrome, and it is reported that DYRK1A alone overexpresses SYRK1A in mice. It has been reported that DYRK1A expression is increased in the brains of Down syndrome patients and Down syndrome model mice. Furthermore, as described in the Examples below, normal development occurs when a compound of formula (I), which is an active ingredient of the pharmaceutical composition of the present invention, is administered to a DYRK1A overexpressing Xenopus model exhibiting abnormal development in the nervous system. Can guide you. Therefore, it can be said that the pharmaceutical composition of the present invention can treat or prevent a neuropsychiatric condition including Down's syndrome.

ＤＹＲＫ１Ａの活性亢進を伴う疾病、及びＤＹＲＫ１Ａの活性阻害が治療又は予防に有効な疾病である精神神経疾患の第二の例が、アルツハイマー病である。アルツハイマー病の発症機構として、まずアミロイド前駆体蛋白質（ＡＰＰ）から産生されたベータアミロイド（Ａβ）が蓄積し、次いでＴａｕの過剰リン酸化が起こり、その結果、神経原線維変化・神経細胞死が誘導されるという機序が最も有力であると考えられている。Ｔａｕの異常リン酸化はＤＹＲＫ１Ａと密接な関係にあり、後述の実施例に記載するとおり、細胞内にＤＹＲＫ１Ａ及びＴａｕを過剰発現させた系において、Ｔａｕの異常リン酸化は、本発明の医薬組成物の有効成分である上記式（Ｉ）の化合物の投与により抑制される。さらに、ＤＹＲＫ１Ａは、Ａβの蓄積を促進するＡＰＰのリン酸化に関与することも示唆されている（Ryoo SR et al. J Neurochem. 2008 Mar;104(5):1333-44. Epub 2007 Nov 14）。したがって、本発明の医薬組成物であれば、アルツハイマー病を含む精神神経症状を治療又は予防できるといえる。また、ダウン症候群の原因である２１番染色体トリソミーのダウン症クリティカル領域（ＤＳＣＲ）には、ＤＹＲＫ１Ａ遺伝子のみならず、ＡＰＰ遺伝子も位置し、ダウン症患者が健常人に比べて早期にかつ高効率にアルツハイマー病を発症することから、本発明の医薬組成物であれば、ダウン症患者におけるアルツハイマー病を治療又は予防できるといえる。   Alzheimer's disease is a second example of a disease associated with increased activity of DYRK1A and a neuropsychiatric disease in which inhibition of DYRK1A activity is effective for treatment or prevention. As the pathogenesis of Alzheimer's disease, beta amyloid (Aβ) produced from amyloid precursor protein (APP) first accumulates, and then Tau hyperphosphorylation occurs, resulting in neurofibrillary tangles and neuronal cell death. It is considered that the mechanism of being done is the most powerful. Abnormal phosphorylation of Tau is closely related to DYRK1A, and as described in Examples below, in a system in which DYRK1A and Tau are overexpressed in cells, abnormal phosphorylation of Tau is a pharmaceutical composition of the present invention. It is suppressed by administration of the compound of the above formula (I) which is an active ingredient of Furthermore, DYRK1A has also been implicated in APP phosphorylation that promotes Aβ accumulation (Ryoo SR et al. J Neurochem. 2008 Mar; 104 (5): 1333-44. Epub 2007 Nov 14). . Therefore, it can be said that the pharmaceutical composition of the present invention can treat or prevent a neuropsychiatric condition including Alzheimer's disease. In addition, not only the DYRK1A gene but also the APP gene is located in the Down syndrome critical region (DSCR) of chromosome 21 trisomy that causes Down syndrome, and Alzheimer's disease is early and highly efficient in patients with Down syndrome compared to healthy individuals. Therefore, the pharmaceutical composition of the present invention can treat or prevent Alzheimer's disease in patients with Down syndrome.

ＤＹＲＫ１Ａの活性亢進を伴う疾病、及びＤＹＲＫ１Ａの活性阻害が治療又は予防に有効な疾病である悪性腫瘍の例は、ヒトパピローマウイルス（ＨＰＶ）によるがんである。ＤＹＲＫ１Ａは、ＨＰＶ１６の蛋白質をリン酸化することにより該ウイルス蛋白質の安定化に働き、それらが子宮頸部がんの細胞癌化に関与することが報告されている（Chang HS et al. Int J Cancer. 2007 Jun 1;120(11):2377-85.; Liang YJ et al. Int J Biochem Cell Biol. 2008 40(11):2431-41.）。したがって、本発明の医薬組成物であれば、ＨＰＶによるがん、好ましくはＨＰＶ１６によるがん、さらに好ましくはＨＰＶによる子宮頸部がん、さらにより好ましくはＨＰＶ１６による子宮頸部がんを治療又は予防できるといえる。   An example of a disease associated with increased activity of DYRK1A and a malignant tumor in which inhibition of DYRK1A activity is an effective treatment or prevention is cancer caused by human papillomavirus (HPV). DYRK1A acts to stabilize the viral protein by phosphorylating the HPV16 protein, and it has been reported that they are involved in cervical cancer cell transformation (Chang HS et al. Int J Cancer). 2007 Jun 1; 120 (11): 2377-85 .; Liang YJ et al. Int J Biochem Cell Biol. 2008 40 (11): 2431-41.). Therefore, the pharmaceutical composition of the present invention treats or prevents cancer caused by HPV, preferably cancer caused by HPV16, more preferably cervical cancer caused by HPV, and even more preferably cervical cancer caused by HPV16. I can say that.

ＤＹＲＫ１Ｂの活性亢進を伴う疾病、及びＤＹＲＫ１Ｂの活性阻害が治療又は予防に有効な疾病としては、悪性腫瘍が挙げられる。上述したとおり、ＤＹＲＫ１Ｂは、膵管がん組織や横紋筋肉腫において発現が増加する。さらに、後述の実施例に記載するとおり、悪性腫瘍由来の細胞系の細胞に本発明の医薬組成物の有効成分である上記式（Ｉ）の化合物を投与すると増殖抑制効果及び／又は抗がん剤作用増強効果を発揮する。よって、本発明の医薬組成物は、悪性腫瘍の治療に有用である。また、本発明は、その他の態様として、ＤＹＲＫ１Ｂの活性亢進を伴う悪性腫瘍において抗がん剤の作用を増強するための医薬組成物であって、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの蛋白質リン酸化活性を阻害する化合物を有効成分とする医薬組成物を提供しうる。さらにその他の態様として、本発明は、ＤＹＲＫ１Ｂの活性亢進を伴う悪性腫瘍において抗がん剤の作用を増強するための医薬組成物であって、上記式（Ｉ）のベンゾチアゾール誘導体、又は、その製薬上許容される塩若しくは溶媒和物を、好ましくは有効成分として含む医薬組成物を提供しうる。   A malignant tumor is mentioned as a disease with the activity enhancement of DYRK1B, and a disease in which the activity inhibition of DYRK1B is effective for treatment or prevention. As described above, DYRK1B has increased expression in pancreatic duct cancer tissue and rhabdomyosarcoma. Further, as described in the examples below, when the compound of the above formula (I), which is an active ingredient of the pharmaceutical composition of the present invention, is administered to cells of a malignant tumor-derived cell line, the growth inhibitory effect and / or anticancer Demonstrate the effect of enhancing drug action. Therefore, the pharmaceutical composition of the present invention is useful for the treatment of malignant tumors. In another aspect, the present invention provides a pharmaceutical composition for enhancing the action of an anticancer agent in a malignant tumor with increased activity of DYRK1B, wherein at least one protein of DYRK1A, DYRK1B, and DYRK2 A pharmaceutical composition comprising a compound that inhibits phosphorylation activity as an active ingredient can be provided. As yet another aspect, the present invention provides a pharmaceutical composition for enhancing the action of an anticancer agent in a malignant tumor associated with enhanced DYRK1B activity, the benzothiazole derivative of the above formula (I), or a A pharmaceutical composition comprising a pharmaceutically acceptable salt or solvate, preferably as an active ingredient may be provided.

［有効成分］
本発明の医薬組成物の有効成分は、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの蛋白質リン酸化活性を阻害する化合物である。本発明において蛋白質リン酸化活性を阻害する化合物とは、インビトロ及びインビボの少なくとも一方における公知の蛋白質リン酸化活性阻害のアッセイ系において、前記化合物を加えた場合の蛋白質リン酸化活性を、前記化合物を加えないコントロールと比べて、例えば６０％以下、好ましくは５０％以下、より好ましくは４０％以下、さらに好ましくは３０％以下、さらにより好ましくは２０％以下、特に好ましくは１０％以下にまで阻害できる化合物をいう。前記アッセイ系において、添加する前記化合物の量は、例えば、０．０１〜１０μＭである。蛋白質リン酸化活性阻害アッセイの具体例としては、後述する実施例におけるインビトロ及びインビボにおけるアッセイが挙げられる。ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの蛋白質リン酸化活性を阻害する化合物の一例として、下記ベンゾチアゾール誘導体が挙げられる。[Active ingredients]
The active ingredient of the pharmaceutical composition of the present invention is a compound that inhibits at least one protein phosphorylation activity of DYRK1A, DYRK1B, and DYRK2. In the present invention, the compound that inhibits the protein phosphorylation activity refers to the protein phosphorylation activity when the compound is added in the known assay system for inhibiting protein phosphorylation activity in at least one of in vitro and in vivo. A compound that can inhibit, for example, 60% or less, preferably 50% or less, more preferably 40% or less, even more preferably 30% or less, even more preferably 20% or less, and particularly preferably 10% or less, compared to a control that is not present Say. In the assay system, the amount of the compound to be added is, for example, 0.01 to 10 μM. Specific examples of the protein phosphorylation activity inhibition assay include in vitro and in vivo assays in Examples described later. Examples of the compound that inhibits at least one protein phosphorylation activity of DYRK1A, DYRK1B, and DYRK2 include the following benzothiazole derivatives.

［ベンゾチアゾール誘導体］
本発明の医薬組成物は、一態様において、下記式（Ｉ）のベンゾチアゾール誘導体、又は、その製薬上許容される塩若しくは溶媒和物を、好ましくは有効成分として含む。

［上記式（Ｉ）において、
Ａは、Ｓ（硫黄原子）又はＳｅ（セレニウム原子）であり、
Ｘは、ハロゲン置換されてもよいＣ１−Ｃ１０アルキル基であり、
Ｙは、Ｈ（水素原子）、ハロゲン置換されてもよいＣ１−Ｃ１０アルキル基、Ｃ２−Ｃ６アルケニル基、−Ｒ１ＯＨ、−ＣＯＯＲ２、及び、−Ｒ１ＯＣＯＲ３から選択され、
Ｚは、ハロゲン置換されてもよいＣ１−Ｃ１０アルキル基、ハロゲン、−ＮＨＣＯＲ２、−ＯＲ２、及び、−ＯＣＯＲ３から選択され、
Ｒ１は、Ｃ１−Ｃ１０アルキレン基であり、
Ｒ２は、Ｈ、又は、Ｃ１−Ｃ１０アルキル基であり、
Ｒ３は、Ｃ１−Ｃ１０アルキル基、Ｃ１−Ｃ３アルコキシ基若しくはＣ１−Ｃ６アルキル基で置換されてもよいアリール基、及び、Ｃ１−Ｃ６アルキル基で置換されてもよいへテロアリール基から選択される。］[Benzothiazole derivatives]
In one aspect, the pharmaceutical composition of the present invention preferably contains a benzothiazole derivative of the following formula (I) or a pharmaceutically acceptable salt or solvate thereof as an active ingredient.

[In the above formula (I),
A is S (sulfur atom) or Se (selenium atom),
X is a halogen-substituted C 1 may be -C 10 alkyl group,
Y is selected from H (hydrogen atom), C 1 -C 10 alkyl group optionally substituted by halogen, C 2 -C 6 alkenyl group, —R 1 OH, —COOR 2 , and —R 1 OCOR 3. ,
Z is a halogen substituted may be C 1 -C 10 alkyl group, halogen, -NHCOR 2, -OR 2, and is selected from -OCOR 3,
R 1 is a C 1 -C 10 alkylene group,
R 2 is H or a C 1 -C 10 alkyl group,
R 3 may be substituted with a C 1 -C 10 alkyl group, an aryl group that may be substituted with a C 1 -C 3 alkoxy group or a C 1 -C 6 alkyl group, and a C 1 -C 6 alkyl group. Selected from good heteroaryl groups. ]

なお、本発明において、「Ｃ１−Ｃｎアルキル基」とは、１〜ｎ個の炭素原子を有する直鎖状又は分枝状のアルキル基をいい、「Ｃ１−Ｃｎアルキレン基」とは、前記定義のＣ１−Ｃｎアルキル基の二価の置換基をいう。また、「Ｃ１−Ｃｍアルコキシ基若しくはＣ１−Ｃｎアルキル基で置換されてもよいアリール基」とは、側鎖のないアリール基（例えば、フェニル基、１−ナフチル基、２−ナフチル基等）及び１以上のＣ１−Ｃｍアルコキシ基（炭素数１〜ｍ個のアルコキシ基）側鎖若しくは１以上のＣ１−Ｃｎアルキル基（炭素原子数１〜ｎ個のアルキル基）側鎖を有するアリール基（例えば、ｏ，ｍ，ｐ−メトキシフェニル基、ジメトキシフェニル基、トリメトキシフェニル基、ｏ，ｍ，ｐ−トリル基、ジメチルフェニル基、メシチル基等）をいう。「へテロアリール基」とはアリール基の環内に１個以上のヘテロ原子を含むものをいい、ヘテロ原子としては、窒素、硫黄、酸素が挙げられる。「ハロゲン置換されてもよい」とは、１以上の水素原子がハロゲン原子により置換される場合を含むことをいい、ハロゲン原子は、フッ素原子（Ｆ）、塩素原子（Ｃｌ）、及び臭素原子（Ｂｒ）を含む。また、「Ｃ２−Ｃｎアルケニル基」とは、２〜ｎ個の炭素原子を有する直鎖状又は分枝状のアルケニル基をいい、ビニル基、アリル基、プロペニル基、ブテニル基等を含む。In the present invention, the “C 1 -C n alkyl group” refers to a linear or branched alkyl group having 1 to n carbon atoms, and is referred to as “C 1 -C n alkylene group”. Means a divalent substituent of the C 1 -C n alkyl group defined above. The “aryl group optionally substituted with a C 1 -C m alkoxy group or a C 1 -C n alkyl group” means an aryl group having no side chain (for example, a phenyl group, a 1-naphthyl group, a 2-naphthyl group). group) and one or more C 1 -C m alkoxy group (1 to m carbon atoms number of alkoxy groups) side chain, or one or more C 1 -C n alkyl group (carbon atom number 1~n number of alkyl group) An aryl group having a side chain (for example, o, m, p-methoxyphenyl group, dimethoxyphenyl group, trimethoxyphenyl group, o, m, p-tolyl group, dimethylphenyl group, mesityl group, etc.). “Heteroaryl group” means an aryl group containing one or more heteroatoms in the ring, and examples of the heteroatoms include nitrogen, sulfur and oxygen. The term “optionally substituted with halogen” means that one or more hydrogen atoms are substituted with a halogen atom. The halogen atom is a fluorine atom (F), a chlorine atom (Cl), or a bromine atom ( Br). The “C 2 -C n alkenyl group” means a linear or branched alkenyl group having 2 to n carbon atoms, and includes a vinyl group, an allyl group, a propenyl group, a butenyl group, and the like. .

また、ベンゾチアゾール化合物としては、上記式（Ｉ）のＡは硫黄（Ｓ）であるが、本発明におけるベンゾチアゾール誘導体は、上記式（Ｉ）のＡがセレニウム（Ｓｅ）に置換された化合物を含みうる。   As the benzothiazole compound, A in the above formula (I) is sulfur (S), but the benzothiazole derivative in the present invention is a compound in which A in the above formula (I) is substituted with selenium (Se). May be included.

上記式（Ｉ）において、前記Ｃ１−Ｃ１０アルキル基は、リン酸化活性に対する阻害能の点から、Ｃ１−Ｃ６アルキル基であることが好ましく、Ｃ１−Ｃ４アルキル基であることがより好ましい。また、前記Ｃ１−Ｃ１０アルキレン基は、リン酸化活性に対する阻害能の点から、Ｃ１−Ｃ６アルキレン基であることが好ましく、Ｃ１−Ｃ４アルキレン基であることがより好ましい。また、前記Ｃ２−Ｃ６アルケニル基は、リン酸化活性に対する阻害能の点から、Ｃ２−Ｃ４アルケニル基が好ましく、Ｃ２−Ｃ３アルケニル基がより好ましい。また、上記式（Ｉ）における前記Ｃ１−Ｃ６アルキル基は、リン酸化活性に対する阻害能の点から、Ｃ１−Ｃ４アルキル基であることが好ましく、Ｃ１−Ｃ３アルキル基であることがより好ましい。In the above formula (I), said C 1 -C 10 alkyl group, in terms of ability to inhibit the phosphorylation activity is preferably C 1 -C 6 alkyl group, a C 1 -C 4 alkyl group Is more preferable. Furthermore, the C 1 -C 10 alkylene group, in terms of ability to inhibit the phosphorylation activity is preferably C 1 -C 6 alkylene group, more preferably a C 1 -C 4 alkylene group. Also, the C 2 -C 6 alkenyl group, in terms of ability to inhibit the phosphorylation activity, preferably C 2 -C 4 alkenyl group, C 2 -C 3 alkenyl group is more preferable. In addition, the C 1 -C 6 alkyl group in the above formula (I) is preferably a C 1 -C 4 alkyl group from the viewpoint of the ability to inhibit phosphorylation activity, and is a C 1 -C 3 alkyl group. It is more preferable.

上記式（Ｉ）において、Ｘは、ハロゲン置換されてもよいＣ１−Ｃ１０アルキル基であって、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２のリン酸化活性に対する阻害能の点から、ハロゲン置換されてもよいＣ１−Ｃ４アルキル基が好ましく、ハロゲン置換されてもよいＣ１−Ｃ３アルキル基がより好ましく、メチル基、エチル基、又はパーフルオロメチル基がさらに好ましい。In the above formula (I), X is a C 1 -C 10 alkyl group which may be halogen-substituted, and may be halogen-substituted from the viewpoint of its ability to inhibit the phosphorylation activity of DYRK1A, DYRK1B, and DYRK2. A preferable C 1 -C 4 alkyl group is preferable, a C 1 -C 3 alkyl group which may be halogen-substituted is more preferable, and a methyl group, an ethyl group, or a perfluoromethyl group is further preferable.

上記式（Ｉ）において、Ｙは、Ｈ、ハロゲン置換されてもよいＣ１−Ｃ１０アルキル基、Ｃ２−Ｃ６アルケニル基、−Ｒ１ＯＨ、−ＣＯＯＲ２、及び、−Ｒ１ＯＣＯＲ３から選択される。ここで、−Ｒ１ＯＨとしては、ヒドロキシＣ１−Ｃ６アルキル基が挙げられる。−Ｒ１ＣＯＯＲ２としては、Ｃ１−Ｃ４アルコキシカルボニルＣ１−Ｃ４アルキル基が挙げられ、−ＣＯＯＲ２としては、カルボキシル基、及びＣ１−Ｃ４アルコキシカルボニル基が挙げられ−Ｒ１ＯＣＯＲ３としては、Ｃ１−Ｃ４アルキルアシルオキシＣ１−Ｃ４アルキル基、（ヘテロ）アリールアシルオキシＣ１−Ｃ４アルキル基、及びＣ１−Ｃ４アルキル（ヘテロ）アリールアシルオキシＣ１−Ｃ４アルキル基が挙げられる。In the above formula (I), Y is H, C 1 -C 10 alkyl group optionally substituted with halogen, C 2 -C 6 alkenyl group, —R 1 OH, —COOR 2 , and —R 1 OCOR 3. Selected from. Here, examples of —R 1 OH include a hydroxy C 1 -C 6 alkyl group. -R 1 COOR 2 includes a C 1 -C 4 alkoxycarbonyl C 1 -C 4 alkyl group, and -COOR 2 includes a carboxyl group and a C 1 -C 4 alkoxycarbonyl group -R 1 OCOR 3 includes C 1 -C 4 alkyl acyloxy C 1 -C 4 alkyl group, (hetero) aryl acyloxy C 1 -C 4 alkyl group, and C 1 -C 4 alkyl (hetero) aryl acyloxy C 1 -C 4. An alkyl group is mentioned.

Ｙは、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２のリン酸化活性に対する阻害能の点から、Ｈ、Ｃ１−Ｃ６アルキル基、Ｃ２−Ｃ４アルケニル基、ヒドロキシＣ１−Ｃ６アルキル基、Ｃ１−Ｃ４アルキルアシルオキシＣ１−Ｃ４アルキル基、及びＣ１−Ｃ４アルコキシカルボニル基が好ましく、Ｈ、メチル基、エチル基、ペンチル基、ビニル基、ヒドロキシメチル基、アセトキシメチル基、メトキシカルボニルブチル基、及びエトキシカルボニル基がより好ましい。Y is H, C 1 -C 6 alkyl group, C 2 -C 4 alkenyl group, hydroxy C 1 -C 6 alkyl group, C 1 from the viewpoint of the ability to inhibit phosphorylation activity of DYRK1A, DYRK1B, and DYRK2. -C 4 alkyl acyloxyalkyl C 1 -C 4 alkyl group, and C 1 -C 4 alkoxycarbonyl group preferably, H, methyl, ethyl, pentyl group, a vinyl group, a hydroxymethyl group, acetoxymethyl group, methoxycarbonyl butyl A group and an ethoxycarbonyl group are more preferable.

上記式（Ｉ）において、Ｚは、ハロゲン置換されてもよいＣ１−Ｃ１０アルキル基、ハロゲン、−ＮＨＣＯＲ２、−ＯＲ２、及び、−ＯＣＯＲ３から選択される。ここで、−ＯＲ２としては、ヒドロキシ基、及びＣ１−Ｃ６アルコキシ基が挙げられる。なお、−ＯＣＯＲ３は、ヒドロキシ基のプロドラッグ形態と考えることができる。この場合、生体内で代謝されヒドロキシ基となるものであれば、−ＯＣＯＲ３におけるＲ３は特に制限されないが、−ＯＣＯＲ３としては、Ｃ１−Ｃ４アルキルアシルオキシ基、（ヘテロ）アリールアシルオキシ基、Ｃ１−Ｃ３アルコキシアリールアシルオキシ基、及びＣ１−Ｃ４アルキル（へテロ）アリールアシルオキシ基が挙げられる。In the above formula (I), Z is selected from a C 1 -C 10 alkyl group that may be halogen-substituted, halogen, —NHCOR 2 , —OR 2 , and —OCOR 3 . Here, examples of —OR 2 include a hydroxy group and a C 1 -C 6 alkoxy group. In addition, -OCOR 3 can be considered as a prodrug form of a hydroxy group. In this case, R 3 in —OCOR 3 is not particularly limited as long as it is metabolized in vivo to form a hydroxy group, but as —OCOR 3 , C 1 -C 4 alkylacyloxy group, (hetero) arylacyloxy group , C 1 -C 3 alkoxy aryl acyl group, and C 1 -C 4 alkyl (hetero) aryl acyloxyalkyl groups.

