JP2016065055A - Myeloid leukemia therapeutic agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a therapeutic agent effective against myeloid leukemia.SOLUTION: The therapeutic agent comprises a quinoline compound having a specific structure comprising 5-amino-7-(cyclohexylamino)-1-ethyl-6-fluoro-4-oxo-1,4-dihydro quinoline-3-carboxylic acid as an active ingredient.SELECTED DRAWING: Figure 4

Description

  The present invention relates to a therapeutic agent for myeloid leukemia comprising a specific compound as an active ingredient.

  In myeloid leukemia, the presence of leukemia stem cells, a group having self-replicating ability and tumor remodeling ability, has been shown (Non-patent Document 3), and it has been shown to cause treatment resistance and malignant progression. In order to cure leukemia, it is necessary to develop a new treatment targeting leukemia stem cells.

  For the treatment of chronic myelogenous leukemia, ABL selective tyrosine kinase inhibitors (ST1571, Imanitiv, etc.) are currently known as molecular targeted therapeutic agents. Imatinib acts by targeting the BCR-ABL1 protein, which causes chronic myeloid leukemia, and reduces leukemic cells. Imatinib inhibits proliferation of leukemia cells by interfering with the ATP binding site of BCR-ABL1 protein, which is important for signal transduction of leukemia cells in chronic myelogenous leukemia, and suppressing signal transduction that occurs when ATP is originally bound. Show antitumor effects.

  However, imatinib clinical trials report that many patients with advanced stage of chronic myeloid leukemia respond well, but leukemia stem cells remain and then relapse. Also in acute myeloid leukemia, leukemia stem cells are known to be resistant to existing treatments. Under such circumstances, a new therapeutic agent for myeloid leukemia stem cells has been desired.

  By the way, FOXO is a transcription factor belonging to the subgroup “O” of the Forkhead family having the DNA binding domain Forkhead box (FOX), and so far, through the induction of expression of many genes, stress response, metabolic control, cell It is known to be an important molecule for maintaining the normal hematopoietic stem cell pool, involved in the cycle, apoptosis, DNA repair, and the like. Furthermore, it is known that it is essential for imatinib resistance of chronic myeloid leukemia stem cells (Non-patent Document 1) and maintenance of undifferentiation of acute myeloid leukemia stem cells (Non-patent Document 2).

  Based on the above knowledge, as a method for screening for a therapeutic agent for myeloid leukemia stem cells, for example, the method disclosed in Patent Document 1 can be mentioned. In the screening method disclosed in Patent Literature 1, forkhead transcription factors (for example, Foxo3a etc.) are required for the survival of leukemia stem cells of chronic myeloid leukemia. A test compound that promotes phosphorylation or a test compound that inhibits the expression of a forkhead transcription factor is screened as a therapeutic or prophylactic agent for chronic myeloid leukemia.

  Non-Patent Document 2 shows that a forkhead transcription factor is essential for maintaining the undifferentiation of acute myeloid leukemia stem cells.

Japanese Patent No. 5555897

Nature, 463, p. 676-680, 2010 Cell 146, p. 697-708, 2011 Nature Medicine, 3, p730-737, 1997

  However, as shown in Patent Document 1, a drug that inactivates a forkhead transcription factor can be expected to have a therapeutic effect on myeloid leukemia, but the degree of effect of various drugs is completely unknown. The current situation is that no effective therapeutic agent for myeloid leukemia has been found. Therefore, in view of such circumstances, an object of the present invention is to provide an effective therapeutic agent for myeloid leukemia.

  As a result of intensive studies by the present inventors in order to achieve the above-mentioned object, it has been found that a compound having a specific structure among forkhead type transcription factor inhibitors has an extremely excellent therapeutic effect on myeloid leukemia. The invention has been completed.

［式中の記号は、以下の意味を示す。
X：C-R⁷、又はN。
Y：C-R⁶、又はN。
R²：それぞれ置換されていてもよい低級アルキル、シクロアルキル、アリール若しくはへテロ環。
R³：ハロゲン、低級アルキル、又は-O-低級アルキル。
R⁴：それぞれ置換されていてもよいシクロアルキル若しくは非芳香族へテロ環、又はシクロアルキルで置換されている低級アルキル。ただし、R⁴が置換されていてもよい非芳香族へテロ環を示す場合、環を構成する炭素原子が隣接するNHと結合するものとする。
R⁵：-H、ハロゲン、シアノ、ニトロ、低級アルキル、ハロゲノ低級アルキル、シクロアルキル、アリール、ヘテロ環、-O-低級アルキル、-OH、-NHCO-低級アルキル、-N(低級アルキル)CO-低級アルキル、低級アルキルで置換されていてもよいアミノ、又は置換されていてもよい環状アミノ。
R⁶：-H、ハロゲン、低級アルキル又はハロゲノ低級アルキル。
R⁷：-H、ハロゲン、低級アルキル又はハロゲノ低級アルキル。
ただし、YがC-R⁶を示す場合、R²とR⁶は一体となって、低級アルキレン、又は低級アルケニレンを形成してもよい。］ The present invention includes the following.
(1) A therapeutic agent for myeloid leukemia comprising a quinoline compound (X) represented by the following formula as an active ingredient.

[The symbols in the formula have the following meanings.
X: CR ⁷ or N.
Y: CR ⁶ or N.
R ² : each optionally substituted lower alkyl, cycloalkyl, aryl or heterocycle.
R ³ : halogen, lower alkyl, or —O-lower alkyl.
R ⁴ : each independently substituted cycloalkyl or non-aromatic heterocyclic ring, or lower alkyl substituted with cycloalkyl. However, when R ⁴ represents an optionally substituted non-aromatic heterocycle, the carbon atoms constituting the ring shall be bonded to the adjacent NH.
R ⁵ : —H, halogen, cyano, nitro, lower alkyl, halogeno lower alkyl, cycloalkyl, aryl, heterocycle, —O-lower alkyl, —OH, —NHCO-lower alkyl, —N (lower alkyl) CO— Lower alkyl, amino optionally substituted with lower alkyl, or cyclic amino optionally substituted.
R ⁶ : —H, halogen, lower alkyl or halogeno lower alkyl.
R ⁷ : —H, halogen, lower alkyl or halogeno lower alkyl.
However, when Y represents CR ⁶ , R ² and R ⁶ may be combined to form lower alkylene or lower alkenylene. ]

(2) In the quinoline compound (X), X and Y are CH, R ² is lower alkyl, R ³ is halogen, and R ⁴ may be substituted cycloalkyl R ⁵ is lower alkyl The therapeutic agent according to (1), which is amino optionally substituted by

(3) The quinoline compound (X) is a 5-amino-7- (cyclohexylamino) -1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid represented by the following formula: The therapeutic agent according to (1), which is

(4) The therapeutic agent according to (1), wherein the myeloid leukemia is acute myeloid leukemia.

  According to the present invention, an effective therapeutic agent for myeloid leukemia including chronic myelogenous leukemia can be provided.

