JP2022188761A - Platelet production method employing nitrogen atom-containing heterocyclic compound - Google Patents

Abstract

To provide a method of promoting production of platelets from proplatelets such as megakaryocytes in vitro.SOLUTION: A platelet production promotor comprises a compound represented by general formula [I], where each symbol is as described in the specification, or a salt thereof.SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to a method for promoting platelet production from platelet progenitor cells such as megakaryocyte cells in vitro using a nitrogen atom-containing heterocyclic compound.

Platelet preparations are administered to patients with bleeding tendencies associated with massive bleeding during surgery or injury, or bleeding tendencies associated with decreased platelets after anticancer drug therapy, for the purpose of treatment of symptoms and prevention of unexpected bleeding. be.
At present, platelet preparations rely on blood donations, but platelet preparations have a very short shelf life of about 4 days. Furthermore, it is predicted that platelet products will be in short supply in the near future if blood supply is dependent on blood donation alone due to a decrease in the blood donor population.
To meet these needs, methods of producing platelets in vitro are being investigated.
As an in vitro platelet production method, a method of differentiating various stem cells to obtain megakaryocyte cells and culturing them to release platelets has been developed. For example, Takayama et al. succeeded in inducing the differentiation of human ES cells into megakaryocyte cells and platelets (Non-Patent Document 1).
Also, as a method for producing platelets from hematopoietic progenitor cells in vitro, there is a method of culturing in the presence of an aromatic hydrocarbon receptor antagonist and a thrombopoietin (TPO) or ROCK (Rho-associated coiled-coil forming kinase) inhibitor. It has been proposed (Patent Documents 1, 2, 3, Non-Patent Documents 2, 3, 4).
Indolyl acrylamide compounds have been disclosed as transcription factor inhibitors (Patent Document 4, Non-Patent Document 5)

WO2014/138485 WO2016/204256 WO2010/059401 WO2019/167973 publication

Takayama et al., Blood, 111, 5298 (2008) Boitano et al., Science, 329, 1345 (2010) Strassel et al., Blood, 127, 2231 (2016) Ito et al., Cell, 174, 636 (2018) Perron et al., J. Biol. Chem., 293, 8285 (2018)

An object of the present invention is to provide a method for promoting platelet production from platelet progenitor cells such as megakaryocyte cells in vitro using a nitrogen atom-containing heterocyclic compound or a salt thereof.

Means for Solving the Problems As a result of extensive studies to solve the above problems, the present inventors have found a method for promoting platelet production by using a nitrogen atom-containing heterocyclic compound represented by the following formula [I]. was completed.

［式中、
R¹¹は、水素原子、ハロゲン、-C_1-6アルキルまたは-O-C_1-6アルキルであり、
R²は、水素原子または-C_1-6アルキルであり、
R³は、ハロゲン、-Q_k-(C_1-6アルキル)_m-Q_p-R³¹、置換基を有していてもよいフェニルまたはフリル、チエニル、オキサゾリル、チアゾリル、ピラゾリル、ピリジル、ピラジル、ピリダジルまたはピリミジルからなる群から選択される置換基を有していてもよいヘテロアリールであり、
R³¹は、-C_1-6アルキルまたは-C_3-8シクロアルキルであり、
Qは各々独立して、同一または異なって酸素原子、硫黄原子、-C(=O)-O-または-NH-であり、
k、m、pは、0または1であり、
nは、0、1または2であり、nが2のときは、R³は各々独立して、同一または異なる置換基である
Wは、炭素原子または窒素原子であり、
Xは、炭素原子、窒素原子またはN-R¹²であり、
Yは、炭素原子または窒素原子であり、
Zは、各々独立して、同一または異なって窒素原子またはC-Hであり、
ただし、XとYは同時に炭素原子ではない
R¹²は、水素原子、-C_1-6アルキル、-C_1-6アルキル-O-C_1-6アルキル、-C(=O)-C_1-6アルキル、-C(=O)-アリールまたは-C(=O)-O-C_1-6アルキルであり、
環Aは、アリールまたはヘテロアリールであり、
---は、単結合または二重結合であり、
ただし、XがN-H、Yが炭素原子、全てのZがCHのとき、環Aは2-(-O-C_1-6アルキル)フェニルまたは2,5-ジ(-O-C_1-6アルキル)フェニルを除く]
で表される化合物またはその塩を含む血小板産生促進剤。
［１－２］
一般式[I]中、

［式中、R¹¹、W、X、Y、Zおよび---は上記定義と同じ］
である［１－１］に記載の促進剤。
［１－３］ 一般式[I]中、

［R³およびnは上記定義と同じ］
である［１－１］に記載の促進剤。
［１－４］ 一般式[I]中、環Aにおけるヘテロアリールがフラン、チオフェン、ピリジンおよびキノリンから選ばれる、
［１－１］に記載の促進剤。
［１－５］ 一般式［Ｉ］中、

［式中、Vは同一または異なって、各々独立して窒素原子またはC-Hであり、R⁴は水素原子、ハロゲン、-C_1-6アルキルまたは-O-C_1-6アルキルである］
である、［１－１］に記載の促進剤。
［１－６］ 一般式[Ia]

[式中、R¹¹は、水素原子、ハロゲン、-C_1-6アルキルまたは-O-C_1-6アルキルであり、

はピリジルベンゼン、（ハロゲン、-C_1-6アルキルまたは-O-C_1-6アルキルで置換されていてもよいピリミジル）ベンゼン、フェニルチオフェン、ピリジルチオフェンまたはピリミジルチオフェンである]
で表される［１－１］に記載の促進剤。
［１－７］ 化合物が下記から選ばれる［１－１］に記載の促進剤

［１－８］ 芳香族炭化水素受容体阻害剤と併用するための、［１－１］から［１－７］のいずれかに記載の促進剤。
［２］ ［１－１］から［１－７］のいずれかに記載の化合物またはその塩を用いる血小板産生方法。
［３］ 血小板産生を促進させるための、［１－１］から［１－７］のいずれかに記載の化合物またはその塩の使用。
［４］ ［１－１］から［１－７］のいずれかに記載の化合物またはその塩の存在下、血小板前駆細胞を培養することを含む、血小板産生を促進させるための方法。
［５］ ［１－１］から［１－７］のいずれかに記載の化合物またはその塩の存在下、血小板前駆細胞を培養することを含む、血小板の製造方法。
［６］ 血小板産生を促進させるための血小板前駆細胞の培養方法であって、［１－１］から［１－７］のいずれかに記載の化合物またはその塩の存在下、血小板前駆細胞を培養する方法。 That is, the present invention includes the following aspects.
[1-1] General formula [I]:

[In the formula,
R ¹¹ is a hydrogen atom, halogen, -C _1-6 alkyl or -OC _1-6 alkyl,
^R2 is a hydrogen atom or _-C1-6 alkyl,
R ³ is halogen, -Q _k -(C _1-6 alkyl) _m -Q _p -R ³¹ , optionally substituted phenyl or furyl, thienyl, oxazolyl, thiazolyl, pyrazolyl, pyridyl, pyrazyl, heteroaryl optionally having a substituent selected from the group consisting of pyridazyl or pyrimidyl,
R ³¹ is -C _1-6 alkyl or -C _3-8 cycloalkyl,
each Q is independently the same or different and is an oxygen atom, a sulfur atom, -C(=O)-O- or -NH-;
k, m, p are 0 or 1,
n is 0, 1 or 2, and when n is 2, each R ³ is independently the same or different substituent
W is a carbon or nitrogen atom,
X is a carbon atom, a nitrogen atom or ^NR12 ,
Y is a carbon or nitrogen atom,
each Z is independently the same or different and is a nitrogen atom or CH;
However, X and Y are not carbon atoms at the same time
R ¹² is a hydrogen atom, -C _1-6 alkyl, -C _1-6 alkyl-OC _1-6 alkyl, -C(=O)-C _1-6 alkyl, -C(=O)-aryl or - C(=O)-OC _1-6 alkyl,
Ring A is aryl or heteroaryl,
--- is a single or double bond,
provided that when X is NH, Y is a carbon atom, and all Zs are CH, ring A excludes 2-(-OC _1-6 alkyl)phenyl or 2,5-di(-OC _1-6 alkyl)phenyl ]
Platelet production promoter comprising a compound represented by or a salt thereof.
[1-2]
In general formula [I],

[Wherein, R ¹¹ , W, X, Y, Z and --- are the same as defined above]
The accelerator according to [1-1].
[1-3] In general formula [I],

[R ³ and n are the same as defined above]
The accelerator according to [1-1].
[1-4] In general formula [I], heteroaryl in ring A is selected from furan, thiophene, pyridine and quinoline,
The accelerator according to [1-1].
[1-5] In general formula [I],

[wherein V is the same or different and each independently is a nitrogen atom or CH, and ^R4 is a hydrogen atom, halogen, -C1-6 _alkyl or -OC1-6 _alkyl ]
The accelerator according to [1-1].
[1-6] general formula [Ia]

[wherein R ¹¹ is a hydrogen atom, halogen, -C _1-6 alkyl or -OC _1-6 alkyl,

is pyridylbenzene, (pyrimidyl optionally substituted by halogen, _-C1-6alkyl or _-OC1-6alkyl )benzene, phenylthiophene, pyridylthiophene or pyrimidylthiophene]
Accelerator according to [1-1] represented by.
[1-7] The accelerator according to [1-1], wherein the compound is selected from the following

[1-8] The accelerator according to any one of [1-1] to [1-7], which is used in combination with an aromatic hydrocarbon receptor inhibitor.
[2] A method for producing platelets using the compound or salt thereof according to any one of [1-1] to [1-7].
[3] Use of the compound or a salt thereof according to any one of [1-1] to [1-7] for promoting platelet production.
[4] A method for promoting platelet production, comprising culturing platelet progenitor cells in the presence of the compound or salt thereof according to any one of [1-1] to [1-7].
[5] A method for producing platelets, comprising culturing platelet progenitor cells in the presence of the compound or salt thereof according to any one of [1-1] to [1-7].
[6] A method for culturing platelet progenitor cells for promoting platelet production, comprising culturing platelet progenitor cells in the presence of the compound or a salt thereof according to any one of [1-1] to [1-7]. how to.

