Nitrogen-containing compounds, compositions containing the nitrogen-containing compounds, and predictive markers for tumor malignancy

Abstract

The present invention addresses the problem of providing a nitrogen-containing compound effective for inhibiting cancer stem cell proliferation.　This problem is solved by a nitrogen-containing compound represented by general formula (I) or a pharmaceutically acceptable salt thereof (wherein: m, n and p are the same or different and represent an integer of 1-3; R1, R2 and R3 are the same or different and represent a hydrogen atom, a C1-6 linear or branched alkyl group optionally having a halogen atom as a substituent, a C1-6 linear or branched alkoxy group, a halogen atom, an amino group, a hydroxyl group, a nitro group, a carboxy group or a cyano group; and R4 represents a C1-6 branched or linear alkyl group optionally having a halogen atom as a substituent).

Description

[Technical Field]

[0001] Disclosed herein are nitrogen-containing compounds, compositions comprising said nitrogen-containing compounds, and predictive markers of tumor malignancy. [Background technology]

[0002] It is said that there are approximately 2,200 glioblastoma patients and 110,000 lung cancer patients in Japan. Glioblastoma in particular often involves infiltration that cannot be surgically removed, and even with the combined use of radiation therapy and the commercially available drug temozolomide, the two-year survival rate is low at 27% (Non-Patent Document 1), and current treatment methods are not sufficiently effective. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] N Engl J Med. 2005 Mar 10;352(10):987-96 Summary of the Invention [Problem to be solved by the invention]

[0004] Cancer stem cells often cannot be removed by surgical treatment and tend to proliferate repeatedly. Therefore, there is a need to develop therapeutic agents that target and kill cancer stem cells. One objective of the present invention is to provide a compound that inhibits the proliferation of cancer stem cells. Another objective of the present invention is to provide a predictive marker for predicting the malignancy of tumors. [Means for solving the problem]

[0005] As a result of extensive research, the present inventors have found that the following nitrogen-containing compounds are effective in inhibiting the proliferation of cancer stem cells, such as glioblastoma cancer stem cells and lung cancer stem cells, and have also found that the expression level of proteins targeted by the nitrogen-containing compounds correlates with the malignancy of tumors. The present invention was completed based on this finding and includes the following aspects. Item 1. A method for detecting at least one selected from mucolipin protein and mucolipin mRNA present in tumor cells in a sample collected from a subject, A method for detecting predictive markers for predicting tumor malignancy. Item 2. A method of using at least one selected from mucolipin protein and mucolipin mRNA present in tumor cells in a sample collected from a subject as a predictive marker for predicting tumor malignancy. Item 3. A detection reagent for detecting mucolipin protein present in tumor cells in a specimen collected from a subject as a predictive marker for predicting tumor malignancy, the test reagent comprising an antibody for detecting mucolipin protein. The detection reagent. Item 4. A detection reagent for detecting mucolipin mRNA present in tumor cells in a specimen collected from a subject as a predictive marker for predicting tumor malignancy, the test reagent comprising an oligonucleotide for detecting mucolipin mRNA. The detection reagent. Item 5. A detection kit for detecting mucolipin protein or mucolipin mRNA present in tumor cells in a sample collected from a subject as a predictive marker for predicting tumor malignancy, comprising the detection reagent according to Item 3. Item 6. A prediction device for predicting tumor malignancy, comprising a processing unit, wherein the processing unit obtains a measurement value of at least one selected from the amount of mucolipin protein and mucolipin mRNA present in tumor cells in a specimen collected from a subject, compares the obtained measurement value with a reference value, and outputs a label indicating that the tumor has a high malignancy when the measurement value exceeds the reference value, or outputs a label indicating that the tumor has a low malignancy when the measurement value is equal to or less than the reference value, and the subject has a malignant tumor. The prediction device. Item 7. A nitrogen-containing compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof: [ka] (where, m, n, and p are the same or different and each represents an integer of 1 to 3. R 1 , R 2 , and R 3 are the same or different and represent a hydrogen atom, a C1-6 linear or branched alkyl group which may have a halogen atom as a substituent, a C1-6 linear or branched alkoxy group which may have a halogen atom as a substituent, a halogen atom, an amino group, a hydroxyl group, a nitro group, a carboxy group or a cyano group. R 4 represents a C1-6 branched or linear alkyl group which may have a halogen atom as a substituent. Section 8.R 4 Item 8. The nitrogen-containing compound according to Item 7, or a pharmaceutically acceptable salt thereof, wherein represents a C3-6 branched alkyl group. Section 9.R 1 , R 2 , and R 3 Item 9. The nitrogen-containing compound according to Item 7 or 8, or a pharmaceutically acceptable salt thereof, wherein is a hydrogen atom. Item 10. The nitrogen-containing compound according to Item 7, wherein the nitrogen-containing compound represented by general formula (I) is represented by the following formula (1), or a pharmaceutically acceptable salt thereof: [ka] . Item 11. A composition comprising the nitrogen-containing compound according to any one of Items 7 to 10, or a pharmaceutically acceptable salt thereof. Item 12. The composition according to Item 11, which is used to treat a malignant tumor. Item 13. The composition according to Item 11, which is used to prevent the recurrence of a malignant tumor or to prevent the metastasis of a malignant tumor. Item 14. The composition according to Item 11, which is used to suppress the proliferation of tumor stem cells of malignant tumors. Item 15. The composition according to any one of Items 11 to 14, wherein the malignant tumor is glioblastoma or lung cancer. Item 16. A composition comprising one nitrogen-containing compound selected from the group consisting of the following general formula (II), the following general formula (III), the following general formula (IV), the following general formula (V), and pharmaceutically acceptable salts thereof: [ka] (In the above general formula (II), n represents 1 or 2. R 21 , R 22 , R 23 , and R 24 are the same or different and represent a C1-3 linear or branched alkyl group which may have a hydrogen atom or a halogen atom as a substituent; [ka] (In general formula (III), R 31 and R 32 are the same or different and represent a halogen atom; [ka] (In the general formula (IV), n represents an integer of 1 to 3. R 41 and R 42 are the same or different and represent a halogen atom. R 43 represents a C1-3 linear or branched alkyl group which may have a hydrogen atom or a halogen atom as a substituent; [ka] (In the general formula (V), n represents 1 or 2. R 51 , R 52 , and R 53 are the same or different and represent a C1-3 linear or branched alkyl group which may have a hydrogen atom or a halogen atom as a substituent. Item 17. A composition comprising the nitrogen-containing compound according to Item 16 or a pharmaceutically acceptable salt thereof. Item 18. The composition according to Item 17, which is used to treat malignant tumors. Item 19. The composition according to Item 17, which is used to prevent the recurrence of a malignant tumor or to prevent the metastasis of a malignant tumor. Item 20. The composition according to Item 17, which is used to suppress the proliferation of tumor stem cells of malignant tumors. Item 21. The composition according to any one of Items 17 to 20, wherein the malignant tumor is glioblastoma or lung cancer. [Effects of the Invention]

[0006] It can suppress the proliferation of cancer stem cells such as glioblastoma cancer stem cells and lung cancer stem cells, and can also predict the malignancy of tumors. [Brief explanation of the drawings]

[0007] [Figure 1] 1 shows the configuration of a prediction system 1000. [Figure 2] 1 shows the hardware configuration of a prediction device 10. [Figure 3] 10 shows the processing flow of the prediction program 104b. [Figure 4] 1 shows an immunostained image of mucolipin protein in a pathological tissue specimen. [Figure 5]A shows the relationship between the expression of mucolipin MCOLN1 and the overall survival rate of patients. B shows the relationship between the expression of mucolipin MCOLN1 and the disease / progression (D / P)-free survival rate of patients. C shows the relationship between the expression of mucolipin MCOLN2 and the overall survival rate of patients. D shows the relationship between the expression of mucolipin MCOLN2 and the disease / progression (D / P)-free survival rate of patients. E shows the relationship between the expression of mucolipin MCOLN3 and the overall survival rate of patients. F shows the relationship between the expression of mucolipin MCOLN3 and the disease / progression (D / P)-free survival rate of patients. [Figure 6] This is a list of nitrogen-containing compounds used in in silico screening. [Figure 7] The analytical conditions for liquid chromatography mass spectrometry (LCMS) are shown below. [Figure 8] The LCMS results are shown below. [Figure 9] Among the novel nitrogen-containing compounds obtained through this screening, the chemical formulas of six compounds (KMU5, KMU12, KMU13, KMU23, KMU7, and KMU9) ​​that were structurally relatively similar to KMU3 are shown below. [Figure 10] Panel A shows the cytostatic activity of KMU3, KMU5, KMU12, KMU13, KMU23, KMU7, and KMU9 on glioblastoma cells when added to the culture medium. Panel B shows the relative proliferation rate (% of control) compared to control cells. "C" indicates the control; "#" indicates KMU5, KMU9, and KMU12 (shown for convenience because the three growth curves overlap); and "&" indicates KMU7, KMU13, and KMU23 (shown for convenience because the three growth curves overlap). "3" indicates KMU3. [Figure 11]1 shows the concentration-dependent cell growth inhibitory activity of KMU3, SR33805, and temozolomide. ● indicates KMU3, ◯ indicates SR33805, and × indicates temozolomide. [Figure 12] 1 shows the change in current through mucolipin in the presence of SR33805. [Figure 13] (A) shows the results of immunostaining using anti-MCOLN3 antibody. (B) shows the results of an antibody absorption test in which an antigen peptide was reacted with anti-MCOLN3 antibody. (C) shows the cell proliferation rate of lung cancer stem cells in the presence of SR33805. [Figure 14] A shows a comparison of overall survival between the KMU3-administered group (K3) and the negative control group (Control). B shows overall survival between the lomerizine dihydrochloride-administered group (Drug L3), the temozolomide-administered group (Tmz), and the negative control group (Control). C shows a comparison of overall survival between the KMU84-administered group (K84) and the negative control group (Control). DETAILED DESCRIPTION OF THE INVENTION

[0008] This specification discloses nitrogen-containing compounds that bind to mucolipin proteins or pharmaceutically acceptable salts thereof, methods for producing the nitrogen-containing compounds, mucolipin proteins as predictive markers for predicting tumor malignancy, methods for detecting the predictive markers, devices for detecting the predictive markers, programs for detecting the predictive markers, and the like.