It is a characteristic figure which shows the FOXO transcriptional activity inhibitory activity (right) by AS1842856, and the FOXO transcriptional activity inhibitory activity (left) by AS1708727. It is a characteristic figure which shows the expression level fluctuation | variation of the FOXO target gene (p27 gene, BIM gene, and Bcl6 gene) expression-induced by imatinib by AS1842856. It is a characteristic figure which shows the expression level fluctuation | variation of the FOXO target gene (Bcl6 gene) induced by imatinib by AS1708727. It is a characteristic figure which shows the cell growth inhibitory effect with respect to a leukemia origin cell line by AS1842856. It is a characteristic view which shows the result of having detected the luminescent signal resulting from the expression of FOXO in a control strain (scrambled) and a shFOXO1 / 3a function suppression cell strain. It is a characteristic figure which shows the cell growth inhibitory effect by AS1842856 in a control strain (scrambled) and a shFOXO1 / 3a function suppression cell strain. It is a photograph which shows the result of having observed the microscope about THP-1 when processing AS1842856. It is a photograph which shows the result of having observed with microscope about U937 when processing AS1842856. It is a characteristic view which shows the histogram regarding CD11b positive cell when AS1708727 or AS1842856 is used by a high concentration (0-25 micromol). It is a characteristic view which shows the histogram regarding CD11b positive cell when AS1842856 is used by a high concentration (0-25 micromol) or a low concentration (0-10 micromol). It is a characteristic view which shows the result of having counted the number of colony formation when AS1842856 was made to act with respect to a human cord blood origin mononuclear cell. It is a characteristic view which shows the result of having measured the tumor volume about AS18 administration group and a control group. It is a photograph which shows the result of having carried out HE staining about the tumor mass of an AS18 administration group and a control group. It is a photograph which shows the result of the IHC dyeing | staining using the anti-CD11b antibody about the tumor mass of an AS18 administration group and a control group. It is a photograph which shows the result of the IHC dyeing | staining using the anti-CD115 antibody about the tumor mass of an AS18 administration group and a control group. It is a photograph which shows the result of the IHC dyeing | staining using an anti-Vimentin antibody about the tumor mass of an AS18 administration group and a control group. It is a photograph which shows the result of the IHC dyeing | staining using an anti- Lysozyme antibody about the tumor mass of an AS18 administration group and a control group. It is a photograph which shows the result of the IHC dyeing | staining using an anti- PCNA antibody about the tumor mass of an AS18 administration group and a control group. It is a photograph which shows the result of the IHC dyeing | staining using an anti- Ki67 antibody about the tumor mass of an AS18 administration group and a control group. It is a photograph which shows the result of the IHC dyeing | staining using an anti- MCM2 antibody about the tumor mass of an AS18 administration group and a control group.

Hereinafter, the present invention will be described in detail.
<Active ingredient>
The therapeutic agent for myeloid leukemia according to the present invention contains a quinoline compound (X) represented by the following formula as an active ingredient.

［式中の記号は、以下の意味を示す。
X：C-R⁷、又はN。
Y：C-R⁶、又はN。
R²：それぞれ置換されていてもよい低級アルキル、シクロアルキル、アリール若しくはへテロ環。
R³：ハロゲン、低級アルキル、又は-O-低級アルキル。
R⁴：それぞれ置換されていてもよいシクロアルキル若しくは非芳香族へテロ環、又はシクロアルキルで置換されている低級アルキル。ただし、R⁴が置換されていてもよい非芳香族へテロ環を示す場合、環を構成する炭素原子が隣接するNHと結合するものとする。
R⁵：-H、ハロゲン、シアノ、ニトロ、低級アルキル、ハロゲノ低級アルキル、シクロアルキル、アリール、ヘテロ環、-O-低級アルキル、-OH、-NHCO-低級アルキル、-N(低級アルキル)CO-低級アルキル、低級アルキルで置換されていてもよいアミノ、又は置換されていてもよい環状アミノ。
R⁶：-H、ハロゲン、低級アルキル又はハロゲノ低級アルキル。
R⁷：-H、ハロゲン、低級アルキル又はハロゲノ低級アルキル。
ただし、YがC-R⁶を示す場合、R²とR⁶は一体となって、低級アルキレン、又は低級アルケニレンを形成してもよい。］

[The symbols in the formula have the following meanings.
X: CR ⁷ or N.
Y: CR ⁶ or N.
R ² : each optionally substituted lower alkyl, cycloalkyl, aryl or heterocycle.
R ³ : halogen, lower alkyl, or —O-lower alkyl.
R ⁴ : each independently substituted cycloalkyl or non-aromatic heterocyclic ring, or lower alkyl substituted with cycloalkyl. However, when R ⁴ represents an optionally substituted non-aromatic heterocycle, the carbon atoms constituting the ring shall be bonded to the adjacent NH.
R ⁵ : —H, halogen, cyano, nitro, lower alkyl, halogeno lower alkyl, cycloalkyl, aryl, heterocycle, —O-lower alkyl, —OH, —NHCO-lower alkyl, —N (lower alkyl) CO— Lower alkyl, amino optionally substituted with lower alkyl, or cyclic amino optionally substituted.
R ⁶ : —H, halogen, lower alkyl or halogeno lower alkyl.
R ⁷ : —H, halogen, lower alkyl or halogeno lower alkyl.
However, when Y represents CR ⁶ , R ² and R ⁶ may be combined to form lower alkylene or lower alkenylene. ]

In particular, as an active ingredient of the therapeutic agent for myeloid leukemia according to the present invention, in the above formula, X and Y are CH, R ² is lower alkyl, R ³ is halogen, and R ⁴ is substituted. It is preferable that R ⁵ is an optionally substituted cycloalkyl and R ⁵ is optionally substituted with lower alkyl.

  Furthermore, as an active ingredient of the therapeutic agent for myeloid leukemia according to the present invention, 5-amino-7- (cyclohexylamino) -1-ethyl-6-fluoro-4-oxo-1,4- represented by the following formula: Dihydroquinoline-3-carboxylic acid (hereinafter, the compound is referred to as AS1842856) is more preferable. AS1842856 does not bind to non-active forkhead transcription factor (FOXO1) protein (phosphorylated) as described in the reference (Mol. Pharmacol. 78: 961-970, 2010). A specific inhibitory action was shown (IC50 = 0.033 uM), and it has been shown that it exhibits a blood glucose-lowering action in db / db mice after oral administration.

Here, the term “lower” means a straight or branched carbon chain having 1 to 6 carbon atoms unless otherwise specified. Thus, “lower alkyl” means C _1-6 alkyl, specifically, for example, methyl, ethyl, propyl, butyl, pentyl or hexyl, or structural isomers such as isopropyl or tert-butyl. And preferably C _1-5 alkyl methyl, ethyl, propyl, isopropyl, butyl, isobutyl, 3-pentyl.

“Lower alkenyl” means C _2-6 alkyl having one or more double bonds at any position, specifically, for example, ethenyl, propenyl, butenyl, pentenyl, hexenyl, butadienyl, etc. Preferred are C _2-3 alkenyl ethenyl, 1-propenyl, 2-propenyl and 3-propenyl.

“Lower alkynyl” means C _2-6 alkyl having one or more triple bonds at any position.

  “Lower alkylene” means a divalent group formed by removing one hydrogen at any position of “lower alkyl”, specifically methylene, methylmethylene, ethylene, trimethylene, propylene, butylene, etc. Preferred are methylene, ethylene and trimethylene.