INDUSTRIAL APPLICABILITY The method according to the present invention has excellent efficacy in promoting platelet production from platelet progenitor cells in vitro.

Phrases and terms used herein are detailed below.

As used herein, "halogen" is fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine. Fluorine or chlorine is more preferred.

As used herein, "C _1-6 alkyl" is straight or branched chain alkyl having 1 to 6 carbon atoms (C _1-6 ), and specific examples thereof include methyl, ethyl, n-propyl, Examples include isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, 3-methylpentyl and the like.
"C _1-6 alkyl" also includes C _1-6 alkyl in which 1 to 7 hydrogen atoms are replaced with deuterium atoms.

As used herein, "C _3-8 cycloalkyl" is cycloalkyl having 3 to 8 carbon atoms (C _3-8 ), and specific examples thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cycloalkyl Octyl and the like can be exemplified.

As used herein, "aryl" refers to monocyclic or polycyclic aromatic rings. Specific examples of "aryl" include benzene, naphthalene, anthracene and the like.

As used herein, "heteroaryl" refers to a heteroaromatic ring containing 1 to 3 heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur as ring-constituting elements. Specific examples of "heteroaryl" include furan, thiophene, oxazole, thiazole, pyrazole, pyridine, pyrimidine, pyridazine, pyrazine, quinoline, isoquinoline, quinazoline and the like.

As used herein, "phenyl optionally having substituent(s)" refers to unsubstituted phenyl or phenyl substituted with 1 to 3 substituents. Examples of substituents include halogen, -C _1-6 alkyl, -OC _1-6 alkyl and the like. Specific examples include phenyl, fluorophenyl, chlorophenyl, bromophenyl, iodophenyl and the like.

In the present specification, "heteroaryl optionally having a substituent selected from furyl, thienyl, oxazolyl, thiazolyl, pyrazolyl, pyridyl, pyrazyl, pyridazyl and pyrimidyl" is unsubstituted or has 1 to 3 substituents Furyl, thienyl, oxazolyl, thiazolyl, pyrazolyl, pyridyl, pyrazyl, pyridazyl or pyrimidyl substituted with . Examples of substituents include halogen, -C _1-6 alkyl, -OC _1-6 alkyl and the like. Specific examples include furyl, fluorofuryl, chlorofuryl, bromofuryl, iodofuryl, methylfuryl, ethylfuryl, methoxyfuryl, ethoxyfuryl, thienyl, fluorothienyl, chlorothienyl, bromothienyl, iodothienyl, methylthienyl, ethylthienyl, methoxythienyl , ethoxythienyl, oxazolyl, fluorooxazolyl, chlorooxazolyl, bromoxazolyl, iodooxazolyl, methyloxazolyl, ethyloxazolyl, methoxyoxazolyl, ethoxyoxazolyl, thiazolyl, fluorothia zolyl, chlorothiazolyl, bromothiazolyl, iodothiazolyl, methylthiazolyl, ethylthiazolyl, methoxythiazolyl, ethoxythiazolyl, pyrazolyl, fluoropyrazolyl, chloropyrazolyl, bromopyrazolyl, iodopyrazolyl, methylpyrazolyl, ethylpyrazolyl, methoxypyrazolyl, ethoxypyrazolyl, pyridyl , fluoropyridyl, chloropyridyl, bromopyridyl, iodopyridyl, methylpyridyl, ethylpyridyl, methoxypyridyl, ethoxypyridyl, pyrazyl, fluoropyridyl, chloropyridyl, bromopyridyl, iodopyrazyl, methylpyrazyl, ethylpyridyl, methoxypyrazyl, ethoxypyridyl, pyridazyl , fluoropyridazyl, chloropyridazyl, bromopyridazyl, iodopyridazyl, methylpyridazyl, ethylpyridazyl, methoxypyridazyl, ethoxypyridazyl, pyrimidyl, fluoropyrimidyl, chloropyrimidyl, bromopyrimidyl, iodopyrimidyl, methylpyrimidyl, ethylpyrimidyl, methoxypyrimidyl, ethoxypyridyl Mijil and the like can be exemplified.

As used herein, “optionally substituted pyrimidyl” refers to unsubstituted pyrimidyl or pyrimidyl substituted with 1 to 3 substituents. Examples of substituents include halogen, -C _1-6 alkyl, -OC _1-6 alkyl and the like. Specific examples include pyrimidyl, fluoropyrimidyl, chloropyrimidyl, bromopyrimidyl, iodopyrimidyl, methylpyrimidyl, ethylpyrimidyl, methoxypyrimidyl, ethoxypyrimidyl and the like.

As used herein, "alkyl halide" includes iodomethane, iodoethane, 1-iodopropane, 2-iodopropane, 1-iodobutane, 2-iodobutane, 1-iodo-2-methylpropane, tert-butyl iodide, 1-iodo Examples include pentane, 2-iodopentane, 1-iodo-2,2-dimethylpropane, 1-iodohexane, 2-iodohexane, and 3-iodomethylpentane.

As used herein, "acid anhydride" includes acetic anhydride, propionic anhydride, n-butyric anhydride, isobutyric anhydride, n-valeric anhydride, isovaleric anhydride, isovaleric anhydride, pivalic anhydride, and anhydride. Examples include n-hexanoic acid, heptanoic anhydride, and benzoic anhydride.

As used herein, "acid halide" includes benzoyl chloride, acetyl chloride, acetyl bromide, propionyl chloride, n-butyryl chloride, isobutyryl chloride, pentanoyl chloride, isopentanoyl chloride, DL-2-methylbutyryl chloride, pivaloyl chloride. , n-hexanoyl chloride, 4-methylpentanoyl chloride, and heptanoyl chloride.

As used herein, "halocarboxylic acid ester" includes methyl chloroformate, ethyl chloroformate, propyl chloroformate, isopropyl chloroformate, butyl chloroformate, sec-butyl chloroformate, isobutyl chloroformate, pentyl chloroformate, and neopentyl chloroformate. , n-hexyl chloroformate.

In the present specification, "boronic acid" or "boronic acid ester" (e.g., compound [V] described later) may be separately produced, isolated and purified, or used, for example, a precursor halogenated compound For example, bispinacol diborane may be reacted in the presence of a palladium compound and used for the coupling reaction without isolation and purification.

As used herein, the term "base" is not particularly limited, and examples thereof include inorganic bases and organic bases. "Inorganic bases" include alkali metal hydroxides (e.g. lithium hydroxide, sodium hydroxide, potassium hydroxide), alkaline earth metal hydroxides (e.g. magnesium hydroxide, calcium hydroxide, barium hydroxide) , alkali metal carbonates (e.g. sodium carbonate, potassium carbonate, cesium carbonate), alkaline earth metal carbonates (e.g. magnesium carbonate, calcium carbonate, barium carbonate), alkali metal hydrogen carbonates (e.g. sodium hydrogen carbonate, carbonate potassium hydrogen), alkali metal phosphates (e.g. sodium phosphate, potassium phosphate, cesium phosphate), alkaline earth metal phosphates (e.g. magnesium phosphate, calcium phosphate), alkali metal alkoxides (e.g. sodium methoxy sodium ethoxide, sodium tert-butoxide, potassium tert-butoxide), alkali metal hydrides (eg, sodium hydride, potassium hydride), sodium hydride and the like. "Organic bases" include trialkylamines (e.g., trimethylamine, triethylamine, N,N-diisopropylethylamine (DIPEA)), dialkylamines (e.g., diethylamine, diisopropylamine), 4-dimethylaminopyridine (DMAP), N- Methylmorpholine, picoline, 1,5-diazabicyclo[4.3.0]non-5-ene, 1,4-diazabicyclo[2.2.2]octane and 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) etc. One or two or more of these can be appropriately selected and mixed for use.

In the present specification, the "condensing agent" is not particularly limited, but specifically includes 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (WSC/HCl), N,N' -dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), N,N'-carbonyldiimidazole (CDI), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl )-4-methylmorpholinium chloride (DMT-MM), benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yloxytripyrrolidinophosphonium hexafluoro Phosphate (PyBOP), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), (1-cyano-2-ethoxy -2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate (COMU) and the like, preferably WSC·HCl, HATU and COMU.

In the present specification, "additives" are not particularly limited, but 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxysuccinimide (HOSu), ethyl ( hydroxyimino)cyanoacetate (Oxyma), 4-dimethylaminopyridine (DMAP), triethylamine (TEA), diisopropylethylamine (DIPEA), N-methylmorpholine and the like, preferably HOBt, TEA and DIPEA.

In the present specification, the "palladium compound" is not particularly limited. 1,1'-Bis(diphenylphosphino)ferrocene]palladium(II) dichloride dichloromethane adduct (Pd(dppf) _Cl2.CH2Cl2 ), ( ₂ -dicyclohexylphosphino- ₂ ',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) methanesulfonate (XPhos Pd G3), palladium(II) chloride, palladium bromide (II), palladium(II) acetate, palladium acetylacetonate(II), dichlorobis(benzonitrile)palladium(II), dichlorobis(acetonitrile)palladium(II), dichlorobis(triphenylphosphine)palladium(II), dichlorotetraammine Palladium(II), dichloro(cycloocta-1,5-diene)palladium(II), palladium trifluoroacetate(II), 1,1'-bis(diphenylphosphino)ferrocenedichloropalladium(II)-dichloromethane complex, etc. Divalent palladium catalysts; tris(dibenzylideneacetone)dipalladium (0) (Pd ₂ (dba) ₃ ), tris(dibenzylideneacetone)dipalladium chloroform complex (0), tetrakis(triphenylphosphine)palladium (0) zerovalent palladium catalysts such as (Pd(PPh ₃ ) ₄ ), and the like. These palladium compounds are used singly or in combination of two or more.

In the present specification, the "leaving group" specifically includes halogen, C _1-18 alkanesulfonyl, lower alkanesulfonyloxy, arylsulfonyloxy, aralkylsulfonyloxy, perhaloalkanesulfonyloxy, sulfonio, toluenesulfoxy etc. Preferred leaving groups in this reaction include halogen.

Examples of the above "C _1-18 alkanesulfonyl" include linear or branched alkanesulfonyl having 1 to 18 carbon atoms (C _1-18 ), specific examples thereof being methanesulfonyl, 1-propanesulfonyl , 2-propanesulfonyl, butanesulfonyl, cyclohexanesulfonyl, dodecanesulfonyl, octadecanesulfonyl, and the like.