[0009] As used herein, mucolipin proteins may include mucolipin-1 (e.g., UniProtKB / Swiss-Prot: Q9GZU1), mucolipin-2 (e.g., UniProtKB / Swiss-Prot: Q8IZK6), and mucolipin-3 (e.g., UniProtKB / Swiss-Prot: Q8TDD5). Mucolipin-1 is a protein encoded by the MCOLN1 gene, registered with NCBI as Gene ID: 57192. Mucolipin-2 is a protein encoded by the MCOLN2 gene, registered with NCBI as Gene ID: 255231. Mucolipin-3 is a protein encoded by the MCOLN3 gene, registered with NCBI as Gene ID: 55283.

[0010] In this specification, the term "tumor" may be either a malignant tumor or a benign tumor, but is preferably a malignant tumor. The term "tumor" includes both non-epithelial and epithelial malignant tumors. Specifically, the term "tumor" includes central nervous system malignant tumors; respiratory system malignant tumors arising from the trachea, bronchi, or lungs; digestive system malignant tumors arising from the nasopharynx, esophagus, stomach, duodenum, jejunum, ileum, cecum, appendix, ascending colon, transverse colon, sigmoid colon, rectum, or anus; liver cancer; pancreatic cancer; urinary system malignant tumors arising from the bladder, ureter, or kidney; female reproductive system malignant tumors arising from the ovaries, fallopian tubes, uterus, and the like; breast cancer; prostate cancer; skin cancer; endocrine system malignant tumors such as the hypothalamus, pituitary gland, thyroid gland, parathyroid gland, and adrenal gland; and malignant tumors arising from bone and soft tissue. Examples of the tumor include solid tumors such as leukemia, and hematopoietic malignancies such as myelodysplastic syndrome, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelomonocytic leukemia, chronic myelomonocytic leukemia, acute monocytic leukemia, chronic monocytic leukemia, acute panmyelogenous leukemia, acute megakaryocytic leukemia, erythroleukemia, eosinophilic leukemia, chronic eosinophilic leukemia, chronic neutrophilic leukemia, adult T-cell leukemia, hairy cell leukemia, plasma cell leukemia, multiple myeloma, and malignant lymphoma; and hematopoietic tumors such as lymphatic malignancies. Solid tumors are more preferred, and brain tumors or lung cancers are even more preferred. As brain tumors, gliomas are most preferred, and as lung cancers, squamous cell carcinomas are most preferred. Gliomas may include glioblastoma, anaplastic oligodendroglioma, anaplastic astrocytoma, diffuse midline glioma, oligodendroglioma, diffuse astrocytoma, pilocytic astrocytoma, subependymal giant cell glioma, pleomorphic xanthoastrocytoma, etc. Glioblastoma, anaplastic oligodendroglioma, anaplastic astrocytoma, diffuse midline glioma, etc. are classified as high-grade gliomas, while oligodendroglioma, diffuse astrocytoma, pilocytic astrocytoma, subependymal giant cell glioma, etc. are classified as low-grade gliomas.

[0011] The malignancy of brain tumors is generally expressed in four stages, from grade 1 to 4, depending on the type of tumor, with grades 3 and 4 being considered highly malignant. The higher the grade, the lower the two-year and five-year survival rates tend to be.

[0012] 1. Nitrogen-containing compound or pharmaceutically acceptable salt thereof, and method for producing nitrogen-containing compound (1) Nitrogen-containing compound The present embodiment relates to a nitrogen-containing compound that can bind to a mucolipin protein and preferably blocks ion flow through an ion channel formed by the mucolipin protein.

[0013] In this specification, "C" in the expressions "C1-6", "C1-4", "C3-6", etc. indicates carbon. "C1-6" indicates that the number of carbon atoms is 1 to 6, "C1-4" indicates that the number of carbon atoms is 1 to 4, and "C3-6" indicates that the number of carbon atoms is 3 to 6. The nitrogen-containing compound according to this embodiment is represented by the following general formula (I).

[0014] [ka]

[0015] In general formula (I), m, n, and p are the same or different and represent an integer of 1 to 3. Preferably, m, n, and p are the same or different and represent an integer of 1 or 2. More preferably, m, n, and p are all 1.

[0016] In general formula (I), R 1 , R 2 , and R 3 are the same or different and represent a hydrogen atom, a C1-6 linear or branched alkyl group which may have a halogen atom as a substituent, a C1-6 linear or branched alkoxy group which may have a halogen atom as a substituent, a halogen atom, an amino group, a hydroxyl group, a nitro group, a carboxy group or a cyano group.

[0017] R 1 , R 2 , and R 3In the formula (I), examples of the C1-6 linear alkyl group include methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl groups. Examples of the C3-6 branched alkyl group include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, neopentyl, 3-methylpentyl, 3,3-dimethylbutyl, and 3-ethylbutyl groups.

[0018] The C1-6 linear or branched alkyl group is preferably a C1-3 linear or branched alkyl group (i.e., methyl, ethyl, n-propyl, or isopropyl group), more preferably a C1-3 linear alkyl group (i.e., methyl, ethyl, or 3-propyl group), even more preferably a C1-2 alkyl group (i.e., methyl or ethyl group), and even more preferably a C1 alkyl group (i.e., methyl group). Examples of halogen atoms as substituents include fluorine, chlorine, bromine, and iodine atoms.

[0019] R 1 , R 2 , and R 3 The alkyl group in the C1-6 linear or branched alkoxy group which may have a halogen atom as a substituent is the same as the C1-6 linear alkyl group and C3-6 branched alkyl group described above. Examples of the halogen atom as a substituent include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. R 1 , R 2 , and R 3 Examples of the halogen atom in include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. R 1 , R 2 , and R 3 is preferably a hydrogen atom.

[0020] R 4represents a C1-6 branched or linear alkyl group which may have a halogen atom as a substituent. The definition of a halogen atom and a C1-6 branched or linear alkyl group are as follows: R 1 , R 2 , and R 3 This is similar to the halogen atoms in

[0021] R 4 is preferably a C3-6 branched alkyl group optionally having a halogen atom as a substituent (i.e., isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, neopentyl, 3-methylpentyl, 3,3-dimethylbutyl, and 3-ethylbutyl), more preferably a C3-4 branched alkyl group optionally having a halogen atom as a substituent (i.e., isopropyl, sec-butyl, isobutyl, or tert-butyl), even more preferably an unsubstituted C3-4 branched alkyl group (i.e., isopropyl, sec-butyl, isobutyl, or tert-butyl), and even more preferably an unsubstituted C3 branched alkyl group (i.e., isopropyl). Examples of halogen atoms as substituents include chlorine, fluorine, bromine, and iodine atoms.

[0022] The nitrogen-containing compound represented by general formula (I) is more preferably a nitrogen-containing compound represented by the following formula (1):

[0023] [ka] . The nitrogen-containing compound represented by general formula (I) is more preferably a nitrogen-containing compound represented by the following formula (11):

[0024] [ka] .

[0025] (2) Pharmaceutically acceptable salts of nitrogen-containing compounds Pharmaceutically acceptable salts of the nitrogen-containing compound represented by the general formula (I) are not particularly limited. Examples of such salts include inorganic acid addition salts such as hydrochloride, hydrobromide, sulfate, and nitrate; and organic acid addition salts such as acetate, tartrate, maleate, succinate, citrate, methanesulfonate, malate, oxalate, and benzenesulfonate. These salts can be prepared, for example, by treating the nitrogen-containing compound represented by the general formula (I) with an acid.

[0026] (3) Method for producing nitrogen-containing compounds The nitrogen-containing compound represented by the general formula (I) is a compound represented by the following general formula (IA): [ka] (m, n, R 1 , and R 2 is the same as above.) The following general formula (IB) and [ka] (p, R 3 , and R 4 is the same as above.) can be produced by amide bonding under known conditions.