Ｚは、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２のリン酸化活性に対する阻害能の点から、ヒドロキシ基、Ｃ１−Ｃ６アルコキシ基、ハロゲン、及びＣ１−Ｃ４アルキルアシルオキシ基が好ましく、ヒドロキシ基、メトキシ基、エトキシ基、フッ素、塩素、臭素、アセトキシ基がより好ましい。また、ＤＹＲＫ２のリン酸化活性に対する阻害能の点からは、Ｚは、−ＮＨＣＯＲ２であることも好ましく、アセチルアミノ基であることがより好ましい。Ｚは、ヒドロキシ基のプロドラッグ形態としては、生体内で代謝されてヒドロキシ基へのなり易さ及びＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２のリン酸化活性に対する阻害能の点から、Ｃ１−Ｃ４アルキルアシルオキシ基、アリールアシルオキシ基、Ｃ１−Ｃ３アルコキシアリールアシルオキシ基、及びヘテロアリールアシルオキシ基が好ましく、アセトキシ基、ピバロイルオキシ基、ベンゾイルオキシ基、（モノ-、ジ-、トリ-）メトキシベンゾイルオキシ基、ニコチノイルオキシ基、及びイソニコチノイルオキシ基がより好ましい。Z is preferably a hydroxy group, a C 1 -C 6 alkoxy group, a halogen, or a C 1 -C 4 alkylacyloxy group from the viewpoint of the ability to inhibit phosphorylation activity of DYRK1A, DYRK1B, and DYRK2, and is preferably a hydroxy group, a methoxy group, A group, ethoxy group, fluorine, chlorine, bromine and acetoxy group are more preferable. From the viewpoint of the ability to inhibit DYRK2 phosphorylation activity, Z is preferably —NHCOR 2 , more preferably an acetylamino group. Z is a prodrug form of the hydroxy groups are metabolized in vivo to become to hydroxy groups ease and DYRK1A, DYRK1B, and, in terms of ability to inhibit the phosphorylation activity of DYRK2, C 1 -C 4 alkyl acyloxy group, arylacyloxy group, C 1 -C 3 alkoxy aryl acyl group, and a heteroaryl acyl group preferably acetoxy, pivaloyloxy group, benzoyloxy group, (mono -, di -, tri -) methoxybenzoyl group, A nicotinoyloxy group and an isonicotinoyloxy group are more preferable.

上記式（Ｉ）のベンゾチアゾール誘導体は、製薬上許容される塩又は溶媒和物の形態であってもよい。塩の形態としては、例えば、無機酸塩、有機酸塩、無機塩基塩、有機塩基塩、酸性または塩基性アミノ酸塩などが挙げられる。無機酸塩の好ましい例としては、塩酸塩、臭化水素酸塩、硫酸塩、硝酸塩、リン酸塩などが挙げられ、有機酸塩の好ましい例としては、酢酸塩、コハク酸塩、フマル酸塩、マレイン酸塩、酒石酸塩、クエン酸塩、乳酸塩、ステアリン酸塩、安息香酸塩、メタンスルホン酸塩、ｐ−トルエンスルホン酸塩などが挙げられる。また、無機塩基塩の好ましい例としては、ナトリウム塩、カリウム塩などのアルカリ金属塩、カルシウム塩、マグネシウム塩などのアルカリ土類金属塩、アルミニウム塩、アンモニウム塩などが挙げられ、有機塩基塩の好ましい例としては、ジエチルアミン塩、ジエタノールアミン塩、メグルミン塩、Ｎ，Ｎ’−ジベンジルエチレンジアミン塩などが挙げられる。酸性アミノ酸塩の好ましい例としては、アスパラギン酸塩、グルタミン酸塩などが挙げられ、塩基性アミノ酸塩の好ましい例としては、アルギニン塩、リジン塩、オルニチン塩などが挙げられる。   The benzothiazole derivative of the above formula (I) may be in the form of a pharmaceutically acceptable salt or solvate. Examples of the salt form include inorganic acid salts, organic acid salts, inorganic base salts, organic base salts, acidic or basic amino acid salts, and the like. Preferable examples of inorganic acid salts include hydrochloride, hydrobromide, sulfate, nitrate, phosphate and the like, and preferable examples of organic acid salts include acetate, succinate, and fumarate. Maleate, tartrate, citrate, lactate, stearate, benzoate, methanesulfonate, p-toluenesulfonate, and the like. In addition, preferable examples of the inorganic base salt include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, aluminum salt, ammonium salt and the like, and preferable organic base salts. Examples include diethylamine salt, diethanolamine salt, meglumine salt, N, N′-dibenzylethylenediamine salt and the like. Preferable examples of the acidic amino acid salt include aspartate and glutamate. Preferable examples of the basic amino acid salt include arginine salt, lysine salt and ornithine salt.

また、上記式（Ｉ）のベンゾチアゾール誘導体は、溶媒和物の形態としては、大気中の水分を吸収しあるいは吸着水が付いた水和物や、他のある種の溶媒を吸収した溶媒和物が挙げられる。   In addition, the benzothiazole derivative of the above formula (I) is in the form of a solvate that absorbs moisture in the atmosphere or hydrates with adsorbed water, or a solvate that absorbs some other solvent. Things.

本発明の医薬組成物の有効成分として好ましい形態の一例は、以下のものが挙げられる。但し、本発明はこれらに限定されない。   Examples of preferred forms as the active ingredient of the pharmaceutical composition of the present invention include the following. However, the present invention is not limited to these.

製造例００９のベンゾチアゾール誘導体は、製造例００１の５位のメトキシ基の脱保護（脱メチル化）あるいは、製造例０１９の５位のアセチル基の脱保護（アセチル基のアルカリ加水分解）により得ることができる。また、製造例０１９のベンゾチアゾール誘導体は、製造例００９の５位のヒドロキシ基がアセチル化されたものであって、製造例００９のプロドラッグ形態として機能しうるベンゾチアゾール誘導体である。したがって、本発明は、その他の態様において、生体内（好ましくはヒトの体内）において製造例００９となりうるベンゾチアゾール誘導体、すなわち、製造例００９のプロドラッグ形態として機能しうるベンゾチアゾール誘導体を有効成分とする医薬組成物である。製造例００９のプロドラッグとしては、以下の形態も挙げることができる。   The benzothiazole derivative of Production Example 009 is obtained by the deprotection (demethylation) of the 5-position methoxy group of Production Example 001 or the deprotection of the acetyl group at the 5-position of Production Example 019 (alkali hydrolysis of the acetyl group). be able to. Further, the benzothiazole derivative of Production Example 019 is a benzothiazole derivative which is obtained by acetylating the hydroxy group at the 5-position of Production Example 009 and can function as a prodrug form of Production Example 009. Therefore, in another aspect, the present invention provides, as an active ingredient, a benzothiazole derivative that can be produced as Production Example 009 in vivo (preferably in the human body), that is, a benzothiazole derivative that can function as a prodrug form of Production Example 009. A pharmaceutical composition. Examples of prodrugs of Production Example 009 can also include the following forms.

［ベンゾチアゾール誘導体の製造方法］
上記式（Ｉ）のベンゾチアゾール誘導体は、例えば、当業者であれば、文献［M Muraki et al. 2004: J Biol Chem 279 (23) 24246-24254］（非特許文献９）に記載に基づき、市販のベンゾチアゾール化合物を用いて製造できる。より詳細には、後述する実施例における製造例を参照でき、製造した化合物以外の化合物についても、実施例を参照して製造できる。[Method for producing benzothiazole derivative]
The benzothiazole derivative of the above formula (I) is, for example, based on the description in the literature [M Muraki et al. 2004: J Biol Chem 279 (23) 24246-24254] (Non-Patent Document 9), It can be produced using a commercially available benzothiazole compound. In more detail, the manufacture example in the Example mentioned later can be referred, and it can manufacture with reference to an Example also about compounds other than the manufactured compound.

［医薬組成物の製造方法］
本発明の医薬組成物は、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの蛋白質リン酸化活性を阻害する化合物、好ましくは、上記式（Ｉ）のベンゾチアゾール誘導体、又は、その製薬上許容される塩若しくは溶媒和物を含むほかは特に制限されない。本発明の医薬組成物の剤形は、投与方法に応じて適宜選択でき、例えば、注射剤、液体製剤、カプセル剤、咀嚼剤、錠剤、懸濁剤、クリーム剤、軟膏等が挙げられる。また、投与方法も特に制限されず、経口投与、非経口投与が挙げられる。本発明の医薬組成物は、投与形態や剤形に応じた従来公知の添加物（例えば、賦形剤又は希釈剤）を含有してもよい。本発明の医薬組成物は、例えば、下記に示す治療・予防方法に使用することができる。[Method for producing pharmaceutical composition]
The pharmaceutical composition of the present invention is a compound that inhibits at least one protein phosphorylation activity of DYRK1A, DYRK1B, and DYRK2, preferably a benzothiazole derivative of the above formula (I), or a pharmaceutically acceptable salt thereof Or there is no particular limitation except that it includes a solvate. The dosage form of the pharmaceutical composition of the present invention can be appropriately selected depending on the administration method, and examples thereof include injections, liquid preparations, capsules, chewing agents, tablets, suspensions, creams, ointments and the like. The administration method is not particularly limited, and examples thereof include oral administration and parenteral administration. The pharmaceutical composition of the present invention may contain conventionally known additives (for example, excipients or diluents) according to the dosage form and dosage form. The pharmaceutical composition of the present invention can be used, for example, in the following treatment / prevention methods.

経口投与に適した剤形としては、錠剤、粒子、液体又は粉末含有カプセル、トローチ、咀嚼剤、多粒子及びナノ粒子、ゲル、フィルムなどの固形製剤；懸濁液、溶液、シロップ及びエリキシルなどの液体製剤が挙げられる。前記賦形剤としては、セルロース、炭酸カルシウム、第二リン酸カルシウム、マンニトール及びクエン酸ナトリウムなどの担体；ポリビニルピロリジン、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース及びゼラチンなどの造粒結合剤；澱粉グリコール酸ナトリウム及びケイ酸塩などの崩壊剤；ステアリン酸マグネシウム及びステアリン酸などの滑沢剤；ラウリル硫酸ナトリウムなどの湿潤剤；保存剤、抗酸化剤、矯味矯臭剤及び着色剤が挙げられる。   Dosage forms suitable for oral administration include solid preparations such as tablets, particles, liquid or powder-containing capsules, troches, chewing agents, multiparticulates and nanoparticles, gels, films, etc .; suspensions, solutions, syrups and elixirs, etc. Liquid formulations are mentioned. Examples of the excipient include carriers such as cellulose, calcium carbonate, dicalcium phosphate, mannitol and sodium citrate; granulated binders such as polyvinylpyrrolidine, hydroxypropylcellulose, hydroxypropylmethylcellulose and gelatin; sodium starch glycolate and silica Disintegrating agents such as acid salts; lubricants such as magnesium stearate and stearic acid; wetting agents such as sodium lauryl sulfate; preservatives, antioxidants, flavoring agents and coloring agents.

本発明の医薬組成物の非経口投与としては、上記式（Ｉ）のベンゾチアゾール誘導体、又は、その製薬上許容される塩若しくは溶媒和物を、血流中、筋肉中又は内部器官中に直接投与することが挙げられる。非経口投与は、静脈内、動脈内、腹腔内、鞘内、心室内、尿道内、胸骨内、頭蓋内、筋肉内及び皮下の投与を含む。非経口投与は、例えば、針注射器、針なし注射器及びその他の注入技術で行える。また、非経口投与に適した剤形としては、例えば、賦形剤及び／又は緩衝剤を含む水溶液が挙げられる。   For parenteral administration of the pharmaceutical composition of the present invention, the benzothiazole derivative of the above formula (I), or a pharmaceutically acceptable salt or solvate thereof, is directly introduced into the bloodstream, muscle or internal organ. Administration. Parenteral administration includes intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous administration. Parenteral administration can be accomplished, for example, with needle syringes, needleless syringes, and other infusion techniques. Examples of the dosage form suitable for parenteral administration include an aqueous solution containing an excipient and / or a buffer.

［治療・予防方法］
本発明は、その態様において、本発明の医薬組成物を用いる治療又は予防方法を提供する。本発明の治療又は予防方法と対象となる疾病は、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴う疾病であって、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性阻害が治療又は予防に有効な疾病であることが好ましく、精神神経疾患及び悪性腫瘍を含むことがより好ましい。前記精神神経疾患は、上述のとおり、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴うもの、好ましくはＤＹＲＫ１Ａの活性亢進を伴うもの、さらに好ましくはＴａｕリン酸化亢進を伴うものであって、代表的には、ダウン症候群、アルツハイマー病、及びダウン症患者におけるアルツハイマー病を含むがこれらには限定されない。前記悪性腫瘍は、上述のとおり、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴うもの、好ましくはＤＹＲＫ１Ａ及びＤＹＲＫ１Ｂの活性亢進を伴う悪性新生物であり、より好ましくはＤＹＲＫ１Ｂの活性亢進を伴う悪性新生物である。代表的には、膵管がん及び横紋筋肉腫、並びに、ＨＰＶによるがんを含むがこれらには限定されない。本発明の医薬組成物により精神神経疾患及び悪性腫瘍治療又は予防できるメカニズムは上述のとおりである。また、本発明の治療又は予防方法の対象はヒト及びヒト以外の動物であってよい。[Treatment and prevention methods]
In its embodiments, the present invention provides a method of treatment or prevention using the pharmaceutical composition of the present invention. The therapeutic or prophylactic method of the present invention and the target disease are diseases accompanied by at least one increased activity of DYRK1A, DYRK1B, and DYRK2, and treatment or inhibition of at least one activity of DYRK1A, DYRK1B, and DYRK2 is treated or It is preferably a disease effective for prevention, and more preferably includes a neuropsychiatric disorder and a malignant tumor. As described above, the neuropsychiatric disorder is accompanied by at least one increased activity of DYRK1A, DYRK1B, and DYRK2, preferably accompanied by enhanced DYRK1A activity, more preferably accompanied by increased Tau phosphorylation. , Typically including, but not limited to, Down's syndrome, Alzheimer's disease, and Alzheimer's disease in patients with Down's syndrome. As described above, the malignant tumor is accompanied by at least one increased activity of DYRK1A, DYRK1B, and DYRK2, preferably a malignant neoplasm with increased activity of DYRK1A and DYRK1B, more preferably increased activity of DYRK1B. Associated malignant neoplasm. Typically, including but not limited to pancreatic duct cancer and rhabdomyosarcoma, and HPV cancer. The mechanism by which the neuropsychiatric disorder and malignant tumor can be treated or prevented by the pharmaceutical composition of the present invention is as described above. The subject of the treatment or prevention method of the present invention may be humans and non-human animals.

本発明の治療又は予防方法は、本発明の医薬組成物を対象に有効量投与することを含むことが好ましい。対象がヒトである場合、例えば、上記式（Ｉ）のベンゾチアゾール誘導体の１日あたりの総量は、一般的には、０．０００１ｍｇ／ｋｇ〜１００ｍｇ／ｋｇの範囲とすることができる。また、一日当たりの総量は、単回又は分割投与で投与することができる。投与方法は、上述した経口／非経口投与を適宜選択できる。なお、使用する医薬組成物に含まれる上記式（Ｉ）のベンゾチアゾール誘導体の好ましい形態は、上述した本発明の医薬組成物の場合と同様である。   The therapeutic or prophylactic method of the present invention preferably includes administering an effective amount of the pharmaceutical composition of the present invention to a subject. When the subject is a human, for example, the total amount per day of the benzothiazole derivative of the above formula (I) can generally be in the range of 0.0001 mg / kg to 100 mg / kg. The total amount per day can be administered in a single dose or divided doses. As the administration method, the above-mentioned oral / parenteral administration can be appropriately selected. In addition, the preferable form of the said benzothiazole derivative of the said formula (I) contained in the pharmaceutical composition to be used is the same as that of the case of the pharmaceutical composition of this invention mentioned above.

本発明の治療又は予防方法は、さらに、対象においてＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つが活性亢進していることを確認する工程を含むことが好ましい。この確認工程は、本発明の医薬組成物を投与する前に行うことが好ましい。この確認工程は、対象から血液、細胞、組織等を採取し、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つのｍＲＮＡの転写量、蛋白質の量、及び、リン酸化活性の少なくとも１つについて測定し、正常細胞又は正常個体と比較することを含んでもよい。具体的な測定方法は、当業者であれば適宜選択できる。あるいは、確認工程は、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、又は、ＤＹＲＫ２に特異的な抗体を含むプローブを使用したイメージング方法を使用して蛋白質の量を測定することを含んでもよい。   The treatment or prevention method of the present invention preferably further includes a step of confirming that at least one of DYRK1A, DYRK1B, and DYRK2 is activated in the subject. This confirmation step is preferably performed before administration of the pharmaceutical composition of the present invention. In this confirmation step, blood, cells, tissues, etc. are collected from a subject and measured for at least one of the transcription amount, protein amount, and phosphorylation activity of at least one mRNA of DYRK1A, DYRK1B, and DYRK2. Comparing with normal cells or normal individuals may be included. A specific measurement method can be appropriately selected by those skilled in the art. Alternatively, the confirmation step may include measuring the amount of protein using an imaging method using a probe containing an antibody specific for DYRK1A, DYRK1B, or DYRK2.

また、本発明の治療又は予防方法が悪性腫瘍の治療又は予防方法である場合、さらに、従来の抗がん剤を投与する工程を含んでもよい。本発明の医薬組成物と従来の抗がん剤とを併用することで、例えば、従来の抗がん剤の抗がん作用を増強できる。したがって、本発明は、その他の態様として、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進、好ましくはＤＹＲＫ１Ｂの活性亢進を伴う悪性腫瘍において抗がん剤の作用を増強する方法であって、本発明の医薬組成物を対象（ヒト、動物、細胞を含む）に有効量投与することを含む方法を提供しうる。この方法においても、対象においてＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つが活性亢進していることを確認する工程を含むことが好ましい。   Moreover, when the treatment or prevention method of this invention is a treatment or prevention method of a malignant tumor, the process of administering the conventional anticancer agent may be further included. By using the pharmaceutical composition of the present invention and a conventional anticancer agent in combination, for example, the anticancer action of the conventional anticancer agent can be enhanced. Therefore, the present invention provides, as another aspect, a method for enhancing the action of an anticancer agent in a malignant tumor with increased activity of at least one of DYRK1A, DYRK1B, and DYRK2, preferably DYRK1B, There may be provided a method comprising administering an effective amount of the pharmaceutical composition of the present invention to a subject (including human, animal and cell). This method also preferably includes a step of confirming that at least one of DYRK1A, DYRK1B, and DYRK2 is activated in the subject.

［阻害試薬キット］
本発明は、さらにその他の態様として、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つのリン酸化活性を阻害する試薬のキットを提供できる。前記キットは、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの蛋白質リン酸化活性を阻害する化合物、好ましくは、上記式（Ｉ）のベンゾチアゾール誘導体、又は、その製薬上許容される塩若しくは溶媒和物を含む試薬を備える。前記キットは、例えば、研究開発において、細胞や個体のＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つのリン酸化活性を阻害するために使用できる。なお、使用する上記式（Ｉ）のベンゾチアゾール誘導体の好ましい形態は、上述した本発明の医薬組成物の場合と同様である。さらに、前記キットは、リン酸化活性を阻害するための前記試薬の使用方法などが記載された取扱い説明書を含むことが好ましい。[Inhibition reagent kit]
As still another aspect, the present invention can provide a kit of a reagent that inhibits at least one phosphorylation activity of DYRK1A, DYRK1B, and DYRK2. The kit is a compound that inhibits at least one protein phosphorylation activity of DYRK1A, DYRK1B, and DYRK2, preferably a benzothiazole derivative of the above formula (I), or a pharmaceutically acceptable salt or solvate thereof A reagent comprising: The kit can be used, for example, in research and development to inhibit the phosphorylation activity of at least one of DYRK1A, DYRK1B, and DYRK2 in cells and individuals. In addition, the preferable form of the benzothiazole derivative of the said formula (I) to be used is the same as that of the pharmaceutical composition of this invention mentioned above. Further, the kit preferably includes an instruction manual describing how to use the reagent for inhibiting phosphorylation activity.

［阻害方法］
本発明は、さらにその他の態様として、ヒト及びヒト以外の動物の細胞又は個体においてＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つのリン酸化活性を阻害する方法であって、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの蛋白質リン酸化活性を阻害する化合物、好ましくは、上記式（Ｉ）のベンゾチアゾール誘導体、又は、その製薬上許容される塩若しくは溶媒和物を細胞又は個体と接触させる方法を提供できる。前記細胞との接触は、ｉｎ ｖｉｖｏ、ｉｎ ｖｉｔｒｏ、ｅｘ ｖｉｖｏの接触を含みうる。また、個体への接触方法は、上述した本発明の医薬組成物の投与方法と同様とすることができる。なお、使用する上記式（Ｉ）のベンゾチアゾール誘導体の好ましい形態は、上述した本発明の医薬組成物の場合と同様である。[Inhibition method]
In still another aspect, the present invention provides a method for inhibiting at least one phosphorylation activity of DYRK1A, DYRK1B, and DYRK2 in cells or individuals of humans and non-human animals, including DYRK1A, DYRK1B, and DYRK2 A method of contacting a cell or an individual with a compound that inhibits at least one protein phosphorylation activity, preferably a benzothiazole derivative of the above formula (I), or a pharmaceutically acceptable salt or solvate thereof . The contact with the cell may include in vivo, in vitro, or ex vivo contact. Moreover, the contact method to an individual | organism | solid can be made to be the same as that of the administration method of the pharmaceutical composition of this invention mentioned above. In addition, the preferable form of the benzothiazole derivative of the said formula (I) to be used is the same as that of the pharmaceutical composition of this invention mentioned above.