Examples of the above "lower alkanesulfonyloxy" include linear or branched alkanesulfonyloxy having 1 to 6 carbon atoms (C _1-6 ), and specific examples thereof include methanesulfonyloxy, ethanesulfonyloxy, 1-propanesulfonyloxy, 2-propanesulfonyloxy, 1-butanesulfonyloxy, 3-butanesulfonyloxy, 1-pentanesulfonyloxy, 1-hexanesulfonyloxy and the like.

Examples of the above "arylsulfonyloxy" include linear or branched alkyl having 1 to 6 (C _{1-6 ) carbon atoms and 1 to 6 (C 1-6 )} carbon atoms as substituents on the phenyl ring. straight or branched chain alkoxy, phenylsulfonyloxy, naphthylsulfonyloxy and the like which may have 1 to 3 groups selected from the group consisting of nitro and halogen. Specific examples of the above "phenylsulfonyloxy which may have a substituent" include phenylsulfonyloxy, 4-methylphenylsulfonyloxy, 2-methylphenylsulfonyloxy, 4-nitrophenylsulfonyloxy, 4-methoxyphenylsulfonyloxy, 2-nitrophenylsulfonyloxy, 3-chlorophenylsulfonyloxy and the like. Specific examples of the above "naphthylsulfonyloxy" include α-naphthylsulfonyloxy, β-naphthylsulfonyloxy and the like.

Examples of the above "aralkylsulfonyloxy" include linear or branched alkyl having 1 to 6 (C _{1-6 ) carbon atoms and 1 to 6 (C 1-6 )} carbon atoms as substituents on the phenyl ring. C 1-6 (C _1-6 ) linear or branched phenyl-substituted phenyl optionally having 1 to 3 groups selected from the group consisting of linear or branched alkoxy, nitro and halogen It includes branched-chain alkanesulfonyloxy, straight-chain or branched-chain alkanesulfonyloxy having 1 to 6 carbon atoms (C _1-6 ) substituted with naphthyl, and the like. Specific examples of the above "phenyl-substituted alkanesulfonyloxy" include benzylsulfonyloxy, 2-phenylethylsulfonyloxy, 4-phenylbutylsulfonyloxy, 4-methylbenzylsulfonyloxy, 2-methylbenzylsulfonyloxy, 4- nitrobenzylsulfonyloxy, 4-methoxybenzylsulfonyloxy, 3-chlorobenzylsulfonyloxy and the like. Specific examples of the above "naphthyl-substituted alkanesulfonyloxy" include α-naphthylmethylsulfonyloxy, β-naphthylmethylsulfonyloxy and the like.

Specific examples of the above-mentioned "perhaloalkanesulfonyloxy" include trifluoromethanesulfonyloxy and the like.

Specific examples of the above "sulfonio" include dimethylsulfonio, diethylsulfonio, dipropylsulfonio, di(2-cyanoethyl)sulfonio, di(2-nitroethyl)sulfonio, di-(aminoethyl)sulfonio, di( 2-methylaminoethyl)sulfonio, di-(2-dimethylaminoethyl)sulfonio, di-(2-hydroxyethyl)sulfonio, di-(3-hydroxypropyl)sulfonio, di-(2-methoxyethyl)sulfonio, di -(2-carbamoylethyl)sulfonio, di-(2-carbamoylethyl)sulfonio, di-(2-carboxyethyl)sulfonio, di-(2-methoxycarbonylethyl)sulfonio, diphenylsulfonio and the like.

As used herein, the "solvent" may be any solvent inert to the reaction, such as water, ethers (e.g., dioxane, tetrahydrofuran, diethyl ether, 1,2-dimethoxyethane, diethylene glycol dimethyl ether, ethylene glycol dimethyl ether). , halohydrocarbons (e.g. methylene chloride, chloroform, 1,2-dichloroethane, carbon tetrachloride), aromatic hydrocarbons (e.g. benzene, toluene, xylene), lower alcohols (e.g. methanol, ethanol, isopropanol), polar Solvents (eg, N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), dimethylsulfoxide (DMSO), hexamethylphosphoric acid triamide, acetonitrile) can be mentioned. These solvents are used singly or in combination of two or more.

Each substituent in the compound represented by general formula [I] in the present specification (hereinafter referred to as "compound [I]") will be described below.

R ¹¹ in compound [I] is hydrogen atom, halogen, -C _1-6 alkyl or -OC _1-6 alkyl, preferably hydrogen atom, fluorine, chlorine, bromine, iodine, methyl, ethyl, n-propyl , isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, 3-methylpentyl, -O-methyl, -O-ethyl, -On-propyl , -O-isopropyl, -On-butyl, -O-isobutyl, -O-sec-butyl, -O-tert-butyl, -On-pentyl, -O-isopentyl, -O-neopentyl, -On-hexyl, -O-isohexyl or -O-3-methylpentyl, more preferably hydrogen atom, chlorine, methyl or -O-methyl.

R ¹² in compound [I] is a hydrogen atom, -C _1-6 alkyl, -C _1-6 alkyl-OC _1-6 alkyl, -C(=O)-C _1-6 alkyl, -C(=O )-aryl or -C(=O)-OC _1-6 alkyl, preferably a hydrogen atom, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n- Pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, 3-methylpentyl, -methyl-O-methyl, -methyl-O-ethyl, -methyl-O-propyl, -ethyl-O-methyl, -ethyl-O- Ethyl, -ethyl-O-propyl, -propyl-O-methyl, -propyl-O-ethyl, -propyl-O-propyl, -C(=O)-methyl, -C(=O)-ethyl, -C (=O)-n-propyl, -C(=O)-isopropyl, -C(=O)-n-butyl, -C(=O)-isobutyl, -C(=O)-sec-butyl, - C(=O)-tert-butyl, -C(=O)-n-pentyl, -C(=O)-isopentyl, -C(=O)-neopentyl, -C(=O)-n-hexyl, -C(=O)-isohexyl, -C(=O)-3-methylpentyl, -C(=O)-phenyl, -C(=O)-naphthyl, -C(=O)-O-methyl, -C(=O)-O-ethyl, -C(=O)-On-propyl, -C(=O)-O-isopropyl, -C(=O)-On-butyl, -C(=O) -O-isobutyl, -C(=O)-O-sec-butyl, -C(=O)-O-tert-butyl, -C(=O)-On-pentyl, -C(=O)-O -isopentyl, -C(=O)-O-neopentyl, -C(=O)-On-hexyl, -C(=O)-O-isohexyl or -C(=O)-O-3-methylpentyl and more preferably a hydrogen atom, methyl, -ethyl-O-methyl, -C(=O)-methyl, -C(=O)-phenyl or -C(=O)-O-methyl.

R ² in compound [I] is a hydrogen atom or -C _1-6 alkyl, preferably a hydrogen atom, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, It is n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl or 3-methylpentyl, more preferably a hydrogen atom or methyl.

R ³ in compound [I] is halogen, -Q _k -(C _1-6 alkyl) _m -Q _p -R ³¹ , optionally substituted phenyl or furyl, thienyl, oxazolyl, thiazolyl, pyrazolyl , pyridyl, pyrazyl, pyridazyl or pyrimidyl, the heteroaryl optionally having a substituent, preferably halogen, -Q _k -(C _1-6 alkyl) _m -Q _p -R ³¹ , optionally substituted phenyl, furyl, thienyl, oxazolyl, thiazolyl, pyrazolyl, pyridyl, pyrazyl, pyridazyl and optionally substituted pyrimidyl, more preferably is fluorine, chlorine, bromine, iodine, -O-methyl, -O-ethyl, -O-propyl, -O-butyl, -O-methyl-O-methyl, -O-ethyl-O-methyl, -O -Ethyl-O-ethyl, -O-methyl-cyclopropyl, -O-methyl-cyclobutyl, -O-methyl-cyclopentyl, -O-ethyl-cyclopropyl, -O-ethyl-cyclobutyl, -O-ethyl-cyclopentyl , -S-methyl, -S-ethyl, -S-propyl, -methyl-S-methyl, -methyl-S-ethyl, -ethyl-S-ethyl, -NH-methyl, -NH-ethyl, -C( =O)-O-methyl, -C(=O)-O-ethyl, -C(=O)-On-propyl, -C(=O)-O-isopropyl, -C(=O)-On- butyl, -C(=O)-O-isobutyl, -C(=O)-O-sec-butyl, -C(=O)-O-tert-butyl, -C(=O)-On-pentyl, -C(=O)-O-Isopentyl, -C(=O)-O-Neopentyl, -C(=O)-On-Hexyl, -C(=O)-O-Isohexyl, -C(=O) -O-3-methylpentyl, phenyl, fluorophenyl, chlorophenyl, bromophenyl, iodophenyl, furyl, thienyl, oxazolyl, thiazolyl, pyrazolyl, pyridyl, pyrazyl, pyrimidyl, fluoropyrimidyl, chloropyrimidyl, bromopyrimidyl, iodopyrimidyl, methylpyrimidyl, ethylpyrimidyl, methoxypyrimidyl, ethoxypyrimidyl or pyridazyl, more preferably fluorine, methyl, -O-methyl, -O-ethyl, -O-ethyl-O-methyl, -O-methyl-cyclopropyl , -S-ethyl, -methyl-S -methyl, -NH-ethyl, -C(=O)-O-methyl, phenyl, fluorophenyl, furyl, thienyl, oxazolyl, thiazolyl, pyrazolyl, pyridyl, pyrazyl, pyrimidyl, fluoropyrimidyl, methylpyrimidyl, methoxypyrimi is di- or pyridazyl;

R ³¹ in compound [I] is -C _1-6 alkyl or -C _3-8 cycloalkyl, preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert -butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, 3-methylpentyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl, more preferably methyl or cyclopropyl.

R ⁴ in compound [I] is a hydrogen atom, halogen, -C _1-6 alkyl or -OC _1-6 alkyl, preferably a hydrogen atom, fluorine, chlorine, bromine, iodine, methyl, ethyl, n- Propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, 3-methylpentyl, -O-methyl, -O-ethyl, -O- It is propyl or -O-butyl, more preferably hydrogen atom, fluorine, methyl or -O-methyl.