[0027] The reaction of compound (IA) with compound (IB) is a method in which compound (IA) is reacted with a carboxylic acid of compound (IB) by a conventional amide bond forming reaction.

[0028] The amide bond forming reaction can be carried out under a wide range of conditions known in the art for amide bond forming reactions. For example, (i) the mixed acid anhydride method, i.e., a method in which compound (IB) is reacted with an alkyl halocarboxylic acid to form a mixed acid anhydride, which is then reacted with compound (IA); (ii) the activated ester method, i.e., a method in which compound (IB) is converted into an activated ester such as p-nitrophenyl ester, N-hydroxysuccinimide ester, 1-hydroxybenzotriazole ester, or an activated amide with benzoxazoline-2-thione, which is then reacted with compound (IA); (iii) the carbodiimide method, i.e., a method in which compound (IA) is reacted with compound (IB) by the addition of dicyclohexyl carboxylic acid; (iv) a method of condensing compound (IB) in the presence of an activator such as carbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (WSC), or carbonyldiimidazole; (v) other methods, such as a method of converting compound (IB) into a carboxylic acid anhydride using a dehydrating agent such as acetic anhydride, and then reacting this with compound (IA); a method of reacting compound (IA) with an ester of compound (IB) and a lower alcohol under high pressure and high temperature; and a method of reacting compound (IA) with an acid halide of compound (IB), i.e., a carboxylic acid halide.

[0029] The mixed acid anhydride used in the above mixed anhydride method (a) is obtained by a conventional Schotten-Baumann reaction, and is usually reacted with compound (IA) without isolation to produce the nitrogen-containing compound of general formula (I). The Schotten-Baumann reaction is carried out in the presence of a basic compound.

[0030] Examples of the basic compound to be used include compounds commonly used in the Schotten-Baumann reaction, such as organic bases such as triethylamine, trimethylamine, pyridine, dimethylaniline, N-ethyldiisopropylamine, dimethylaminopyridine, N-methylmorpholine, 1,5-diazabicyclo[4.3.0]nonene-5 (DBN), 1,8-diazabicyclo[5.4.0]undecene-7 (DBU), and 1,4-diazabicyclo[2.2.2]octane (DABCO); carbonates such as sodium carbonate, potassium carbonate, sodium bicarbonate, and potassium bicarbonate; metal hydroxides such as sodium hydroxide, potassium hydroxide, and calcium hydroxide; and metal alcoholates such as potassium hydride, sodium hydride, potassium, sodium, sodium amide, sodium methylate, and sodium ethylate.

[0031] The reaction is usually carried out at about −20 to 100° C., preferably about 0 to 50° C., for a reaction time of about 5 minutes to 10 hours, preferably about 5 minutes to 2 hours.

[0032] The reaction of the resulting mixed acid anhydride with compound (IA) is generally carried out at about -20 to 150°C, preferably about 10 to 50°C, for a reaction time of about 5 minutes to 10 hours, preferably about 5 minutes to 5 hours. The mixed acid anhydride method is generally carried out in a solvent.

[0033] The solvent used may be any solvent commonly used in the mixed acid anhydride method, and specific examples thereof include halogenated hydrocarbons such as chloroform, dichloromethane, dichloroethane, and carbon tetrachloride; aromatic hydrocarbons such as benzene, toluene, and xylene; ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, and dimethoxyethane; esters such as methyl acetate, ethyl acetate, and isopropyl acetate; aprotic polar solvents such as N,N-dimethylacetamide, N,N-dimethylformamide, dimethyl sulfoxide, and hexamethylphosphoric triamide; and mixed solvents thereof.

[0034] Examples of alkyl halocarboxylic acids used in the mixed acid anhydride method include methyl chloroformate, methyl bromoformate, ethyl chloroformate, ethyl bromoformate, isobutyl chloroformate, etc. In this method, the carboxylic acid (IB), alkyl halocarboxylic acid, and compound (IA) are usually used in equimolar amounts, but the alkyl halocarboxylic acid and compound (IB) can each be used in an amount within the range of about equimolar to 1.5 times the molar amount of compound (IA). In the method (iii) of carrying out the condensation reaction in the presence of the activator, the reaction is carried out in a suitable solvent in the presence or absence of a basic compound.

[0035] As the solvent and basic compound used here, any of the solvents used in the method of reacting a carboxylic acid halide with compound (IA) in Other Method (iv) below can be used.

[0036] The amount of the activator used is at least equimolar to compound (IA), preferably equimolar to 5 times the molar amount. When WSC is used as the activator, the reaction proceeds more favorably if 1-hydroxybenzotriazole is added to the reaction system.

[0037] The reaction is usually carried out at about −20 to 180° C., preferably about 0 to 150° C., and is generally completed in about 5 minutes to 90 hours.

[0038] Furthermore, when the method of reacting a carboxylic acid halide with compound (IA) is adopted among the other methods (d) above, the reaction is carried out in a suitable solvent in the presence of a basic compound.

[0039] A wide range of known basic compounds can be used as the basic compound, and for example, any of the basic compounds used in the Schotten-Baumann reaction can be used.

[0040] Examples of the solvent to be used include, in addition to the solvents used in the mixed acid anhydride method, alcohols such as methanol, ethanol, isopropanol, propanol, butanol, 3-methoxy-1-butanol, ethyl cellosolve, and methyl cellosolve, acetonitrile, pyridine, acetone, and water.

[0041] The ratio of compound (IA) to carboxylic acid halide used is not particularly limited and may be appropriately selected within a wide range, but it is usually good to use at least an equimolar amount, preferably an equimolar to 5-fold molar amount, of the latter relative to the former. The reaction is usually carried out at about -20 to 180°C, preferably about 0 to 150°C, and is generally completed in about 5 minutes to 50 hours.

[0042] Furthermore, the above-mentioned amide bond forming reaction can also be carried out by a method in which compound (IB) and compound (IA) are reacted in the presence of a condensing agent which is a phosphorus compound such as diphenylphosphinic chloride, phenyl-N-phenylphosphoramidochloridate, diethylchlorophosphate, diethyl cyanophosphate, diphenylphosphoric azide, or bis(2-oxo-3-oxazolidinyl)phosphinic chloride.

[0043] The reaction is carried out in the presence of the solvent and basic compound used in the above method of reacting carboxylic acid halide with compound (IA), usually at about -20 to 150°C, preferably about 0 to 100°C, and is generally completed in about 5 minutes to 30 hours. The condensing agent and compound (IB) are used in at least equimolar amounts, preferably equimolar to 2 times the molar amount, of compound (IA).

[0044] The compounds produced by any of the above methods can be isolated and purified by known methods.

[0045] 2. New uses for other nitrogen-containing compounds This embodiment relates to the use of the following nitrogen-containing compounds. The nitrogen-containing compounds, which are different from the nitrogen-containing compounds represented by general formula (I), can bind to mucolipin proteins. Preferably, the nitrogen-containing compounds shown below can block ion flow through ion channels formed by mucolipin proteins.

[0046] (1) Nitrogen-containing compound: a nitrogen-containing compound represented by general formula (II) The nitrogen-containing compound is a nitrogen-containing compound represented by the following general formula (II) or a pharmaceutically acceptable acid salt thereof: [ka] . In the general formula (II), n represents 1 or 2. Preferably, n represents 2.

[0047] In general formula (II), R 21 , R 22 , R 23 , and R 24 are the same or different and each represents a C1-3 linear or branched alkyl group which may have a hydrogen atom or a halogen atom as a substituent.

[0048] R 21 , R 22 , R 23 , and R 24 In the formula, examples of the C1-3 linear alkyl group include a methyl group, an ethyl group, and an n-propyl group. Examples of the C3 branched alkyl group include an isopropyl group. More preferably, R 21 , R 22 , and R 24 is a C1-3 linear alkyl group, and R 23 is a C3 branched alkyl group. Examples of the halogen atom as a substituent include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0049] The nitrogen-containing compound represented by the general formula (II) can be produced, isolated and purified by known methods.

[0050] The nitrogen-containing compound represented by the general formula (II) is preferably a nitrogen-containing compound represented by the following formula (21): [ka] . The nitrogen-containing compound represented by general formula (II) is more preferably SR33805.

[0051] Pharmaceutically acceptable salts of the nitrogen-containing compound represented by the general formula (II) are not particularly limited. Examples of such salts include inorganic acid addition salts such as hydrochloride, hydrobromide, sulfate, and nitrate; and organic acid addition salts such as acetate, tartrate, maleate, succinate, citrate, methanesulfonate, malate, oxalate, and benzenesulfonate. These salts can be prepared, for example, by treating the nitrogen-containing compound represented by the general formula (II) with an acid.

[0052] The oxalate salt of the nitrogen-containing compound represented by formula (II) is preferred as a pharmaceutically acceptable salt, and the oxalate salt of SR33805 is most preferred in this embodiment.

[0053] (2) Nitrogen-containing compound: a nitrogen-containing compound represented by general formula (III) The nitrogen-containing compound is a nitrogen-containing compound represented by the following general formula (III) or a pharmaceutically acceptable acid salt thereof: [ka] .