［スクリーニング方法］
本発明は、さらにその他の態様として、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴う疾病の治療又は予防のための医薬組成物の有効成分のスクリーニング方法であって、モデル動物の初期胚にＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つのｍＲＮＡを誘導して発生異常を起こしうる状態とすること、前記初期胚を候補化合物と接触させること、及び、候補化合物を接触させなかった場合と比較して候補化合物による発生異常の抑制効果を評価することを含むスクリーニング方法を提供しうる。ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つのｍＲＮＡに誘導される発生異常は、神経発生異常及び／又は形態的発生異常を含む。前記モデル生物としては特に制限されないが、例えば、ショウジョウバエ、アフリカツメガエル、メダカ、ゼブラフィッシュ、ラット、マウスなどが挙げられ、これらの中でも、後述の実施例で示すようにアフリカツメガエルが好ましい。ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つのｍＲＮＡを初期胚に導入してＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つを過剰発現させる技術は、当業者であれば容易である。候補化合物は、例えば、市販のライブラリを使用してもよい。本発明のスクリーニング方法によれば、例えば、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの蛋白質リン酸化活性を阻害する化合物を有効に探索でき、本発明の医薬組成物の有効成分の候補化合物の絞り込みを含む、本発明の医薬組成物の有効成分の開発につなげることができる。[Screening method]
In still another aspect, the present invention provides a method for screening an active ingredient of a pharmaceutical composition for the treatment or prevention of a disease accompanied by at least one increased activity of DYRK1A, DYRK1B, and DYRK2, comprising the initial stage of a model animal. Inducing embryos with at least one mRNA of DYRK1A, DYRK1B, and DYRK2 to cause a developmental abnormality, contacting the early embryo with a candidate compound, and not contacting a candidate compound A screening method can be provided that includes evaluating the effect of suppressing developmental abnormalities caused by candidate compounds. The developmental abnormality induced by at least one mRNA of DYRK1A, DYRK1B, and DYRK2 includes a neurogenesis abnormality and / or a morphological development abnormality. Although it does not restrict | limit especially as said model organism, For example, Drosophila, Xenopus, medaka, zebrafish, a rat, a mouse | mouth etc. are mentioned, Among these, Xenopus is preferable as shown in the below-mentioned Example. A person skilled in the art can easily perform a technique for overexpressing at least one of DYRK1A, DYRK1B, and DYRK2 by introducing at least one mRNA of DYRK1A, DYRK1B, and DYRK2 into an early embryo. As the candidate compound, for example, a commercially available library may be used. According to the screening method of the present invention, for example, a compound that inhibits at least one protein phosphorylation activity of DYRK1A, DYRK1B, and DYRK2 can be effectively searched, and candidate compounds as active ingredients of the pharmaceutical composition of the present invention can be narrowed down. It can lead to development of the active ingredient of the pharmaceutical composition of this invention containing this.

［新規なベンゾチアゾール誘導体］
本発明は、さらなるその他の態様として、新規なベンゾチアゾール誘導体を提供できる。すなわち、本発明のベンゾチアゾール誘導体は、上記式（Ｉ）化合物、又は、その製薬上許容される塩若しくは溶媒和物であって、上記式（Ｉ）において、さらに下記条件ａ又はｂを満たすものである。
ａ）：ＡがＳかつＸ及びＹがメチル基の場合、Ｚは、フッ素原子（Ｆ）、ヒドロキシ基、メトキシ基、エトキシ基、及び、アセトキシ基を除く上記範囲から選択される。
ｂ）：ＡがＳかつＸがエチル基かつＹがメチル基の場合、Ｚは、メトキシ基を除く上記範囲から選択される。[New benzothiazole derivatives]
As still another embodiment, the present invention can provide a novel benzothiazole derivative. That is, the benzothiazole derivative of the present invention is a compound of the above formula (I), or a pharmaceutically acceptable salt or solvate thereof, and further satisfies the following conditions a or b in the above formula (I). It is.
a): When A is S and X and Y are methyl groups, Z is selected from the above range excluding the fluorine atom (F), hydroxy group, methoxy group, ethoxy group, and acetoxy group.
b): When A is S, X is an ethyl group and Y is a methyl group, Z is selected from the above range excluding the methoxy group.

すなわち、上記条件ａは、上記式（Ｉ）においてＡ＝Ｓ，Ｘ＝Ｙ＝−ＣＨ３の場合には、Ｚは、ハロゲン置換されてもよいＣ１−Ｃ６アルキル基、Ｃｌ、Ｂｒ、−ＮＨＣＯＲ２、Ｃ３−Ｃ６アルコキシ基、Ｃ２−Ｃ４アルキルアシルオキシ基、（ヘテロ）アリールアシルオキシ基、Ｃ１−Ｃ３アルコキシアリールアシルオキシ基、及びＣ１−Ｃ４アルキル（へテロ）アリールアシルオキシ基から選択されることを規定することが好ましい。また、上記条件ｂは、上記式（Ｉ）においてＡ＝Ｓ，Ｘ＝−Ｃ２Ｈ５，Ｙ＝−ＣＨ３の場合には、Ｚは、ハロゲン置換されてもよいＣ１−Ｃ６アルキル基、ハロゲン、−ＮＨＣＯＲ２、ヒドロキシ基、Ｃ１−Ｃ６アルコキシ基、Ｃ２−Ｃ４アルキルアシルオキシ基、（ヘテロ）アルキルアリールアシルオキシ基、Ｃ１−Ｃ３アルコキシアリールアシルオキシ基、及びＣ１−Ｃ４アルキル（へテロ）アリールアシルオキシ基から選択されることを規定することが好ましい。本発明のベンゾチアゾール誘導体の好ましい形態は、上記特許文献１及び非特許文献９と重複するものを除き、上述した本発明の医薬組成物の場合と同様である。That is, the condition a is that when A = S and X = Y = —CH 3 in the above formula (I), Z is a C 1 -C 6 alkyl group optionally substituted with halogen, Cl, Br, —NHCOR 2 , C 3 -C 6 alkoxy group, C 2 -C 4 alkylacyloxy group, (hetero) aryl acyloxy group, C 1 -C 3 alkoxyaryl acyloxy group, and C 1 -C 4 alkyl (hetero) aryl It is preferred to prescribe that it is selected from acyloxy groups. In the above-mentioned condition b, when A = S, X = —C 2 H 5 , Y = —CH 3 in the above formula (I), Z is C 1 -C 6 alkyl which may be halogen-substituted. group, halogen, -NHCOR 2, hydroxy groups, C 1 -C 6 alkoxy group, C 2 -C 4 alkyl acyl group, (hetero) alkylaryl acyl group, C 1 -C 3 alkoxy aryl acyl group, and C 1 - It is preferred to prescribe that it is selected from C 4 alkyl (hetero) aryl acyloxy groups. The preferable form of the benzothiazole derivative of the present invention is the same as that of the above-described pharmaceutical composition of the present invention except for those overlapping with Patent Document 1 and Non-Patent Document 9.

次に、実施例により本発明をさらに説明するが、本発明はこれらに限定されない。   EXAMPLES Next, although an Example demonstrates this invention further, this invention is not limited to these.

［ベンゾチアゾール誘導体の製造例］
ベンゾチアゾール誘導体を以下の製造例００１〜０２５のように製造した。[Production example of benzothiazole derivative]
The benzothiazole derivatives were produced as in the following Production Examples 001 to 025.

［製造例００１］
１−（３−エチル−５−メトキシ−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オンの合成

市販の５−メトキシ−２−メチルベンゾチアゾール（２０２ｍｇ、１．１２ｍｍｏｌ）とヨウ化エチル（２．７０ｍｌ、３３．７ｍｍｏｌ）を混合して、２４．５時間還流させた。漏斗を用いて沈殿物を濾しとり、エチルアセテートで洗浄後、減圧乾燥し、淡緑色固体の３−エチル−５−メトキシ−２−メチルベンゾチアゾリウムのヨウ化物を得た（２７０ｍｇ、８０５μｍｏｌ、収率７１．９％）。２ｍｌのアセトニトリル中に３−エチル−５−メトキシ−２−メチルベンゾチアゾリウムのヨウ化物（５０２ｍｇ、１．４９ｍｍｏｌ）を含むサスペンションに、無水酢酸（３３０μｌ、３．４９ｍｍｏｌ）及びトリエチルアミン（４９０μｌ、３．５１ｍｍｏｌ）を常温で連続的に添加して、２時間還流させた。その後、混合物を常温まで冷却し、減圧下で濃縮した。その残留物に水（５０ｍｌ）を添加し、その混合物を１５ｍｌのエチルアセテートで３回抽出した。１つにまとめた有機抽出液をブライン（３０ｍｌ）で洗浄し、Ｎａ２ＳＯ４上で乾燥し、ろ過し、減圧濃縮した。その残留物をシリカゲルカラムクロマトグラフィー（１８ｇ、ＣＨ２Ｃｌ２／エチルアセテート、４：１）を用いて精製し、１−（３−エチル−５−メトキシ−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オン（製造例００１の化合物）を淡黄色固体として得た（２０１ｍｇ、８０６μｍｏｌ、収率５４．１％）。ＮＭＲとＭＳの結果を下記に示す。
1H NMR (CDCl3, 400 MHz) δ 1.37 (t, 3H, J = 7.3 Hz, CH3), 2.24 (s, 3H, CH3), 3.88 (s, 3H, OCH3), 4.02 (q, 2H, J = 7.3 Hz, CH2), 5.87 (s, 1H, olefinic), 6.65 (d,1H, J = 2.3 Hz, aromatic), 6.75 (dd, 1H, J = 2.3, 8.5 Hz, aromatic), 7.45 (d, 1H, J = 8.5 Hz, aromatic).
HRMS (FAB+) m/z 250.901 ((M+H)+, C13H16NO2S requires 250.0902).[Production Example 001]
Synthesis of 1- (3-ethyl-5-methoxy-2,3-dihydrobenzothiazol-2-ylidene) propan-2-one

Commercially available 5-methoxy-2-methylbenzothiazole (202 mg, 1.12 mmol) and ethyl iodide (2.70 ml, 33.7 mmol) were mixed and refluxed for 24.5 hours. The precipitate was filtered using a funnel, washed with ethyl acetate, and then dried under reduced pressure to obtain a pale green solid 3-ethyl-5-methoxy-2-methylbenzothiazolium iodide (270 mg, 805 μmol, Yield 71.9%). To a suspension of 3-ethyl-5-methoxy-2-methylbenzothiazolium iodide (502 mg, 1.49 mmol) in 2 ml of acetonitrile was added acetic anhydride (330 μl, 3.49 mmol) and triethylamine (490 μl, 3 .51 mmol) was continuously added at room temperature and refluxed for 2 hours. The mixture was then cooled to room temperature and concentrated under reduced pressure. Water (50 ml) was added to the residue and the mixture was extracted 3 times with 15 ml of ethyl acetate. The combined organic extract was washed with brine (30 ml), dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified using silica gel column chromatography (18 g, CH 2 Cl 2 / ethyl acetate, 4: 1) to give 1- (3-ethyl-5-methoxy-2,3-dihydrobenzothiazole-2- Iridene) propan-2-one (the compound of Production Example 001) was obtained as a pale yellow solid (201 mg, 806 μmol, yield 54.1%). The results of NMR and MS are shown below.
1 H NMR (CDCl 3 , 400 MHz) δ 1.37 (t, 3H, J = 7.3 Hz, CH 3 ), 2.24 (s, 3H, CH 3 ), 3.88 (s, 3H, OCH 3 ), 4.02 (q, 2H, J = 7.3 Hz, CH 2 ), 5.87 (s, 1H, olefinic), 6.65 (d, 1H, J = 2.3 Hz, aromatic), 6.75 (dd, 1H, J = 2.3, 8.5 Hz, aromatic), 7.45 (d, 1H, J = 8.5 Hz, aromatic).
HRMS (FAB + ) m / z 250.901 ((M + H) + , C 13 H 16 NO 2 S requires 250.0902).

［製造例００２］
１−（３−エチル−５−メトキシ−２，３−ジヒドロベンゾチアゾール−２−イリデン）ブタン−２−オンの合成

無水酢酸にかえて無水プロピオン酸を使用したほかは、［製造例００１］と同様に製造し、黄色油状物の１−（３−エチル−５−メトキシ−２，３−ジヒドロベンゾチアゾール−２−イリデン）ブタン−２−オン（製造例００２の化合物）を得た。
1H NMR (CDCl3, 400 MHz) δ 1.20 (t, 3H, J = 7.3 Hz, CH3), 1.37 (t, 3H, J = 7.2 Hz, CH3), 2.50 (q, 2H, J = 7.3 Hz, CH2), 3.86 (s, 3H, OCH3), 4.02 (q, 2H, J = 7.2 Hz, CH2), 5.87 (s, 1H, olefin), 6.64 (d, 1H, J = 2.3 Hz, aromatic), 6.74 (dd, 1H, J = 2.3, 8.5 Hz, aromatic), 7.44 (d, 1H, J = 8.5 Hz, aromatic).
HRMS (FAB+) m/z 264.1051 ((M+H)+, C14H18NO2S requires 264.1058).[Production Example 002]
Synthesis of 1- (3-ethyl-5-methoxy-2,3-dihydrobenzothiazol-2-ylidene) butan-2-one

This was prepared in the same manner as in [Preparation Example 001] except that propionic anhydride was used instead of acetic anhydride, and 1- (3-ethyl-5-methoxy-2,3-dihydrobenzothiazole-2- Iridene) butan-2-one (Compound of Production Example 002) was obtained.
1 H NMR (CDCl 3 , 400 MHz) δ 1.20 (t, 3H, J = 7.3 Hz, CH 3 ), 1.37 (t, 3H, J = 7.2 Hz, CH 3 ), 2.50 (q, 2H, J = 7.3 Hz, CH 2 ), 3.86 (s, 3H, OCH 3 ), 4.02 (q, 2H, J = 7.2 Hz, CH 2 ), 5.87 (s, 1H, olefin), 6.64 (d, 1H, J = 2.3 Hz , aromatic), 6.74 (dd, 1H, J = 2.3, 8.5 Hz, aromatic), 7.44 (d, 1H, J = 8.5 Hz, aromatic).
HRMS (FAB + ) m / z 264.1051 ((M + H) + , C 14 H 18 NO 2 S requires 264.1058).

［製造例００３］
１−（３−エチル−５−フルオロ−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オンの合成

５−メトキシ−２−メチルベンゾチアゾールにかえて５−フルオロ−２−メチルベンゾチアゾールを使用したほかは、［製造例００１］と同様に製造し、淡黄色固体の１−（３−エチル−５−フルオロ−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オン（製造例００３の化合物）を得た。
1H NMR (CDCl3, 400 MHz) δ 1.38 (t, 3H, J = 7.3 Hz, CH3), 2.53 (s, 3H, CH3), 4.01 (q, 2H, J = 7.3 Hz, CH2), 5.90 (s, 1H, olefinic), 6.81 (dd, 1H, J = 2.4, 3JH-F= 9.2 Hz, aromatic), 6.89 (ddd, 1H, J = 2.4, 8.4, 3JH-F = 9.2 Hz, aromatic), 7.48 (dd, 1H, J = 8.4, 4JH-F = 5.4 Hz, aromatic).
HRMS (FAB+) m/z 238.0697 ((M+H)+, C12H13FNOS requires 238.0702).[Production Example 003]
Synthesis of 1- (3-ethyl-5-fluoro-2,3-dihydrobenzothiazol-2-ylidene) propan-2-one

The compound was prepared in the same manner as in [Production Example 001] except that 5-fluoro-2-methylbenzothiazole was used instead of 5-methoxy-2-methylbenzothiazole, and 1- (3-ethyl-5 -Fluoro-2,3-dihydrobenzothiazol-2-ylidene) propan-2-one (the compound of Production Example 003) was obtained.
1 H NMR (CDCl 3 , 400 MHz) δ 1.38 (t, 3H, J = 7.3 Hz, CH 3 ), 2.53 (s, 3H, CH 3 ), 4.01 (q, 2H, J = 7.3 Hz, CH 2 ) , 5.90 (s, 1H, olefinic), 6.81 (dd, 1H, J = 2.4, 3 J HF = 9.2 Hz, aromatic), 6.89 (ddd, 1H, J = 2.4, 8.4, 3 J HF = 9.2 Hz, aromatic ), 7.48 (dd, 1H, J = 8.4, 4 J HF = 5.4 Hz, aromatic).
HRMS (FAB + ) m / z 238.0697 ((M + H) + , C 12 H 13 FNOS requires 238.0702).

［製造例００４］
１−（５−エトキシ−３−エチル−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オンの合成

市販の５−ヒドロキシ−２−メチルベンゾチアゾール（２００ｍｇ、１．２１ｍｍｏｌ）を１．５ｍｌのアセトンに溶解させた後、炭酸カリウム（１６９ｍｇ、１．２２ｍｍｏｌ）およびヨウ化エチル（３００μｌ、３．７４ｍｍｏｌ）を加え、２日間還流させた。続いて減圧濃縮により溶媒を除去した後、水（１０ｍｌ）を添加し、その混合物を１０ｍｌのエチルアセテートで３回抽出した。１つにまとめた有機抽出液をブライン（２０ｍｌ）で洗浄し、Ｎａ２ＳＯ４上で乾燥し、ろ過し、減圧濃縮した。その残留物をシリカゲルカラムクロマトグラフィー（２０ｇ、ｎ−ヘキサン／エチルアセテート、２：１）を用いて精製し、褐色油状物の５−エトキシ−２−メチルベンゾチアゾールを得た（２１６ｍｇ、１．１１ｍｍｏｌ、収率９２．３％）。５−メトキシ−２−メチルベンゾチアゾールにかえて５−エトキシ−２−メチルベンゾチアゾールを使用したほかは、［製造例００１］と同様に製造し、淡黄色固体の１−（５−エトキシ−３−エチル−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オン（製造例００４の化合物）を得た。
1H NMR (CDCl3, 400 MHz) δ 1.37 (t, 3H, J = 7.4 Hz, CH3), 1.44 (t, 3H, J = 7.0 Hz, CH3), 2.23 (s, 3H, CH3), 4.01 (q, 2H, J = 7.4 Hz, CH2), 4.08 (q, 2H, J = 7.0 Hz, CH2), 5.86 (s, 1H, olefinic), 6.65 (d, 1H, J = 2.3 Hz, aromatic), 6.74 (dd, 1H, J = 2.3, 8.5 Hz, aromatic), 7.43 (d, 1H, J = 8.5 Hz, aromatic)
HRMS (FAB+) m/z 264.1051 ((M+H)+, C14H18NO3S requires 264.1058).[Production Example 004]
Synthesis of 1- (5-ethoxy-3-ethyl-2,3-dihydrobenzothiazol-2-ylidene) propan-2-one

Commercially available 5-hydroxy-2-methylbenzothiazole (200 mg, 1.21 mmol) was dissolved in 1.5 ml of acetone followed by potassium carbonate (169 mg, 1.22 mmol) and ethyl iodide (300 μl, 3.74 mmol). Was added and refluxed for 2 days. Subsequently, the solvent was removed by concentration under reduced pressure, water (10 ml) was added, and the mixture was extracted three times with 10 ml of ethyl acetate. The combined organic extract was washed with brine (20 ml), dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified using silica gel column chromatography (20 g, n-hexane / ethyl acetate, 2: 1) to give 5-ethoxy-2-methylbenzothiazole as a brown oil (216 mg, 1.11 mmol). Yield 92.3%). This was prepared in the same manner as in [Preparation Example 001] except that 5-ethoxy-2-methylbenzothiazole was used instead of 5-methoxy-2-methylbenzothiazole, and 1- (5-ethoxy-3 -Ethyl-2,3-dihydrobenzothiazol-2-ylidene) propan-2-one (the compound of Production Example 004) was obtained.
1 H NMR (CDCl 3 , 400 MHz) δ 1.37 (t, 3H, J = 7.4 Hz, CH 3 ), 1.44 (t, 3H, J = 7.0 Hz, CH 3 ), 2.23 (s, 3H, CH 3 ) , 4.01 (q, 2H, J = 7.4 Hz, CH 2 ), 4.08 (q, 2H, J = 7.0 Hz, CH 2 ), 5.86 (s, 1H, olefinic), 6.65 (d, 1H, J = 2.3 Hz , aromatic), 6.74 (dd, 1H, J = 2.3, 8.5 Hz, aromatic), 7.43 (d, 1H, J = 8.5 Hz, aromatic)
HRMS (FAB + ) m / z 264.1051 ((M + H) + , C 14 H 18 NO 3 S requires 264.1058).

［製造例００５］
１−（３−ヘキシル−５−メトキシ−２，３−ジヒドロベンゾチアゾール−２−イリデン）ブタン−２−オンの合成

ヨウ化エチルにかえて臭化ヘキシル、および無水酢酸にかえて無水プロピオン酸を使用したほかは、［製造例００１］と同様に製造し、淡黄色固体の１−（３−ヘキシル−５−メトキシ−２，３−ジヒドロベンゾチアゾール−２−イリデン）ブタン−２−オン（製造例００５の化合物）を得た。
1H NMR (CDCl3, 400 MHz) δ 0.91 (t, 3H, J = 7.2 Hz, CH3), 1.21 (t, 3H, J = 7.5 Hz, CH3), 1.29-1.48 (m, 6H, CH2 x 3), 1.78 (m, 2H, J = 7.6 Hz, CH2), 2.50 (q, 2H,J = 7.5 Hz, CH2), 3.86 (s, 3H, OCH3), 3.94 (t, 2H, J = 7.6 Hz, CH2), 5.86 (s, 1H, olefinic), 6.64 (d, 1H, J = 2.3 Hz, aromatic), 6.75 (dd, 1H, J = 2.3, 8.6 Hz,aromatic), 8.55 (d, 1H, J = 8.6 Hz, aromatic).
HRMS (FAB+) m/z 320.1683 ((M+H)+, C18H25NO3S requires 320.1684).[Production Example 005]
Synthesis of 1- (3-hexyl-5-methoxy-2,3-dihydrobenzothiazol-2-ylidene) butan-2-one

Prepared in the same manner as in [Preparation Example 001] except that hexyl bromide was used instead of ethyl iodide, and propionic anhydride was used instead of acetic anhydride, and 1- (3-hexyl-5-methoxy of pale yellow solid was obtained. -2,3-Dihydrobenzothiazol-2-ylidene) butan-2-one (Compound of Production Example 005) was obtained.
1 H NMR (CDCl 3 , 400 MHz) δ 0.91 (t, 3H, J = 7.2 Hz, CH 3 ), 1.21 (t, 3H, J = 7.5 Hz, CH 3 ), 1.29-1.48 (m, 6H, CH 2 x 3), 1.78 (m, 2H, J = 7.6 Hz, CH 2 ), 2.50 (q, 2H, J = 7.5 Hz, CH 2 ), 3.86 (s, 3H, OCH 3 ), 3.94 (t, 2H , J = 7.6 Hz, CH 2 ), 5.86 (s, 1H, olefinic), 6.64 (d, 1H, J = 2.3 Hz, aromatic), 6.75 (dd, 1H, J = 2.3, 8.6 Hz, aromatic), 8.55 (d, 1H, J = 8.6 Hz, aromatic).
HRMS (FAB + ) m / z 320.1683 ((M + H) + , C 18 H 25 NO 3 S requires 320.1684).