  “Lower alkenylene” means a divalent group formed by removing one hydrogen at an arbitrary position of “lower alkenyl”, specifically vinylene, 1-propenylene, 2-propenylene, 1-butenylene, 2- Butenylene, 3-butenylene and the like, preferably vinylene, 1-propenylene and 2-propenylene.

  “Lower alkylidene” means a group in which a free valence formed by removing one hydrogen from a carbon atom having a bond of “lower alkyl” becomes part of a double bond.

“Cycloalkyl” means a monovalent group of a C _3-10 non-aromatic hydrocarbon ring, which may form a bridged ring or a spiro ring, and has a partially unsaturated bond. It may be. Specific examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, cyclohexenyl, cyclooctanedienyl, adamantyl, and norbornyl, with cyclopentyl or cyclohexyl being preferred.

“Aryl” means a monovalent group of a monocyclic to tricyclic C _6-14 aromatic hydrocarbon ring, and specific examples include phenyl, naphthyl, etc., preferably phenyl. .

  The term “non-aromatic heterocycle” refers to a heteroatom such as nitrogen, oxygen, or sulfur that may have a saturated or partially unsaturated bond and may be condensed with an aryl or aromatic heterocycle. It means a monovalent group having 3 to 10 members, preferably 5 to 7 members, having 4 to 4 members. However, the bond is on a saturated or partially unsaturated ring. Specific examples include pyrrolidinyl, piperidinyl, piperazinyl, azepinyl, morpholinyl, thiomorpholinyl, pyrazolidinyl, dihydropyrrolyl, tetrahydropyranyl, tetrahydrofuryl, dioxanyl, tetrahydrothiopyranyl, tetrahydrothienyl, etc., preferably pyrrolidinyl, piperidinyl Piperazinyl, azepinyl, morpholinyl, thiomorpholinyl, tetrahydropyranyl, dioxanyl, tetrahydrothiopyranyl.

  “Heterocycle” is a general term in which “aromatic heterocycle” is added to the above “non-aromatic heterocycle”, and “aromatic heterocycle” is the same selected from the group consisting of nitrogen, oxygen and sulfur. Or a monovalent group of an aromatic heterocycle which may be condensed with a benzene ring, containing 1 to 4 different heteroatoms, specifically, for example, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, triazolyl , Oxazolyl, thiazolyl, furazanyl, pyridyl, pyranyl, thiopyranyl, pyridazinyl, pyrimidinyl, pyrazyl, indolyl, isoindolyl, indolizinyl, benzofuryl, benzothienyl, benzoimidazolyl, indazolyl, benzotriazolyl, benzoxazolyl, benzothiazolyl, benzooxadiazonyl , Quinolyl, isoquinolyl, chrome , Benzothiopyranyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, benzodioxolyl, benzodioxinyl, benzodioxepinyl, carbazolyl, etc., and the nitrogen atom constituting these rings and / or The sulfur atom may be oxidized. These rings may be partially saturated. However, the bond is on the aromatic ring. Pyridyl, furyl, thienyl, indolyl, indazolyl, and benzotriazolyl are preferable.

  “Halogen” means a monovalent group of a halogen atom, and specific examples thereof include fluoro, chloro, bromo, iodo and the like, preferably fluoro and chloro.

  The “halogeno lower alkyl group” means a group in which one or more arbitrary hydrogen atoms of the “lower alkyl group” are substituted with one or more “halogen”, specifically, for example, trifluoromethyl, trifluoro Ethyl etc. are mentioned, Preferably it is trifluoromethyl.

  “Cyclic amino” is a monovalent heterocyclic group having at least one nitrogen atom and having a bond to the nitrogen atom, and may further contain oxygen or sulfur as a hetero hetero atom. Specific examples include pyrrolidino, piperidino, piperazino, homopiperazino, morpholino, thiomorpholino, 3,4-dihydroisoquinolin-2 (1H) -yl, and preferably pyrrolidino, piperidino, piperazino, 3,4-dihydroisoquinoline. -2 (1H) -yl.

  The “reactive derivative” of the carboxylic acid derivative in the production method for condensing the carboxylic acid derivative and the amine compound is not particularly limited as long as it is a derivative that increases the reactivity in the reaction. For example, it corresponds to the carboxylic acid derivative. Acid halide derivatives or mixed anhydrides, or carboxylic acid derivatives and carbonyldiimidazole (CDI), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (WSC.HCl), dicyclohexylcarbodiimide, diphenyl Derivatives prepared from a condensing agent such as phosphoryl azide and diethylphosphoryl cyanide.

  In the present specification, the permissible substituent of the word “which may be substituted” may be any substituent as long as it is a commonly used substituent in the technical field. In addition, 1 to 5 or more of the same or different substituents may be present in each group.

“Optionally substituted cycloalkyl”, “optionally substituted aryl”, “optionally substituted non-aromatic heterocycle”, “optionally substituted aromatic heterocycle” for R ² ; R ⁴ "cycloalkyl which may be substituted" in, "optionally substituted non-aromatic heterocycle"; substituents allowed in and the "cyclic amino which may be substituted" in R ⁵ Examples of these include groups shown in the following (a) to (h). Further, examples of the substituent allowed in “lower alkyl which may be substituted” and “lower alkenyl which may be substituted” in R ² include groups shown in the following (a) to (g). It is done. R ^Z is —OH, —O-lower alkyl, amino optionally substituted with one or two lower alkyls, —CO ₂ H, —CO ₂ R ^Z , one or two lower Optionally substituted with one or more groups selected from the group consisting of carbamoyl, aryl (which may be substituted with halogen), aromatic heterocycles and halogen which may be substituted with alkyl Lower alkyl is shown.

(A) Halogen.
(B) -OH, -OR ^Z , -O-aryl, -OCO-R ^Z , oxo (= O), -OSO ₃ H, -OP (O) (O-RZ) ₂ , -P (O) ( O-RZ) ₂ , -OP (O) (OH) (OR ^Z ), -P (O) (OH) (OR ^Z ), -OP (O) (OH) ₂ , -P (O) (OH) ₂ .
(C) —SH, —SR ^Z , —S-aryl, —SO—R ^Z , —SO-aryl, —SO ₂ —R ^Z , —SO ₃ H, —SO ₂ -aryl, one or two R Sulfamoyl optionally substituted with ^Z.
(D) amino, —NHCO—R ^Z , —NHCO-aryl, —NHSO ₂ —R ^Z , —NHSO ₂ -aryl, nitro, imino optionally substituted with one or two R ^Z (= NR ^Z ).
(E) —CHO, —CO—R ^Z , —CO ₂ H, —CO ₂ —R ^Z , carbamoyl optionally substituted with one or two R ^Z or aryl, —CO—non-aromatic heterocycle (This non-aromatic heterocycle may be substituted with —CO ₂ H or —CO ₂ —R ^Z ), cyano.
(F) Aryl or cycloalkyl. These groups are --OH, --O-lower alkyl, amino optionally substituted with one or two lower alkyls, --CO ₂ H, --CO ₂ R ^Z , one or two lower alkyls. Each of which may be substituted with one or more groups selected from the group consisting of carbamoyl, aryl, aromatic heterocycle, halogen and R ^Z which may be substituted with
(G) An aromatic heterocycle or a non-aromatic heterocycle. These groups are —OH, —O-lower alkyl, oxo (═O), amino optionally substituted with one or two lower alkyls, —CO ₂ H, —CO ₂ R ^Z , 1 It may be substituted with one or more groups selected from the group consisting of carbamoyl, aryl, aromatic heterocycle, halogen and R ^Z which may be substituted with one or two lower alkyls.
(H) A lower alkyl which may be substituted with one or more groups selected from the substituents shown in the above (a) to (g).