Each Q in compound [I] is independently the same or different and is an oxygen atom, a sulfur atom, -C(=O)-O- or -NH-.

k, m and p in compound [I] are the same or different and each independently 0 or 1;

n in compound [I] is 0, 1 or 2, and when n is 2, each R ³ is independently the same or different substituent.

V in compound [I] are the same or different and each independently represents a nitrogen atom or C-H.

W in compound [I] is a carbon atom or a nitrogen atom, preferably a carbon atom.

X in compound [I] is a carbon atom, a nitrogen atom or ^NR12 .

Y in compound [I] is a carbon atom or a nitrogen atom.

Each Z in compound [I] is independently the same or different and each independently is a nitrogen atom or C-H.

Ring A in compound [I] is aryl or heteroaryl, examples of which include benzene, naphthalene, and anthracene, and examples of heteroaryl include furan, thiophene, oxazole, thiazole, pyrazole, Examples include pyridine, pyrimidine, pyridazine, pyrazine, quinoline, isoquinoline, quinazoline and the like.

である。 In addition, in compound [I]

is.

の具体例は、エトキシベンゼン、メトキシエトキシベンゼン、シクロプロピルメトキシベンゼン、エチルスルファニルベンゼン、メチルスルファニルメチルベンゼン、エチルアミノベンゼン、安息香酸メチル、ビフェニル、フルオロビフェニル、メトキシビフェニル、ピリジルベンゼン、ピリミジルベンゼン、(フルオロピリミジル)ベンゼン、(メチルピリミジル)ベンゼン、(メトキシピリミジル)ベンゼン、ピラジルベンゼン、ピリダジルベンゼン、フリルベンゼン、チエニルベンゼン、オキサゾリルベンゼン、チアゾリルベンゼン、ピラゾリルベンゼン、フェニルフラン、エトキシチオフェン、フェニルチオフェン、フリルチオフェン、チエニルチオフェン、ピリジルチオフェン、ピリミジルチオフェン、メチルキノリン、メトキシキノリン、エトキシピリジンであり、好ましくは、ピリジルベンゼン、ピリミジルベンゼン、(フルオロピリミジル)ベンゼン、(メチルピリミジル)ベンゼン、(メトキシピリミジル)ベンゼン、フェニルチオフェン、ピリジルチオフェン、ピリミジルチオフェンであり、さらに好ましくは、2-ピリジルベンゼン、2-ピリミジルベンゼン、2-(5-フルオロピリミジル)ベンゼン、2-(5-メチルピリミジル)ベンゼン、2-(5-メトキシピリミジル)ベンゼン、3-フェニルチオフェン、3-(2-ピリジル)チオフェン、3-(2-ピリミジル)チオフェンである。 in compound [I]

Specific examples of are ethoxybenzene, methoxyethoxybenzene, cyclopropylmethoxybenzene, ethylsulfanylbenzene, methylsulfanylmethylbenzene, ethylaminobenzene, methyl benzoate, biphenyl, fluorobiphenyl, methoxybiphenyl, pyridylbenzene, pyrimidylbenzene, (fluoropyrimidyl)benzene, (methylpyrimidyl)benzene, (methoxypyrimidyl)benzene, pyrazylbenzene, pyridazylbenzene, furylbenzene, thienylbenzene, oxazolylbenzene, thiazolylbenzene, pyrazolylbenzene, phenyl furan, ethoxythiophene, phenylthiophene, furylthiophene, thienylthiophene, pyridylthiophene, pyrimidylthiophene, methylquinoline, methoxyquinoline, ethoxypyridine, preferably pyridylbenzene, pyrimidylbenzene, (fluoropyrimidyl) benzene, (methylpyrimidyl)benzene, (methoxypyrimidyl)benzene, phenylthiophene, pyridylthiophene, pyrimidylthiophene, more preferably 2-pyridylbenzene, 2-pyrimidylbenzene, 2-(5-fluoro pyrimidyl)benzene, 2-(5-methylpyrimidyl)benzene, 2-(5-methoxypyrimidyl)benzene, 3-phenylthiophene, 3-(2-pyridyl)thiophene, 3-(2-pyrimidyl)thiophene be.

である。 In addition, in compound [I]

is.

in compound [I]
---
is a single or double bond.

Preferred compounds [I] include, for example, in the general formula [I]
R ¹¹ is a hydrogen atom, halogen, -C _1-6 alkyl or -OC _1-6 alkyl,
^R2 is a hydrogen atom,
R3 is phenyl ^, pyridyl, pyrimidyl (optionally substituted with halogen, _-C1-6alkyl or _-OC1-6alkyl ),
X is NH;
Y is a carbon atom,
Z is a nitrogen atom or CH,
Compounds in which ring A is benzene or thiophene can be mentioned.

[式中、R¹¹は、水素原子、ハロゲン、-C_1-6アルキルまたは-O-C_1-6アルキルであり、

は、ピリジルベンゼン、（ハロゲン、-C_1-6アルキルまたは-O-C_1-6アルキルで置換されていてもよいピリミジル）ベンゼン、フェニルチオフェン、ピリジルチオフェンまたはピリミジルチオフェンである]で表される化合物が挙げられ、
特に、一般式[Ia]中、
R¹¹は、水素原子、メチルまたは-O-メチルであり、

は、ピリジルベンゼン、ピリミジルベンゼン、（フルオロピリミジル）ベンゼン、（メチルピリミジル）ベンゼン、（メトキシピリミジル）ベンゼン、フェニルチオフェン、ピリジルチオフェンまたはピリミジルチオフェンである化合物が挙げられる。 More preferred compounds [I] include, for example, general formula [Ia]

[wherein R ¹¹ is a hydrogen atom, halogen, -C _1-6 alkyl or -OC _1-6 alkyl,

is pyridylbenzene, (pyrimidyl optionally substituted with halogen, _-C1-6alkyl or _-OC1-6alkyl )benzene, phenylthiophene, pyridylthiophene or pyrimidylthiophene] compound represented by are mentioned,
In particular, in general formula [Ia],
R ¹¹ is a hydrogen atom, methyl or -O-methyl,

includes compounds that are pyridylbenzene, pyrimidylbenzene, (fluoropyrimidyl)benzene, (methylpyrimidyl)benzene, (methoxypyrimidyl)benzene, phenylthiophene, pyridylthiophene or pyrimidylthiophene.

Further preferred compounds [I] include compounds selected from the following.

Compound [I] or a salt thereof is useful as a platelet production promoter. Therefore, one aspect of the present invention is a platelet production promoter containing compound [I] or a salt thereof.
Also included in this aspect are platelet production-enhancing agents for use in combination with aromatic hydrocarbon receptor inhibitors.

One aspect of the present invention is the use of compound [I] or a salt thereof for promoting platelet production.
This aspect also includes uses with aromatic hydrocarbon receptor inhibitors.

One aspect of the present invention is a method for promoting platelet production, comprising culturing platelet progenitor cells in the presence of compound [I] or a salt thereof.
This embodiment also includes a method comprising culturing platelet progenitor cells in the coexistence of an aromatic hydrocarbon receptor inhibitor.

One aspect of the present invention is a method for producing platelets, which comprises culturing platelet progenitor cells in the presence of compound [I] or a salt thereof.
This embodiment also includes a method comprising culturing platelet progenitor cells in the coexistence of an aromatic hydrocarbon receptor inhibitor.

One aspect of the present invention is a method for culturing proplatelet cells for promoting platelet production, which method comprises culturing proplatelet cells in the presence of compound [I] or a salt thereof.
This embodiment also includes a method comprising culturing platelet progenitor cells in the coexistence of an aromatic hydrocarbon receptor inhibitor.

Throughout the specification, presentations of preferred embodiments and options for different features of compound [I] or salts thereof, uses, methods and compositions that can be used in the methods of the present invention are intended to be combinable and contradictory. Unless otherwise specified, it also includes presentation of preferred embodiments and alternative combinations for such different features.

The method for producing compound [I] is described below. Compound [I] can be produced based on the production method shown below. In addition, compound [I] can be produced, for example, by the method described in WO2019/167973. These production methods are examples, and the production method of compound [I] is not limited to these.

In the following reaction schemes, when alkylation reaction, hydrolysis reaction, amination reaction, esterification reaction, amidation reaction, etherification reaction, nucleophilic substitution reaction, addition reaction, oxidation reaction, reduction reaction, etc. are carried out, these The reaction is carried out according to a method known per se. Examples of such methods include Jikken Kagaku Koza (5th Edition, The Chemical Society of Japan, Maruzen Co., Ltd.), ORGANIC FUNCTIONAL GROUP PREPARATIONS, 2nd Edition, Academic Press ( ACADEMIC PRESS, INC., 1989; Comprehensive Organic Transformations, VCH Publishers Inc., 1989, P.G.M. Wuts and T.W. Greene, "Greene's Protective Groups in Organic Synthesis" (4th edition, 2006).

（式中、各記号は上記に定義したとおりである） General synthetic route of compound [I] (1)

(where each symbol is as defined above)

Compound [I] that can be used in the method of the present invention can be produced by the reaction represented by the above reaction scheme. Specifically, compound [I] can be produced by condensing compound [II] and compound [III].

Other reaction conditions (reaction temperature, reaction time, etc.) can be appropriately determined based on condensation reactions known per se.

（式中、R^12aは-C_1-6アルキルであり、その他の各記号は上記に定義したとおりである） General synthetic route of compound [I] (2)

(wherein R ^12a is -C _1-6 alkyl and each other symbol is as defined above)

Compound [Ic] that can be used in the method of the present invention can be produced by the reaction represented by the above reaction scheme. Specifically, compound [Ic] can be produced by reacting compound [Ib] with an alkyl halide.

Other reaction conditions (reaction temperature, reaction time, etc.) can be appropriately determined based on condensation reactions known per se.

（式中、R^12bは-C(=O)-C_1-6アルキル、-C(=O)-アリールまたは-C(=O)-O-C_1-6アルキルであり、その他の各記号は上記に定義したとおりである） General synthetic route of compound [I] (3)

(wherein R ^12b is -C(=O)-C _1-6 alkyl, -C(=O)-aryl or -C(=O)-OC _1-6 alkyl and each other symbol is (as defined in

Compound [Id] that can be used in the method of the present invention can be produced by the reaction represented by the above reaction scheme. Specifically, the compound [Id] can be produced by reacting the compound [Ib] with an acid anhydride, an acid halide or a halocarboxylic acid ester.