[0054] In general formula (III), R 31 and R 32 are the same or different and represent a halogen atom. Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Preferably, both halogen atoms are fluorine atoms.

[0055] The nitrogen-containing compound represented by the general formula (III) can be produced, isolated and purified by known methods.

[0056] The general formula (III) is preferably a nitrogen-containing compound represented by the following formula (31): [ka] .

[0057] Pharmaceutically acceptable salts of the nitrogen-containing compound represented by the general formula (III) are not particularly limited. Examples of such salts include inorganic acid addition salts such as hydrochloride, hydrobromide, sulfate, and nitrate; and organic acid addition salts such as acetate, tartrate, maleate, succinate, citrate, methanesulfonate, malate, oxalate, and benzenesulfonate. These salts can be prepared, for example, by treating the nitrogen-containing compound represented by the general formula (III) with an acid.

[0058] The pharmaceutically acceptable salt of the nitrogen-containing compound represented by general formula (III) is preferably a hydrochloride salt, and the dihydrochloride salt of flunarizine is most preferred in this embodiment.

[0059] (3) Nitrogen-containing compound: a nitrogen-containing compound represented by general formula (IV) The nitrogen-containing compound is a nitrogen-containing compound represented by the following general formula (IV) or a pharmaceutically acceptable acid salt thereof: [ka] . In general formula (IV), n represents an integer of 1 to 3. Preferably, n represents 2 or 3. More preferably, n represents 3.

[0060] In general formula (IV), R 41 and R 42are the same or different and represent a halogen atom. Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Preferably, both halogen atoms are fluorine atoms.

[0061] In general formula (IV), R 43 represents a C1-3 linear or branched alkyl group which may have a hydrogen atom or a halogen atom as a substituent. 43 In the formula (I), examples of the C1-3 linear alkyl group include a methyl group, an ethyl group, and an n-propyl group. An example of the C3 branched alkyl group is an isopropyl group. Examples of the halogen atom as a substituent include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0062] The nitrogen-containing compound represented by general formula (IV) can be produced, isolated and purified by known methods.

[0063] The nitrogen-containing compound represented by the general formula (IV) is preferably a nitrogen-containing compound represented by the following formula (41): [ka] .

[0064] Pharmaceutically acceptable salts of the nitrogen-containing compound represented by the general formula (IV) are not particularly limited. Examples of such salts include inorganic acid addition salts such as hydrochloride, hydrobromide, sulfate, and nitrate; and organic acid addition salts such as acetate, tartrate, maleate, succinate, citrate, methanesulfonate, malate, oxalate, and benzenesulfonate. These salts can be prepared, for example, by treating the nitrogen-containing compound represented by the general formula (IV) with an acid.

[0065] The pharmaceutically acceptable salt of the nitrogen-containing compound represented by general formula (IV) is preferably a hydrochloride salt, and the dihydrochloride salt of lomerizine is most preferred in this embodiment.

[0066] (4) Nitrogen-containing compound: a nitrogen-containing compound represented by general formula (V) The nitrogen-containing compound is a nitrogen-containing compound represented by the following general formula (V) or a pharmaceutically acceptable acid salt thereof: [ka] . In the general formula (V), n represents 1 or 2. Preferably, n represents 2.

[0067] In general formula (V), R 51 , R 52 , and R 53 are the same or different and each represents a C1-3 linear or branched alkyl group which may have a hydrogen atom or a halogen atom as a substituent.

[0068] R 51 , R 52 , and R 53 In the formula (I), examples of the C1-3 linear alkyl group include a methyl group, an ethyl group, and an n-propyl group. Examples of the C3 branched alkyl group include an isopropyl group. Preferably, R 51 , and R 52 is a linear alkyl group, and R 53 is a branched alkyl group. Examples of the halogen atom as a substituent include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0069] The nitrogen-containing compound represented by general formula (V) can be produced, isolated and purified by known methods.

[0070] The nitrogen-containing compound represented by general formula (V) is preferably a nitrogen-containing compound represented by the following formula (51): [ka] .

[0071] Pharmaceutically acceptable salts of the nitrogen-containing compound represented by the general formula (V) are not particularly limited. Examples of such salts include inorganic acid addition salts such as hydrochloride, hydrobromide, sulfate, and nitrate; and organic acid addition salts such as acetate, tartrate, maleate, succinate, citrate, methanesulfonate, malate, oxalate, and benzenesulfonate. These salts can be produced, for example, by treating the nitrogen-containing compound represented by the general formula (V) with an acid. The pharmaceutically acceptable salt of the nitrogen-containing compound represented by general formula (V) is preferably the hydrochloride salt.

[0072] 3. Composition This embodiment relates to a composition comprising the nitrogen-containing compound described in 1. and 2. above. The composition can be used to treat malignant tumors. The composition can be used to prevent the recurrence of malignant tumors. The composition can be used to inhibit the proliferation of tumor stem cells in malignant tumors. By inhibiting the proliferation of tumor stem cells in malignant tumors, the composition can prevent the recurrence and metastasis of malignant tumors.

[0073] In this embodiment, "prevention" includes preventing and / or delaying the onset or recurrence of tumors. "Treatment" includes shrinking and / or eliminating existing tumors. "Treatment" may include treatment by administering the composition alone, as well as combined use with radiation therapy or surgical treatment. The maximum daily dose of the composition is 0.001 to 10 mg per kg of body weight in terms of the nitrogen-containing compound represented by formula (I).

[0074] The composition may be administered once a day at the above dosage, or if necessary, the above dosage may be divided into 2, 3, 4, or 5 doses, preferably 2 or 3 doses, per day.

[0075] The composition can be prepared by combining the nitrogen-containing compound represented by the general formula (I) with a suitable carrier or additive for formulation. The carrier or additive used in preparing the composition can be, depending on the dosage form of the composition, various types commonly used in conventional pharmaceuticals, such as excipients, binders, disintegrants, lubricants, colorants, flavoring agents, odorants, surfactants, etc.

[0076] When the composition is administered orally (including sublingually), the dosage form is not particularly limited, and examples thereof include tablets, powders, granules, capsules (including hard capsules and soft capsules), liquids, pills, suspensions, jelly preparations, emulsions, etc. When the compounded preparation or formulation is administered parenterally, examples thereof include injections, drip infusions, suppositories, nasal drops, and pulmonary preparations.

[0077] When the composition is prepared as an oral solid preparation such as a tablet, powder, granule, pill, or capsule, carriers may be used, for example, excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, methylcellulose, glycerin, sodium alginate, or gum arabic; or binders such as simple syrup, glucose solution, starch solution, gelatin solution, polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, carboxymethylcellulose, shellac, methylcellulose, ethylcellulose, water, ethanol, or potassium phosphate. Disintegrants such as mixtures, dry starch, sodium alginate, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, and lactose; disintegration inhibitors such as sucrose, stearic acid, cocoa butter, and hydrogenated oil; absorption promoters such as sodium lauryl sulfate; humectants such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite, and colloidal silicic acid; and lubricants such as purified talc, stearates, boric acid powder, and polyethylene glycol.

[0078] Here, tablets include oral tablets (plain tablets, sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double-layered tablets, multi-layered tablets, etc.), chewable tablets (including those that are taken by chewing in the oral cavity), lozenges (including those that are taken after dissolving in the oral cavity, such as lozenges), sublingual tablets, and buccal tablets.

[0079] When the composition is prepared as a solid oral preparation in the form of a pill, carriers that can be used include excipients such as glucose, lactose, starch, cacao butter, hardened vegetable oil, kaolin, and talc; binders such as powdered gum arabic, powdered tragacanth, and gelatin; and disintegrants such as laminaran and agar.

[0080] When the composition is prepared as a solid oral preparation in the form of a capsule, the capsule is prepared by mixing the active ingredient with the various carriers exemplified above and filling the mixture into a hard capsule, a soft capsule, or the like.

[0081] When the composition is a liquid, it may be in a liquid form, such as an aqueous or oily suspension, solution, syrup, elixir, or drink. The liquid is prepared using conventional additives in a conventional manner. The container into which the liquid is filled is not limited as long as it can be sealed, and may be a glass container, an aluminum container, or a plastic container.

[0082] When preparing an injectable composition, carriers that can be used include diluents such as water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters; pH adjusters such as sodium citrate, sodium acetate, and sodium phosphate; buffers such as dipotassium phosphate, trisodium phosphate, sodium hydrogen phosphate, and sodium citrate; stabilizers such as sodium metabisulfite, EDTA, thioglycolic acid, and thiolactic acid; and sugars such as mannitol, inositol, maltose, sucrose, and lactose as molding agents for lyophilization. In this case, glucose or glycerin may be added to the pharmaceutical preparation in an amount sufficient to adjust the solution to isotonicity, and conventional solubilizers, soothing agents, local anesthetics, and the like may also be added. By adding these carriers, subcutaneous, intramuscular, and intravenous injections can be prepared by conventional methods. When the composition is in the form of an infusion, it can be prepared by dissolving the nitrogen-containing compound to be administered in an isotonic electrolyte infusion preparation based on physiological saline, Ringer's solution or the like.