  “Amino optionally substituted with an optionally substituted lower alkyl group” means an amino mono- or di-substituted with the above-mentioned “optionally substituted lower alkyl group” and is disubstituted lower alkyl The groups may be the same or different from each other.

  The quinolone derivative represented by the formula (X) may form a salt and is included in the target compound according to the production method of the present invention as long as the salt is a pharmaceutically acceptable salt. Specifically, inorganic salts such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid Acid addition salts with organic acids such as lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, aspartic acid, glutamic acid, metals such as sodium, potassium, calcium, magnesium Examples thereof include inorganic bases, addition salts with organic bases such as methylamine, ethylamine, ethanolamine, lysine, ornithine, and ammonium salts.

  In addition, the quinolone derivative represented by the formula (X) may contain an asymmetric carbon atom depending on the type of substituent, and optical isomers based on this may exist. The production method of the present invention includes all production methods for producing a mixture or an isolated product of these optical isomers. In addition, the quinolone derivative represented by the formula (X) may exist as a tautomer, but the therapeutic agent for myeloid leukemia of the present invention includes a mixture of these isomers or a mixture thereof. . Further, a label, that is, a compound in which one or more atoms of the quinolone derivative represented by the formula (X) are substituted with a radioactive isotope or a non-radioactive isotope is also encompassed in the present invention.

  Furthermore, the present invention also includes various hydrates and solvates of the quinolone derivative represented by the formula (X) and substances having crystal polymorphs. That is, the therapeutic agent for myeloid leukemia according to the present invention includes all of the derivative represented by the formula (X) and pharmaceutically acceptable salts thereof. Of course, the therapeutic agent for myeloid leukemia according to the present invention is not limited to the production method of the quinolone derivative represented by the formula (X), and may be produced by any production method. In addition, although it does not specifically limit as a manufacturing method of the quinolone derivative represented by Formula (X), For example, the method disclosed by patent 4858683 can be mentioned.

<Myeloid leukemia>
In the present invention, “myeloid leukemia” is meant to include chronic myeloid leukemia and acute myeloid leukemia.

  Chronic myelogenous leukemia (hereinafter sometimes abbreviated as “CML”) is a chromosomal abnormality in which part of chromosomes 9 and 22 are replaced, that is, a BCR / ABL chimeric gene formed as a result of the Philadelphia translocation. Is a myeloproliferative disorder that develops from The BCR / ABL chimeric protein has significantly enhanced tyrosine kinase activity possessed by ABL, and by stimulating cell proliferation signals such as RAS signal, PI-3K / Akt signal and Stat signal constantly, the growth stimulating effect And exerts anti-apoptotic effect.

  Diagnosis of chronic myelogenous leukemia is usually based on a blood count test of the collected blood and a bone marrow test to check the hematopoietic status in the bone marrow. In the bone marrow examination, the proliferation state of leukocyte cells in the bone marrow is examined, chromosome / gene analysis of leukemia cells is performed, and the diagnosis is made based on the results.

  When the therapeutic agent according to the present invention is applied to chronic myelogenous leukemia, the stage of chronic myelogenous leukemia may be in the chronic phase, in the transitional phase, or in the acute transformation phase. That is, the therapeutic agent according to the present invention can be applied regardless of the degree of progression of chronic myelogenous leukemia.

  Acute myeloid leukemia (hereinafter sometimes abbreviated as “AML”) is an acute leukemia that is a neoplastic disease caused by abnormal proliferation of white blood cells, which are blood cells. This is a myeloproliferative disease in which abnormally proliferating cells are myeloid cells.

  Diagnosis of acute myeloid leukemia is usually performed by combining the results of symptom confirmation, blood test, and bone marrow test (bone marrow biopsy, bone marrow puncture). That is, in blood tests and bone marrow tests, leukemia cells (blasts) are higher than a predetermined value in bone marrow or peripheral blood, and increasing blasts are more than a predetermined value by myeloperoxidase (MPO) staining. Diagnose with AML.

  In addition, when the therapeutic agent according to the present invention is applied to acute myeloid leukemia, it may be applied to any disease type (M0 to M7) regardless of the FAB classification (French-American-British Classification) of acute myeloid leukemia. .

<Dosage form and dose>
The quinolone derivative represented by the above formula (X), which is an active ingredient of the therapeutic agent according to the present invention, is a powder, a granule, a tablet, a capsule, an intestinal solvent, a liquid, an injection (liquid, It can be prepared according to a conventional method in various forms such as a suspension. The therapeutic agent according to the present invention is usually preferably prepared as a pharmaceutical composition using one or more pharmaceutical carriers.

  Further, the dose or intake of the therapeutic agent according to the present invention is not particularly limited as long as the effect of the present invention can be obtained, and the effectiveness of the components contained, the dosage form, the administration route, It is appropriately selected according to the type of disease, the nature of the subject (weight, age, medical condition, presence or absence of use of other medicines, etc.), judgment of the doctor in charge, and the like. The therapeutic agent according to the present invention can be administered in 1 to several times a day, and may be administered intermittently at a rate of once every several days or weeks.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, the technical scope of this invention is not limited to a following example.

<Forkhead transcription factor (FOXO) transcription activity inhibition experiment>
In this example, FOXO transcriptional activity inhibition experiments (FOXO1, FOXO3 and FOXO4) were performed on two types of compounds known to be FOXO inhibitors (AS1842856 and AS1708727). AS1708727 is 2-cyclopentyl-N- {2,4-dichloro-3-[(isoquinolin-5-yloxy) methyl] phenyl} -N-methylacetamide, European Journal of Pharmacology 645 (2010) 185- 191 is a compound in which FOXO1 transcription factor inhibitory activity was confirmed.

In this example, HEK293 cells were seeded in a DMEM (Dulbecco's Modified Eagle's Medium) containing 10% fetal bovine serum (FBS) in a 2x10 ⁶ cells / 10 cm plate and cultured at 5% CO ₂ at 37 ° C for 24 hours. .

Gene transfer was performed according to the protocol using 10 μg of FOXO expression plasmid using X-tremeGENE HP DNA Transfection Reagent (Roche). As a FOXO expression plasmid per plate, 2.8 μg FOXO1 (pcDNA3 Flag-FKHR: purchased from addgene, product number: # 8360), FOXO3 (Flag-FOXO3a WT (product number: # 13507) purchased from addgene) are restriction enzymes Plasmids constructed by cleaving with Xba I and Hind III, and inserting FOXO3a WT cDNA into the Xba I and Hind III sites of pcDNA3 Flag: pcDNA3 Flag-FOXO3a) and 5.7μg 6xDBE / pGL basic Luciferase reporter vector Provided by Dr. Noboru Motoyama (Reference Center: Kobayashi Int J Mol Med 2005). In addition, 1.4 μg pZeoSVO2 / lacZ vector (manufactured by lifetechnologies) was added to each as an internal standard. Five hours after gene introduction, cells were collected by trypsin treatment and washed once with 10 mL of PBS. A cell suspension was prepared so as to be 5,000 cells / 30 μL (1.67 × 10 ⁵ cells / mL) in 1% FBS-containing DMEM.