Other reaction conditions (reaction temperature, reaction time, etc.) can be appropriately determined based on condensation reactions known per se.

（式中、環Ｂは置換基を有していてもよいベンゼンまたはチオフェンであり、Uは脱離基であり、他の記号は上記に定義したとおりである） General synthetic route for compound [I] (4)

(wherein ring B is optionally substituted benzene or thiophene, U is a leaving group, and other symbols are as defined above)

Compound [Ie] that can be used in the method of the present invention can be produced by the reaction represented by the above reaction scheme. Specifically, compound [Ie] can be produced by coupling compound [IV] having a leaving group (U) with compound [V] in the presence of a palladium compound.

Other reaction conditions (reaction temperature, reaction time, etc.) can be appropriately determined based on known coupling reactions.

In each reaction in the above reaction scheme, the product can be used as a reaction solution or as a crude product in the next reaction, but it can also be isolated from the reaction mixture according to a conventional method, and can be easily separated by a conventional separation means. It can also be refined. Common separation means include, for example, recrystallization, distillation, chromatography.

Geometric isomers, stereoisomers, optical isomers and tautomers are included in the starting material compounds, intermediate compounds and target compounds in each of the above steps and the compounds or salts thereof that can be used in the method of the present invention. . Various isomers can be separated by a general optical resolution method. It can also be produced from a suitable optically active raw material compound.

The compounds or salts thereof that can be used in the method of the present invention can be produced by the synthetic methods shown in the above reaction schemes or methods analogous thereto.

Unless a specific manufacturing method is described as a raw material compound for the production of a compound or a salt thereof that can be used in the method of the present invention, a commercially available one may be used, and one produced according to a method known per se or a method analogous thereto may be used. may

The starting material compounds and target compounds in each of the above steps can be used in appropriate salt forms. Such salts include those similar to those exemplified below as salts of compound [I] that can be used in the method of the present invention.

In addition, the compound or its salt that can be used in the method of the present invention includes its salt form, and depending on the type of acid addition salt or substituent, it may form a salt with a base. Examples of such "acids" include inorganic acids (e.g. hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc.); and organic acids (e.g. methanesulfonic acid, p-toluenesulfonic acid, acetic acid, citric acid , tartaric acid, maleic acid, fumaric acid, malic acid, lactic acid, etc.). Examples of such "bases" include inorganic bases such as sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, etc.; and organic bases such as methylamine , diethylamine, trimethylamine, triethylamine, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, tris(hydroxymethyl)methylamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, guanidine, pyridine, picoline, choline, etc.); and ammonium Salt etc. are mentioned. In addition, salts with amino acids such as lysine, arginine, aspartic acid and glutamic acid may be formed.

Compounds or salts thereof that can be used in the method of the present invention include compounds in which one or more atoms are replaced with one or more isotopic atoms. Examples of isotopic atoms include deuterium ( ² H), tritium ( ³ H), ¹³ C, ¹⁵ N, ¹⁸ O, and the like.

The compound or salt thereof that can be used in the method of the present invention has the function of promoting platelet production from platelet progenitor cells in vitro.

A method for producing platelets from platelet progenitor cells using a compound or a salt thereof that can be used in the method of the present invention will be described below.

Platelets can be produced by culturing platelet progenitor cells (e.g., megakaryocyte cells or progenitor cells thereof) in the presence of one or more compounds or salts thereof that can be used in the method of the present invention. can. The concentration in the medium of the compound or its salt that can be used in the method of the present invention is not particularly limited, and can be appropriately determined by those skilled in the art according to the platelet production promoter. , preferably 10 nM to 100 μM, more preferably 100 nM to 10 μM, but the amount may be outside this range as long as the desired effect is achieved.

A compound or a salt thereof that can be used in the method of the present invention can increase the amount of platelets produced from megakaryocyte cells. Compounds or salts thereof that can be used in the method of the present invention are not limited, but, for example, can increase the platelet count by 200% or more, preferably 300% or more, more preferably 400% or more compared to controls. can.

The timing of adding (presence) the compound or salt thereof that can be used in the method of the present invention to the medium is not particularly limited as long as the desired effect is exhibited. For example, compounds or salts thereof that can be used in the methods of the present invention are added to megakaryocyte cells or progenitor cells thereof. Megakaryocytic cells may be pre-multinucleated megakaryocytic cells or multinucleated megakaryocytic cells, and multinucleated megakaryocytic cells may be platelet-producing megakaryocytic cells. As described below, immortalized megakaryocyte cells are produced by forcibly expressing at least one gene selected from the group consisting of oncogenes, polycomb genes, and apoptosis-suppressing genes in cells undifferentiated from megakaryocytes, If forced expression is then canceled to promote multinucleation of immortalized megakaryocyte cells, compound [I] or a salt thereof that can be used in the method of the present invention can be added to the medium after the forced expression is canceled. preferable. Compounds or salts thereof that can be used in the method of the present invention are administered on the 1st, 2nd, 3rd, 4th, 5th, and 6th days after the initiation of culture for platelet production and at the same time. It may be added to the medium later.

Known cells can be used for megakaryocyte cells that can be used in the present invention, for example, immortalized megakaryocyte cells can be prepared using the method disclosed in WO2016/204256.

The origin of megakaryocyte cells or their progenitor cells is not particularly limited as long as they have the ability to produce platelets, and examples thereof include pluripotent stem cells, particularly induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells). be able to. Although the origin of iPS cells and ES cells is not particularly limited, human-derived cells can be mentioned, for example.

Compounds or salts thereof that can be used in the method of the present invention include one or more aromatic hydrocarbon receptor inhibitors (AhR antagonists), one or more thrombopoietin (TPO) or TPO receptor agonists, Use as a platelet production promoter in combination with one or two or more ROCK (Rho-associated coiled-coil forming kinase) inhibitors and/or one or two or more ADAM (a disintegrin and metalloprotease) inhibitors, etc. can be done.

The compound or its salt that can be used in the method of the present invention exhibits a superior effect of promoting platelet production by culturing platelet progenitor cells in the presence of an aromatic hydrocarbon receptor inhibitor.

・N-[2-(1H-インドル-3-イル)エチル]-9-(1-メチルエチル-2-(5-メチル-3-ピリジニル)-9H-プリン-6-アミン（N-[2-(1H-indol-3-yl)ethyl]-9-(1-methylethyl)-2-(5-methyl-3-pyridinyl)-9H-Purin-6-amine）（化合物A2）

・4-(2-メチル-4-ピリジニル)-N-[4-(3-ピリジニル)フェニル]-ベンゼンアセタミド（4-(2-Methyl-4-pyridinyl)-N-[4-(3-pyridinyl)phenyl]-benzeneacetamide）（化合物A3）

・1-メチル-N-[2-メチル-4-[2-(2-メチルフェニル)ジアゼニル]フェニル]-1H-ピラゾル-5-カルボキサミド（1-Methyl-N-[2-methyl-4-[2-(2-methylphenyl)diazenyl]phenyl]-1H-pyrazole-5-carboxamide）（化合物A4）

・3-[5-[2-[[2-(5-フルオロピリジン-3-イル)-8,8-ジメチル-7H-プリノ[8,9-b][1,3]オキサゾル-4-イル]アミノ]エチル]-2-ヒドロキシフェニル]ベンゾニトリル（3-[5-[2-[[2-(5-Fluoropyridin-3-yl)-8,8-dimethyl-7H-purino[8,9-b][1,3]oxazol-4-yl]amino]ethyl]-2-hydroxyphenyl]benzonitrile）（化合物A5）

The aromatic hydrocarbon receptor inhibitor that can be used together with the compound or salt thereof that can be used in the method of the present invention is not particularly limited as long as it has a platelet production promoting effect. mentioned. These compounds can be synthesized, for example, by the method described in WO2020/050409.
・4-[2-[[2-benzo[b]thien-3-yl-9-(1-methylethyl-9H-purin-6-yl]amino]ethyl]phenol (4-[2-[[2 -benzo[b]thien-3-yl-9-(1-methylethyl)-9H-purin-6-yl]amino]ethyl]phenol) (Compound A1)

・N-[2-(1H-indol-3-yl)ethyl]-9-(1-methylethyl-2-(5-methyl-3-pyridinyl)-9H-purin-6-amine (N-[2 -(1H-indol-3-yl)ethyl]-9-(1-methylethyl)-2-(5-methyl-3-pyridinyl)-9H-Purin-6-amine) (compound A2)

・4-(2-Methyl-4-pyridinyl)-N-[4-(3-pyridinyl)phenyl]-benzeneacetamide (4-(2-Methyl-4-pyridinyl)-N-[4-(3 -pyridinyl)phenyl]-benzeneacetamide) (compound A3)

・1-Methyl-N-[2-methyl-4-[2-(2-methylphenyl)diazenyl]phenyl]-1H-pyrazole-5-carboxamide (1-Methyl-N-[2-methyl-4-[ 2-(2-methylphenyl)diazenyl]phenyl]-1H-pyrazole-5-carboxamide) (compound A4)

・3-[5-[2-[[2-(5-fluoropyridin-3-yl)-8,8-dimethyl-7H-purino[8,9-b][1,3]oxazol-4-yl ]amino]ethyl]-2-hydroxyphenyl]benzonitrile (3-[5-[2-[[2-(5-Fluoropyridin-3-yl)-8,8-dimethyl-7H-purino[8,9- b][1,3]oxazol-4-yl]amino]ethyl]-2-hydroxyphenyl]benzonitrile) (compound A5)

The concentration of the aromatic hydrocarbon receptor inhibitor is not particularly limited, and can be appropriately determined by those skilled in the art according to the compound. The concentration of the aromatic hydrocarbon receptor inhibitor can be, for example, 1.0 nM to 1000 μM, 10 nM to 100 μM, 100 nM to 100 μM or 100 nM to 10 μM, but may be outside this range as long as the desired effect is achieved. may be the amount of

ROCK inhibitors include, for example, Y27632, Y39983, Fasudil hydrochloride, Ripasudil, SLX-2119, RKI-1447, Azaindole 1, SR-3677, Staurosporine, H1152 dihydrochloride (H1152 Dihydrochloride), AR-12286, INS-117548, etc., but not limited to these. The concentration of the ROCK inhibitor is not particularly limited, and can be appropriately determined by those skilled in the art according to the compound. The concentration of the ROCK inhibitor can be, for example, 1.0 nM to 1.0 mM, 10 nM to 0.1 mM, 100 nM to 0.1 mM or 100 nM to 0.01 mM, although concentrations outside this range can be used as long as the desired effect is achieved. It can be the amount.