[0083] 4. Methods for detecting predictive markers One aspect of the present disclosure relates to a predictive marker for predicting tumor malignancy and a detection method thereof. The detection method includes detecting at least one selected from mucolipin protein and mucolipin mRNA present in tumor cells in a sample collected from a subject. That is, mucolipin protein, cells expressing mucolipin protein, and mucolipin mRNA present in a sample collected from a subject can be used as predictive markers for predicting tumor malignancy. The number of cells expressing mucolipin protein can also be used as a predictive marker for predicting tumor malignancy.

[0084] Detection of mucolipin protein or mucolipin mRNA in tumor cells is performed in vitro using a sample collected from a subject. There are no limitations on the sample, as long as it contains tumor cells. Examples of samples include tumor tissue, body fluids that may contain tumor cells, and blood. When tumor tissue is used as the sample, the sample can be collected, for example, by surgical resection or biopsy from a primary or metastatic lesion, or by endoscopic resection or biopsy. Body fluids may include cerebrospinal fluid, ascites, pleural effusion, etc. Examples of blood include peripheral blood, venous blood, and arterial blood. It is preferable to collect blood using an anticoagulant.

[0085] Determination of whether tissue is tumorous or normal can be performed by macroscopic observation, microscopic observation, etc. Alternatively, cell proliferation or tumor marker expression may be used as an indicator to determine whether the tissue is tumorous. In the case of glioma, examples of tumor markers include deletion of chromosome 1p / 19q, mutation of isocitrate dehydrogenase (IDH) 1 and 2, methylation of the O(6)-MGMT promoter, loss of nuclear ATRX expression, and CDKN2A homozygous deletion. When cell proliferation is used as an indicator to determine whether the tissue is tumorous, for example, a higher BrdU or Ki-67 protein labeling index in the tissue being examined compared to normal tissue can be used to determine whether the tissue is tumorous or normal. When a tumor marker is used as an indicator, tissue positive for tumor marker expression can be determined to be tumorous. Similarly, determination of whether a cell is tumorous or normal can be performed using the tumor markers, cell proliferation, etc. Tumor cells or tumor tissue collected from a subject is pretreated according to the method for detecting mucolipin protein or mucolipin mRNA.

[0086] Methods for detecting mucolipin protein as a protein include known methods such as immunostaining, Western blotting, and flow cytometry. Methods for detecting mucolipin protein as mRNA include known methods such as in situ hybridization, RT-PCR (including quantitative RT-PCR), microarrays, and RNA-Seq. Cells expressing mucolipin protein can be fluorescently labeled by immunostaining and then detected using a flow cytometer. Alternatively, cells expressing mucolipin protein can be fluorescently labeled by in situ hybridization and then detected using a flow cytometer.

[0087] When immunostaining or in situ hybridization is performed using tumor tissue, the tumor tissue is pretreated by fixing it with a known fixative such as formalin or paraformaldehyde, followed by preparation of a paraffin-embedded block. Alternatively, the tumor tissue is embedded in a resin for preparing frozen blocks, such as OCT Compound (registered trademark), either with or without fixation, to prepare a frozen block. The prepared paraffin-embedded block or frozen block is then thinly sliced ​​to prepare tissue sections, which are then subjected to immunostaining or in situ hybridization. Here, the tumor tissue embedded in the block may consist of only the tumor tissue, but may also include, for example, normal tissue.

[0088] When immunostaining or in situ hybridization is performed using tumor cells, the cells are smeared or collected on a slide glass and fixed with formalin, paraformaldehyde, ethanol, or the like as a pretreatment. Here, when mucolipin protein is immunostained, a positive signal appears on the cell surface, i.e., on the cell membrane.

[0089] When mucolipin protein is detected as a protein by Western blotting or the like, tumor cells or tumor tissues are lysed in a predetermined lysis buffer as a pretreatment. The sample lysed in the lysis buffer is used as a test sample. When mucolipin protein is detected as a protein by flow cytometry or the like, the sample is hemolyzed with a predetermined buffer as a pretreatment.

[0090] When detecting mucolipin protein as mRNA using RT-PCR, microarray, RNA-Seq, etc., total RNA or mRNA is extracted from tumor cells or tumor tissue as a pretreatment. If necessary, the extracted total RNA or mRNA may be used as a template for reverse transcription to synthesize complementary DNA (cDNA). The total RNA, mRNA, or cDNA is used as the test sample.

[0091] The primary antibody used to detect mucolipin proteins by immunostaining, Western blotting, or flow cytometry is not limited as long as it can detect mucolipin proteins. Examples include anti-MCOLN1 antibody (HPA031763; Atlas Antibodies), anti-MCOLN2 antibody (HPA019114; Atlas Antibodies), and anti-MCOLN3 antibody (HPA018106; Atlas Antibodies). Primary antibodies bound to mucolipin proteins can be detected by a reaction between an enzyme-labeled secondary antibody that binds to the primary antibody and the enzyme and its substrate. When immunostaining is performed, tissue sections may be deparaffinized and immersed in water, followed by treatment with a proteolytic enzyme such as trypsin, prior to immunostaining. Methods for preparing probes used in in situ hybridization are known, and commercially available probes may also be used.

[0092] Commercially available primers (which may include a probe in the case of quantitative RT-PCR) can be used for RT-PCR, and commercially available microarrays can also be used. RNA-Seq can obtain the number of mucolipin mRNA reads using a next-generation sequencer (for example, manufactured by Illumina).

[0093] When detecting mucolipin protein by immunostaining or in situ hybridization, the presence or absence of mucolipin protein can be detected by human observation of the immunostained or in situ hybridized tissue specimen using a microscope, slide scanner, or the like. When a signal from immunostaining or in situ hybridization is confirmed in tumor cells in the specimen, it can be determined (confirmed) that mucolipin protein has been detected. When even one tumor cell containing mucolipin protein is detected in the specimen, it can be determined that "mucolipin protein has been detected" or "expression of mucolipin protein is positive." Alternatively, for example, when the number of tumor cells present in a predetermined section of a microscope or slide scanner is taken as 100%, it can be determined that "mucolipin protein has been detected" or "expression of mucolipin protein is positive" when 1% or more, preferably 5% or more, and more preferably 10% or more of tumor cells contain mucolipin protein.

[0094] When mucolipin protein is detected by Western blotting, flow cytometry, RT-PCT, or RNA-Seq, if mucolipin protein is detected in a sample extracted from tumor cells or tumor tissue in a specimen, it may be determined that "mucolipin protein has been detected" or "expression of mucolipin protein is positive."

[0095] Alternatively, by comparing the amount of mucolipin protein in a sample containing tumor cells or tumor tissue with the amount of mucolipin protein in a sample derived from normal cells or normal tissue, if the amount of mucolipin protein in the sample derived from tumor cells or tumor tissue is higher than the amount of mucolipin protein derived from normal cells or normal tissue, it may be determined that "mucolipin protein has been detected" or "expression of mucolipin protein is positive." By comparing the number of cells expressing mucolipin protein in a sample containing tumor cells or tumor tissue with the number of cells expressing mucolipin protein in a sample derived from normal cells or normal tissue, it may be determined that "mucolipin protein has been detected" or "expression of mucolipin protein is positive" if the number of cells expressing mucolipin protein in the sample containing tumor cells or tumor tissue is higher than the number of cells expressing mucolipin protein in a sample derived from normal cells or normal tissue. The amount of mucolipin mRNA derived from a sample containing tumor cells or tumor tissue is compared with the amount of mucolipin mRNA in a sample derived from normal cells or normal tissue, and if the amount of mucolipin mRNA in the sample containing tumor cells or tumor tissue is higher than the amount of mucolipin mRNA in a sample derived from normal cells or normal tissue, it may be determined that "mucolipin protein has been detected" or "mucolipin protein expression is positive."

[0096] Furthermore, when the amount of mucolipin protein in a specimen containing tumor cells or tumor tissue is similar to the amount of mucolipin protein in a specimen derived from normal cells or normal tissue, it may be determined that "mucolipin protein is not detected" or that "mucolipin protein expression is negative." When the number of cells expressing mucolipin protein in a specimen containing tumor cells or tumor tissue is similar to the number of cells expressing mucolipin protein in a specimen derived from normal cells or normal tissue, it may be determined that "mucolipin protein is not detected" or that "mucolipin protein expression is negative." When the amount of mucolipin mRNA in a specimen containing tumor cells or tumor tissue is similar to the amount of mucolipin mRNA in a specimen derived from normal cells or normal tissue, it may be determined that "mucolipin protein is not detected" or that "mucolipin protein expression is negative."