For the two types of compounds described above, a plurality of reagents having different concentrations were prepared in DMEM containing 1% FBS so that the final concentrations were 0.25, 0.5, 1, 5, 10, 25, or 50 μM, respectively. The reagent amount was 10 μl and added to each well of a plate (Product No. 781182, manufactured by Greiner). A cell suspension (5,000 cells / 30 μl) was dispensed into each well. The total reaction volume was 40 μl. After culturing at 5% CO ₂ and 37 ° C. for 20 hours, 40 μL of Glo Lysis buffer was added and left for 10 minutes to lyse the cells (total amount: 80 μL). The cell lysate was divided into two parts (30 μl each), transferred to a plate (Product No. 781080, manufactured by Greiner), and Steady-Glo (30 μl) was added to one, and luciferase activity was measured. On the other side, Beta-Glo: Promega (30 μl) was added and b-gal activity was measured.

Activity values were measured with a microplate reader (infinite 200 pro). Internal correction was performed using the luciferase activity value / b-gal activity value to obtain the activity value of each well. The inhibitory effect (%) for the two types of compounds described above was calculated with the average value of the measured values of 0 μM as 100%. IC50 was calculated according to the following formula.
IC50 = 10 ^ (LOG (A / B) * (50-C) / (DC) + LOG (B))
A: High concentration across 50%
B: Low concentration across 50%
Inhibition rate at C: B
Inhibition rate with D: A

  The results are shown in FIG. The right graph in FIG. 1 shows the FOXO transcription activity inhibitory activity by AS1842856, and the left graph shows the FOXO transcription activity inhibitory activity by AS1708727. As shown in FIG. 1, the two compounds evaluated in this example showed transcription inhibitory activity against Foxo1 as previously reported, and also showed transcription inhibitory activity against Foxo3. It was.

<Foxo target gene expression suppression experiment>
Next, the expression levels of genes (p27, BIM and Bcl6) that are transcriptionally induced by the FOXO transcription factor in the presence of the compound AS1842856 were evaluated by quantitative PCR. For comparison, the expression level of Bcl6 was evaluated by quantitative PCR in the presence of compound AS1708727.

Specifically, first, KCL22 cells, a cell line derived from chronic myeloid leukemia, were seeded on 1.2 × 10 ⁶ cells / 10 cm plate. Then, the compound was added so that the final concentration was 0, 0.1, 1, or 10 μM, and after pretreatment for 30 minutes, 1 μM of Imatinib mesylate (biovision) was added, and 5% CO ₂ at 37 ° C. for 48 hours. Cultured. After culturing, the supernatant was removed, washed once with PBS, and the cells were collected, and then total RNA was prepared.

  The total RNA was prepared using a QIAquick PCR Purification Kit (Quagen) according to the attached experimental instructions. After measuring the RNA concentration with absorbance at a wavelength of 260 nm, reverse transcription reaction was performed using Advantage cDNA PCR Kit (manufactured by Clonetech) to synthesize cDNA. Downstream gene expression was evaluated by quantitative PCR using SYBR Premix Ex Taq (Tli RNase H Plus) (TaKaRa) using cDNA as a template.

  In the system using the compound AS1842856, amplification was performed using specific primers that recognize downstream genes of FOXO (p27, BIM and Bcl6), and the mRNA expression level was quantified. The GAPDH expression level was corrected as an internal control, and the effect of AS1842856 on expression induction was evaluated. Further, in the system using the compound AS1708727, the effect of AS1708727 on the induction of Bcl6 expression was similarly evaluated.

  As specific primers for amplifying the GAPDH gene, forward primer: 5'-GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 1) and reverse primer: 5'-GAAGATGGTGATGGGATTTC (SEQ ID NO: 2) were used. As specific primers for amplifying the p27 gene, forward primer: 5'-GCCCTCCCCAGTCTCTCTTA (SEQ ID NO: 3) and reverse primer: 5'-TCAAAACTCCCAAGCACCTC (SEQ ID NO: 4) were used. As specific primers for amplifying the BIM gene, forward primer: 5'-TATGAGAAGATCCTCCCTGC (SEQ ID NO: 5) and reverse primer: 5'-ATATCTGCAGGTTCAGCCTG (SEQ ID NO: 6) were used. As specific primers for amplifying the Bcl6 gene, forward primer: 5'-CTCAGATTCTAGCTGTGAGAACG (SEQ ID NO: 7) and reverse primer: 5'-GTCACACTTGTAGGGTTTGTCAC (SEQ ID NO: 8) were used.

  The effect of compound AS1842856 on the induction of p27 gene, BIM gene and Bcl6 gene expression is shown in FIG. 2, and the effect of compound AS1708727 on the induction of Bcl6 gene expression is shown in FIG. As shown in FIG. 2, it was revealed that the expression of the FOXO target gene whose expression is induced by imatinib in KCL22 cells is suppressed by AS1842856. On the other hand, as shown in FIG. 3, AS1708727, which is a Foxo inhibitor, similar to AS1842856, was found to be unable to suppress the expression of genes induced by imatinib in KCL22 cells.

  Bcl6 protein is considered as one of the causes of imatinib resistance in chronic myelogenous leukemia, and it is known that the expression of BcL6 gene is increased by imatinib administration. As shown in FIG. 2, it can be seen that in the presence of AS1842856, the effect of enhancing implication of Bcl6 gene by imatinib is reduced or lost. This result suggests that AS1842856 is effective against chronic myeloid leukemia that is resistant to imatinib.

<Cell growth inhibitory effect of imatinib in combination with chronic myeloid leukemia cell line>
Next, the cell growth inhibitory effect of the chronic myeloid leukemia cell line by the combined use of AS1842856 and imatinib was examined. Specifically, a cell suspension of a chronic myelogenous leukemia cell line (K562) was prepared so as to be 10,000 cells / 75 μl. The reaction was started by adding 25 μl of AS1842856 and 75 μl of cell suspension to a 96-well U-bottom multiwell plate (FALCON). 5% CO ₂ in the presence of AS1842856 and Imatinib mesylate (biovision) at 37 ° C. Cultured for days. After culturing, the cell suspension (40 μl) was collected and transferred to a Greiner 781080 plate. An equal amount (40 μl) of CellTiter-Glo Luminescent Cell Viability Assay (manufactured by Promega) was added, and the cells were lysed by reaction for 10 minutes or more, and then the luminescence signal was measured with a microplate reader (infinite 200 pro). The cell growth rate was calculated with the luminescence value of AS1842856 untreated cells as 100%. The cell growth inhibition rate was calculated when imatinib was used alone or when AS1842856 was used in combination with imatinib.