Thrombopoietins include thrombopoietin (TPO) and human recombinant thrombopoietin. Examples of TPO receptor agonists include, but are not limited to, TA-316. The concentrations of TPO and human recombinant TPO are not particularly limited and can be determined appropriately by those skilled in the art. The concentration of TPO and human recombinant TPO can be, for example, 0.5 ng/mL to 5 μg/mL, preferably 5 to 500 ng/mL, more preferably 50 ng/mL, as long as the desired effect is achieved. , may be an amount outside this range. The concentration of the TPO receptor agonist is not particularly limited, and can be appropriately determined by those skilled in the art according to the compound. The concentration of the TPO receptor agonist can be, for example, 0.1 ng/mL to 1 mg/mL, preferably 1 ng/mL to 100 μg/mL, more preferably 10 ng/mL to 10 μg/mL. Amounts outside this range may be used as long as the desired effect is achieved.

Examples of ADAM inhibitors include, but are not limited to, KP-457. The concentration of the ADAM inhibitor is not particularly limited, and can be appropriately determined by those skilled in the art according to the compound. The concentration of the ADAM inhibitor can be, for example, 1.0 nM to 1.0 mM, preferably 10 nM to 0.1 mM, more preferably 100 nM to 0.1 mM, although amounts outside this range can be used as long as the desired effect is achieved. may be

Compounds or salts thereof that can be used in the method of the present invention include one or more aromatic hydrocarbon receptor antagonists, one or more TPO or TPO receptor agonists, one or more ROCK It can be a kit in combination with an inhibitor and/or one or more ADAM inhibitors and the like.

The timing of adding the compounds to be used in combination (coexisting the compounds that can be used in the method of the present invention or salts thereof in the medium) is not particularly limited as long as the desired effects are exhibited. The compounds used in combination can be added to the medium before, after, or simultaneously with the addition of the compounds or salts thereof that can be used in the method of the present invention to the medium. Immortalized megakaryocyte cells are produced by forcibly expressing at least one gene selected from the group consisting of oncogenes, polycomb genes, and apoptosis-suppressing genes in cells undifferentiated from megakaryocytes, and then forced expression is released. When promoting multinucleation of immortalized megakaryocyte cells, it is preferable to add to the medium after the release of forced expression (including simultaneous release).

The forced expression period is not particularly limited, and can be determined as appropriate by those skilled in the art. After forced expression, the cells may be subcultured, and the period from the last passage to the date of release of forced expression is not particularly limited, but may be, for example, 1 day, 2 days, or 3 days or more. .

When the compound or salt thereof that can be used in the method of the present invention is added to the medium after the forced expression is canceled, the period until the compound that can be used in the method of the present invention or the salt thereof is added to the medium after the forced expression is canceled Although the period is not particularly limited, the culture may be started within 1, 2, 3, 4, 5, or 6 days, for example. The period of culturing the cells in the presence of the compound or its salt that can be used in the method of the present invention is also not particularly limited. Usually, the compound or its salt that can be used in the method of the present invention is added to the medium, and functional platelets are gradually released from around day 1, and the number increases with the number of culture days. go. The period for culturing the cells in the presence of the compound or salt thereof that can be used in the method of the present invention is, for example, 5 to 10 days, but the number of days for culturing may be shorter or longer. The compound or salt thereof that can be used in the method of the present invention may be additionally added to the medium one or more times during the culture period.

Cell culture conditions can be normal conditions. For example, the temperature can be from about 35°C to about 42°C, preferably from about 36°C to about 40°C, more preferably from about 37°C to about 39°C, with 5% _CO2 and/or 20% _O2 . good too. It may be stationary culture or shaking culture. The shaking speed in shaking culture is not particularly limited either, and can be, for example, 10 rpm to 200 rpm, preferably 30 rpm to 150 rpm.

When megakaryocyte cells and / or progenitor cells thereof are contacted with a compound or a salt thereof that can be used in the method of the present invention and cultured, mature megakaryocyte cells are obtained and platelets are produced from the cytoplasm. Here, maturation of megakaryocyte cells means that the megakaryocyte cells become multinucleated and become able to release platelets.

The medium for culturing megakaryocyte cells is not particularly limited, and a known medium suitable for producing platelets from megakaryocyte cells or a medium based thereon can be used as appropriate. For example, a medium used for culturing animal cells can be prepared as a basal medium. Examples of basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI1640 medium, Fischer's medium, and Neurobasal Medium (Life Technologies). and mixed media thereof.

The medium may contain serum or plasma, or may be serum-free. When serum is used, fetal bovine serum (FBS) and human serum can be used. Optionally, the medium contains e.g. albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, monothioglycerol (MTG), lipids, amino acids (e.g. L-glutamine), ascorbic acid , heparin, non-essential amino acids, vitamins, growth factors, small compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, cytokines, and the like. Cytokines are proteins that promote blood cell lineage differentiation, and examples thereof include VEGF, TPO, TPO receptor agonists, SCF, ITS (insulin-transferrin-selenite) supplements, ADAM inhibitors, and the like.

The agents and amounts used, the timing of their addition to the medium, platelet progenitor cells, their culture methods and culture conditions, etc. described for the platelet production promoting agent and platelet manufacturing method are the same as other aspects of the present invention (agents, use, method, etc.).

The disclosures of all patent and non-patent literature cited herein are hereby incorporated by reference in their entirety.

The present invention will be further described in detail by the following Reference Examples, Production Examples and Examples, but these are not intended to limit the present invention and may be changed without departing from the scope of the present invention.
In this specification, the following abbreviations may be used.

"Room temperature" in the following Reference Examples, Production Examples and Examples generally means about 10°C to about 35°C. The ratios shown for mixed solvents are volume ratios unless otherwise specified. % indicates % by weight unless otherwise specified.
¹ HNMR (proton nuclear magnetic resonance spectrum) was measured by Fourier transform NMR (either Bruker AVANCE III 400 (400 MHz) or Bruker AVANCE III HD (500 MHz)).
MS (mass spectrum) was measured by LC/MS (ACQUITY UPLC H-Class). As the ionization method, the ESI method was used, and the data described are actually measured values (found). Molecular ion peaks ([M+H] ⁺ , [MH] ⁻ , etc.) are usually observed. In the case of salts, a molecular ion peak or fragment ion peak of the free form is usually observed.
In silica gel column chromatography, aminopropylsilane-bonded silica gel was used when it was described as basic.
The absolute configuration of the compound is determined by a known X-ray crystal structure analysis method (for example, Shigeru Ohba and Shigenobu Yano, "Basic Course for Chemists 12 X-ray Crystal Structure Analysis" (1st edition, 1999)), or Waldemar Adam, Rainer T. Fell, Chantu R. Saha-Moller and Cong-Gui Zhao : Tetrahedron: Asymmetry 1998, 9, 397-401. Yuanming Zhu, Yong Tu, Hongwu Yu, Yian Shi: Tetrahedron Lett. 1988, 29, 2437-2440).

[Reference example]
Reference example 1
Preparation of (E)-N-[2-(2-bromophenyl)ethyl]-3-(7-methoxy-1H-indol-3-yl)prop-2-enamide
DIPEA (40.2 μl) and COMU (59.1 mg) were added, and the mixture was stirred overnight at room temperature. The reaction solution was concentrated, and the residue was purified by column chromatography (Hexane/AcOEt) to obtain the desired product (28 mg).

Reference example 2
Production of 2-(2-aminoethyl)-N-ethylaniline dihydrochloride
4N HCl/AcOEt (1 ml) was added to a solution of tert-butyl N-[2-(2-aminoethyl)phenyl]-N-ethylcarbamate (180 mg) in EtOH (2 ml) and Stirred for 1.5 hours. After concentrating the reaction solution, the residue was dispersed and washed with AcOEt to obtain the desired product (170 mg).

Reference example 3
Preparation of tert-butyl N-[2-(2-aminoethyl)phenyl]-N-ethylcarbamate
10% Pd/C (50 mg) was added to a solution of tert-butyl N-[2-(2-azidoethyl)phenyl]-N-ethylcarbamate (300 mg) in EtOH (3 ml), Stir at room temperature for 3 hours. The solid matter was filtered through celite, and the filtrate was concentrated to obtain the desired product (208 mg).

Reference example 4
Preparation of tert-butyl N-[2-(2-azidoethyl)phenyl]-N-ethylcarbamate
NaH (0.18 g) and iodoethane (0.37 ml) were added to a solution of tert-butyl N-[2-(2-azidoethyl)phenyl]carbamate (1.0 g) in DMF (3 ml), and the mixture was stirred overnight at room temperature. Water was added to the reaction mixture, and the mixture was extracted with AcOEt. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. Filter and concentrate the filtrate. The residue was purified by column chromatography (Hexane/AcOEt) to obtain the desired product (940 mg).

Reference example 6
Preparation of 2-(3-ethoxythiophen-2-yl)ethanamine hydrochloride To a solution of tris(pentafluorophenyl)borane (14.7 mg) in DCM (2 ml) was added diethylsilane (310 µl) at 0°C under a nitrogen atmosphere. ), a solution of 2-(3-ethoxythiophen-2-yl)acetonitrile (160 mg) in DCM (1 ml) was added and stirred at room temperature for 1 hour. The reaction mixture was concentrated, and 4N HCl/AcOEt (718 μl) was added to the residue. The precipitated solid was collected by filtration to obtain the desired product (38 mg).

Reference example 7
Preparation of 2-(3-ethoxythiophen-2-yl)acetonitrile
To a suspension of KOtBu (524 mg) in DME (4 ml) was added dropwise a solution of TosMIC (502 mg) in DME (3 ml) at -50°C under a nitrogen atmosphere. 365 mg) in DME (3 ml) was added dropwise and stirred for 1 hour. The temperature was brought to room temperature, MeOH (10 ml) was added, and the mixture was stirred under reflux with heating for 1 hour. Water was added to the reaction mixture, and the mixture was extracted with AcOEt. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. It was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (Hexane/AcOEt) to obtain the desired product (162 mg).