[0097] Here, "high" refers to a value that is 1.2 times or more, preferably 1.5 times or more, more preferably 2 times or more, and even more preferably 5 times or more higher. "Similar" refers to a value that is approximately 0.8 to less than 1.2 times higher. Furthermore, before comparing the amounts of mucolipin protein or mucolipin mRNA, the amount of protein or RNA in each test sample may be normalized to the amount of protein or mRNA derived from a housekeeping gene such as GAPDH, β2-microglobulin, or β-actin. Protein amount may be expressed in terms of mass or concentration, or may be expressed in terms of the luminescence intensity of a substrate, etc. mRNA amount may be expressed in terms of the number of mRNA copies or reads, or may be expressed in terms of fluorescence intensity, etc.

[0098] In another embodiment, reference values ​​may be determined in advance for the measured amount of mucolipin protein, the measured number of cells expressing mucolipin protein, or the measured amount of mucolipin mRNA, and each measured value in a specimen containing tumor cells or tumor tissue is compared with the corresponding reference value. If the measured value is higher than the reference value, it may be determined that "mucolipin protein has been detected" or "expression of mucolipin protein is positive." Alternatively, each measured value in a specimen containing tumor cells or tumor tissue may be compared with the corresponding reference value. If the measured value is lower than the reference value, it may be determined that "mucolipin protein is not detected" or "expression of mucolipin protein is negative." There are no limitations on the reference value, so long as it is a value that can determine whether mucolipin protein, cells expressing mucolipin protein, or mucolipin mRNA has been detected or is positive, and it can be determined by known methods. A value that can distinguish whether mucolipin protein, cells expressing mucolipin protein, or mucolipin mRNA is detected or whether expression is positive can also be determined by ROC (receiver operating characteristic curve), discriminant analysis, mode method, Kittler method, 3σ method, p-tile method, etc. Examples of reference values ​​include sensitivity, specificity, negative predictive value, positive predictive value, and first quartile.

[0099] The method for detecting a predictive marker may further comprise determining that the tumor is of low malignancy if mucolipin protein is not detected, or determining that the tumor is of high malignancy if mucolipin protein is detected.

[0100] The method for detecting a predictive marker may include a step of presenting a label indicating that the subject's tumor has a low malignancy when mucolipin protein is not detected in the sample, or a step of presenting a label indicating that the subject's tumor has a high malignancy when mucolipin protein is detected in the sample.

[0101] The method for detecting a predictive marker may include a step of presenting a label indicating that the subject's tumor is of low malignancy when the amount of mucolipin protein, the number of cells expressing mucolipin protein, or the amount of mucolipin mRNA in a sample is below a reference value. Alternatively, the method for detecting a predictive marker may include a step of presenting a label indicating that the subject's tumor is of high malignancy when the amount of mucolipin protein, the number of cells expressing mucolipin protein, or the amount of mucolipin mRNA in a sample is above a reference value.

[0102] Alternatively, instead of presenting a label indicating that the subject's tumor is low in malignancy, a label indicating that the subject's prognosis is good or that the subject's survival rate is high may be presented. Alternatively, instead of presenting a label indicating that the subject's tumor is high in malignancy, a label indicating that the subject's prognosis is poor or that the subject's survival rate is low may be presented. The survival rate refers to the overall survival rate or disease / progression-free survival rate. Preferably, it is the 5-year survival rate.

[0103] 5. Testing reagents Another aspect of the present disclosure relates to a test reagent for detecting mucolipin protein or mucolipin mRNA present in tumor cells collected from a subject as a predictive marker for predicting the malignancy of the above-mentioned tumors. The test reagents may include mucolipin protein detection reagents and / or mucolipin mRNA detection reagents.

[0104] The mucolipin protein detection reagent contains one or more antibodies (e.g., primary antibodies) capable of binding to at least a portion of the mucolipin protein. The "antibody" may be a polyclonal antibody, a monoclonal antibody, or a fragment thereof (e.g., Fab, F(ab'), F(ab)2, etc.). The immunoglobulin class and subclass are not particularly limited. Furthermore, the antibody may be one screened from an antibody library, or may be a chimeric antibody, scFv, etc. Furthermore, the antibody does not necessarily have to be purified, and may be an antiserum containing the antibody, ascites fluid, or an immunoglobulin fraction fractionated therefrom.

[0105] The antibody contained in the test reagent may be in a dry state or dissolved in a buffer such as phosphate-buffered saline. Furthermore, the test reagent may contain at least one of a stabilizer such as β-mercaptoethanol or DTT, a protectant such as albumin, a surfactant such as polyoxyethylene (20) sorbitan monolaurate or polyoxyethylene (10) octylphenyl ether, or a preservative such as sodium azide.

[0106] The antibody that binds to the mucolipin protein may be labeled with an enzyme or a fluorescent dye, or may be immobilized on a microplate, magnetic beads, or the like.

[0107] The mucolipin protein detection reagent may be provided as a test kit containing the test reagent and a package insert describing how to use the reagent or providing a URL for a web page describing how to use the reagent. Furthermore, when the antibody that binds to the mucolipin protein is an unlabeled primary antibody, the test kit may contain a secondary antibody labeled with an enzyme or a fluorescent dye. Furthermore, the test kit may contain a substrate that reacts with the enzyme.

[0108] The mucolipin mRNA detection reagent contains a nucleic acid that hybridizes with all or part of mucolipin mRNA or mucolipin mRNA cDNA. The nucleic acid is preferably a detection nucleic acid (DNA or RNA) that functions as a primer and / or probe. There are no particular limitations on the length of the detection nucleic acid.

[0109] If the detection nucleic acid is a primer used in a PCR reaction, the sequence that hybridizes with mucolipin mRNA or mucolipin mRNA cDNA is preferably 50 mer or less, more preferably 30 mer or less, and even more preferably about 15 to 25 mer. The primer may contain a sequence that does not hybridize with mucolipin or mucolipin mRNA cDNA. Furthermore, the primer may be labeled with a fluorescent dye or the like.

[0110] In addition to primers, RT-PCR can also use a quantification probe that is degraded during the PCR reaction and is used for real-time quantification of PCR products. The quantification probe is also not limited as long as it hybridizes with mucolipin mRNA or mucolipin protein cDNA. The quantification probe is preferably a nucleic acid of approximately 5 to 20 mer that contains a sequence that hybridizes with mucolipin mRNA or mucolipin mRNA cDNA. Furthermore, it is preferable that one end of the quantification probe is labeled with a fluorescent dye and the other end is labeled with a quencher for the fluorescent dye.

[0111] If the detection nucleic acid is used as a capture probe in a microarray or the like, the sequence that hybridizes with mucolipin mRNA or mucolipin mRNA cDNA is preferably about 100-mer, more preferably about 60-mer, and even more preferably about 20-30-mer. The capture probe may contain a sequence that does not hybridize with mucolipin mRNA or mucolipin mRNA cDNA. Furthermore, the capture probe is preferably immobilized on a chip.

[0112] When the detecting nucleic acid is a probe for in situ hybridization, the sequence of the detecting nucleic acid that hybridizes with mucolipin mRNA may be an oligonucleotide of about 15 to 100 mer, or a polynucleotide of more than 100 mer. The polynucleotide may be DNA or RNA. A labeling substance such as digoxigenin or a fluorescent dye may be bound to the in situ hybridization probe. The probe may also contain a sequence that does not hybridize with mucolipin mRNA.

[0113] Mucolipin mRNA detection reagents may be provided as test kits containing the test reagent and a package insert describing how to use the reagent or a URL for a web page describing how to use the reagent. Furthermore, when detecting mucolipin mRNA or mucolipin protein cDNA by RT-PCR, the test kit may contain a nucleic acid amplification reagent (including polymerase, buffer, dNTPs, etc., even if it is heat-stable DNA), reverse transcriptase, etc. The nucleic acid amplification reagent may contain a dye such as SYBER GREEN (registered trademark), as needed. When detecting mucolipin mRNA or mucolipin protein cDNA by microarray, the test kit may contain a hybridization buffer, a washing buffer, etc. When detecting mucolipin mRNA by in situ hybridization, the test kit may contain a protease such as proteinase K, a hybridization buffer, a washing buffer, etc.

[0114] 6. Tumor malignancy prediction device 6-1. Configuration of the prediction system One embodiment of the present disclosure relates to a prediction system 1000 for predicting tumor malignancy (hereinafter abbreviated as "prediction system 1000") and a prediction device 10 for predicting tumor malignancy (hereinafter abbreviated as "prediction device 10"). Here, "predicting tumor malignancy" can be replaced with predicting the prognosis of a subject and / or predicting the survival rate of a subject.

[0115] FIG. 1 is a schematic diagram of a prediction system 1000, and in one embodiment, the prediction system 1000 may include an analysis device 5a or an analysis device 5b in addition to a prediction device 10. 2 shows a block diagram of the prediction device 10. The prediction device 10 may be connected to an input unit 111, an output unit 112, and a storage medium 113.

[0116] In the prediction device 10, a processing unit 101, a main memory unit 102, a ROM (read only memory) 103, an auxiliary memory unit 104, a communication interface (I / F) 105, an input interface (I / F) 106, an output interface (I / F) 107, and a media interface (I / F) 108 are connected to each other via a bus 109 so as to be able to communicate data with each other. The main memory unit 102 and the auxiliary memory unit 104 may collectively be simply referred to as a memory unit. The memory unit stores measured values, reference values, etc. in a volatile or non-volatile manner.