  According to this experiment, the chronic myelogenous leukemia strain can obtain a result that the cell growth inhibitory effect is higher when AS1842856 and imatinib are used in combination than when imatinib is used alone. From this result, it can be understood that the cell growth inhibitory effect of imatinib against chronic myeloid leukemia strain is enhanced by AS1842856.

<Cell growth inhibition test with AS1842856>
Next, in order to verify the cell growth inhibitory effect of AS1842856, the cell growth rate in the presence of AS1842856 was calculated using various cancer cell lines.

  Specifically, in this experimental example, THP1 and HL60 are used as acute myeloid leukemia cell lines (AML), Molt4 and Jurkat are used as T cell leukemia cell lines, K562 and Chronic myeloid leukemia cell lines (CML) are used. KCL22 was used and BV-173 was used as the B cell leukemia cell line.

Cell suspensions were prepared so that each cell line was 10,000 cells / 75 μL. In the leukemia-derived cell line, 25 μL of the compound solution and 75 μL of the cell suspension were added to a 96-well U-bottom multi-well plate to start the reaction, followed by culturing at 5% CO ₂ and 37 ° C. for 4 days. After culturing, the cell suspension was reacted with an equal amount of CellTiter-Glo Luminescent Cell Viability Assay (Promega) for 10 minutes or more to lyse the cells, and the luminescence signal was measured with a microplate reader (infinite 200 pro). . The cell proliferation rate was calculated with the luminescence value of the cells not treated with the compound as 100%.

  From the results shown in FIG. 4, AS1842856 was found to have a cell growth inhibitory effect on chronic myeloid leukemia-derived cell lines and acute myeloid leukemia-derived cell lines. In particular, AS1842856 has been found to have an extremely excellent cell growth-inhibiting effect on cell lines derived from acute myeloid leukemia.

<Verification of FOXO dependence of cell growth inhibition>
As described above, it was confirmed that AS1842856 has a cell growth inhibitory effect on the myeloid leukemia-derived cell line. In this experiment, it was verified whether this cell growth inhibitory effect was FOXO-dependent. Specifically, a FOXO1 / 3a knockdown leukemia cell line was prepared using a recombinant lentivirus, and it was confirmed that AS1842856 reduced the cell growth inhibitory effect on the cell line.

The recombinant lentivirus was prepared as follows. First, 293T cells were seeded in DMEM containing 10% FBS so as to be 2 × 10 ⁶ cells / 10 cm dish and cultured at 37 ° C. for 5 hours at 5% CO ₂ . Gene transfer was performed using X-tremeGENE HP DNA Transfection Reagent (Roche) according to the protocol. A packaging plasmid and 5ug of shFOXO1 / 3a (pLKO.1 vector: GATCTACGAGTGGATGGTCAA (SEQ ID NO: 1)) were introduced into the cells. As a control for shRNA, scrambled DNA plasmid was introduced. The culture medium was changed 16 hours after the gene introduction, and the medium was changed and collected 24, 48 and 72 hours later. The collected culture solution was filtered through a 0.45 μm PVDF membrane (Millipore), and 8 μg / ml polybrene (Sigma) was added to obtain a virus solution.

Using the resulting virus solution, a shFOXO1 / 3a function-suppressing cell line was prepared as follows. First, THP1 cells were seeded at 1.2 × 10 ⁶ cells / 10 cm dish and contacted with the virus solution obtained above for 32 hours to infect the THP1 cells with the virus. After infection, the cells were cultured in normal RPMI1640 medium (Wako) for 24 hours. Virus-infected cells were selected by adding 1.5 μg / ml puromycin (Invivogen) to the culture and culturing for 72 hours. The selected cell line was designated as a shFOXO1 / 3a function inhibitory cell line.

Using the obtained shFOXO1 / 3a function-suppressed cell line, a cell suspension was prepared so as to be 10,000 cells / 75 μL. The reaction was started by adding 25 μL of AS1842856 solution and 75 μL of cell suspension to a 96-well U-bottom multi-well plate, and cultured for 4 days at 37 ° C. with 5% CO ₂ . After culturing, the cell suspension was reacted with an equal amount of CellTiter-Glo Luminescent Cell Viability Assay (Promega) for 10 minutes or longer to lyse the cells, and then the luminescence signal was measured with a microplate reader (infinite 200 pro). The cell viability was calculated with the luminescence value of the untreated cells as 100%.

  The detection result of the luminescent signal is shown in FIG. 5, and the result of calculating the cell viability is shown in FIG. In the graphs shown in FIGS. 5 and 6, “scrambled” is a cell line introduced with scrambled DNA plasmid as a control, and “shFOXO1 / 3” is a cell line introduced with shFOXO1 / 3a. As can be seen from FIG. 5, in the fFOXO1 / 3a function-suppressed cell line, the expression of FOXO is suppressed regardless of the presence or absence of AS1842856. As can be seen from FIG. 6, the cell growth inhibitory effect of AS1842856 is greatly reduced. I understand that. From these results, it was revealed that the cell growth inhibitory effect of AS1842856 on the myeloid leukemia-derived cell line is a condition (FOXO-dependent) that FOXO is functioning.

<Induction of leukemia differentiation of AS1842856>
As described above, AS1842856 was confirmed to have a cell growth inhibitory effect in a FOXO-dependent manner against a myeloid leukemia-derived cell line. In this experiment, AS1842856 was examined to induce differentiation of a cell line derived from myeloid leukemia.

Specifically, the cells were reacted with leukemia cell lines (THP1 and U937: one of acute myeloid leukemia cell lines), and cell shape changes due to differentiation induction by AS1842856 were evaluated by May-Grunwald Giemsa staining. The leukemia cell line and AS1842856 (1 μM) were cultured for 48 hours at 37 ° C. with 5% CO ₂ . After culturing, the cells were washed with PBS, and cell specimens were prepared by cytospin. The cell specimen was stained and fixed with May Grünwald solution (Mudo Chemical), and then washed with a phosphate buffer (Mudo Chemical). Then, it dye | stained with Giemsa dyeing liquid (Mudo Chemical), washed with water, and air-dried. Cell morphological changes were observed under a microscope.

  The results are shown in FIGS. FIG. 7 shows the results of microscopic observation of THP-1, which is an acute myeloid leukemia cell line, and FIG. 8 shows the results of microscopic observation of U937, which is an acute monocytic leukemia cell line. As can be seen from FIG. 7, THP-1 which is an acute myeloid leukemia cell line has been induced to differentiate into monocyte-like cells by AS1842856, and as can be seen from FIG. 8, U937, which is an acute monocytic leukemia cell line. Was found to be induced to differentiate into macrophage-like cells by AS1842856.

<Differentiation of leukemia cells by AS1842856>
In this experiment, AS1842856 and AS1708727 were analyzed for their ability to induce differentiation of acute myeloid leukemia cell lines (THP1 and HL60) into macrophages.

First, THP1 or HL60 cells were collected, and a cell suspension was prepared with RPMI medium containing 10% FBS so as to be 50,000 cells / 75 μL. As for AS1842856 and AS1708727 dilution lines, a high concentration line with final concentrations of 0, 1, 10, 25 μM and a low concentration line with final concentrations of 0, 1, 10, 100 nM were diluted in 25 μl of medium. The total reaction volume was 100 μL. The reaction was started by adding 25 μL of the compound solution and 75 μL of the cell suspension to a 96-well U-bottom multi-well plate (FALCON), and cultured at 5% CO ₂ and 37 ° C. for 72 hours.