［製造例００６］
１−（３−エチル−５−メトキシ−２，３−ジヒドロベンゾチアゾール−２−イリデン）−３，３，３−トリフルオロプロパン−２−オンの合成

無水酢酸にかえて無水トリフルオロ酢酸を使用したほかは、［製造例００１］と同様に製造し、白色固体の１−（３−エチル−５−メトキシ−２，３−ジヒドロベンゾチアゾール−２−イリデン）−３，３，３−トリフルオロプロパン−２−オン（製造例００６の化合物）を得た。
1H NMR (CDCl3, 400 MHz) δ 1.46 (t, 3H, J = 7.3 Hz, CH3), 3.91 (s, 3H, OCH3), 4.20 (q, 2H, J = 7.3 Hz, CH2), 6.17 (s, 1H, olefinic), 6.82 (d, 1H, J = 2.1 Hz, aromatic), 6.93 (dd, 1H, J = 2.1, 8.5 Hz, aromatic), 7.60 (d, 1H, J = 8.5 Hz, aromatic).
HRMS (FAB+) m/z 304.0618 ((M+H)+, C13H13F3NO2S requires 304.0619).[Production Example 006]
Synthesis of 1- (3-ethyl-5-methoxy-2,3-dihydrobenzothiazol-2-ylidene) -3,3,3-trifluoropropan-2-one

This was prepared in the same manner as in [Preparation Example 001] except that trifluoroacetic anhydride was used instead of acetic anhydride, and 1- (3-ethyl-5-methoxy-2,3-dihydrobenzothiazole-2- Iridene) -3,3,3-trifluoropropan-2-one (Compound of Production Example 006) was obtained.
1 H NMR (CDCl 3 , 400 MHz) δ 1.46 (t, 3H, J = 7.3 Hz, CH 3 ), 3.91 (s, 3H, OCH 3 ), 4.20 (q, 2H, J = 7.3 Hz, CH 2 ) , 6.17 (s, 1H, olefinic), 6.82 (d, 1H, J = 2.1 Hz, aromatic), 6.93 (dd, 1H, J = 2.1, 8.5 Hz, aromatic), 7.60 (d, 1H, J = 8.5 Hz , aromatic).
HRMS (FAB + ) m / z 304.0618 ((M + H) + , C 13 H 13 F 3 NO 2 S requires 304.0619).

［製造例００７］
１−（５−メトキシ−３−メチル−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オンの合成

ヨウ化エチルにかえてヨウ化メチルを使用したほかは、［製造例００１］と同様に製造し、淡黄色固体の１−（５−メトキシ−３−メチル−２，３−ジヒドロベンゾチアゾール−２−イリデン）−プロパン−２−オン（製造例００７の化合物）を得た。
1H NMR (CDCl3, 400 MHz) δ 2.24 (s, 3H, CH3), 3.52 (s, 3H, CH3), 3.87 (s, 3H, OCH3), 5.86 (s, 1H, olefinic), 6.66 (d, 1H, J = 2.1 Hz, aromatic), 6.75 (dd, 1H, J= 2.1, 8.5 Hz, aromatic), 7.44 (d, 1H, J = 8.5 Hz, aromatic).
HRMS (FAB+) m/z 236.0746 ((M+H)+, C12H14NO2S requires 236.0745).[Production Example 007]
Synthesis of 1- (5-methoxy-3-methyl-2,3-dihydrobenzothiazol-2-ylidene) propan-2-one

1- (5-Methoxy-3-methyl-2,3-dihydrobenzothiazole-2 was prepared in the same manner as in [Production Example 001] except that methyl iodide was used instead of ethyl iodide. -Iridene) -propan-2-one (the compound of Production Example 007) was obtained.
1 H NMR (CDCl 3 , 400 MHz) δ 2.24 (s, 3H, CH 3 ), 3.52 (s, 3H, CH 3 ), 3.87 (s, 3H, OCH 3 ), 5.86 (s, 1H, olefinic), 6.66 (d, 1H, J = 2.1 Hz, aromatic), 6.75 (dd, 1H, J = 2.1, 8.5 Hz, aromatic), 7.44 (d, 1H, J = 8.5 Hz, aromatic).
HRMS (FAB + ) m / z 236.0746 ((M + H) + , C 12 H 14 NO 2 S requires 236.0745).

［製造例００８］
１−（５−フルオロ−３−メチル−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オンの合成

５−メトキシ−２−メチルベンゾチアゾールにかえて５−フルオロ−２−メチルベンゾチアゾール、およびヨウ化エチルにかえてヨウ化メチル使用したほかは、［製造例００１］と同様に製造し、無色固体の１−（５−フルオロ−３−メチル−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オン（製造例００８の化合物）を得た。
1H NMR (CDCl3, 400 MHz) δ 2.23 (s, 3H, CH3), 3.52 (s, 3H, CH3), 5.89 (s, 1H, olefinic), 6.84 (dd, 1H, J = 2.3, 3JH-F = 9.6 Hz, aromatic), 6.89 (ddd, 1H, J = 2.3, 8.6, 3JH-F = 8.9 Hz, aromatic), 7.48 (dd, 1H, J = 8.6, 4JH-F = 5.2 Hz, aromatic).
HRMS (FAB+) m/z 224.0547 ((M+H)+, C11H11FNOS requires 224.0545).[Production Example 008]
Synthesis of 1- (5-fluoro-3-methyl-2,3-dihydrobenzothiazol-2-ylidene) propan-2-one

A colorless solid was prepared in the same manner as in [Production Example 001] except that 5-fluoro-2-methylbenzothiazole was used instead of 5-methoxy-2-methylbenzothiazole, and methyl iodide was used instead of ethyl iodide. Of 1- (5-fluoro-3-methyl-2,3-dihydrobenzothiazol-2-ylidene) propan-2-one (Compound of Production Example 008) was obtained.
1 H NMR (CDCl 3 , 400 MHz) δ 2.23 (s, 3H, CH 3 ), 3.52 (s, 3H, CH 3 ), 5.89 (s, 1H, olefinic), 6.84 (dd, 1H, J = 2.3, 3 J HF = 9.6 Hz, aromatic), 6.89 (ddd, 1H, J = 2.3, 8.6, 3 J HF = 8.9 Hz, aromatic), 7.48 (dd, 1H, J = 8.6, 4 J HF = 5.2 Hz, aromatic ).
HRMS (FAB + ) m / z 224.0547 ((M + H) + , C 11 H 11 FNOS requires 224.0545).

［製造例００９］
１−（３−エチル−５−ヒドロキシ−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オンの合成

アルゴン雰囲気下、８０ｍｌのＣＨ２Ｃｌ２中に１−（３−エチル−５−メトキシ−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オン（製造例００１の化合物）（５．４８ｇ、２２．０ｍｍｏｌ）を含むサスペンションに、三臭化ホウ素 １．０Ｍ ＣＨ２Ｃｌ２溶液（４４．０ｍｌ、４４．０ｍｍｏｌ）を０℃にて添加して、室温にて２３時間撹拌した。その後、混合物へ飽和炭酸水素ナトリウム溶液（１００ｍｌ）を添加し、減圧濃縮によりＣＨ２Ｃｌ２を除去した。漏斗を用いて沈殿物を濾しとり、エチルアセテートで洗浄後、減圧乾燥した後、エタノール（５５０ｍｌ）を用いて再結晶により精製し、乳白色固体の１−（３−エチル−５−ヒドロキシ−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オン（製造例００９の化合物）を得た（３．２１ｇ、１３．６ｍｍｏｌ、収率６２．１％）。
1H NMR (CDCl3, 400 MHz) δ 1.37 (t, 3H, J = 6.9 Hz, CH3), 2.07 (s, 3H, CH3), 4.06 (q, 2H, J = 6.9 Hz, CH2), 6.05 (s, 1H, olefinic), 6.63 (d, 1H, J = 8.4 Hz, aromatic), 6.77 (br s, 1H, aromatic), 7.46 (d, 1H, J = 8.4 Hz, aromatic), 9.72 (s, 1H, OH).
HRMS (FAB+) m/z 236.0744 ((M+H)+, C12H14NO2S requires 236.0745).[Production Example 009]
Synthesis of 1- (3-ethyl-5-hydroxy-2,3-dihydrobenzothiazol-2-ylidene) propan-2-one

1- (3-Ethyl-5-methoxy-2,3-dihydrobenzothiazol-2-ylidene) propan-2-one (compound of Preparation Example 001) in 80 ml of CH 2 Cl 2 under an argon atmosphere (5. 48 g, 22.0 mmol) was added boron tribromide 1.0M CH 2 Cl 2 solution (44.0 ml, 44.0 mmol) at 0 ° C. and stirred at room temperature for 23 hours. Thereafter, saturated sodium hydrogen carbonate solution (100 ml) was added to the mixture, and CH 2 Cl 2 was removed by concentration under reduced pressure. The precipitate was filtered using a funnel, washed with ethyl acetate, dried under reduced pressure, purified by recrystallization using ethanol (550 ml), 1- (3-ethyl-5-hydroxy-2, 3-Dihydrobenzothiazol-2-ylidene) propan-2-one (Compound of Preparation Example 009) was obtained (3.21 g, 13.6 mmol, yield 62.1%).
1 H NMR (CDCl 3 , 400 MHz) δ 1.37 (t, 3H, J = 6.9 Hz, CH 3 ), 2.07 (s, 3H, CH 3 ), 4.06 (q, 2H, J = 6.9 Hz, CH 2 ) , 6.05 (s, 1H, olefinic), 6.63 (d, 1H, J = 8.4 Hz, aromatic), 6.77 (br s, 1H, aromatic), 7.46 (d, 1H, J = 8.4 Hz, aromatic), 9.72 ( s, 1H, OH).
HRMS (FAB + ) m / z 236.0744 ((M + H) + , C 12 H 14 NO 2 S requires 236.0745).

［製造例０１０］
１−（５−ブロモ−３−エチル−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オンの合成

５−メトキシ−２−メチルベンゾチアゾールにかえて５−ブロモ−２−メチルベンゾチアゾールを使用したほかは、［製造例００１］と同様に製造し、無色固体の１−（５−ブロモ−３−エチル−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オン（製造例０１０の化合物）を得た。
1H NMR (CDCl3, 400 MHz) δ 1.39 (t, 3H, J = 7.3 Hz, CH3), 2.26 (s, 3H, CH3), 4.02 (q, 2H, J = 7.3 Hz, CH2), 5.90 (s, 1H, olefinic), 7.22 (d, 1H, J = 1.7 Hz, aromatic), 7.27 (dd, 1H, J = 1.7, 8.2 Hz, aromatic), 7.43 (d, 1H, J = 8.2 Hz, aromatic).
HRMS (FAB+) m/z 297.9899 ((M+H)+, C12H13NOSBr requires 297.9901).[Production Example 010]
Synthesis of 1- (5-bromo-3-ethyl-2,3-dihydrobenzothiazol-2-ylidene) propan-2-one

This was prepared in the same manner as in [Preparation Example 001] except that 5-bromo-2-methylbenzothiazole was used instead of 5-methoxy-2-methylbenzothiazole, and colorless solid 1- (5-bromo-3- Ethyl-2,3-dihydrobenzothiazol-2-ylidene) propan-2-one (Compound of Production Example 010) was obtained.
1 H NMR (CDCl 3 , 400 MHz) δ 1.39 (t, 3H, J = 7.3 Hz, CH 3 ), 2.26 (s, 3H, CH 3 ), 4.02 (q, 2H, J = 7.3 Hz, CH 2 ) , 5.90 (s, 1H, olefinic), 7.22 (d, 1H, J = 1.7 Hz, aromatic), 7.27 (dd, 1H, J = 1.7, 8.2 Hz, aromatic), 7.43 (d, 1H, J = 8.2 Hz , aromatic).
HRMS (FAB + ) m / z 297.9899 ((M + H) + , C 12 H 13 NOSBr requires 297.9901).

［製造例０１１］
１−（５−クロロ−３−エチル−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オンの合成

５−メトキシ−２−メチルベンゾチアゾールにかえて５−クロロ−２−メチルベンゾチアゾールを使用したほかは、［製造例００１］と同様に製造し、無色固体の１−（５−クロロ−３−エチル−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オン（製造例０１１の化合物）を得た。
1H NMR (CDCl3, 400 MHz) δ 1.39 (t, 3H, J = 7.3 Hz, CH3), 2.26 (s, 3H, CH3), 4.03 (q, 2H, J = 7.3 Hz, CH2), 5.90 (s, 1H, olefinic), 7.08 (d, 1H, J = 1.8 Hz, aromatic), 7.13 (dd, 1H, J = 1.8, 8.2 Hz, aromatic), 7.48 (d, 1H, J = 8.2 Hz, aromatic).
HRMS (FAB+) m/z 254.0411 ((M+H)+, C12H13NOSCl requires 254.0406).[Production Example 011]
Synthesis of 1- (5-chloro-3-ethyl-2,3-dihydrobenzothiazol-2-ylidene) propan-2-one

This was prepared in the same manner as in [Preparation Example 001] except that 5-chloro-2-methylbenzothiazole was used instead of 5-methoxy-2-methylbenzothiazole, and colorless solid 1- (5-chloro-3- Ethyl-2,3-dihydrobenzothiazol-2-ylidene) propan-2-one (the compound of Production Example 011) was obtained.
1 H NMR (CDCl 3 , 400 MHz) δ 1.39 (t, 3H, J = 7.3 Hz, CH 3 ), 2.26 (s, 3H, CH 3 ), 4.03 (q, 2H, J = 7.3 Hz, CH 2 ) , 5.90 (s, 1H, olefinic), 7.08 (d, 1H, J = 1.8 Hz, aromatic), 7.13 (dd, 1H, J = 1.8, 8.2 Hz, aromatic), 7.48 (d, 1H, J = 8.2 Hz , aromatic).
HRMS (FAB + ) m / z 254.0411 ((M + H) + , C 12 H 13 NOSCl requires 254.0406).

［製造例０１２］
Ｎ−［３−エチル−２−（２−オキソプロピリデン）−２，３−ジヒドロベンゾチアゾール−５−イル］アセトアミドの合成

５−ヒドロキシ−２−メチルベンゾチアゾールにかえて５−アミノ−２−メチルベンゾチアゾール２塩酸塩を使用したほかは、後述する［製造例０１９−Ｂ］と同様に製造し、無色固体のＮ−［３−エチル−２−（２−オキソプロピリデン）−２，３−ジヒドロベンゾチアゾール−５−イル］アセトアミド（製造例０１２の化合物）を得た。
1H NMR (CDCl3, 400 MHz) δ 1.37 (t, 3H, J = 7.3 Hz, CH3), 2.23 (s, 3H, CH3), 2.25 (s, 3H, CH3), 4.06 (q, 2H, J = 7.3 Hz, CH2), 5.89 (s, 1H, olefinic), 6.90 (d, 1H, J = 8.3 Hz, aromatic), 7.45 (d, 1H, J = 8.3 Hz, aromatic), 7.66 (s, 1H, NH), 7.96 (s, 1H, aromatic).
HRMS (FAB+) m/z 277.1013 ((M+H)+, C14H17N2O2S requires 277.1011).[Production Example 012]
Synthesis of N- [3-ethyl-2- (2-oxopropylidene) -2,3-dihydrobenzothiazol-5-yl] acetamide

This was prepared in the same manner as in [Production Example 019-B] to be described later, except that 5-amino-2-methylbenzothiazole dihydrochloride was used instead of 5-hydroxy-2-methylbenzothiazole. [3-Ethyl-2- (2-oxopropylidene) -2,3-dihydrobenzothiazol-5-yl] acetamide (Compound of Production Example 012) was obtained.
1 H NMR (CDCl 3 , 400 MHz) δ 1.37 (t, 3H, J = 7.3 Hz, CH 3 ), 2.23 (s, 3H, CH 3 ), 2.25 (s, 3H, CH 3 ), 4.06 (q, 2H, J = 7.3 Hz, CH 2 ), 5.89 (s, 1H, olefinic), 6.90 (d, 1H, J = 8.3 Hz, aromatic), 7.45 (d, 1H, J = 8.3 Hz, aromatic), 7.66 ( s, 1H, NH), 7.96 (s, 1H, aromatic).
HRMS (FAB + ) m / z 277.1013 ((M + H) + , C 14 H 17 N 2 O 2 S requires 277.1011).

［製造例０１３］
１−（５−メトキシ−３−プロピル−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オンの合成

ヨウ化エチルにかえてヨウ化プロピルを使用したほかは、［製造例００１］と同様に製造し、無色固体の１−（５−メトキシ−３−プロピル−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オン（製造例０１３の化合物）を得た。
1H NMR (CDCl3, 400 MHz) δ 1.05 (t, 3H, J = 7.5 Hz, CH3), 1.83 (m, 2H, J = 7.5 Hz, CH2), 2.24 (s, 3H, CH3), 3.86 (s, 3H, OCH3), 3.91 (q, 2H, J = 7.5 Hz, CH2), 5.86 (s, 1H, olefinic), 6.65 (d, 1H, J = 2.3 Hz, aromatic), 6.75 (dd, 1H, J = 2.3, 8.5 Hz, aromatic), 7.45 (d, 1H, J = 8.5 Hz, aromatic).
HRMS (FAB+) m/z 264.1065 ((M+H)+, C14H18NO2S requires 264.1058).[Production Example 013]
Synthesis of 1- (5-methoxy-3-propyl-2,3-dihydrobenzothiazol-2-ylidene) propan-2-one

This was prepared in the same manner as in [Production Example 001] except that propyl iodide was used instead of ethyl iodide, and colorless solid 1- (5-methoxy-3-propyl-2,3-dihydrobenzothiazole-2- Iridene) propan-2-one (the compound of Production Example 013) was obtained.
1 H NMR (CDCl 3 , 400 MHz) δ 1.05 (t, 3H, J = 7.5 Hz, CH 3 ), 1.83 (m, 2H, J = 7.5 Hz, CH 2 ), 2.24 (s, 3H, CH 3 ) , 3.86 (s, 3H, OCH 3 ), 3.91 (q, 2H, J = 7.5 Hz, CH 2 ), 5.86 (s, 1H, olefinic), 6.65 (d, 1H, J = 2.3 Hz, aromatic), 6.75 (dd, 1H, J = 2.3, 8.5 Hz, aromatic), 7.45 (d, 1H, J = 8.5 Hz, aromatic).
HRMS (FAB + ) m / z 264.1065 ((M + H) + , C 14 H 18 NO 2 S requires 264.1058).

［製造例０１４］
１−［３−（２−プロペニル）−５−メトキシ−２，３−ジヒドロベンゾチアゾール−２−イリデン］プロパン−２−オンの合成

ヨウ化エチルにかえて臭化アリルを使用したほかは、［製造例００１］と同様に製造し、淡黄色固体の１−［３−（２−プロペニル）−５−メトキシ−２，３−ジヒドロベンゾチアゾール−２−イリデン］プロパン−２−オン（製造例０１４の化合物）を得た。
1H NMR (CDCl3, 400 MHz) δ 2.22 (s, 3H, CH3), 3.85 (s, 3H, OCH3), 4.57 (ddd, 2H,J = 1.8, 2.1, 4.6 Hz, CH2), 5.13 (dd, 1H, J = 2.1, 17.2 Hz, olefinic), 5.29 (dd, 1H, J = 1.8, 10.4 Hz, olefinic), 5.89 (ddt, 1H, J = 4.6, 10.4, 17.2 Hz, olefinic), 6.60 (d, 1H, J = 2.1 Hz, aromatic), 6.76 (dd, 1H, J = 2.1, 8.6 Hz, aromatic), 7.45 (d, 1H, J = 8.6 Hz, aromatic).
HRMS (FAB+) m/z 262.0902 ((M+H)+, C14H16NO2S requires 262.0902).[Production Example 014]
Synthesis of 1- [3- (2-propenyl) -5-methoxy-2,3-dihydrobenzothiazol-2-ylidene] propan-2-one

1- [3- (2-propenyl) -5-methoxy-2,3-dihydro was prepared in the same manner as in [Production Example 001] except that allyl bromide was used instead of ethyl iodide. Benzothiazol-2-ylidene] propan-2-one (Compound of Production Example 014) was obtained.
1 H NMR (CDCl 3 , 400 MHz) δ 2.22 (s, 3H, CH 3 ), 3.85 (s, 3H, OCH 3 ), 4.57 (ddd, 2H, J = 1.8, 2.1, 4.6 Hz, CH 2 ), 5.13 (dd, 1H, J = 2.1, 17.2 Hz, olefinic), 5.29 (dd, 1H, J = 1.8, 10.4 Hz, olefinic), 5.89 (ddt, 1H, J = 4.6, 10.4, 17.2 Hz, olefinic), 6.60 (d, 1H, J = 2.1 Hz, aromatic), 6.76 (dd, 1H, J = 2.1, 8.6 Hz, aromatic), 7.45 (d, 1H, J = 8.6 Hz, aromatic).
HRMS (FAB + ) m / z 262.0902 ((M + H) + , C 14 H 16 NO 2 S requires 262.0902).

［製造例０１５］
１−（３−エチル−５−メトキシ−２，３−ジヒドロベンゾセレナゾール−２−イリデン）プロパン−２−オンの合成

５−メトキシ−２−メチルベンゾチアゾールにかえて５−メトキシ−２−メチルベンゾセレナゾールを使用したほかは、［製造例００１］と同様に製造し、淡黄色固体の１−（３−エチル−５−メトキシ−２，３−ジヒドロベンゾセレナゾール−２−イリデン）プロパン−２−オン（製造例０１５の化合物）を得た。
1H NMR (CDCl3, 400 MHz) δ 1.39 (t, 3H, J = 7.2 Hz, CH3), 2.27 (s, 3H, CH3), 3.87 (s, 3H), 4.08 (q, 2H, J = 7.2 Hz, CH2), 6.19 (s, 1H, olefinic), 6.70 (d, 1H, J= 2.3 Hz, aromatic), 6.76 (dd, 1H, J = 2.3, 8.4 Hz, aromatic), 7.54 (d, 1H, J =8.4 Hz, aromatic).
HRMS (FAB+) m/z 298.0347 ((M+H)+, C13H16NO2Se requires 298.0346).[Production Example 015]
Synthesis of 1- (3-ethyl-5-methoxy-2,3-dihydrobenzoselenazol-2-ylidene) propan-2-one

This was prepared in the same manner as in [Preparation Example 001] except that 5-methoxy-2-methylbenzoselenazole was used instead of 5-methoxy-2-methylbenzothiazole, and 1- (3-ethyl- 5-Methoxy-2,3-dihydrobenzoselenazol-2-ylidene) propan-2-one (the compound of Production Example 015) was obtained.
1 H NMR (CDCl 3 , 400 MHz) δ 1.39 (t, 3H, J = 7.2 Hz, CH 3 ), 2.27 (s, 3H, CH 3 ), 3.87 (s, 3H), 4.08 (q, 2H, J = 7.2 Hz, CH 2 ), 6.19 (s, 1H, olefinic), 6.70 (d, 1H, J = 2.3 Hz, aromatic), 6.76 (dd, 1H, J = 2.3, 8.4 Hz, aromatic), 7.54 (d , 1H, J = 8.4 Hz, aromatic).
HRMS (FAB + ) m / z 298.0347 ((M + H) + , C 13 H 16 NO 2 Se requires 298.0346).