Reference example 8
Preparation of (E)-N-[2-(2-bromo-5-fluorophenyl)ethyl]-3-(7-methoxy-1H-indol-3-yl)prop-2-enamide
(E)-3-(7-Methoxy-1H-indol-3-yl)prop-2-enoic acid (25.0 mg), 2-bromo-5-fluorophenethylamine (30.1 mg) in DCM (2 ml) , DIPEA (40.2 μl) and HATU (52.5 mg) were added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated, and the residue was purified by column chromatography (Hexane/AcOEt) to obtain the desired product (43 mg).

Reference example 13
Production of 2-(2-pyrimidin-2-ylphenyl)ethanamine hydrochloride
In a toluene (4 ml) solution of tert-butyl N-[2-(2-bromophenyl)ethyl]carbamate (200 mg), 2-tributylstannylpyrimidine (232 μl) and Pd(PPh ₃ ) ₄ were added under an argon atmosphere. (77.0 mg) was added, and the mixture was heated to reflux and stirred overnight. The reaction solution was concentrated and the residue was purified by column chromatography (Hexane/AcOEt). 4N HCl/AcOEt (0.5 ml) was added to an EtOH (1 ml) solution of the purified product, and the mixture was stirred at 50° C. for 1.5 hours. The reaction solution was concentrated to obtain the desired product (76.0 mg).

Reference example 14
Preparation of (E)-N-[2-(3-bromothiophen-2-yl)ethyl]-3-(7-methoxy-1H-indol-3-yl)prop-2-enamide
A THF (3 ml) solution of 3-bromo-2-[(E)-2-nitroethenyl]thiophene (400 mg) was added to a suspension of LAH (0.084 g) in THF (4 ml) at 0°C under a nitrogen atmosphere. It was added dropwise and stirred at room temperature for 2 hours. Water (0.15 ml), 15% NaOH aqueous solution (0.15 ml) and water (0.45 ml) were added, filtered through Celite, and the filtrate was concentrated. To a solution of the residue in DCM (1 ml) were added (E)-3-(7-methoxy-1H-indol-3-yl)prop-2-enoic acid (40.0 mg), DIPEA (0.048 ml), HATU (91.0 mg). ) was added and stirred overnight at room temperature. The reaction solution was purified by column chromatography (Hexane/AcOEt) to obtain the desired product (0.032 g).

Reference example 15
Production of 2-(3-thiophen-2-ylthiophen-2-yl)ethanamine hydrochloride
tert-butyl N-[2-(3-bromothiophen-2-yl)ethyl]carbamate (57.0 mg), 2-thiopheneboronic acid (40.5 mg), _PdCl2 (dppf)DCM (7.6 mg), _K3PO A mixture of ₄ (79.0 mg) and 1,4-dioxane/water (4/1) (1 ml) was stirred under a nitrogen atmosphere at 90°C for 2 hours. The reaction solution was purified by column chromatography (Hexane/AcOEt). 4N HCl/AcOEt (0.5 ml) was added to an EtOH (0.5 ml) solution of the purified product, and the mixture was stirred overnight at room temperature. The reaction solution was concentrated to obtain the desired product (38.2 mg).

Reference example 19
Production of 2-(2-pyrimidin-4-ylphenyl)ethanamine hydrochloride
tert-butyl N-[2-[2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]ethyl]carbamate (150 mg), 4-chloropyrimidine hydrochloride A mixture of salt (98.0 mg), PdCl ₂ (dppf)DCM (35.3 mg), K ₃ PO ₄ (183 mg), DME/water (4/1) (2 ml) was stirred under nitrogen atmosphere overnight at reflux. did. The reaction solution was concentrated and the residue was purified by column chromatography (Hexane/AcOEt). 4N HCl/AcOEt (0.5 ml) was added to an EtOH (1 ml) solution of the purified product, and the mixture was stirred at 50° C. for 1.5 hours. The reaction solution was concentrated to obtain the desired product (55.0 mg).

Reference example 20
Preparation of (E)-3-(7-methoxy-1H-pyrrolo[2,3-c]pyridin-3-yl)prop-2-enoic acid
Hexamethylenetetramine (265 mg) was added to an AcOH (3 ml) solution of 7-methoxy-1H-pyrrolo[2,3-c]pyridine (420 mg), and the mixture was stirred at 100°C for 6 hours. A saturated NaHCO ₃ aqueous solution was added to the reaction mixture, and the mixture was extracted with AcOEt. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. Filter and concentrate the filtrate. The residue was suspended in DCM (3 ml), DIBOC (439 μl) and DMAP (23.1 mg) were added and stirred for 30 minutes. The reaction solution was concentrated and the residue was purified by column chromatography (Hexane/AcOEt).
NaH (22.7 mg) was added to a solution of ethyl diethylphosphonoacetate (113 μl) in THF (3 ml) and stirred for 30 minutes. and stirred for 1 hour. Water was added to the reaction mixture, and the mixture was extracted with AcOEt. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. Filter and concentrate the filtrate. The residue was purified by column chromatography (Hexane/AcOEt).
5N NaOH aqueous solution (240 μl) was added to a solution of the purified product (104 mg) in THF-MeOH-water (1:1:1) (6 ml), and the mixture was heated under reflux and stirred overnight. After concentration, 1N HCl aqueous solution was added to the residue to neutralize it, and the solid matter was collected by filtration to obtain the desired product (48.0 mg).

Reference example 22
Preparation of (E)-3-(4-methoxyindol-1-yl)prop-2-enoic acid
Cs ₂ CO ₃ (996 mg) and ethyl propiolate (248 μl) were added to a solution of 4-methoxyindole (300 mg) in DMF (3 ml), and the mixture was stirred at room temperature for 1 hour. Water was added to the reaction mixture, and the mixture was extracted with AcOEt. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. Filter and concentrate the filtrate. The residue was purified by column chromatography (Hexane/AcOEt). A 5N NaOH aqueous solution (636 μl) was added to a solution of the purified product in THF-tert-butanol-water (1:1:0.5) (9 ml), and the mixture was heated under reflux with stirring for 3 hours. After concentration, 1N HCl aqueous solution was added to the residue, and the solid matter was collected by filtration to obtain the desired product (212 mg).

Reference example 23
Preparation of (E)-3-(8-methoxyimidazo[1,2-a]pyridin-3-yl)prop-2-enoic acid To a solution of ethyl diethylphosphonoacetate (378 μl) in THF (5 ml), After adding NaH (76.0 mg) and stirring for 1 hour, a solution of 8-methoxyimidazo[1,2-a]pyridine-3-carbaldehyde (280 mg) in THF (10 ml) was added dropwise, and the mixture was stirred at room temperature for 1 hour. . Water was added to the reaction mixture, and the mixture was extracted with AcOEt. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. Filter and concentrate the filtrate. The residue was washed with IPE. 5N NaOH aqueous solution (804 μl) was added to a solution of the purified product in THF-MeOH-water (1:1:1) (6 ml), and the mixture was heated under reflux and stirred overnight. After concentration, 5N HCl aqueous solution was added to the residue to make it weakly acidic, and the solid matter was collected by filtration to obtain the desired product (212 mg).

Reference example 26
Preparation of 2-[2-(5-fluoropyrimidin-2-yl)phenyl]ethanamine hydrochloride
tert-butyl N-[2-[2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]ethyl]carbamate (222 mg), 2-chloro-5 - A mixture of fluoropyrimidine (118 μl), Pd(tBu ₃ P) ₂ (16.3 mg), K ₃ PO ₄ (271 mg), 1,4-dioxane/water (4/1) (2.5 ml) was placed under nitrogen atmosphere. The mixture was stirred at 90° C. for 7 hours. The reaction solution was concentrated and the residue was purified by column chromatography (Hexane/AcOEt). 4N HCl/AcOEt (0.5 ml) was added to an EtOH (1 ml) solution of the purified product, and the mixture was stirred at 50° C. for 1.5 hours. The reaction solution was concentrated to obtain the desired product (128 mg).

In the same manner as in Reference Examples 1-4, 6-8, 13-15, 19, 20, 22, 23 and 26, Reference Examples 5, 9-12, 16-18, 21, 24, 25, 27 and 28 were prepared respectively. The structural formulas and physical property data of the compounds of Reference Examples 1 to 28 are shown in Tables 1-1 to 1-5, respectively.

[Manufacturing example]
Production example 10
Preparation of (E)-N-[2-[2-(cyclopropylmethoxy)phenyl]ethyl]-3-(7-methoxy-1-methylindol-3-yl)-N-methylprop-2-enamide
(E)-N-[2-[2-(cyclopropylmethoxy)phenyl]ethyl]-3-(7-methoxy-1H-indol-3-yl)-N-methylprop-2-enamide (25.0 mg), Iodomethane (5.80 μl) and Cs ₂ CO ₃ (40.3 mg) were added to a DMF (1 ml) solution, and the mixture was stirred at room temperature for 5 hours. Water was added to the reaction solution and extracted with AcOEt. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. Filter and concentrate the filtrate. The residue was purified by column chromatography (Hexane/AcOEt) to obtain the desired product (23.0 mg).

Production Example 17
Preparation of (E)-3-(7-methoxy-1H-indol-3-yl)-N-[2-(2-pyrimidin-2-ylphenyl)ethyl]prop-2-enamide
(E)-3-(7-Methoxy-1H-indol-3-yl)prop-2-enoic acid (30.0 mg), 2-(2-pyrimidin-2-ylphenyl)ethanamine hydrochloride (71.6 mg) DIPEA (96.0 μl) and COMU (71.0 mg) were added to a DCM (3 ml) solution and stirred at room temperature for 2 hours. A saturated NaHCO ₃ aqueous solution was added to the reaction solution, and the mixture was extracted with AcOEt. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. Filter and concentrate the filtrate. The residue was purified by column chromatography (Hexane/AcOEt) to obtain the desired product (40 mg).