[0117] The processing unit 101 is a CPU of the prediction device 10. The processing unit 101 may work in cooperation with a GPU. The processing unit 101 executes an operation system 104a and a prediction program 104b stored in the auxiliary storage unit 104, and processes acquired data, thereby causing the prediction device 10 to function.

[0118] The ROM 103 is configured by a mask ROM, PROM, EPROM, EEPROM, or the like, and stores computer programs executed by the processing unit 101 and data used therefor. The processing unit 101 may be an MPU 101. The ROM 103 stores a boot program executed by the processing unit 101 when the prediction device 10 is started up, as well as programs and settings related to the operation of the hardware of the prediction device 10.

[0119] The main memory unit 102 is configured by a RAM (Random Access Memory) such as an SRAM or a DRAM. The main memory unit 102 is used to read out computer programs stored in the ROM 103 and the auxiliary memory unit 104. The main memory unit 102 is also used as a working area when the processing unit 101 executes these computer programs.

[0120] The auxiliary storage unit 104 is configured by a hard disk, a semiconductor memory element such as a flash memory, an optical disk, etc. The auxiliary storage unit 104 stores an operation system 104a, a prediction program 104b (described later), and a reference value database (DB) 104c that stores reference values. The prediction program 104b performs prediction processing in cooperation with the operation system 104a.

[0121] The communication I / F 105 is composed of a serial interface such as USB, IEEE1394, or RS-232C, a parallel interface such as SCSI, IDE, or IEEE1284, an analog interface including a D / A converter or an A / D converter, a network interface controller (NIC), etc. Under the control of the processing unit 101, the communication I / F 105 receives data from the analysis devices 5a, 5b or other external devices, and transmits or displays information stored or generated by the prediction device 10 to the analysis devices 5a, 5b or externally as needed. The communication I / F 105 may communicate with the analysis devices 5a, 5b or other external devices via a network.

[0122] The input I / F 106 is configured from, for example, a serial interface such as USB, IEEE1394, or RS-232C, a parallel interface such as SCSI, IDE, or IEEE1284, and an analog interface including a D / A converter or an A / D converter. The input I / F 106 accepts character input, clicks, voice input, etc. from the input unit 111. The accepted input content is stored in the main memory unit 102 or the auxiliary memory unit 104.

[0123] The input unit 111 is composed of a touch panel, a keyboard, a mouse, a pen tablet, a microphone, etc., and is used to input text or voice to the prediction device 10. The input unit 111 may be connected to the prediction device 10 from outside, or may be integrated with the prediction device 10.

[0124] The output I / F 107 is configured, for example, from an interface similar to the input I / F 106. The output I / F 107 outputs information generated by the processing unit 101 to the output unit 112. The output I / F 107 outputs information generated by the processing unit 101 and stored in the auxiliary storage unit 104 to the output unit 112.

[0125] The output unit 112 is configured by, for example, a display, a printer, etc., and displays the measurement results sent from the analysis devices 5a and 5b, various operation windows in the prediction device 10, analysis results, etc.

[0126] The media I / F 108 reads, for example, application software stored in the storage medium 113. The read application software is stored in the main memory unit 102 or the auxiliary memory unit 104. The media I / F 108 also writes information generated by the processing unit 101 to the storage medium 113. The media I / F 108 writes information generated by the processing unit 101 and stored in the auxiliary memory unit 104 to the storage medium 113.

[0127] The storage medium 113 is configured with a flexible disk, a CD-ROM, a DVD-ROM, etc. The storage medium 113 is connected to the media I / F 108 by a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, etc. The storage medium 113 may store application programs and the like for the computer to execute operations.

[0128] The processing unit 101 may acquire the prediction program 104b and various settings necessary for controlling the prediction device 10 via a network instead of reading them from the ROM 103 or the auxiliary storage unit 104. The application program may be stored in the auxiliary storage unit of a server computer on the network, and the prediction device 10 may access this server computer to download the computer program and store it in the ROM 103 or the auxiliary storage unit 104.

[0129] An operating system that provides a graphical user interface environment, such as Windows (registered trademark) manufactured and sold by Microsoft Corporation, is installed in the ROM 103 or the auxiliary storage unit 104. The application program according to the second embodiment runs on the operating system. In other words, the prediction device 10 may be a personal computer or the like.

[0130] The prediction system 1000 does not need to be installed in one place, and the prediction device 10 and the analysis devices 5a and 5b may be located in different places and connected via a network. Furthermore, the prediction device 10 may be a device that does not require an operator by omitting the input unit 111 and the output unit 112.

[0131] Referring to FIG. 1, the analytical device 5a is a device for measuring the amount or concentration of a protein, and includes a sample storage area 51, a reaction area 52, and a detection area 53. The cell lysate placed in the sample storage area 51 is dispensed into a microplate on which an antigen capture antibody is immobilized, which is placed in the reaction area 52, and incubated. After removing unreacted antigens as needed, a detection antibody is dispensed into the microplate and incubated. After removing unreacted antigens as needed, a substrate for detecting the detection antibody is dispensed into the microplate, the microplate is moved to the detection area 53, and a signal generated by the reaction of the substrate is measured.

[0132] Referring to Figure 1, another embodiment of the analytical device 5a is a device for measuring the expression level of mRNA by microarray analysis, in which the reverse transcription reaction product set in the sample storage area 51 is dispensed onto a microarray chip set in the reaction section 52, hybridization is performed, and after washing, it is moved to the detection section 53 where the signal is detected.

[0133] 1, another embodiment of the analytical device 5a is a device for measuring the expression level of mRNA by RT-PCR, in which a reverse transcription reaction product set in a sample storage area 51 is dispensed into a microtube set in a reaction section 52, and then quantitative PCR reagents are dispensed into the microtube. While the PCR reaction is carried out in the reaction section 52, a signal in the tube is detected in a detection section 53.

[0134] 1, the analysis device 5b is an apparatus for measuring the expression level of mRNA by the RNA-Seq method, and includes a sequence analysis unit 54a. A sample that has undergone a reaction for RNA-Seq is set in the sequence analysis unit 54, and base sequence analysis is performed within the sequence analysis unit 54a.

[0135] 1, the analysis device 5b is a fully automated Western blotting device for measuring protein amounts by Western blotting, and is equipped with a chemiluminescence signal detection unit 54b. A sample of tumor cells or tumor tissue lysed in a lysis buffer is placed in a predetermined position in the automated Western blotting device, and SDS-PAGE, blotting onto a membrane, antibody reaction, and chemiluminescence are performed. Analysis is then performed by the chemiluminescence signal detection unit 54b, and the signal intensity is quantified.

[0136] 1, the analytical device 5b is a flow cytometer for detecting mucolipin protein-positive cells by flow cytometry or for quantifying mucolipin protein on the cell surface, and includes a cell detection unit 54c equipped with a flow cell. Mucolipin protein present in cells is fluorescently labeled by immunostaining, and the fluorescence intensity of the cells is measured in the cell detection unit 54c.

[0137] The analysis devices 5a and 5b are connected to the prediction device 10 by wire or wirelessly. The analysis device 5a A / D converts the measurement value of the protein or the measurement value of the mRNA and transmits it as digital data to the prediction device 10. Similarly, the analysis device 5b A / D converts the measurement value of the mRNA and transmits it as digital data to the prediction device 10. This allows the prediction device 10 to acquire the measurement value of the protein or the measurement value of the mRNA as digital data that can be processed by calculation.

[0138] 6-2.Prediction program processing FIG. 3 shows an example of a flowchart of the process executed by the prediction program 104b. When an operator inputs a processing start request from the input unit 111, the processing unit 101 of the prediction device 10 starts processing for predicting the malignancy of a tumor.

[0139] In step S11, the processing unit 101 acquires at least one of the amount of mucolipin protein and mucolipin mRNA present in tumor cells in a specimen collected from a subject, which the operator inputs via the input unit 111. Alternatively, the processing unit 101 acquires a measurement value of at least one of the amount of mucolipin protein and mucolipin mRNA present in tumor cells in a specimen collected from a subject from the analysis device 5a or 5b. Alternatively, the operator may input via the input unit 111 whether the expression of mucolipin protein by immunostaining or in situ hybridization is positive or negative (or whether mucolipin protein is detected), and the processing unit 101 acquires this input data as a measurement value.

[0140] Next, in step S12, the processing unit 101 acquires, from the storage unit, a reference value corresponding to the measurement value acquired in step S11, and then compares the measurement value with the corresponding reference value.

[0141] If the acquired measurement value is lower than the reference value in step S13, processing unit 101 proceeds to step S14 (YES), determines that the tumor in the subject has low malignancy, and outputs a label indicating the determination result to output unit 112 (step S16).If the acquired measurement value is equal to or higher than the reference value in step S13, processing unit 101 proceeds to step S15 (NO), determines that the tumor in the subject has high malignancy, and outputs a label indicating the determination result to output unit 112 (step S16). The label may be a mark such as an X, a circle, or an exclamation mark. For details of the reference values, comparison methods, and determination methods, the explanation in 2. above is incorporated herein by reference.