［製造例０１６］
２−［５−メトキシ−２−（２−オキソプロピリデン）−２，３−ジヒドロベンゾチアゾール−３−イル］エチルアセテートの合成

ヨウ化エチルにかえて２−ブロモエタノールを使用したほかは、［製造例００１］と同様に製造し、黄土色固体の２−［５−メトキシ−２−（２−オキソプロピリデン）−２，３−ジヒドロベンゾチアゾール−３−イル］エチルアセテート（製造例０１６の化合物）を得た。
1H NMR (DMSO-d6, 400 MHz) δ 1.87 (s, 3H, CH3), 2.09 (s, 3H, CH3), 3.81 (s, 3H, OCH3), 4.35 (d, 2H, J = 4.3 Hz, CH2), 4.39 (d, 2H, J = 4.3 Hz, CH2), 6.14 (s, 1H, olefinic), 6.78 (dd, 1H, J = 2.0, 8.6 Hz, aromatic), 7.04 (d, 1H, J = 2.0 Hz, aromatic), 7.58 (d, 1H, J = 8.6 Hz, aromatic).
HRMS (FAB+) m/z 308.0964 ((M+H)+, C15H18NO4S requires 308.0957).[Production Example 016]
Synthesis of 2- [5-methoxy-2- (2-oxopropylidene) -2,3-dihydrobenzothiazol-3-yl] ethyl acetate

This was prepared in the same manner as in [Preparation Example 001] except that 2-bromoethanol was used instead of ethyl iodide, and an ocherous solid 2- [5-methoxy-2- (2-oxopropylidene) -2, 3-Dihydrobenzothiazol-3-yl] ethyl acetate (the compound of Production Example 016) was obtained.
1 H NMR (DMSO-d 6 , 400 MHz) δ 1.87 (s, 3H, CH 3 ), 2.09 (s, 3H, CH 3 ), 3.81 (s, 3H, OCH 3 ), 4.35 (d, 2H, J = 4.3 Hz, CH 2 ), 4.39 (d, 2H, J = 4.3 Hz, CH 2 ), 6.14 (s, 1H, olefinic), 6.78 (dd, 1H, J = 2.0, 8.6 Hz, aromatic), 7.04 ( d, 1H, J = 2.0 Hz, aromatic), 7.58 (d, 1H, J = 8.6 Hz, aromatic).
HRMS (FAB + ) m / z 308.0964 ((M + H) + , C 15 H 18 NO 4 S requires 308.0957).

［製造例０１７］
１−［３−（２−ヒドロキシエチル）−５−ヒドロキシ−２，３−ジヒドロベンゾチアゾール−２−イリデン］プロパン−２−オンの合成

１−（３−エチル−５−メトキシ−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オン（製造例００１の化合物）にかえて２−［５−メトキシ−２−（２−オキソプロピリデン）−２，３−ジヒドロベンゾチアゾール−３−イル］エチルアセテート（製造例０１６の化合物）を使用したほかは、［製造例００９］と同様に製造し、淡黄色固体の１−［３−（２−ヒドロキシエチル）−５−ヒドロキシ−２，３−ジヒドロベンゾチアゾール−２−イリデン］プロパン−２−オン（製造例０１７の化合物）を得た。
1H NMR (DMSO-d6, 400 MHz) δ 2.06 (s, 3H, CH3), 3.70 (q, 2H, J = 5.7 Hz, CH2), 4.07 (t, 2H, J = 5.7 Hz, CH2), 4.97 (t, 1H, J = 5.7 Hz, OH), 6.05 (s, 1H, olefinic), 6.62 (dd, 1H, J = 2.0, 8.4 Hz, aromatic), 6.81 (d, 1H, J = 2.0 Hz, aromatic), 7.44 (d, 1H, J = 8.4 Hz, aromatic), 9.67 (s, 1H, OH).
HRMS (FAB+) m/z 252.0693 ((M+H)+, C12H14NO3S requires 252.0694).[Production Example 017]
Synthesis of 1- [3- (2-hydroxyethyl) -5-hydroxy-2,3-dihydrobenzothiazol-2-ylidene] propan-2-one

Instead of 1- (3-ethyl-5-methoxy-2,3-dihydrobenzothiazol-2-ylidene) propan-2-one (the compound of Preparation Example 001), 2- [5-methoxy-2- (2- Oxopropylidene) -2,3-dihydrobenzothiazol-3-yl] ethyl acetate (Compound of Production Example 016) was used in the same manner as in [Production Example 009] to produce a pale yellow solid of 1- [ 3- (2-Hydroxyethyl) -5-hydroxy-2,3-dihydrobenzothiazol-2-ylidene] propan-2-one (the compound of Production Example 017) was obtained.
1 H NMR (DMSO-d 6 , 400 MHz) δ 2.06 (s, 3H, CH 3 ), 3.70 (q, 2H, J = 5.7 Hz, CH 2 ), 4.07 (t, 2H, J = 5.7 Hz, CH 2 ), 4.97 (t, 1H, J = 5.7 Hz, OH), 6.05 (s, 1H, olefinic), 6.62 (dd, 1H, J = 2.0, 8.4 Hz, aromatic), 6.81 (d, 1H, J = 2.0 Hz, aromatic), 7.44 (d, 1H, J = 8.4 Hz, aromatic), 9.67 (s, 1H, OH).
HRMS (FAB + ) m / z 252.0693 ((M + H) + , C 12 H 14 NO 3 S requires 252.0694).

［製造例０１８］
エチル ２−［５−メトキシ−２−（２−オキソプロピリデン）−２，３−ジヒドロベンゾチアゾール−３−イル］アセテートの合成

ヨウ化エチルにかえてエチルブロモアセテートを使用したほかは、［製造例００１］と同様に製造し、黄土色固体のエチル２−［５−メトキシ−２−（２−オキソプロピリデン）−２，３−ジヒドロベンゾチアゾール−３−イル］アセテート（製造例０１８の化合物）を得た。
1H NMR (CDCl3, 400 MHz) δ 1.29 (t, 3H, J = 7.2 Hz), 2.23 (s, 3H, CH3), 3.84 (s,3H, OCH3), 4.27 (q, 2H, J = 7.2 Hz, CH2), 4.64 (s, 2H, CH2), 5.78 (s, 1H, olefinic), 6.55 (d, 1H, J = 2.2 Hz, aromatic), 6.77 (dd, 1H, J = 2.2, 8.5 Hz, aromatic), 7.45 (d, 1H, J = 8.5 Hz, aromatic).
HRMS (FAB+) m/z 308.0960 ((M+H)+, C15H18NO4S requires 308.0957).[Production Example 018]
Synthesis of ethyl 2- [5-methoxy-2- (2-oxopropylidene) -2,3-dihydrobenzothiazol-3-yl] acetate

Except that ethyl bromoacetate was used instead of ethyl iodide, it was prepared in the same manner as in [Preparation Example 001], and an ocherous solid ethyl 2- [5-methoxy-2- (2-oxopropylidene) -2, 3-Dihydrobenzothiazol-3-yl] acetate (the compound of Production Example 018) was obtained.
1 H NMR (CDCl 3 , 400 MHz) δ 1.29 (t, 3H, J = 7.2 Hz), 2.23 (s, 3H, CH 3 ), 3.84 (s, 3H, OCH 3 ), 4.27 (q, 2H, J = 7.2 Hz, CH 2 ), 4.64 (s, 2H, CH 2 ), 5.78 (s, 1H, olefinic), 6.55 (d, 1H, J = 2.2 Hz, aromatic), 6.77 (dd, 1H, J = 2.2 , 8.5 Hz, aromatic), 7.45 (d, 1H, J = 8.5 Hz, aromatic).
HRMS (FAB + ) m / z 308.0960 ((M + H) + , C 15 H 18 NO 4 S requires 308.0957).

［製造例０１９−Ａ］
１−（５−アセトキシ−３−エチル−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オンの合成

５ｍｌのピリジン中に１−（３−エチル−５−ヒドロキシ−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オン（製造例００９の化合物）（２３５ｍｇ、９９９μｍｏｌ）を含むサスペンションに、４−ジメチルアミノピリジン（７．０ｍｇ、５７μｍｏｌ）及び無水酢酸（１９０μｌ、２．０１ｍｍｏｌ）を０℃で連続的に添加して、０℃にて３０分間撹拌した。その後、混合物を常温にした後に水を加え、エチルアセテートで３回抽出した。１つにまとめた有機抽出液を蒸留水にて３回、引き続きブラインで１回洗浄し、Ｎａ２ＳＯ４上で乾燥し、ろ過し、減圧濃縮した。その残留物をシリカゲルカラムクロマトグラフィー（３０ｇ、ＣＨ２Ｃｌ２／エチルアセテート、４：１）を用いて精製し、無色固体の１−（５−アセトキシ−３−エチル−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オン（製造例０１９の化合物）を得た（２７４ｍｇ、９８８μｍｏｌ、収率９８．９％）。
1H NMR (CDCl3, 400 MHz) δ 1.37 (t, 3H, J = 7.4 Hz, CH3), 2.24 (s, 3H, CH3), 2.33 (s, 3H, CH3), 4.01 (q, 2H, J = 7.4 Hz, CH2), 5.88 (s, 1H, olefinic), 6.87-6.90 (m, 2H, aromatic), 7.53 (d, 1H, J = 8.4 Hz, aromatic)
HRMS (FAB+) m/z 278.0846 ((M+H)+, C14H16NO3S requires 278.0851).[Production Example 019-A]
Synthesis of 1- (5-acetoxy-3-ethyl-2,3-dihydrobenzothiazol-2-ylidene) propan-2-one

In a suspension containing 1- (3-ethyl-5-hydroxy-2,3-dihydrobenzothiazol-2-ylidene) propan-2-one (compound of Preparation 009) (235 mg, 999 μmol) in 5 ml of pyridine, 4-Dimethylaminopyridine (7.0 mg, 57 μmol) and acetic anhydride (190 μl, 2.01 mmol) were successively added at 0 ° C., and the mixture was stirred at 0 ° C. for 30 minutes. Thereafter, the mixture was brought to room temperature, water was added, and the mixture was extracted three times with ethyl acetate. The combined organic extracts were washed three times with distilled water and then once with brine, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified using silica gel column chromatography (30 g, CH 2 Cl 2 / ethyl acetate, 4: 1) to give colorless solid 1- (5-acetoxy-3-ethyl-2,3-dihydrobenzothiazole. -2-ylidene) propan-2-one (the compound of Preparation Example 019) was obtained (274 mg, 988 μmol, yield 98.9%).
1 H NMR (CDCl 3 , 400 MHz) δ 1.37 (t, 3H, J = 7.4 Hz, CH 3 ), 2.24 (s, 3H, CH 3 ), 2.33 (s, 3H, CH 3 ), 4.01 (q, 2H, J = 7.4 Hz, CH 2 ), 5.88 (s, 1H, olefinic), 6.87-6.90 (m, 2H, aromatic), 7.53 (d, 1H, J = 8.4 Hz, aromatic)
HRMS (FAB + ) m / z 278.0846 ((M + H) + , C 14 H 16 NO 3 S requires 278.0851).

［製造例０１９−Ｂ］
１−（５−アセトキシ−３−エチル−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オンの合成
３．０ｍｌのピリジン中に市販の５−ヒドロキシ−２−メチルベンゾチアゾール（１．００ｇ、６．０５ｍｍｏｌ）を溶解し、無水酢酸（１．５０ｍｌ、１５．９ｍｍｏｌ）及び触媒量のＤＭＡＰを０℃で連続的に添加して、室温にて１６時間撹拌した。その後、混合物に２Ｎ塩酸（５．０ｍｌ）および水（２．５ｍｌ）を加え、エチルアセテートで３回抽出した。一つにまとめた有機抽出液をブラインで１回洗浄し、Ｎａ２ＳＯ４上で乾燥し、ろ過し、減圧濃縮した。その残留物をエチルアセテートを用いて再結晶により精製し、淡赤色結晶の５−アセトキシ−２−メチルベンゾチアゾールを得た（６２１ｍｇ、２．９９ｍｍｏｌ、収率４９．５％）。
1H NMR (CDCl3, 400 MHz) δ 2.36 (s, 3H, CH3), 2.85 (s, 3H, CH3), 7.12 (dd, 1H, J= 2.3, 8.5 Hz, aromatic), 7.67 (d, 1H, J = 2.3 Hz, aromatic), 7.80 (d, 1H, J = 8.5 Hz, aromatic).
５−メトキシ−２−メチルベンゾチアゾールにかえて上記５−アセトキシ−２−メチルベンゾチアゾールを使用したほかは、［製造例００１］と同様に製造し、無色固体の１−（５−アセトキシ−３−エチル−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オン（製造例０１９の化合物）を得た。[Production Example 019-B]
Synthesis of 1- (5-acetoxy-3-ethyl-2,3-dihydrobenzothiazol-2-ylidene) propan-2-one Commercially available 5-hydroxy-2-methylbenzothiazole (1 0.000 g, 6.05 mmol) was dissolved, acetic anhydride (1.50 ml, 15.9 mmol) and a catalytic amount of DMAP were added successively at 0 ° C. and stirred at room temperature for 16 hours. Thereafter, 2N hydrochloric acid (5.0 ml) and water (2.5 ml) were added to the mixture, and the mixture was extracted three times with ethyl acetate. The combined organic extracts were washed once with brine, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified by recrystallization using ethyl acetate to obtain 5-acetoxy-2-methylbenzothiazole as light red crystals (621 mg, 2.99 mmol, yield 49.5%).
1 H NMR (CDCl 3 , 400 MHz) δ 2.36 (s, 3H, CH 3 ), 2.85 (s, 3H, CH 3 ), 7.12 (dd, 1H, J = 2.3, 8.5 Hz, aromatic), 7.67 (d , 1H, J = 2.3 Hz, aromatic), 7.80 (d, 1H, J = 8.5 Hz, aromatic).
A colorless solid 1- (5-acetoxy-3) was prepared in the same manner as in [Preparation Example 001] except that 5-acetoxy-2-methylbenzothiazole was used instead of 5-methoxy-2-methylbenzothiazole. -Ethyl-2,3-dihydrobenzothiazol-2-ylidene) propan-2-one (Compound of Production Example 019) was obtained.

［製造例０２０］
２−［５−アセトキシ−２−（２−オキソプロピリデン）−２，３−ジヒドロベンゾチアゾール−３−イル］エチルアセテートの合成

１．５ｍｌのピリジン中に１−［３−（２−ヒドロキシエチル）−５−ヒドロキシ−２，３−ジヒドロベンゾチアゾール−２−イリデン］プロパン−２−オン（製造例０１７の化合物）（８５．３ｍｇ、３３９μｍｏｌ）を含むサスペンションに、４−ジメチルアミノピリジン（２．０ｍｇ、１６μｍｏｌ）及び無水酢酸（７１．０μｌ、７５１μｍｏｌ）を０℃で連続的に添加して、室温にて２時間撹拌した。その後、混合物に水を加え、エチルアセテートで３回抽出した。１つにまとめた有機抽出液を水にて１回、引き続きブラインで１回洗浄し、Ｎａ２ＳＯ４上で乾燥し、ろ過し、減圧濃縮した。その残留物をシリカゲルカラムクロマトグラフィー（１０ｇ、ＣＨ２Ｃｌ２／エチルアセテート、４：１）を用いて精製し、淡黄色固体の２−［５−アセトキシ−２−（２−オキソプロピリデン）−２，３−ジヒドロベンゾチアゾール−３−イル］エチルアセテート（製造例０２０の化合物）を得た（１０４ｍｇ、３１０μｍｏｌ、収率９１．４％）。
1H NMR (CDCl3, 400 MHz) δ 2.02 (s, 3H, CH3), 2.26 (s, 3H, CH3), 2.34 (s, 3H, CH3), 4.24 (t, 2H, J = 6.0 Hz, CH2), 4.42 (t, 2H, J = 6.0 Hz, CH3), 5.93 (s, 1H, olefinic), 6.90 (dd, 1H, J = 2.0, 8.2 Hz, aromatic), 6.95 (d, 1H, J = 2.0 Hz, aromatic), 7.53 (d, 1H,), 7.96 (s, 1H, J = 8.2 Hz, aromatic).
HRMS (FAB+) m/z 336.0905 ((M+H)+, C16H18NO5S requires 336.0906).[Production Example 020]
Synthesis of 2- [5-acetoxy-2- (2-oxopropylidene) -2,3-dihydrobenzothiazol-3-yl] ethyl acetate

1- [3- (2-Hydroxyethyl) -5-hydroxy-2,3-dihydrobenzothiazol-2-ylidene] propan-2-one (compound of Preparation Example 017) (85. 4-Dimethylaminopyridine (2.0 mg, 16 μmol) and acetic anhydride (71.0 μl, 751 μmol) were continuously added to the suspension containing 3 mg, 339 μmol) at 0 ° C. and stirred at room temperature for 2 hours. Thereafter, water was added to the mixture and extracted three times with ethyl acetate. The combined organic extracts were washed once with water and then once with brine, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified using silica gel column chromatography (10 g, CH 2 Cl 2 / ethyl acetate, 4: 1) to give 2- [5-acetoxy-2- (2-oxopropylidene)- 2,3-Dihydrobenzothiazol-3-yl] ethyl acetate (the compound of Production Example 020) was obtained (104 mg, 310 μmol, yield 91.4%).
1 H NMR (CDCl 3 , 400 MHz) δ 2.02 (s, 3H, CH 3 ), 2.26 (s, 3H, CH 3 ), 2.34 (s, 3H, CH 3 ), 4.24 (t, 2H, J = 6.0 Hz, CH 2 ), 4.42 (t, 2H, J = 6.0 Hz, CH 3 ), 5.93 (s, 1H, olefinic), 6.90 (dd, 1H, J = 2.0, 8.2 Hz, aromatic), 6.95 (d, 1H, J = 2.0 Hz, aromatic), 7.53 (d, 1H,), 7.96 (s, 1H, J = 8.2 Hz, aromatic).
HRMS (FAB + ) m / z 336.0905 ((M + H) + , C 16 H 18 NO 5 S requires 336.0906).

［製造例０２１］
３−エチル−２−（２−オキソプロピリデン）−２，３−ジヒドロベンゾチアゾール−５−イル ベンゾエートの合成

２．５ｍｌのピリジン中に１−（３−エチル−５−ヒドロキシ−２，３−ジヒドロベンゾチアゾール−２−イリデン）プロパン−２−オン（製造例００９の化合物）（１５０ｍｇ、６３７μｍｏｌ）を含むサスペンションに、塩化ベンゾイル（９０．０μｌ、７７５μｍｏｌ）を０℃で添加して、０℃にて１時間撹拌した。その後、混合物を常温にした後に水を加え、ＣＨ２Ｃｌ２で３回抽出した。１つにまとめた有機抽出液を水にて１回洗浄し、Ｎａ２ＳＯ４上で乾燥し、ろ過し、減圧濃縮した。その残留物をシリカゲルカラムクロマトグラフィー（３０ｇ、ＣＨ２Ｃｌ２／エチルアセテート、４：１）を用いて精製し、無色固体の３−エチル−２−（２−オキソプロピリデン）−２，３−ジヒドロベンゾチアゾール−５−イルベンゾエート（製造例０２１の化合物）を得た（２１４ｍｇ、６３１μｍｏｌ、収率９９．１％）。
1H NMR (DMSO-d6, 400 MHz) δ 1.21 (t, 3H, J = 7.0 Hz, CH3), 2.11 (s, 3H, CH3), 4.14 (q, 2H, J = 7.0 Hz, CH2), 6.15 (s, 1H, olefinic), 7.11 (dd, 1H, J = 2.0, 8.4Hz, aromatic), 7.50 (d, 1H, J = 2.0 Hz, aromatic), 7.60-7.64 (m, 1H, aromatic),7.74-7.75 (m, 1H, aromatic), 7.79 (d, 1H, J = 8.4 Hz, aromatic), 8.14-8.17 (m, 1H, aromatic).
HRMS (FAB+) m/z 340.1008 ((M+H)+, C19H18NO3S requires 339.0929).[Production Example 021]
Synthesis of 3-ethyl-2- (2-oxopropylidene) -2,3-dihydrobenzothiazol-5-yl benzoate

Suspension containing 1- (3-ethyl-5-hydroxy-2,3-dihydrobenzothiazol-2-ylidene) propan-2-one (Compound of Preparation 009) (150 mg, 637 μmol) in 2.5 ml pyridine To the solution, benzoyl chloride (90.0 μl, 775 μmol) was added at 0 ° C., and the mixture was stirred at 0 ° C. for 1 hour. Thereafter, the mixture was brought to room temperature, water was added, and the mixture was extracted 3 times with CH 2 Cl 2 . The combined organic extracts were washed once with water, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified using silica gel column chromatography (30 g, CH 2 Cl 2 / ethyl acetate, 4: 1) to give 3-ethyl-2- (2-oxopropylidene) -2,3- Dihydrobenzothiazol-5-ylbenzoate (the compound of Production Example 021) was obtained (214 mg, 631 μmol, yield 99.1%).
1 H NMR (DMSO-d 6 , 400 MHz) δ 1.21 (t, 3H, J = 7.0 Hz, CH 3 ), 2.11 (s, 3H, CH 3 ), 4.14 (q, 2H, J = 7.0 Hz, CH 2 ), 6.15 (s, 1H, olefinic), 7.11 (dd, 1H, J = 2.0, 8.4Hz, aromatic), 7.50 (d, 1H, J = 2.0 Hz, aromatic), 7.60-7.64 (m, 1H, aromatic), 7.74-7.75 (m, 1H, aromatic), 7.79 (d, 1H, J = 8.4 Hz, aromatic), 8.14-8.17 (m, 1H, aromatic).
HRMS (FAB + ) m / z 340.1008 ((M + H) + , C 19 H 18 NO 3 S requires 339.0929).