Production Example 19
Preparation of (E)-3-(7-methoxy-1H-indol-3-yl)-N-[2-(2-thiophen-2-ylphenyl)ethyl]prop-2-enamide
(E)-N-[2-(2-bromophenyl)ethyl]-3-(7-methoxy-1H-indol-3-yl)prop-2-enamide (30.0 mg), 2-thiopheneboronic acid (12.5 mg), PdCl ₂ (dppf) DCM (3.1 mg), K ₃ PO ₄ (31.9 mg), 1,4-dioxane/water (4/1) (1 ml) at 90° C. for 6 hours under a nitrogen atmosphere. Stirred for an hour. The reaction solution was purified by column chromatography (Hexane/AcOEt) to obtain the desired product (24.7 mg).

Production example 24
Preparation of (E)-3-(1-acetyl-7-methoxyindol-3-yl)-N-[2-(2-phenylphenyl)ethyl]prop-2-enamide
(E)-3-(7-Methoxy-1H-indol-3-yl)-N-[2-(2-phenylphenyl)ethyl]prop-2-enamide (25.0 mg) in DCE (0.6 ml) , TEA (0.050 ml), DMAP (7.2 mg) and acetic anhydride (0.011 ml) were added, and the mixture was stirred overnight at room temperature. The reaction solution was purified by column chromatography (Hexane/AcOEt). TEA (0.050 ml), DMAP (3.0 mg) and acetic anhydride (0.011 ml) were added to a DCE (0.6 ml) solution of the purified product, and the mixture was stirred at room temperature for 1 hour. The reaction solution was purified by column chromatography (Hexane/AcOEt) to obtain the desired product (20.2 mg).

Production example 25
Preparation of (E)-3-(1-benzoyl-7-methoxyindol-3-yl)-N-[2-(2-phenylphenyl)ethyl]prop-2-enamide
(E)-3-(7-Methoxy-1H-indol-3-yl)-N-[2-(2-phenylphenyl)ethyl]prop-2-enamide (19.7 mg) in DCE (0.6 ml) , TEA (0.039 ml), DMAP (5.7 mg) and benzoyl chloride (0.011 ml) were added and stirred at room temperature for 3 hours. The reaction solution was purified by column chromatography (Hexane/AcOEt) to obtain the desired product (22.2 mg).

Production example 29
Preparation of (E)-N-[2-(2-ethoxypyridin-3-yl)ethyl]-3-(7-methoxy-1H-indol-3-yl)prop-2-enamide
TFA (0.28 ml) was added to a mixture of 2-(2-ethoxypyridin-3-yl)acetonitrile (140 mg), NaBH ₄ (140 mg) and THF (3 ml) at 0°C, and the mixture was stirred at room temperature for 1 hour. . Water, saturated NaHCO ₃ aqueous solution were added, extracted with AcOEt, and the organic layer was concentrated. To a solution of the residue in DCM (1 ml) was added (E)-3-(7-methoxy-1H-indol-3-yl)prop-2-enoic acid (30.0 mg), DIPEA (0.036 ml), HATU (68.3 mg). ) was added and stirred at room temperature for 1 hour. The reaction solution was purified by column chromatography (Hexane/AcOEt) to obtain the desired product (10.8 mg).

Production example 45
Preparation of (E)-3-(7-methoxy-1H-indol-3-yl)-N-[2-(3-pyrimidin-2-ylthiophen-2-yl)ethyl]prop-2-enamide
DIPEA (82.0 µl), (E)-3-(7-methoxy -1H-indol-3-yl)prop-2-enoic acid (15.0 mg) and HATU (33.1 mg) were added, and the mixture was stirred overnight at room temperature. The reaction solution was purified by column chromatography (Hexane/AcOEt) to obtain the desired product (11.8 mg).

Production Example 47
Preparation of (E)-3-(1H-indol-3-yl)-N-[2-(2-pyrimidin-2-ylphenyl)ethyl]prop-2-enamide
DIPEA (128 µl), (E)-3-(1H-indol-3-yl ) Prop-2-enoic acid (27.5 mg) and HATU (72.6 mg) were added and stirred overnight at room temperature. The reaction solution was purified by column chromatography (Hexane/AcOEt/MeOH) to obtain the desired product (36.1 mg).

In the same manner as in Production Examples 10, 17, 19, 24, 25, 29, 45 and 47, Production Examples 1 to 9, 11 to 16, 18, 20 to 23, 26 to 28, 30 to 44, 46 and 48 ~56 compounds were each prepared. The structural formulas and physical property data of the compounds of Production Examples 1 to 56 are shown in Tables 2-1 to 2-13, respectively.

[Example]
Example 1 (Platelet Production: Shaking Culture)
The immortalized megakaryocyte cell line obtained by the method described in WO 2016/204256 was washed twice with D-PBS (-) and cultured in a doxycycline-free medium to release forced expression (gene expression OFF culture). The cells were seeded in a 125 mL polycarbonate Erlenmeyer flask (Corning #431143) at a seeding density of 1×10 ⁵ cells/mL, 25 mL/flask, and cultured in the following medium with shaking at 100 rpm. Culture conditions were 37° C. and 5% CO ₂ .
IMDM was used as a basal medium, and the following components were added (concentrations are final concentrations).
FBS 15%
L-Glutamine 2 mM
ITS 100-fold dilution
MTG 450 μM
Ascorbic acid 50 μg/mL
SCF 50 ng/mL
TA-316 0.1 μg/mL
ADAM inhibitor 15 μM
ROCK inhibitor 0.5 μM
At the same time as cell seeding, an aromatic hydrocarbon receptor inhibitor (compound A5, final concentration 0.1 μM) or DMSO (control) was added to initiate culture. Compounds that can be used in the method of the present invention (Production Examples 1 to 56, final concentration 10 μM) or DMSO (control) were added on the third day after culturing, and after culturing for a total of 6 days, the number of platelets was determined. was measured. The measurement method is as follows. The same operation was performed for the control.
After 6 days of gene expression OFF culture, a portion of the culture supernatant was collected, suspended with the following antibody and Flow-Count Fluorospheres (Beckman Coulter #7547053), and stained.
Anti-CD41 antibody APC-labeled (BioLegend #303710)
Anti-CD42a antibody eFluor 450 conjugated (eBioscience #48-0428-42)
Anti-CD42b antibody PE-labeled (BioLegend #303906)

Thirty minutes after the staining reaction, the platelet count (CD41, CD42a, CD42b positive cells) was calculated using Flow-Count Fluorospheres using FACSVerse manufactured by BD, Japan. The platelet count was described as a ratio to the control.
Table 3 shows the results of culturing by adding DMSO at the same time as the cells were seeded, and Table 4 shows the results of culturing by adding the aromatic hydrocarbon receptor inhibitors at the same time as the cells were seeded.
+ and ++ indicate an increase in platelet production of 1.5 times or more and less than 6.5 times and 6.5 times or more, respectively, compared to the control.

Claims (13)

General formula [I]:

[In the formula,
R ¹¹ is a hydrogen atom, halogen, -C _1-6 alkyl or -OC _1-6 alkyl,
^R2 is a hydrogen atom or _-C1-6 alkyl,
R ³ is halogen, -Q _k -(C _1-6 alkyl) _m -Q _p -R ³¹ , optionally substituted phenyl or furyl, thienyl, oxazolyl, thiazolyl, pyrazolyl, pyridyl, pyrazyl, heteroaryl optionally having a substituent selected from the group consisting of pyridazyl or pyrimidyl,
R ³¹ is -C _1-6 alkyl or -C _3-8 cycloalkyl,
each Q is independently the same or different and is an oxygen atom, a sulfur atom, -C(=O)-O- or -NH-;
k, m, p are 0 or 1,
n is 0, 1 or 2, and when n is 2, each R ³ is independently the same or different substituent
W is a carbon or nitrogen atom,
X is a carbon atom, a nitrogen atom or ^NR12 ,
Y is a carbon or nitrogen atom,
each Z is independently the same or different and is a nitrogen atom or CH;
However, X and Y are not carbon atoms at the same time
R ¹² is a hydrogen atom, -C _1-6 alkyl, -C _1-6 alkyl-OC _1-6 alkyl, -C(=O)-C _1-6 alkyl, -C(=O)-aryl or - C(=O)-OC _1-6 alkyl,
Ring A is aryl or heteroaryl,
--- is a single or double bond,
provided that when X is NH, Y is a carbon atom, and all Zs are CH, ring A excludes 2-(-OC _1-6 alkyl)phenyl or 2,5-di(-OC _1-6 alkyl)phenyl ]
Platelet production promoter comprising a compound represented by or a salt thereof. In general formula [I],

[Wherein, R ¹¹ , W, X, Y, Z and --- are the same as defined above]
The accelerator according to claim 1, which is In general formula [I],

[wherein R ³ and n are the same as defined above]
The accelerator according to claim 1, which is In general formula [I], heteroaryl in ring A is selected from furan, thiophene, pyridine and quinoline,
Accelerator according to claim 1. In general formula [I],

[wherein V is the same or different and each independently is a nitrogen atom or CH, and ^R4 is a hydrogen atom, halogen, -C1-6 _alkyl or -OC1-6 _alkyl ]
The accelerator of claim 1, which is General formula [Ia]

[wherein R ¹¹ is a hydrogen atom, halogen, -C _1-6 alkyl or -OC _1-6 alkyl,

is pyridylbenzene, (pyrimidyl optionally substituted by halogen, _-C1-6alkyl or _-OC1-6alkyl )benzene, phenylthiophene, pyridylthiophene or pyrimidylthiophene]
The accelerator according to claim 1, represented by Accelerator according to claim 1, wherein the compound is selected from:

8. A enhancer according to any one of claims 1 to 7 for use in combination with an aromatic hydrocarbon receptor inhibitor. A method for producing platelets using the compound according to any one of claims 1 to 7 or a salt thereof. Use of a compound or a salt thereof according to any one of claims 1 to 7 for promoting platelet production. A method for promoting platelet production, comprising culturing platelet progenitor cells in the presence of the compound or salt thereof according to any one of claims 1 to 7. A method for producing platelets, comprising culturing platelet progenitor cells in the presence of the compound or salt thereof according to any one of claims 1 to 7. A method for culturing proplatelet cells for promoting platelet production, the method comprising culturing proplatelet cells in the presence of the compound according to any one of claims 1 to 7 or a salt thereof.