After reacting for 72 hours, 5% FBS / PBS was added to the cells, washed 3 times, and resuspended in 20 μL of 5% FBS / PBS (containing 0.5% NaN ₃ ). 5 μL of PE mouse Anti-Human CD11b / Mac1 (BD biosciences) was added and allowed to react on ice for 90 minutes. After the reaction, 1 mL of 5% FBS / PBS was added and washed three times. Cells were resuspended in 50 μL 5% FBS / PBS. 0.5 μL of 7-AAD (BD biosciences) was added, and 7-AAD negative CD11b positive cells were detected using MACS Quant (Milteny biotech). The CD11b positive cells at each compound concentration were graphed with a histogram and analyzed for their ability to differentiate into macrophages. In addition, the sensitive cell rate of positive cells (> 500) was measured.

  The results are shown in FIGS. The upper part of FIG. 9 is an analysis result when AS1708727 is used at a high concentration (0-25 μM), and the lower part is an analysis result when AS1842856 is used at a high concentration (0-25 μM). The upper part of FIG. 10 shows the analysis results when AS1842856 is used at a low concentration (0-10 μM), and the lower part shows the analysis results when AS1842856 is used at a high concentration (0-25 μM). As shown in FIGS. 9 and 10, it was revealed that the acute myeloid leukemia cell line is induced to differentiate into macrophages by AS1842856. In contrast, AS1708727, a Foxo inhibitor, as in AS1842856, was found to be unable to induce differentiation of acute myeloid leukemia cell lines into macrophages.

<Influence of AS1842856 on normal stem cells>
As described above, the effect on normal cells of AS1842856, which was shown to have a cell growth-inhibiting effect in a FOXO-dependent manner and a differentiation-inducing ability against cell lines derived from myeloid leukemia, was examined.

  First, human umbilical cord blood-derived mononuclear cells (purchased from RIKEN) were stained with an anti-CD34 antibody. Differentiation markers (CD4, CD8, B220, TER119, Gr-1, Mac1) negative (Lin-) and CD34 positive cells were isolated from the stained cells using flow cytometry (FACSaria, BD). In order to evaluate the ability of isolated cells to form colonies in vitro, 500 cells of methylcellulose semi-solid medium containing growth factors (EPO, SCF, GM-CSF, IL3, G-CSF) (Methocult H4034, Stem cell technology) The number of colonies formed on day 13 was counted. In addition, the number of colonies formed when AS1842856 was added to the medium was counted, and the effect on normal stem cells was evaluated.

  The results are shown in FIG. As shown in FIG. 11, it can be seen that there is no effect on normal cells at a concentration of AS1842856 of 0.1 μM or less. As described above, AS1842856 has a cell growth inhibitory effect on myeloid leukemia strains at a concentration of 0.1 μM or less. Thus, it was shown that AS1842856 can be used as a novel therapeutic agent for myeloid leukemia in a concentration range that does not show a cytostatic effect on normal cells.

<Evaluation of drug efficacy by frequent administration of AS1842856>
In this experiment, we analyzed the tumor regression effect of AS1842856 administered to mice transplanted with acute myeloid leukemia cell line (HL60).

First, the acute myeloid leukemia cell line HL60 was transplanted subcutaneously into the back of NOD scid mice (7 weeks old, female) (1 × 10 ⁷ cells / mouse) and AS1842856 was administered frequently. Were divided into control groups (12 mice in each group). For the AS18 administration group, after transplantation of HL60 cells, at 10:00 am on the 13th and 21st, and at 10am and 3pm on the 14, 15, 16, 17, 18, 19 and 20th AS1842856, respectively. Was intraperitoneally administered 16 times in total at 20 mg / kg body weight. On the other hand, a solvent (PBS containing 0.5% Cremophor (Cremophor EL, Sigma, C5135-500G) and 10% DMSO) was intraperitoneally administered 16 times to the control group at the same administration timing as the AS18 administration group.

  For each group of mice, tumor volumes were measured 3, 7, 10, 13, 17 and 21 days after HL60 cell transplantation. Tumor volume was measured as follows. Using a digital caliper, the major axis and minor axis of the tumor formed subcutaneously in the back were measured, and the value of (major axis mm × minor axis mm × minor axis mm) / 2 was calculated as the tumor volume. The results of calculating the tumor volume are shown in FIG. In addition, the statistical program ystat2013 was used for the comparison between tumor volumes between groups. As a result of the analysis, a t-test with no correspondence between the control group and the AS18 administration group was performed on the tumor volume value on the last measurement day (21 days after transplantation), and P <0.01 level was significant in the two-sided test Showed the difference.

  From this experimental result, it was clarified that the tumor mass of myeloid leukemia cell line can be regenerated in vivo by intraperitoneal administration of AS1842856, and it was shown that it can be used as a novel therapeutic agent for myeloid leukemia.

<Immunohistological analysis after frequent administration of AS1842856>
In the AS18 administration group and the control group described above, the tumor mass formed subcutaneously on the back 3 hours after the final administration was removed, fixed with 4% paraformaldehyde (4 ° C., overnight), and embedded in paraffin according to a standard method. . Using these tumor samples, tumor tissues were analyzed by hematoxylin and eosin staining (HE staining) and immunohistochemical staining (IHC staining).

  Specifically, first, a 4 μm-thick sliced specimen was prepared from a paraffin-embedded tumor sample, deparaffinized with xylene, and then xylene was removed with ethanol. Then, running water washing using tap water was performed for 10 minutes, followed by washing for 5 minutes using 0.01M PBS three times. Then, HE staining and IHC staining were performed according to a standard protocol.

In IHC staining, first, activation treatment was performed for 10 minutes using 0.01 M citrate buffer (pH 6), and washing was performed twice for 5 minutes using distilled water. Thereafter, endogenous peroxidase activation treatment using 0.3% H ₂ O ₂ methanol was performed for 10 minutes, followed by washing with running water and washing with 0.01M PBS. Then, after blocking with 20% goat normal serum (DAKO), various primary antibodies were reacted. In this experiment, as primary antibodies, CD11b (manufactured by abcam), CD115 (manufactured by abcam), Vimentin [V9] (manufactured by DAKO), Lysozyme (manufactured by Nichirei), PCNA [PC10} (manufactured by DAKO), Ki67 (DAKO) and MCM2 [D7G11] (CST) were used. Of these, primary antibodies other than Vimentin (Ready-to-use) were diluted 100 times with 10% normal goat serum and incubated (4 ° C / ON).