［製造例０２２］
３−エチル−２−（２−オキソプロピリデン）−２，３−ジヒドロベンゾチアゾール−５−イル ピバレートの合成

塩化ベンゾイルにかえて塩化ピバロイルを使用したほかは、［製造例０２１］と同様に製造し、無色固体の３−エチル−２−（２−オキソプロピリデン）−２，３−ジヒドロベンゾチアゾール−５−イルピバレート（製造例０２２の化合物）を得た。
1H NMR (DMSO-d6, 400 MHz) δ 1.20 (t, 3H, J = 7.0 Hz, CH3), 1.32 (s, 9H, CH3 x 3), 2.11 (s, 3H, CH3), 4.13 (q, 2H, J = 7.0 Hz, CH2), 6.12 (s, 1H, olefinic), 6.89 (dd, 1H, J = 1.8, 8.4 Hz, aromatic), 7,26 (d, 1H, J = 1.8Hz, aromatic), 7.72 (d, 1H, J = 8.4 Hz, aromatic).
HRMS (FAB+) m/z 320.1321 ((M+H)+, C17H22NO3S requires 320.1320).[Production Example 022]
Synthesis of 3-ethyl-2- (2-oxopropylidene) -2,3-dihydrobenzothiazol-5-yl pivalate

A colorless solid 3-ethyl-2- (2-oxopropylidene) -2,3-dihydrobenzothiazole-5 was prepared in the same manner as in [Preparation Example 021] except that pivaloyl chloride was used instead of benzoyl chloride. -Obtained yl pivalate (the compound of Production Example 022).
1 H NMR (DMSO-d 6 , 400 MHz) δ 1.20 (t, 3H, J = 7.0 Hz, CH 3 ), 1.32 (s, 9H, CH 3 x 3), 2.11 (s, 3H, CH 3 ), 4.13 (q, 2H, J = 7.0 Hz, CH 2 ), 6.12 (s, 1H, olefinic), 6.89 (dd, 1H, J = 1.8, 8.4 Hz, aromatic), 7,26 (d, 1H, J = 1.8Hz, aromatic), 7.72 (d, 1H, J = 8.4 Hz, aromatic).
HRMS (FAB + ) m / z 320.1321 ((M + H) + , C 17 H 22 NO 3 S requires 320.1320).

［製造例０２３］
３−エチル−２−（２−オキソプロピリデン）−２，３−ジヒドロベンゾチアゾール−５−イル イソニコチネートの合成

塩化ベンゾイルにかえてイソニコチン酸クロライド塩酸塩を使用したほかは［製造例０２１］と同様に製造し、無色固体の３−エチル−２−（２−オキソプロピリデン）−２，３−ジヒドロベンゾチアゾール−５−イルイソニコチネート（製造例０２３の化合物）を得た。
1H NMR (DMSO-d6, 400 MHz) δ 1.22 (t, 3H, J = 7.0 Hz, CH3), 2.12 (s, 3H, CH3), 4.13 (q, 2H, J = 7.0 Hz, CH2), 6.15 (s, 1H, olefinic), 7.15 (dd, 1H, J = 2.0, 8.2Hz, aromatic), 7.54 (d, 1H, J = 2.0 Hz, aromatic), 7.81 (d, 1H, J = 8.2 Hz, aromatic), 8.03 (dd, 2H, J = 1.7, 4.4 Hz, aromatic), 8.90 (dd, 2H, J = 1.7, 4.4 Hz,aromatic).
HRMS (FAB+) m/z 341.0961 ((M+H)+, C18H17N2O3S requires 341.0960).[Production Example 023]
Synthesis of 3-ethyl-2- (2-oxopropylidene) -2,3-dihydrobenzothiazol-5-yl isonicotinate

A colorless solid 3-ethyl-2- (2-oxopropylidene) -2,3-dihydrobenzo was prepared in the same manner as in [Production Example 021] except that isonicotinic acid chloride hydrochloride was used instead of benzoyl chloride. Thiazol-5-ylisonicotinate (Compound of Production Example 023) was obtained.
1 H NMR (DMSO-d 6 , 400 MHz) δ 1.22 (t, 3H, J = 7.0 Hz, CH 3 ), 2.12 (s, 3H, CH 3 ), 4.13 (q, 2H, J = 7.0 Hz, CH 2 ), 6.15 (s, 1H, olefinic), 7.15 (dd, 1H, J = 2.0, 8.2Hz, aromatic), 7.54 (d, 1H, J = 2.0 Hz, aromatic), 7.81 (d, 1H, J = 8.2 Hz, aromatic), 8.03 (dd, 2H, J = 1.7, 4.4 Hz, aromatic), 8.90 (dd, 2H, J = 1.7, 4.4 Hz, aromatic).
HRMS (FAB + ) m / z 341.0961 ((M + H) + , C 18 H 17 N 2 O 3 S requires 341.0960).

［製造例０２４］
３−エチル−２−（２−オキソプロピリデン）−２，３−ジヒドロベンゾチアゾール−５−イル ニコチネートの合成

塩化ベンゾイルにかえてニコチン酸クロライド塩酸塩を使用したほかは、［製造例０２１］と同様に製造し、無色固体の３−エチル−２−（２−オキソプロピリデン）−２，３−ジヒドロベンゾチアゾール−５−イルニコチネート（製造例０２４の化合物）を得た。
1H NMR (DMSO-d6, 400 MHz) δ 1.22 (t, 3H, J = 7.2 Hz, CH3), 2.12 (s, 3H, CH3), 4.13 (q, 2H, J = 7.2 Hz, CH2), 6.15 (s, 1H, olefinic), 7.15 (dd, 1H, J = 2.0, 8.4Hz, aromatic), 7.53 (d, 1H, J = 2.0 Hz, aromatic), 7.67 (ddd, 1H, J = 0.8, 4.9,8.1 Hz, aromatic), 7.81 (d, 1H, J = 8.4 Hz, aromatic), 8.49 (ddd, 1H, J = 1.8, 2.1, 8.1 Hz, aromatic), 8.91 (dd, 1H, J = 1.8, 4.9 Hz, aromatic), 9.28 (dd, 1H, J = 0.8, 2.1 Hz, aromatic).
HRMS (FAB+) m/z 341.0961 ((M+H)+, C18H17N2O3S requires 341.0960).[Production Example 024]
Synthesis of 3-ethyl-2- (2-oxopropylidene) -2,3-dihydrobenzothiazol-5-yl nicotinate

A colorless solid 3-ethyl-2- (2-oxopropylidene) -2,3-dihydrobenzo was prepared in the same manner as in [Preparation Example 021] except that nicotinic acid chloride hydrochloride was used instead of benzoyl chloride. Thiazol-5-ylnicotinate (the compound of Production Example 024) was obtained.
1 H NMR (DMSO-d 6 , 400 MHz) δ 1.22 (t, 3H, J = 7.2 Hz, CH 3 ), 2.12 (s, 3H, CH 3 ), 4.13 (q, 2H, J = 7.2 Hz, CH 2 ), 6.15 (s, 1H, olefinic), 7.15 (dd, 1H, J = 2.0, 8.4Hz, aromatic), 7.53 (d, 1H, J = 2.0 Hz, aromatic), 7.67 (ddd, 1H, J = 0.8, 4.9, 8.1 Hz, aromatic), 7.81 (d, 1H, J = 8.4 Hz, aromatic), 8.49 (ddd, 1H, J = 1.8, 2.1, 8.1 Hz, aromatic), 8.91 (dd, 1H, J = 1.8, 4.9 Hz, aromatic), 9.28 (dd, 1H, J = 0.8, 2.1 Hz, aromatic).
HRMS (FAB + ) m / z 341.0961 ((M + H) + , C 18 H 17 N 2 O 3 S requires 341.0960).

［製造例０２５］
３−エチル−２−（２−オキソプロピリデン）−２，３−ジヒドロベンゾチアゾール−５−イル ２，４，６−トリメトキシベンゾエートの合成

市販の２，４，６−トリメトキシ安息香酸（３．００ｇ、１４．１ｍｍｏｌ）および２−メチル−５−ベンゾチアゾール（１．９５ｇ、１１．８ｍｍｏｌ）を１００ｍＬのＣＨ２Ｃｌ２に溶解させた後、トリエチルアミン（７．４０ｍＬ、５３．１ｍｍｏｌ）、４−ジメチルアミノピリジン（１４４ｍｇ、１．１８ｍｍｏｌ）および１−エチル−３−（３−ジメチルアミノプロピル）カルボジイミド塩酸塩（４．９８ｇ、２６．０ｍｍｏｌ）を常温で連続的に添加して、４９時間室温にて攪拌した。その後、混合溶液を水（３００ｍＬ）へ注ぎ込み、混合物を１５０ｍＬのＣＨ２Ｃｌ２で３回抽出した。１つにまとめた有機抽出液を水（１００ｍＬ）、ブライン（１００ｍＬ）で順次洗浄し、Ｎａ２ＳＯ４上で乾燥し、ろ過し、減圧濃縮した。その残留物をシリカゲルクロマトグラフィー（１２０ｇ、ｎ−ヘキサン／エチルアセテート／ＣＨ２Ｃｌ２、２：１：１）を用いて精製し、無色固体の２−メチルベンゾチアゾール−５−イル ２，４，６−トリメトキシベンゾエートを得た（２．１８ｇ、６．０７ｍｍｏｌ、収率５１．４％）。
そして、５−メトキシ−２−メチルベンゾチアゾールにかえて２−メチルベンゾチアゾール−５−イル ２，４，６−トリメトキシベンゾエートを使用したほかは、［製造例００１］と同様に製造し、３−エチル−２−（２−オキソプロピリデン）−２，３−ジヒドロベンゾチアゾール−５−イル ２，４，６−トリメトキシベンゾエート（製造例０２５の化合物）を得た。
1H NMR (CDCl3, 300 MHz) δ 1.39 (t, 3H, J = 7.2 Hz, CH3), 2.25 (s, 3H, CH3), 3.86 (s, 3H, OCH3), 3.88 (s, 6H, OCH3x 2), 4.05 (q, 2H, J = 7.2 Hz, CH2), 5.90 (s, 1H, oleffin), 6.16 (s, 2H, aromaric x 2), 7.01-7.08 (m, 2H, aromatic x 2), 7.56 (d, 1H, J = 9.0 Hz, aromatic).[Production Example 025]
Synthesis of 3-ethyl-2- (2-oxopropylidene) -2,3-dihydrobenzothiazol-5-yl 2,4,6-trimethoxybenzoate

After dissolving commercially available 2,4,6-trimethoxybenzoic acid (3.00 g, 14.1 mmol) and 2-methyl-5-benzothiazole (1.95 g, 11.8 mmol) in 100 mL CH 2 Cl 2. , Triethylamine (7.40 mL, 53.1 mmol), 4-dimethylaminopyridine (144 mg, 1.18 mmol) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (4.98 g, 26.0 mmol) Was continuously added at room temperature and stirred at room temperature for 49 hours. Thereafter, the mixed solution was poured into water (300 mL), and the mixture was extracted with 150 mL of CH 2 Cl 2 three times. The combined organic extracts were washed sequentially with water (100 mL), brine (100 mL), dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The residue was purified using silica gel chromatography (120 g, n-hexane / ethyl acetate / CH 2 Cl 2 , 2: 1: 1) to give colorless solid 2-methylbenzothiazol-5-yl 2,4, 6-trimethoxybenzoate was obtained (2.18 g, 6.07 mmol, yield 51.4%).
Then, this was prepared in the same manner as in [Production Example 001] except that 2-methylbenzothiazol-5-yl 2,4,6-trimethoxybenzoate was used instead of 5-methoxy-2-methylbenzothiazole. -Ethyl-2- (2-oxopropylidene) -2,3-dihydrobenzothiazol-5-yl 2,4,6-trimethoxybenzoate (Compound of Production Example 025) was obtained.
1 H NMR (CDCl 3 , 300 MHz) δ 1.39 (t, 3H, J = 7.2 Hz, CH 3 ), 2.25 (s, 3H, CH 3 ), 3.86 (s, 3H, OCH 3 ), 3.88 (s, 6H, OCH 3 x 2), 4.05 (q, 2H, J = 7.2 Hz, CH 2 ), 5.90 (s, 1H, oleffin), 6.16 (s, 2H, aromaric x 2), 7.01-7.08 (m, 2H , aromatic x 2), 7.56 (d, 1H, J = 9.0 Hz, aromatic).

［製造したベンゾチアゾール誘導体のリン酸化酵素の活性阻害］
製造例００１〜０２４で製造したベンゾチアゾール誘導体について、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２のリン酸化活性に対する阻害能を測定した。[Inhibition of phosphorylase activity of the manufactured benzothiazole derivatives]
About the benzothiazole derivative manufactured in manufacture example 001-024, the inhibitory ability with respect to the phosphorylation activity of DYRK1A, DYRK1B, and DYRK2 was measured.

大腸菌で発現させて精製した０．２７μｇの組み換えＤＹＲＫ１Ａ蛋白質と８．２５μｇのＭＢＰ（Ｕｐｓｔａｔｅ社製）とを、反応バッファー（１０ｍＭ ＭｇＣｌ、１０μＭ ＡＴＰ、１μＣｉ［γ−３２Ｐ］ＡＴＰ、製造例００１〜０２４の化合物終濃度１０μＭ）中で３０℃、１０分間インキュベートした。製造例００１〜０２４の化合物は、ＤＭＳＯに溶解し、必要に応じてＤＭＳＯにて段階希釈を行った。このリン酸化酵素の活性測定におけるＤＹＲＫ１ＡとＭＢＰの量、及び反応時間の条件は、予め反応の直線性を検討し、直線性が成立する条件を選択した。ＤＹＲＫ１ＡとＭＢＰの反応を１０分行った後、反応液をＰ８１フォスフォセルロースメンブレン（Ｐ８１；Ｗｈａｔｍａｎ）に滴下し、乾燥後、５％リン酸溶液で洗浄した。洗浄後、Ｐ８１メンブレンの３２Ｐ放射活性を液体シンチレーションカウンター（ＡＬＯＫＡ ＬＩＱＵＩＤ ＳＣＩＮＴＩＬＬＡＴＩＯＮ ＣＯＵＮＴＥＲ ＬＳＣ−５１００）により測定した。製造例００１〜０２４の化合物非添加のコントロールとして、化合物の溶解・希釈に用いたＤＭＳＯを反応バッファーに加えることにより、全てのサンプルでＤＭＳＯの最終濃度を統一化した。製造例の化合物、及び、コントロールにつき３個のサンプルで測定を行い、測定結果をコントロールの数値に対する割合（％）及び標準偏差として表した。また、同様の手法により、ＤＹＲＫ１Ｂ及びＤＹＲＫ２についても化合物１０μＭにおけるリン酸化活性の阻害効果を測定した。これらの結果を下記表１に示す。0.27 μg recombinant DYRK1A protein expressed and purified in E. coli and 8.25 μg MBP (manufactured by Upstate) were mixed with reaction buffer (10 mM MgCl, 10 μM ATP, 1 μCi [γ- 32 P] ATP, Production Example 001 The compound was incubated at 30 ° C. for 10 minutes in a final concentration of 024 (10 μM). The compounds of Production Examples 001 to 024 were dissolved in DMSO and serially diluted with DMSO as necessary. As the conditions for the amount of DYRK1A and MBP and the reaction time in this phosphorylase activity measurement, the linearity of the reaction was examined in advance, and the conditions for establishing the linearity were selected. After reacting DYRK1A and MBP for 10 minutes, the reaction solution was dropped onto a P81 phosphocellulose membrane (P81; Whatman), dried, and washed with a 5% phosphoric acid solution. After washing, 32 P-radioactivity of P81 membrane was determined by liquid scintillation counter (ALOKA LIQUID SCINTILLATION COUNTER LSC-5100 ). As a control for adding no compound in Production Examples 001 to 024, DMSO used for dissolving and diluting the compound was added to the reaction buffer to unify the final concentration of DMSO in all samples. The measurement was carried out using three samples per compound of the production example and the control, and the measurement results were expressed as a percentage (%) and standard deviation with respect to the numerical value of the control. Moreover, the inhibitory effect of the phosphorylation activity in 10 micromol of compounds was measured also about DYRK1B and DYRK2 by the same method. These results are shown in Table 1 below.

上記表１に示すとおり、製造例００１〜０１８のベンゾチアゾール誘導体は、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つのリン酸化活性を少なくとも５０％以上阻害できた。また、製造例００１〜０１８のベンゾチアゾール誘導体は、チロシンキナーゼであるＭｅｔやセリン・トレオニンキナーゼであるＡＳＫ１などのリン酸化活性に対して、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２と比べて大きな阻害能を示さなかった（データ示さず）。したがって、製造例００１〜０１８のベンゾチアゾール誘導体は、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２に対して選択的にリン酸化活性を阻害する傾向が示された。   As shown in Table 1 above, the benzothiazole derivatives of Production Examples 001 to 018 were able to inhibit at least 50% or more of the phosphorylation activity of DYRK1A, DYRK1B, and DYRK2. In addition, the benzothiazole derivatives of Production Examples 001 to 018 show a greater inhibitory ability than DYRK1A, DYRK1B, and DYRK2 with respect to phosphorylation activities such as Met, which is a tyrosine kinase, and ASK1, which is a serine / threonine kinase. None (data not shown). Therefore, the benzothiazole derivatives of Production Examples 001 to 018 showed a tendency to selectively inhibit phosphorylation activity with respect to DYRK1A, DYRK1B, and DYRK2.

なお、上記表１における製造例０１９〜０２４の化合物の中には、リン酸化活性阻害能を発揮しないものもあったが、これらは製造例００９の化合物又は製造例０１７の化合物のプロドラッグ形態であるため、インビトロでのリン酸化活性阻害能を示さない場合がある。なお、後述する実施例で、プロドラッグ形態の製造例０１９〜０２４のベンゾチアゾール誘導体が生体内又は細胞内でリン酸化活性を阻害できることが示されている。   In addition, some of the compounds of Production Examples 019 to 024 in Table 1 above did not exhibit the phosphorylation activity inhibitory ability, but these are prodrug forms of the compound of Production Example 009 or the compound of Production Example 017. Therefore, it may not show the ability to inhibit phosphorylation activity in vitro. In the examples described later, it is shown that the benzothiazole derivatives of Production Examples 019 to 024 in prodrug form can inhibit phosphorylation activity in vivo or in cells.

［培養細胞内におけるタウ蛋白質（Ｔａｕ）のリン酸化阻害］
タウ蛋白質のリン酸化は、アルツハイマー病の発症機序と密接な関連性を有することが知られている。そこで、培養細胞内にＤＹＲＫ１Ａ及びタウ蛋白質を発現させて、タウ蛋白質がリン酸化される系を構築し、ベンゾチアゾール誘導体によりタウ蛋白質のリン酸化を阻害できることを確認した。[Inhibition of tau protein (Tau) phosphorylation in cultured cells]
It is known that tau protein phosphorylation is closely related to the pathogenesis of Alzheimer's disease. Thus, DYRK1A and tau protein were expressed in cultured cells, a system in which tau protein was phosphorylated was constructed, and it was confirmed that phosphorylation of tau protein can be inhibited by a benzothiazole derivative.

ＣＯＳ−７細胞（ＣＲＬ−１６５１）をＤＭＥＭ（１０％ＦＢＳ、ペニシリンストレプトマイシン添加）培地にて培養し、ｐＥＧＦＰ−ＤＹＲＫ１ＡとｐＥＧＦＰ−ＭＡＰＴをＧｅｎｅＪｕｉｃｅ Ｔｒａｎｓｆｅｃｔｉｏｎ Ｒｅａｇｅｎｔ（Ｎｏｖａｇｅｎ）を用いて強制発現させた。３時間後にベンゾチアゾール誘導体（製造例００１、００９、０１９〜０２５の化合物）をそれぞれ終濃度１０μＭにて添加し、２１時間、３７℃、５％ＣＯ２培養条件下で培養した。なお、製造例０２５の化合物は、ラット血清中でインキュベーション（３７℃６０分）を行って５位のエステル結合を加水分解（エステラーゼ反応）させ、その代謝産物をＨＰＬＣにより回収して濃縮後、濃度測定、フィルター滅菌を行ったものを添加した。また、ベンゾチアゾール非添加群として前記ベンゾチアゾール誘導体の溶媒であるＤＭＳＯを培地に添加した。その後、プロテアーゼ阻害剤カクテル（Ｒｏｃｈｅ）とホスファターゼ阻害剤カクテル（Ｎａｃａｌａｉ）を加えたＲＩＰＡバッファー（５０ｍＭ Ｔｒｉｓ−ＨＣｌ ｐＨ８．０、１５０ｍＭ ＮａＣｌ、２ｍＭ ＥＧＴＡ、１％ ＮｏｎｉｄｅｔＰ−４０、０．５％ ＤＯＣ、０．１％ ＳＤＳ）で細胞を溶解し、ＳＤＳ−ＰＡＧＥサンプルバッファーで希釈して蛋白質サンプルとした。このサンプルを２０μｇ／レーンにてＳＤＳアクリルアミドゲルに電気泳動し、ＰＶＤＦメンブレンにトランスファー後、抗ＧＦＰ抗体（ＭＢＬ）、抗Ｔａｕ［ｐＴ２１２］抗体（ＢＩＯＳＯＵＲＣＥ）にて抗体反応を行い、Ｓｕｐｅｒ Ｓｉｇｎａｌ Ｗｅｓｔ Ｄｕｒａ（Ｔｈｅｒｍｏ ＳＣＩＥＮＴＩＦＩＣ）によるケミルミ発光にてリン酸化Ｔａｕのバンド、並びに、総Ｔａｕを示すＧＦＰのバンドを検出した。それら検出されたバンドをデンシトメトリー（ＡＴＴＯ ＣＳ Ａｎａｌｙｚｅｒ ｖｅｒ ３．０）により数値化し、化合物非添加群のリン酸化Ｔａｕのバンドの発光を１００％とし、各群におけるバンドの発光を数値化した。なお、それぞれのリン酸化Ｔａｕのバンドは、総Ｔａｕにより補正を行った。その結果を下記表２及び図１に示す。COS-7 cells (CRL-1651) were cultured in DMEM (10% FBS, penicillin streptomycin added) medium, and pEGFP-DYRK1A and pEGFP-MAPT were forcibly expressed using GeneJuice Transfection Reagent (Novagen). Three hours later, benzothiazole derivatives (Compounds of Production Examples 001, 009 and 019 to 025) were added at final concentrations of 10 μM, respectively, and cultured for 21 hours at 37 ° C. under 5% CO 2 culture conditions. The compound of Production Example 025 was incubated in rat serum (37 ° C., 60 minutes) to hydrolyze the ester bond at the 5-position (esterase reaction), and its metabolite was collected by HPLC and concentrated. What was measured and filter sterilized was added. Moreover, DMSO which is a solvent of the said benzothiazole derivative was added to the culture medium as a benzothiazole non-addition group. Then, RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EGTA, 1% Nonidet P-40, 0.5% DOC, 0 with protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Nacalai) The cells were lysed with 1% SDS) and diluted with SDS-PAGE sample buffer to obtain a protein sample. This sample was electrophoresed on an SDS acrylamide gel at 20 μg / lane, transferred to a PVDF membrane, and then subjected to an antibody reaction with an anti-GFP antibody (MBL) and an anti-Tau [pT212] antibody (BIOSOURCE), and Super Signal West Dura ( The phosphorylated Tau band and the GFP band indicating total Tau were detected by chemirmi luminescence by Thermo SCIENTIFIC. The detected bands were quantified by densitometry (ATTO CS Analyzer ver 3.0), the luminescence of the phosphorylated Tau band in the group not added with the compound was taken as 100%, and the luminescence of the band in each group was quantified. Each phosphorylated Tau band was corrected by the total Tau. The results are shown in Table 2 below and FIG.

上記表２及び図１に示すとおり、ベンゾチアゾール誘導体非添加群（コントロール）において観察されたタウ蛋白質のリン酸化は、ベンゾチアゾール誘導体の添加により抑制されることが示された。製造例０１９〜０２４は、上述のインビトロのリン酸化活性阻害能の測定（上記表１）では阻害活性が低かったが、細胞内においては良好なＤＹＲＫ１Ａの活性阻害を示した（上記表２及び図１）。製造例０２５の化合物は、血清中でのインキュベーションを行わない場合にはタウ蛋白質のリン酸化阻害活性は低かった（data not shown）。しかし、製造例０２５の化合物は、血清中でのインキュベーションを行うことで細胞内において良好なＤＹＲＫ１Ａの活性阻害を示した（上記表２及び図１）。   As shown in Table 2 and FIG. 1, it was shown that phosphorylation of tau protein observed in the benzothiazole derivative non-added group (control) was suppressed by addition of the benzothiazole derivative. Production Examples 019 to 024 showed low DYRK1A activity inhibition in cells although the inhibition activity was low in the above-described measurement of the in vitro phosphorylation activity inhibition ability (Table 1 above) (Table 2 and Figure above). 1). When the compound of Production Example 025 was not incubated in serum, tau protein phosphorylation inhibitory activity was low (data not shown). However, the compound of Production Example 025 showed good inhibition of DYRK1A activity in cells by incubation in serum (Table 2 above and FIG. 1).

なお、図２は、ベンゾチアゾール誘導体（製造例００１、０１９、００９）の添加量とタウ蛋白質のリン酸化阻害との関係を示す図である。同図に示すように、培養細胞内におけるタウ蛋白質のリン酸化阻害は、ベンゾチアゾール誘導体の添加量に依存した。   FIG. 2 is a graph showing the relationship between the added amount of benzothiazole derivatives (Production Examples 001, 019, 009) and inhibition of tau protein phosphorylation. As shown in the figure, inhibition of tau protein phosphorylation in cultured cells depended on the amount of benzothiazole derivative added.

［ＤＹＲＫ１Ａ過剰発現モデルにおける神経発生異常の抑制］
アフリカツメガエルの初期胚で、ＤＹＲＫ１Ａのアフリカツメガエルホモログを過剰発現させると神経発生に異常が生じ、形態的異常（発生異常）の表現型を示すことが知られている。そこで、上記製造例００１及び製造例０１９のベンゾチアゾール誘導体を用い、下記のようにして、ＤＹＲＫ１Ａ過剰発現による発生異常に対する抑制効果の有無を確認した。[Inhibition of neurogenesis abnormality in DYRK1A overexpression model]
It is known that when Xenopus homologue of DYRK1A is overexpressed in early stage of Xenopus laevis, an abnormality occurs in neurogenesis and exhibits a phenotype of morphological abnormality (developmental abnormality). Therefore, using the benzothiazole derivatives of Production Example 001 and Production Example 019, the presence or absence of an inhibitory effect on abnormal development due to DYRK1A overexpression was confirmed as follows.

［ＤＹＲＫ１Ａ過剰発現によるアフリカツメガエルの発生異常モデル］
ＤＹＲＫ１Ａのアフリカツメガエルホモログ（ＭＧＣ：１３２２８４）のｃＤＮＡのＮ末にＦＬＡＧタグ（ＭＹＤＫＤＤＤＤＫ）を融合した配列をＰＣＲ法で増幅し、ｐＣＳ２（＋）ベクターのＢａｍＨＩ／ＸｂａＩサイトにサブクローニングした。作製したプラスミドを直鎖化したものをテンプレートとして、ｐＣＳ２（＋）ベクター内のＳＰ６プロモーターを利用してｉｎ ｖｉｔｒｏ転写を行い、ｍＲＮＡを合成した（ＳＰ６ ｍＭＥＳＳＡＧＥ ｍＭＡＣＨＩＮＥ Ｋｉｔ， Ａｍｂｉｏｎ）。アフリカツメガエル胚の前頭部にＤＹＲＫ１Ａを過剰発現させる目的で、このｍＲＮＡを８細胞期胚の動物極背側２割球に注入した（使用量２５０、５００ｐｇ／ｅｍｂｒｙｏ）。胚への注入には、電動マイクロインジェクター（ＩＭ−３００， ＮＡＲＩＳＨＩＧＥ）を用い、実体顕微鏡下において微小硝子管により１割球あたり５ｎｌのＤＹＲＫ１ＡのｍＲＮＡを含む溶液を注入した（１０ｎｌ／ｅｍｂｒｙｏ）。注入の際、胚は３％ Ｆｉｃｏｌｌを含んだ０．１ｘＳｔｅｉｎｂｅｒｇ’ｓ ｂｕｆｆｅｒ中に置き、注入後も同じ溶液中で培養した。培養はすべて暗下で行い、ｓｔａｇｅ４０／４１の時点で形態的異常（発生異常）の有無を観察した。なお、発生段階（ｓｔａｇｅ）はＮｉｅｕｗｋｏｏｐとＦａｂｅｒに準じた（Nieuwkoop,P.D.,and Faber,J.,1956, Normal Table of Xenopus laevis (Daudin), Elsevier/North-Holland Biomedical Press,Amsterdam）。[Model for abnormal development of Xenopus laevis due to overexpression of DYRK1A]
A sequence in which a FLAG tag (MYDKDDDDDK) was fused to the N-terminus of the Xenopus homologue of DYRK1A (MGC: 132284) was amplified by the PCR method and subcloned into the BamHI / XbaI site of the pCS2 (+) vector. Using the prepared plasmid linearized as a template, in vitro transcription was performed using the SP6 promoter in the pCS2 (+) vector, and mRNA was synthesized (SP6 mMESSAGE mMACHINE Kit, Ambion). In order to overexpress DYRK1A in the forehead of Xenopus embryos, this mRNA was injected into the animal's dorsal blastomere of 8-cell stage embryos (use amount 250, 500 pg / embryo). For injection into the embryo, an electric microinjector (IM-300, NARISHIGE) was used, and a solution containing 5 nl of DYRK1A mRNA per 10 blastomers was injected with a micro glass tube under a stereomicroscope (10 nl / embryo). At the time of injection, embryos were placed in 0.1 × Steinberg's buffer containing 3% Ficoll and cultured in the same solution after injection. All the cultures were performed in the dark, and the presence or absence of morphological abnormalities (developmental abnormalities) was observed at stage 40/41. In addition, the stage of development (Nieuwkoop, PD, and Faber, J., 1956, Normal Table of Xenopus laevis (Daudin), Elsevier / North-Holland Biomedical Press, Amsterdam) according to Nieuwkoop and Faber.

［ベンゾチアゾール誘導体によるアフリカツメガエルの発生異常に対する抑制効果］
上記製造例のベンゾチアゾール誘導体の発生異常に対する抑制効果を検討するため、上記ＤＹＲＫ１Ａ過剰発現モデルの作製の際に、以下の作業を行った。すなわち、アフリカツメガエルから受精卵を調製後、胚をただちに製造例００１の化合物（１０μＭ）、製造例０１９の化合物（２．５μＭ）、又は、コントロールとして溶媒（ＤＭＳＯ）を含んだ飼育水で培養し、８細胞期胚においてＤＹＲＫ１Ａ合成ｍＲＮＡを動物極背側２割球に注入した後、上記２種のベンゾチアゾール誘導体及びＤＭＳＯのいずれかを含んだ培地にて２３℃で３日間培養し、ｓｔａｇｅ４０／４１の時点で形態的異常の有無を観察した。その結果を、図３〜５に示す。[Inhibitory effects of benzothiazole derivatives on abnormal development of Xenopus laevis]
In order to examine the inhibitory effect on the abnormal occurrence of the benzothiazole derivative in the above production example, the following work was performed when the DYRK1A overexpression model was prepared. That is, after preparing a fertilized egg from Xenopus laevis, the embryo is immediately cultured in breeding water containing the compound of Production Example 001 (10 μM), the compound of Production Example 019 (2.5 μM), or a solvent (DMSO) as a control. In the 8-cell embryo, DYRK1A synthetic mRNA was injected into the animal's dorsal blastomere, then cultured at 23 ° C. for 3 days in a medium containing either of the above two benzothiazole derivatives and DMSO. The presence or absence of morphological abnormality was observed at 41 time points. The results are shown in FIGS.

図３は、上記ＤＹＲＫ１Ａ過剰発現モデルの発生異常に対する抑制効果実験の結果を示す観察写真である。ａ）、ｄ）及びｇ）はＤＹＲＫ１ＡのｍＲＮＡ非投与群であり、ｂ）、ｅ）及びｈ）は２５０ｐｇのＤＹＲＫ１Ａ ｍＲＮＡ投与群であり、ｃ）、ｆ）及びｉ）は５００ｐｇのＤＹＲＫ１Ａ ｍＲＮＡ投与群である。また、ａ）、ｂ）及びｃ）はベンゾチアゾール誘導体非投与群であり、ｄ）、ｅ）及びｆ）は製造例００１のベンゾチアゾール誘導体投与群であり、ｇ）、ｈ）及びｉ）は製造例０１９のベンゾチアゾール誘導体投与群である。   FIG. 3 is an observation photograph showing the results of an inhibitory effect experiment on the abnormal development of the DYRK1A overexpression model. a), d) and g) are DYRK1A mRNA non-administered groups, b), e) and h) are 250 pg DYRK1A mRNA administered groups, and c), f) and i) are 500 pg DYRK1A mRNA administered. A group. Further, a), b) and c) are benzothiazole derivative non-administered groups, d), e) and f) are benzothiazole derivative administered groups of Production Example 001, and g), h) and i) are It is a benzothiazole derivative administration group of Production Example 019.

図４は、上記ＤＹＲＫ１Ａ過剰発現モデルにおける抑制効果を調べた個体の発生異常（眼形成異常）について、正常、軽度及び重度の３つに分類し、各実験群におけるそれぞれの割合を百分率で示したグラフである。また、図５は、上記ＤＹＲＫ１Ａ過剰発現モデルにおける抑制効果を調べた個体の発生異常（頭部縮小）について、各実験群における割合を百分率で示したグラフである。図４ａはｍＲＮＡ非投与・ベンゾチアゾール非投与群のグラフであり、図４ｂは２５０ｐｇ ｍＲＮＡ投与群のグラフであり、図４ｃは５００ｐｇ ｍＲＮＡ投与群のグラフであり、図５ａはｍＲＮＡ非投与・ベンゾチアゾール非投与群及び２５０ｐｇ ｍＲＮＡ投与群のグラフであり、図５ｂはｍＲＮＡ非投与・ベンゾチアゾール非投与群及び５００ｐｇ ｍＲＮＡ投与群のグラフである。なお、図４及び５の各群におけるサンプル数は以下のとおりである。ｍＲＮＡ非投与・ベンゾチアゾール非投与群（ｎ＝２９）、２５０ｐｇ ｍＲＮＡ投与・ベンゾチアゾール非投与群（ｎ＝３６）、２５０ｐｇ ｍＲＮＡ投与・製造例００１投与群（ｎ＝２９）、２５０ｐｇ ｍＲＮＡ投与・製造例０１９投与群（ｎ＝２３）、５００ｐｇ ｍＲＮＡ投与・ベンゾチアゾール非投与群（ｎ＝５５）、５００ｐｇ ｍＲＮＡ投与・製造例００１投与群（ｎ＝３７）、５００ｐｇ ｍＲＮＡ投与・製造例０１９投与群（ｎ＝３５）。   FIG. 4 categorizes the abnormal development (individual ocular dysfunction) of individuals whose suppression effect in the DYRK1A overexpression model was classified into normal, mild, and severe, and the percentages in each experimental group are shown as percentages. It is a graph. FIG. 5 is a graph showing the percentage of each experimental group as a percentage of the abnormal development of the individual (head reduction) whose inhibitory effect in the DYRK1A overexpression model was examined. 4a is a graph of a group not administered with mRNA / benzothiazole, FIG. 4b is a graph of a group administered with 250 pg mRNA, FIG. 4c is a graph of a group administered with 500 pg mRNA, and FIG. FIG. 5b is a graph of the non-administration group and the 250 pg mRNA administration group, and FIG. 5b is a graph of the mRNA non-administration / benzothiazole non-administration group and the 500 pg mRNA administration group. The number of samples in each group in FIGS. 4 and 5 is as follows. No mRNA administration / benzothiazole non-administration group (n = 29), 250 pg mRNA administration / benzothiazole non-administration group (n = 36), 250 pg mRNA administration / production example 001 administration group (n = 29), 250 pg mRNA administration / production Example 019 administration group (n = 23), 500 pg mRNA administration / benzothiazole non-administration group (n = 55), 500 pg mRNA administration / production example 001 administration group (n = 37), 500 pg mRNA administration / production example 019 administration group ( n = 35).

図３〜５に示すとおり、対照群（ｍＲＮＡ非投与・溶媒のみ、図３ａ、図４ａ、図５ａ及びｂ）では正常な形態形成が認められたのに対し、ベンゾチアゾール誘導体を投与しないＤＹＲＫ１Ａ過剰発現胚では、眼形成異常及び頭部縮小を特徴とする発生異常が認められた（図３ｂ及びｃ、図４ｂ及びｃ、図５）。しかし、ベンゾチアゾール誘導体（製造例００１及び０１９）を投与すると、ＤＹＲＫ１Ａ過剰発現による発生異常は顕著に抑制された（図３ｅ、ｆ、ｈ、ｉ、図４ｂ及びｃ、図５）。すなわち、ベンゾチアゾール誘導体の投与によって、ＤＹＲＫ１Ａ過剰発現に起因する異常を個体レベルで抑制できることが示された。なお、製造例０１９は、上述のインビトロのリン酸化活性阻害能の測定（上記表１）では阻害活性が低かったが、生体内においては良好なＤＹＲＫ１の活性阻害を示した。   As shown in FIGS. 3 to 5, normal morphogenesis was observed in the control group (no mRNA administration / solvent only, FIGS. 3a, 4a, 5a and b), while DYRK1A excess without administration of the benzothiazole derivative was observed. In the expressed embryo, developmental abnormalities characterized by ocular dysplasia and head reduction were observed (FIGS. 3b and c, FIGS. 4b and c, FIG. 5). However, when benzothiazole derivatives (Production Examples 001 and 019) were administered, abnormal development due to DYRK1A overexpression was significantly suppressed (FIGS. 3e, f, h, i, FIGS. 4b and c, FIG. 5). That is, it was shown that the abnormality caused by DYRK1A overexpression can be suppressed at the individual level by administration of the benzothiazole derivative. In addition, Production Example 019 showed a low DYRK1 activity inhibition in vivo, although the inhibition activity was low in the above-described measurement of the in vitro phosphorylation activity inhibition ability (Table 1 above).

ＤＹＲＫ１Ａ過剰発現及びベンゾチアゾール誘導体投与時での発生段階における分子レベルの変化を確認するため、ＤＹＲＫ１Ａ ｍＲＮＡを５００ｐｇ投与した時の各種頭部神経マーカーの発現変化とベンゾチアゾール誘導体投与時の効果を検討した。その結果を図６に示す。   To confirm DYRK1A overexpression and changes in the molecular level in the developmental stage after administration of benzothiazole derivatives, the expression changes of various head nerve markers and the effects of administration of benzothiazole derivatives were examined when 500 pg of DYRK1A mRNA was administered. . The result is shown in FIG.

図６は、アフリカツメガエル初期胚でのＤＹＲＫ１Ａ過剰発現モデル（ｍＲＮＡ（＋）レーン）における神経発生マーカーの発現異常とベンゾチアゾール誘導体投与によるその発現異常回復効果を示す図である。ｍＲＮＡ（−）レーンは、正常個体における神経発生マーカーの発現レベルを示す。まず、アフリカツメガエルの８細胞期胚動物極背側２割球に０又は５００ｐｇのアフリカツメガエルＤＹＲＫ１Ａ合成ｍＲＮＡを注入し、同時に製造例００１（１０μＭ）、製造例０１９（２．５μＭ）、又はコントロールとして溶媒（ＤＭＳＯ）のみを飼育水に添加し、ベンゾチアゾール誘導体による回復効果を検討した。ＤＹＲＫ１Ａ合成ｍＲＮＡ注入後、胚を暗下にて２３℃で１日間培養し、ｓｔａｇｅ１７の時点で頭部を収穫し、ＲＮＡを回収してＲＴ−ＰＣＲによる発現解析を行った。神経全般、後脳、中脳、及び前脳の頭部神経マーカーであるＮＣＡＭ、Ｋｒｏｘ２０、Ｅｎ２、Ｏｔｘ２、並びに、眼形成マーカーであるＬｈｘ２及びＲｘ１について、ｍＲＮＡの発現量の解析を行った。なお、内部標準として、ｘＯｄｃを利用した。同図において、−、＋で示したレーンはそれぞれＤＹＲＫ１Ａ ｍＲＮＡを添加しない個体のコントロール、及びＤＹＲＫ１Ａ ｍＲＮＡを注入した個体のサンプルの結果を示す。   FIG. 6 is a graph showing abnormal expression of a neurogenesis marker in a DYRK1A overexpression model (mRNA (+) lane) in Xenopus early embryos and the effect of restoring abnormal expression by administration of a benzothiazole derivative. The mRNA (−) lane shows the expression level of the neurogenesis marker in normal individuals. First, Xenopus DYRK1A synthetic mRNA was injected into the Xenopus 8-cell embryonic dorsal blastomere, and at the same time, Production Example 001 (10 μM), Production Example 019 (2.5 μM), or as a control Only the solvent (DMSO) was added to the breeding water, and the recovery effect by the benzothiazole derivative was examined. After injecting DYRK1A synthetic mRNA, embryos were cultured in the dark at 23 ° C. for 1 day, the head was harvested at stage 17, the RNA was recovered, and expression analysis by RT-PCR was performed. The expression levels of mRNA were analyzed for NCAM, Krox20, En2, Otx2 which are head nerve markers of general nerves, hindbrain, midbrain, and forebrain, and Lhx2 and Rx1 which are eye formation markers. Note that xOdc was used as an internal standard. In the same figure, the lanes indicated by − and + show the results of the control of the individual to which DYRK1A mRNA was not added and the sample of the individual injected with DYRK1A mRNA, respectively.

図６に示すとおり、ＤＹＲＫ１Ａの過剰発現により、神経全般（ＮＣＡＭ）、後脳（Ｋｒｏｘ２０）、中脳（Ｅｎ２）、及び前脳（Ｏｔｘ２）マーカー、並びに、眼形成マーカー（Ｌｈｘ２及びＲｘ１）の発現低下が認められた（図６、ＤＭＳＯのｍＲＮＡ（＋）レーン）。そして、これらの神経マーカーの発現低下は、製造例００１及び０１９のベンゾチアゾール誘導体を投与することによって改善された（図６、製造例００１及び０１９のｍＲＮＡ（＋）レーン）。したがって、ベンゾチアゾール誘導体投与によりＤＹＲＫ１Ａ過剰発現に起因する発生異常を個体レベルで抑制できることが、分子レベルで示された。   As shown in FIG. 6, expression of DYRK1A overexpresses neural (NCAM), hindbrain (Krox20), midbrain (En2), and forebrain (Otx2) markers, and ocular formation markers (Lhx2 and Rx1). A decrease was observed (FIG. 6, DMSO mRNA (+) lane). The decrease in expression of these neuronal markers was improved by administering the benzothiazole derivatives of Production Examples 001 and 019 (FIG. 6, mRNA (+) lanes of Production Examples 001 and 019). Therefore, it was shown at the molecular level that developmental abnormalities caused by DYRK1A overexpression can be suppressed at the individual level by administration of the benzothiazole derivative.

［ＤＹＲＫ１Ｂ発現悪性腫瘍に対する増殖抑制効果／抗がん剤作用増強効果］
ＤＹＲＫ１Ｂを高発現する代表的な悪性腫瘍である横紋筋肉腫細胞株（ＲＭＳ−ＹＭ）を用いて、ベンゾチアゾール誘導体の細胞増殖抑制効果、及び、抗がん剤作用増強効果を確認した。[Proliferation inhibitory effect on DYRK1B-expressing malignant tumor / anticancer agent action enhancing effect]
Using a rhabdomyosarcoma cell line (RMS-YM), which is a typical malignant tumor that highly expresses DYRK1B, the cell growth inhibitory effect and the anticancer agent enhancing effect of the benzothiazole derivative were confirmed.

まず、横紋筋肉腫細胞株ＲＭＳ−ＹＭ（理研バイオリソースセンター ＲＣＢ１６９５）をＲＰＭＩ１６４０（１０％ＦＢＳ、１００μＭ ｎｏｎｅ ｅｓｓｅｎｓｉａｌ ａｍｉｎｏ ａｃｉｄ、１０ｍＭ ＨＥＰＥＳ、ペニシリンストレプトマイシン添加）培地にて培養し、ベンゾチアゾール誘導体（製造例０１９、００９）及び上記式（Ｉ）に属さない下記化学式で表される化合物（以下、「陰性対照化合物」ともいう）を終濃度２０、５０、１００μＭで添加し、横紋筋肉腫細胞株ＲＭＳ−ＹＭに対するベンゾチアゾール誘導体の細胞増殖抑制効果を検討した。また、ベンゾチアゾール誘導体非添加群をコントロールとして、ベンゾチアゾール誘導体の溶媒であるＤＭＳＯを同じ最終濃度となるよう添加した。ベンゾチアゾール誘導体及び／又はＤＭＳＯ添加後、細胞を２日間培養した。培養後、ＷＳＴ−８（２−（ｍｅｔｈｏｘｙ−４−ｎｉｔｒｏｐｈｅｎｙｌ）−５−（２．４−ｄｉｓｕｌｆｏｐｈｅｎｙｌ）−２Ｈ−ｔｅｔｒａｚｏｌｉｕｍ， ｍｏｎｏｓｏｄｉｕｍ ｓａｌｔ）（Ｎａｃａｌａｉ）法により細胞生存率を測定し、ベンゾチアゾール誘導体非添加群を１００％としたときの細胞生存率を算出した。その結果を図７ａに示す。
First, rhabdomyosarcoma cell line RMS-YM (RIKEN BioResource Center RCB1695) was cultured in RPMI1640 (10% FBS, 100 μM none essential amino acid, 10 mM HEPES, penicillin streptomycin added) medium, and benzothiazole derivative (Production Example 019). , 009) and a compound represented by the following chemical formula not belonging to the above formula (I) (hereinafter also referred to as “negative control compound”) at a final concentration of 20, 50, 100 μM, rhabdomyosarcoma cell line RMS- The cell growth inhibitory effect of benzothiazole derivatives on YM was examined. Moreover, DMSO which is a solvent of a benzothiazole derivative was added so as to have the same final concentration, with the benzothiazole derivative non-added group as a control. After addition of the benzothiazole derivative and / or DMSO, the cells were cultured for 2 days. After culturing, the cell viability was measured by the WST-8 (2- (methoxy-4-nitrophenyl) -5- (2.4-disulphophenyl) -2H-tetrazolium, monosodium salt) (Nacalai) method, and the benzothiazole derivative non- The cell viability when the addition group was 100% was calculated. The result is shown in FIG.