  After the primary antibody reaction, EnVision + Dual Link System-HRP [mouse / rabbit primary antibody dual-use type] (manufactured by DAKO) was dropped on the section to react the secondary antibody. After the secondary antibody reaction, washing was performed, and a color reaction was performed with a color developing solution prepared using DAB tablet (manufactured by Wako Pure Chemical Industries, Ltd.). The CD11b color reaction time was 1 minute and 40 seconds, the CD115 color reaction time was 2 minutes and 15 seconds, the Vimentin color reaction time was 4 minutes, the Lysozyme color reaction time was 1 minute and 40 seconds, and PCNA The color reaction time was 1 minute 15 seconds, the color reaction time of Ki67 was 2 minutes 30 seconds, and the color reaction time of MCM2 was 1 minute 15 seconds. After the color development reaction, it was washed, counterstained with a hematoxylin solution (equal mixture of Cat. No. 109249 manufactured by Millipore and Cat. No. 3002 manufactured by Muto Chemical Co., Ltd.), dehydrated and clarified. Then, it encapsulated using marinol (made by Muto Chemical Co., Ltd.).

  The result of HE staining is shown in FIG. The results of IHC staining using CD11b as the primary antibody are shown in FIG. 14, the results of IHC staining using CD115 are shown in FIG. 15, the results of IHC staining using Vimentin are shown in FIG. 16, and Lysozyme is used. FIG. 17 shows the result of IHC staining, FIG. 18 shows the result of IHC staining using PCNA, FIG. 19 shows the result of IHC staining using Ki67, and FIG. 20 shows the result of IHC staining using MCM2. It was shown to.

  As can be seen from FIG. 13, as a result of HE staining, the AS18 administration group shows a cluster of cells with smaller nuclei compared to the control group (within x400 image: A).

  Further, as shown in FIG. 14, as a result of IHC staining using an antibody against CD11b, which is a differentiation antigen to macrophages, the positive rate was increased in the AS18 administration group, indicating a differentiation tendency due to administration of AS1842856. In addition, most of the positive cells coincided with the cell region in which the nucleus was small, which is considered to be an effect of AS1842856 administration from the result of HE staining (A in the figure).

  Similarly, as shown in FIG. 15, from the results of IHC staining using an antibody against CD115, which is a marker of monocytes / macrophages, which has been reported to be expressed in the process of differentiation into monocytes, The positive rate increased, indicating a tendency to differentiate with AS1842856 administration. As in the case of CD11b, most of the positive cells coincided with the cell region having a smaller nucleus, which is considered to be an effect of AS1842856 administration from the result of HE staining (A in the figure).

  Further, as shown in FIG. 16, from the result of IHC staining using an antibody against Vimentin, which is a marker such as monocyte / macrophage, which is reported to increase in expression level upon differentiation into monocytes, CD11b and CD115 are used. Like the differentiation marker, the positive rate increased in the AS18 administration group, indicating a tendency for differentiation by administration of AS1842856. Most of the positive cells for the antibody against Vimentin were also consistent with the cell region with a smaller nucleus, which is considered to be the effect of AS1842856 administration from the result of HE staining (A in the figure).

  Furthermore, as shown in FIG. 17, from the results of IHC staining using an antibody against Lysozyme, which is reported to increase the expression level upon differentiation into monocytes / macrophages, as in other differentiation markers, in the AS18 administration group. The positive rate increased, indicating a tendency to differentiate with AS1842856 administration. Most of the positive cells against the antibody against Lysozyme were also consistent with the cell region with a smaller nucleus, which is considered to be an effect of AS1842856 administration from the result of HE staining (A in the figure).

  On the other hand, as shown in FIG. 18, from the result of IHC staining using an antibody against PCNA which is a cell proliferation marker, the positive rate was decreased in the AS18 administration group, and the cell growth suppression tendency by AS1842856 administration was shown. Most of the negative cells against the antibody against PCNA were also consistent with the area of cells with smaller nuclei, which was considered to be the effect of AS1842856 administration from the results of HE staining (A in the figure).

  Further, as shown in FIG. 19, from the results of IHC staining using an antibody against Ki67, which is a cell proliferation marker, the positive rate was decreased in the AS18 administration group, and a tendency to suppress cell proliferation by AS1842856 administration was shown. The majority of negative cells for antibodies against Ki67 were also consistent with the area of cells with smaller nuclei, which was thought to be affected by AS1842856 administration from the results of HE staining, as well as IHC staining using antibodies against PCNA. (A in the figure).

  Further, as shown in FIG. 20, from the result of IHC staining using an antibody against MCM2, which is a cell proliferation marker, the positive rate was decreased in the AS18 administration group, and the tendency of cell proliferation suppression by AS1842856 administration was shown. As with IHC staining using antibodies against other cell proliferation markers, most of the negative cells for antibodies against MCM2 also have cell regions with smaller nuclei that are considered to be affected by AS1842856 administration from the results of HE staining. It was in agreement (A in the figure).

<Summary>
From the above experimental results, it can be understood that the quinolone derivative represented by the formula (X) including AS1842856 has an action of inhibiting the proliferation of a cell line derived from myeloid leukemia in a FOXO-dependent manner. Therefore, it was shown that the quinolone derivative represented by the formula (X) containing AS1842856 is a novel therapeutic agent for myeloid leukemia.

  As with AS1842856, compounds known as FOXO inhibitors (for example, AS1708727) did not show growth-inhibiting effects on cell lines derived from myeloid leukemia and proved to be ineffective as a therapeutic agent for myeloid leukemia. did. Thus, according to this example, the therapeutic effect on myeloid leukemia cannot be predicted only by the presence or absence of inhibitory activity against FOXO, and the quinolone derivative represented by the formula (X) including AS1842856 is a cell line derived from myeloid leukemia. It was revealed that there is a very excellent growth inhibitory effect on

Claims (4)

A therapeutic agent for myeloid leukemia comprising a quinoline compound (X) represented by the following formula as an active ingredient.

[The symbols in the formula have the following meanings.
X: CR ⁷ or N.
Y: CR ⁶ or N.
R ² : each optionally substituted lower alkyl, cycloalkyl, aryl or heterocycle.
R ³ : halogen, lower alkyl, or —O-lower alkyl.
R ⁴ : each independently substituted cycloalkyl or non-aromatic heterocyclic ring, or lower alkyl substituted with cycloalkyl. However, when R ⁴ represents an optionally substituted non-aromatic heterocycle, the carbon atoms constituting the ring shall be bonded to the adjacent NH.
R ⁵ : —H, halogen, cyano, nitro, lower alkyl, halogeno lower alkyl, cycloalkyl, aryl, heterocycle, —O-lower alkyl, —OH, —NHCO-lower alkyl, —N (lower alkyl) CO— Lower alkyl, amino optionally substituted with lower alkyl, or cyclic amino optionally substituted.
R ⁶ : —H, halogen, lower alkyl or halogeno lower alkyl.
R ⁷ : —H, halogen, lower alkyl or halogeno lower alkyl.
However, when Y represents CR ⁶ , R ² and R ⁶ may be combined to form lower alkylene or lower alkenylene. ] In the quinoline compound (X), X and Y are CH, R ² is lower alkyl, R ³ is halogen, and R ⁴ may be substituted cycloalkyl R ⁵ is substituted with lower alkyl. The therapeutic agent according to claim 1, which may be amino. The quinoline compound (X) is 5-amino-7- (cyclohexylamino) -1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid represented by the following formula The therapeutic agent of Claim 1 characterized by these.

The therapeutic agent according to claim 1, wherein the myeloid leukemia is acute myeloid leukemia.

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