図７ａは、横紋筋肉腫細胞株ＲＭＳ−ＹＭに対するベンゾチアゾール誘導体の細胞増殖抑制効果を調べた結果のグラフである。図７ａに示すとおり、ベンゾチアゾール誘導体のみを使用した場合において濃度依存的な悪性腫瘍の細胞増殖抑制効果が確認できた。   FIG. 7 a is a graph showing the results of examining the cell growth inhibitory effect of a benzothiazole derivative on rhabdomyosarcoma cell line RMS-YM. As shown in FIG. 7a, the concentration-dependent cell growth inhibitory effect of malignant tumor was confirmed when only the benzothiazole derivative was used.

つぎに、横紋筋肉腫細胞株ＲＭＳ−ＹＭを培養し、抗がん剤非添加・ベンゾチアゾール添加群、抗がん剤非添加・ベンゾチアゾール非添加群、抗がん剤添加・ベンゾチアゾール非添加群、抗がん剤添加・ベンゾチアゾール添加群にて横紋筋肉腫細胞株ＲＭＳ−ＹＭに対するベンゾチアゾール誘導体の細胞増殖抑制効果を検討した。抗がん剤非添加・ベンゾチアゾール添加群には、終濃度２０μＭのベンゾチアゾール誘導体（製造例０１９、００９）又は陰性対照化合物を添加した。抗がん剤非添加・ベンゾチアゾール非添加群には、ベンゾチアゾールの溶媒であるＤＭＳＯを添加した。抗がん剤を添加した群（抗がん剤添加・ベンゾチアゾール非添加群及び抗がん剤添加・ベンゾチアゾール添加群）には、ビンクリスチン（ｖｉｎｃｒｉｓｔｉｎｅ）を終濃度０．５、３ｎＭ、又はアクチノマイシンＤ（Ａｃｔｉｎｏｍｙｃｉｎ Ｄ）を終濃度５ｎＭで添加した。抗がん剤添加・ベンゾチアゾール添加群には、抗がん剤に加えて、終濃度２０μＭのベンゾチアゾール誘導体（製造例０１９、００９）又は陰性対照化合物を添加した。抗がん剤及び／又はベンゾチアゾール誘導体及び／又は陰性対照化合物及び／又はＤＭＳＯ添加後、細胞を２日間培養した。培養後、ＷＳＴ−８法により細胞生存率を測定し、抗がん剤非添加・ベンゾチアゾール非添加群を１００％としたときの各群における細胞生存率を算出した。その結果を図７ｂ及びｃに示す。   Next, the rhabdomyosarcoma cell line RMS-YM is cultured, and the anticancer agent-free group, benzothiazole-added group, the anticancer agent-free group, the benzothiazole-free group, the anticancer agent-added group, benzothiazole-free The cell growth inhibitory effect of the benzothiazole derivative on the rhabdomyosarcoma cell line RMS-YM was examined in the addition group, the anticancer agent addition / benzothiazole addition group. A benzothiazole derivative (Production Examples 019, 009) having a final concentration of 20 μM or a negative control compound was added to the anti-cancer agent-free / benzothiazole-added group. DMSO, which is a solvent for benzothiazole, was added to the group to which no anticancer agent was added and no benzothiazole was added. In the group to which the anticancer agent is added (the anticancer agent added / benzothiazole non-added group and the anticancer agent added / benzothiazole added group), the final concentration of vincristine (vincristine) is 0.5, 3 nM, or actino Mycin D (Actinomycin D) was added at a final concentration of 5 nM. In addition to the anticancer agent, a benzothiazole derivative (Production Examples 019, 009) having a final concentration of 20 μM or a negative control compound was added to the anticancer agent addition / benzothiazole addition group. Cells were cultured for 2 days after addition of anticancer agent and / or benzothiazole derivative and / or negative control compound and / or DMSO. After the culture, the cell viability was measured by the WST-8 method, and the cell viability in each group was calculated when the anticancer agent-free / benzothiazole-free group was taken as 100%. The results are shown in FIGS. 7b and c.

図７ｂ及びｃは、横紋筋肉腫細胞株ＲＭＳ−ＹＭに対するベンゾチアゾール誘導体と抗がん剤（ビンクリスチン：図７ｂ、アクチノマイシンＤ：図７ｃ）との併用の効果を調べた結果のグラフである。図７ｂに示すとおり、ビンクリスチン３ｎＭと各種ベンゾチアゾール誘導体の併用効果を検討すると、ビンクリスチン単独使用時（６５．６％）に比べて細胞生存率が低下し、製造例０１９及び００９ではそれぞれ３５．１％、２６．７％まで低下した。また、図７ｃに示すとおり、同様の効果がアクチノマイシンＤと各種ベンゾチアゾール誘導体の併用時においても認められた。アクチノマイシンＤ ５ｎＭで併用効果を検討すると、単独使用時（６６．４％）に比べて細胞生存率が低下し、製造例０１９及び００９ではそれぞれ３３．３％、３３．２％まで低下した。したがって、ベンゾチアゾール誘導体による抗がん剤作用の増強効果が確認できた。   FIGS. 7b and c are graphs showing the results of examining the effect of combined use of a benzothiazole derivative and an anticancer drug (vincristine: FIG. 7b, actinomycin D: FIG. 7c) on rhabdomyosarcoma cell line RMS-YM. . As shown in FIG. 7b, when the combined effect of vincristine 3nM and various benzothiazole derivatives was examined, the cell viability decreased compared to when vincristine was used alone (65.6%), and in Production Examples 019 and 009, 35.1 respectively. %, 26.7%. In addition, as shown in FIG. 7c, the same effect was observed when actinomycin D was used in combination with various benzothiazole derivatives. When the combined effect was examined with actinomycin D 5 nM, the cell viability decreased compared to the single use (66.4%), and decreased to 33.3% and 33.2% in Production Examples 019 and 009, respectively. Therefore, the enhancement effect of the anticancer agent action by the benzothiazole derivative was confirmed.

上記増殖抑制効果及び抗がん剤作用増強効果が、上記以外のＤＹＲＫ１Ｂが高発現する癌細胞に対しても発現するかどうかを確認するため、その他のＤＹＲＫ１Ｂ高発現癌細胞株（Ｐａｎｃ−１）を用いて上述と同じように実験を行った。   In order to confirm whether the above-mentioned growth inhibitory effect and anti-cancer drug enhancing effect are also expressed on cancer cells in which DYRK1B other than the above is highly expressed, other DYRK1B-high-expressing cancer cell lines (Panc-1) The experiment was conducted in the same manner as described above.

ＤＹＲＫ１Ｂを発現するヒト膵管癌細胞株であるＰａｎｃ−１（ＡＴＣＣ Ｎｏ．ＣＲＬ−１４６９）をＲＰＭＩ１６４０（１０％ＦＢＳ、ペニシリンストレプトマイシン添加）培地にて培養し、抗がん剤非添加群・ベンゾチアゾール非添加群には、ベンゾチアゾールの溶媒であるＤＭＳＯを添加した。抗がん剤非添加・ベンゾチアゾール添加群には、終濃度２０μＭのベンゾチアゾール誘導体（製造例００１、０１９、００９）及び陰性対照化合物を添加した。抗がん剤を添加した群には、ゲムシタビン（ｇｅｍｃｉｔａｂｉｎｅ）をＰａｎｃ−１細胞株に対して終濃度５ｎＭで添加した。抗がん剤添加群・ベンゾチアゾール添加群には、抗がん剤に加えて、終濃度２０μＭのベンゾチアゾール誘導体（製造例００１、０１９、００９）又は陰性対照化合物を添加した。抗がん剤及び／又はベンゾチアゾール誘導体及び／又は陰性対照化合物及び／又はＤＭＳＯ添加後、細胞を３日間培養した。培養後、ＷＳＴ−８法により細胞生存率を測定し、抗がん剤非添加群・ベンゾチアゾール非添加群を１００％としたときの各群における細胞生存率を算出した。その結果を図８に示す。   Panc-1 (ATCC No. CRL-1469), a human pancreatic duct cancer cell line that expresses DYRK1B, is cultured in RPMI1640 (10% FBS, penicillin streptomycin added) medium, anticancer agent non-added group / benzothiazole non-added group DMSO, which is a solvent for benzothiazole, was added to the addition group. A benzothiazole derivative (Production Examples 001, 019, 009) having a final concentration of 20 μM and a negative control compound were added to the anticancer agent-free / benzothiazole-added group. To the group to which the anticancer agent was added, gemcitabine was added at a final concentration of 5 nM to the Panc-1 cell line. In addition to the anticancer agent, a benzothiazole derivative (Production Examples 001, 019, 009) having a final concentration of 20 μM or a negative control compound was added to the anticancer agent addition group and the benzothiazole addition group. Cells were cultured for 3 days after addition of anticancer agent and / or benzothiazole derivative and / or negative control compound and / or DMSO. After the culture, the cell viability was measured by the WST-8 method, and the cell viability in each group was calculated when the anticancer agent-free group and benzothiazole-free group were taken as 100%. The result is shown in FIG.

図８に示すとおり、Ｐａｎｃ−１細胞株のゲムシタビン５ｎＭ単独投与の場合、細胞生存率は５８．４％であった。一方、ベンゾチアゾール誘導体をゲムシタビンと併用すると、製造例００１、０１９及び００９の場合、それぞれ、４１．４％、３６．３％及び３７．６％まで低下した。   As shown in FIG. 8, when the Panc-1 cell line was administered with gemcitabine 5 nM alone, the cell survival rate was 58.4%. On the other hand, when the benzothiazole derivative was used in combination with gemcitabine, in the case of Production Examples 001, 019 and 009, they decreased to 41.4%, 36.3% and 37.6%, respectively.

これらの実験結果から、ベンゾチアゾール誘導体がＤＹＲＫ１Ｂを発現する悪性細胞に対して選択的に他の抗がん剤の作用を増強し、増殖抑制効果を持つことが示唆された。   From these experimental results, it was suggested that the benzothiazole derivative selectively enhances the action of other anticancer agents against malignant cells expressing DYRK1B and has a growth inhibitory effect.

Claims (26)

ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの蛋白質リン酸化活性を阻害する化合物を有効成分とする、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴う疾病の治療又は予防のための医薬組成物。 Pharmaceutical composition for the treatment or prevention of a disease associated with at least one enhanced activity of DYRK1A, DYRK1B, and DYRK2, comprising a compound that inhibits at least one protein phosphorylation activity of DYRK1A, DYRK1B, and DYRK2 as an active ingredient object. 前記疾病が、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性阻害が治療又は予防に有効な疾病である、請求項１記載の医薬組成物。 The pharmaceutical composition according to claim 1, wherein the disease is a disease in which inhibition of at least one activity of DYRK1A, DYRK1B, and DYRK2 is effective for treatment or prevention. 前記疾病が、精神神経疾患及び悪性腫瘍を含む、請求項１又は２に記載の医薬組成物。 The pharmaceutical composition according to claim 1 or 2, wherein the disease comprises a neuropsychiatric disorder and a malignant tumor. 前記精神神経疾患が、ダウン症候群及びアルツハイマー病を含む、請求項３記載の医薬組成物。 The pharmaceutical composition according to claim 3, wherein the neuropsychiatric disorder includes Down's syndrome and Alzheimer's disease. 前記悪性腫瘍が、膵管がん及び横紋筋肉腫を含む、請求項３記載の医薬組成物。 The pharmaceutical composition according to claim 3, wherein the malignant tumor includes pancreatic duct cancer and rhabdomyosarcoma. 前記化合物が、ベンゾチアゾール誘導体である、請求項１から５のいずれかに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 5, wherein the compound is a benzothiazole derivative. ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴う疾病の治療又は予防のための医薬組成物であって、
有効成分として下記式（Ｉ）の化合物、又は、その製薬上許容される塩若しくは溶媒和物を含む、医薬組成物。

［上記式（Ｉ）において、
Ａは、Ｓ（硫黄原子）又はＳｅ（セレニウム原子）であり、
Ｘは、ハロゲン置換されてもよいＣ１−Ｃ１０アルキル基であり、
Ｙは、Ｈ（水素原子）、ハロゲン置換されてもよいＣ１−Ｃ１０アルキル基、Ｃ２−Ｃ６アルケニル基、−Ｒ１ＯＨ、−ＣＯＯＲ２、及び、−Ｒ１ＯＣＯＲ３から選択され、
Ｚは、ハロゲン置換されてもよいＣ１−Ｃ１０アルキル基、ハロゲン、−ＮＨＣＯＲ２、−ＯＲ２、及び、−ＯＣＯＲ３から選択され、
Ｒ１は、Ｃ１−Ｃ１０アルキレン基であり、
Ｒ２は、Ｈ、又は、Ｃ１−Ｃ１０アルキル基であり、
Ｒ３は、Ｃ１−Ｃ１０アルキル基、Ｃ１−Ｃ３アルコキシ基若しくはＣ１−Ｃ６アルキル基で置換されてもよいアリール基、及び、Ｃ１−Ｃ６アルキル基で置換されてもよいへテロアリール基から選択される。］A pharmaceutical composition for treating or preventing a disease associated with at least one increased activity of DYRK1A, DYRK1B, and DYRK2, comprising:
A pharmaceutical composition comprising a compound of the following formula (I) or a pharmaceutically acceptable salt or solvate thereof as an active ingredient.

[In the above formula (I),
A is S (sulfur atom) or Se (selenium atom),
X is a halogen-substituted C 1 may be -C 10 alkyl group,
Y is selected from H (hydrogen atom), C 1 -C 10 alkyl group optionally substituted by halogen, C 2 -C 6 alkenyl group, —R 1 OH, —COOR 2 , and —R 1 OCOR 3. ,
Z is a halogen substituted may be C 1 -C 10 alkyl group, halogen, -NHCOR 2, -OR 2, and is selected from -OCOR 3,
R 1 is a C 1 -C 10 alkylene group,
R 2 is H or a C 1 -C 10 alkyl group,
R 3 may be substituted with a C 1 -C 10 alkyl group, an aryl group that may be substituted with a C 1 -C 3 alkoxy group or a C 1 -C 6 alkyl group, and a C 1 -C 6 alkyl group. Selected from good heteroaryl groups. ] 前記疾病が、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性阻害が治療又は予防に有効な疾病である、請求項７記載の医薬組成物。 The pharmaceutical composition according to claim 7, wherein the disease is a disease in which inhibition of the activity of at least one of DYRK1A, DYRK1B, and DYRK2 is effective for treatment or prevention. 前記疾病が、精神神経疾患及び悪性腫瘍を含む、請求項７又は８に記載の医薬組成物。 The pharmaceutical composition according to claim 7 or 8, wherein the disease includes a neuropsychiatric disorder and a malignant tumor. 前記精神神経疾患が、ダウン症候群及びアルツハイマー病を含む、請求項９記載の医薬組成物。 The pharmaceutical composition according to claim 9, wherein the neuropsychiatric disorder includes Down's syndrome and Alzheimer's disease. 前記悪性腫瘍が、膵管がん及び横紋筋肉腫を含む、請求項９記載の医薬組成物。 The pharmaceutical composition according to claim 9, wherein the malignant tumor includes pancreatic duct cancer and rhabdomyosarcoma. ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴う疾病の治療又は予防の方法であって、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの蛋白質リン酸化活性を阻害する化合物を有効成分とする医薬組成物を対象に有効量投与することを含む、治療又は予防方法。 DYRK1A, DYRK1B, and DYRK2, a method for treating or preventing a disease associated with at least one increased activity, comprising as an active ingredient a compound that inhibits at least one protein phosphorylation activity of DYRK1A, DYRK1B, and DYRK2 A therapeutic or prophylactic method comprising administering an effective amount of a pharmaceutical composition to a subject. 前記対象又はその一部においてＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つが活性亢進していることを確認することを含む、請求項１２記載の治療又は予防方法。 The method for treatment or prevention according to claim 12, comprising confirming that at least one of DYRK1A, DYRK1B, and DYRK2 is activated in the subject or a part thereof. 前記疾病が、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性阻害が治療又は予防に有効な疾病である、請求項１２又は１３記載の治療又は予防方法。 The method of treatment or prevention according to claim 12 or 13, wherein the disease is a disease in which inhibition of the activity of at least one of DYRK1A, DYRK1B, and DYRK2 is effective for treatment or prevention. 前記疾病が、精神神経疾患及び悪性腫瘍を含む、請求項１２から１４のいずれかに記載の治療又は予防方法。 The treatment or prevention method according to any one of claims 12 to 14, wherein the disease includes a neuropsychiatric disorder and a malignant tumor. 前記精神神経疾患が、ダウン症候群及びアルツハイマー病を含む、請求項１５記載の治療又は予防方法。 The method for treatment or prevention according to claim 15, wherein the neuropsychiatric disorder includes Down's syndrome and Alzheimer's disease. 前記悪性腫瘍が、膵管がん及び横紋筋肉腫を含む、請求項１５記載の治療又は予防方法。 The method for treatment or prevention according to claim 15, wherein the malignant tumor includes pancreatic duct cancer and rhabdomyosarcoma. 前記化合物が、ベンゾチアゾール誘導体である、請求項１２から１７のいずれかに記載の治療又は予防方法。 The method for treatment or prevention according to any one of claims 12 to 17, wherein the compound is a benzothiazole derivative. ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴う疾病の治療又は予防の方法であって、請求項７から１１のいずれかに記載の医薬組成物を対象に有効量投与することを含む、治療又は予防方法。 A method for treating or preventing a disease associated with at least one increased activity of DYRK1A, DYRK1B, and DYRK2, comprising administering an effective amount of the pharmaceutical composition according to any one of claims 7 to 11 to a subject , Treatment or prevention methods. 前記対象又はその一部においてＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つが活性亢進していることを確認することを含む、請求項１９記載の治療又は予防方法。 The method for treatment or prevention according to claim 19, comprising confirming that at least one of DYRK1A, DYRK1B, and DYRK2 is activated in the subject or a part thereof. 前記疾病が、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性阻害が治療又は予防に有効な疾病である、請求項１９又は２０記載の治療又は予防方法。 21. The treatment or prevention method according to claim 19 or 20, wherein the disease is a disease in which inhibition of at least one activity of DYRK1A, DYRK1B, and DYRK2 is effective for treatment or prevention. 前記疾病が、精神神経疾患及び悪性腫瘍を含む、請求項１９から２１のいずれかに記載の治療又は予防方法。 The method for treatment or prevention according to any one of claims 19 to 21, wherein the disease includes a neuropsychiatric disorder and a malignant tumor. 前記精神神経疾患が、ダウン症候群及びアルツハイマー病を含む、請求項２２記載の治療又は予防方法。 The method for treatment or prevention according to claim 22, wherein the neuropsychiatric disorder includes Down's syndrome and Alzheimer's disease. 前記悪性腫瘍が、膵管がん及び横紋筋肉腫を含む、請求項２２記載の治療又は予防方法。 The method for treatment or prevention according to claim 22, wherein the malignant tumor includes pancreatic duct cancer and rhabdomyosarcoma. 下記式（Ｉ）化合物、又は、その製薬上許容される塩若しくは溶媒和物。

［上記式（Ｉ）において、
Ａは、Ｓ（硫黄原子）又はＳｅ（セレニウム原子）であり、
Ｘは、ハロゲン置換されてもよいＣ１−Ｃ１０アルキル基であり、
Ｙは、Ｈ（水素原子）、ハロゲン置換されてもよいＣ１−Ｃ１０アルキル基、Ｃ２−Ｃ６アルケニル基、−Ｒ１ＯＨ、−ＣＯＯＲ２、及び、−Ｒ１ＯＣＯＲ３から選択され、
Ｚは、ハロゲン置換されてもよいＣ１−Ｃ１０アルキル基、ハロゲン、−ＮＨＣＯＲ２、−ＯＲ２、及び、−ＯＣＯＲ３から選択され、
Ｒ１は、Ｃ１−Ｃ１０アルキレン基であり、
Ｒ２は、Ｈ、又は、Ｃ１−Ｃ１０アルキル基であり、
Ｒ３は、Ｃ１−Ｃ１０アルキル基、Ｃ１−Ｃ３アルコキシ基若しくはＣ１−Ｃ６アルキル基で置換されてもよいアリール基、及び、Ｃ１−Ｃ６アルキル基で置換されてもよいへテロアリール基から選択され、
但し、
ＡがＳかつＸ及びＹがメチル基の場合、Ｚは、フッ素原子（Ｆ）、ヒドロキシ基、メトキシ基、エトキシ基、及び、アセトキシ基を除く上記範囲から選択され、
ＡがＳかつＸがエチル基かつＹがメチル基の場合、Ｚは、メトキシ基を除く上記範囲から選択される。］A compound of the following formula (I), or a pharmaceutically acceptable salt or solvate thereof.

[In the above formula (I),
A is S (sulfur atom) or Se (selenium atom),
X is a halogen-substituted C 1 may be -C 10 alkyl group,
Y is selected from H (hydrogen atom), C 1 -C 10 alkyl group optionally substituted by halogen, C 2 -C 6 alkenyl group, —R 1 OH, —COOR 2 , and —R 1 OCOR 3. ,
Z is a halogen substituted may be C 1 -C 10 alkyl group, halogen, -NHCOR 2, -OR 2, and is selected from -OCOR 3,
R 1 is a C 1 -C 10 alkylene group,
R 2 is H or a C 1 -C 10 alkyl group,
R 3 may be substituted with a C 1 -C 10 alkyl group, an aryl group that may be substituted with a C 1 -C 3 alkoxy group or a C 1 -C 6 alkyl group, and a C 1 -C 6 alkyl group. Selected from good heteroaryl groups,
However,
When A is S and X and Y are methyl groups, Z is selected from the above range excluding the fluorine atom (F), hydroxy group, methoxy group, ethoxy group, and acetoxy group,
When A is S and X is an ethyl group and Y is a methyl group, Z is selected from the above range excluding the methoxy group. ] モデル動物の初期胚にＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つのｍＲＮＡを誘導して発生異常を起こしうる状態とすること、
前記初期胚を候補化合物と接触させること、及び、
候補化合物を接触させなかった場合と比較して候補化合物による発生異常の抑制効果を評価することを含む、ＤＹＲＫ１Ａ、ＤＹＲＫ１Ｂ、及び、ＤＹＲＫ２の少なくとも１つの活性亢進を伴う疾病の治療又は予防のための医薬組成物の有効成分のスクリーニング方法。Inducing at least one mRNA of DYRK1A, DYRK1B, and DYRK2 to an early embryo of a model animal so as to cause developmental abnormality;
Contacting the early embryo with a candidate compound; and
For the treatment or prevention of a disease associated with an increased activity of at least one of DYRK1A, DYRK1B, and DYRK2, including evaluating the inhibitory effect on developmental abnormalities caused by the candidate compound as compared with the case where the candidate compound is not contacted A method for screening an active ingredient of a pharmaceutical composition.
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