[0142] 7. Storage media storing computer programs Furthermore, one embodiment of this embodiment relates to a program product, such as a storage medium, that stores the prediction program 104b. That is, the computer program can be stored in a storage medium, such as a hard disk, a semiconductor memory device such as a flash memory, or an optical disk. The format of the program stored in the storage medium is not limited as long as the prediction device 10 can read the program. It is preferable that the storage in the storage medium be non-volatile. [Example]

[0143] The present disclosure will be described in more detail below by showing examples, but the present disclosure should not be construed as being limited to the examples.

[0144] The animal experiments in this example were conducted with the approval of the Kansai Medical University Animal Experiment Committee and the president of the university. Furthermore, collection of cells from patients was conducted with the approval of the Medical Ethics Committee of the Kansai Medical University in accordance with the Declaration of Helsinki.

[0145] I. Bioassay 1.Cancer stem-like cells The cancer stem-like cells used were cancer stem-like cells established from high-grade glioma (four types: MD13, Me83, 1123, 30R) [Reference: Neuro Oncol 22(3): 333-44, 2020; Cancer Cell 24(3): 331-46, 2013], cancer stem-like cells derived from lung squamous cell carcinoma (two types: L1, L14), and cancer stem-like cells derived from metastatic brain tumors (two types: B34, B67).

[0146] <Method for establishing cancer stem cell-like cells> Surgically removed cancer tissue (0.1-1 g) was finely minced with scissors. The minced tissue was transferred to a test tube containing 2 mL of cell detachment solution (Accumax™; Nacalai Tesque) and shaken (20 rpm) in a 37°C incubator for 5 minutes. 8 mL of cell culture medium was added, mixed, and centrifuged (40 × g, 5 minutes). The supernatant was discarded, and 10 mL of cell culture medium was added, mixed, and cultured in an ultra-low attachment surface dish (100 mm; Corning). Culture was carried out in a humid environment of 5% CO2 / 95% air at 37°C. The cell culture medium used was D-MEM / Ham's F-12 (Wako Pure Chemical Industries, Ltd.) supplemented with NaHCO3 (49 mM), glucose (26 mM), L-glutamine (3 mM), MACS NeuroBrew-21 (5 mL; Miltenyi Biotec), epidermal growth factor (EGF, 20 ng / mL; PeproTech), fibroblast growth factor (bFGF, 20 ng / mL; PeproTech), penicillin (100 U / mL), and streptomycin (0.1 mg / mL).

[0147] 2. Cell Proliferation Assay The cancer stem-like cells were transferred to a test tube and centrifuged (40 × g, 5 minutes). The supernatant was discarded, and 2 mL of trypsin-EDTA solution (Sigma-Aldrich) was added. The cells were then incubated in a 37°C incubator for 5 minutes. Cell culture medium was added, mixed, and centrifuged. The supernatant was discarded, and cell culture medium was added. The cells were counted, and 2,000 cells were transferred to a 96-well ultra-low attachment surface plate (Corning). The cells were cultured for 4 days in 0.1 mL of cell culture medium containing each test drug. The number of viable cells was counted using the viable cell count reagent SF (Nacalai Tesque).

[0148] 3. Current Measurement The mucolipin permeation current was measured using the patch clamp technique. Patch electrodes were fabricated from glass tubes (G-1.5, Narishige Scientific Instruments Laboratory) using a micropipette maker (PP-83, Narishige Scientific Instruments Laboratory). The patch electrodes had an electrical resistance of 3-7 MΩ when filled with the electrode solution. The electrode solution consisted of CsOH (110 mM), gluconic acid (100 mM), hydrochloric acid (10 mM), glucose (10 mM), HEPES (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid; 10 mM), glycerol ether (GETA) (10 mM), ethylenediaminetetraacetic acid (EDTA) disodium salt (0.5 mM), and adenosine triphosphate (ATP) disodium salt (2 mM), adjusted to pH 7.4 with CsOH.

[0149] Cancer stem-like cells (MD13) were fixed on cover glasses, which were then mounted in a chamber on an inverted microscope (Olympus). The experimental perfusion solution consisted of 150 mM NaCl and 5 mM HEPES, pH adjusted to 7.4 with NaOH. Experiments were performed at 23–30°C. Current measurements were performed using an EPC 800 patch-clamp amplifier (HEKA Elektronik). The experimental protocol and data acquisition were controlled using a data acquisition system (Digidata 1322A; Axon Instruments) and software (Clampex 9; Axon Instruments).

[0150] 4. Cell staining The primary antibody used to detect mucolipin was anti-MCOLN3 antibody (HPA018106; Atlas Antibodies) diluted 200-fold. The secondary antibody used was Alexa Fluor 488-conjugated anti-rabbit immunoglobulin antibody (A11008; Invitrogen). Antibody absorption tests were performed using the MCOLN3 antigen peptide (APrEST73203; Atlas Antibodies). Cell nuclei were stained with DAPI (4',6-Diamidino-2-phenylindole, 1 μg / ml; Dojindo Laboratories). Fluorescence was observed and images were acquired using a confocal laser scanning microscope (LSM510 META; Carl Zeiss).

[0151] 5. Immunohistochemistry An automated immunohistochemistry system (Discovery ULTRA System; Roche Diagnostics) was used. Anti-MCOLN1 antibody (HPA031763; Atlas Antibodies) was used at a 1:100 dilution as the primary antibody to detect mucolipin. Peroxidase-conjugated anti-rabbit immunoglobulin antibody was used as the secondary antibody, and diaminobenzidine (DAB) was used for color development.

[0152] 6.Survival curve The relationship between mRNA expression and prognosis in patients with low-grade glioma was analyzed using cBioPortal from the database (The Cancer Genome Atlas) [Cancer Discov 2(5): 401-404, 2012, Sci Signal 6(269): pl1, 2013]. Five hundred thirty cases were divided into high and low mRNA expression groups (Z-score > 2.0). Kaplan-Meier curves were constructed for overall survival or progression-free survival, and the log-rank test was used.

[0153] II. Mucolipin Expression and Viability Mucolipin protein was expressed in the pathological tissue specimens of four out of five cases of glioblastoma, a high-grade glioma (Figure 4). This suggests that mucolipin expression is characteristic of glioblastoma.

[0154] Survival curves are also shown in Figure 5. In Figure 5, A and B show the relationship between the expression of the mucolipin MCOLN1 and patient survival, C and D show the relationship between the expression of the mucolipin MCOLN2 and patient survival, and E and F show the relationship between the expression of the mucolipin MCOLN3 and patient survival. A, C, and E show the overall survival rate, and B, D, and F show the disease / progression (D / P)-free survival rate.

[0155] Regardless of the type of mucolipin, the overall survival rate and D / P-free survival rate were poor in the group with high mucolipin expression, indicating that mucolipin expression is closely related to tumor malignancy and survival rate.

[0156] III. Compound Screening In silico screening was performed to search for novel compounds that target mucolipin. We measured the cytostatic activity of temozolomide, a treatment for glioblastoma, and existing drugs used to treat diseases other than glioblastoma. We selected 14 existing drugs that had stronger cytostatic activity than temozolomide. The 14 existing drugs are shown in Figure 6. The cytostatic activity and blood-brain barrier permeability of each compound are also shown in Figure 6. The cells used were glioblastoma cells (MD13, 30R), cancer stem cell-like cells derived from metastatic brain tumors (B34), and cancer stem cell-like cells derived from lung squamous cell carcinoma (L1). Furthermore, from the 14 existing drugs selected, we selected SR33805, which had the strongest cytostatic activity, and lomerizine and flunarizine, calcium channel blockers known to permeate the blood-brain barrier.

[0157] The drug-binding region of the mucolipin protein (MCOLN1; NG_015806, PDB:5WJ9) was searched for using the Molsite method (Fukunishi Y and Nakamura H, Protein Sci, 2011, PMID: 21064162). The 3D structures of lomerizine (PubChem ID: 122125), flunarizine (PubChem ID: 941361), and SR33805 (PubChem ID: 129426) were then obtained from PubChem, and docking calculations were performed on the predicted drug-binding region using myPresto software (https: / / www.mypresto5.jp). The drug binding region was selected from the docking model using ΔG and docking score as indicators, and screening was performed from 2 million drugs registered in LigandBox (http: / / www.mypresto5.com / ligandbox / cgi-bin / index.cgi?LANG=ja) using the ML-MST method, which used supervised learning of the three existing drugs mentioned above. The top 100 drugs based on the obtained MTS scores were docked to the protein in question.

[0158] As a result of in silico screening, the structures of 31 virtual compounds were obtained. Based on the structures of the obtained new virtual compounds, each virtual compound was actually synthesized to obtain new nitrogen-containing compounds. The synthesis of the nitrogen-containing compounds was commissioned to Kishida Chemical.

[0159] The efficacy of each of the 31 novel nitrogen-containing compounds was confirmed by measuring their cell growth inhibitory activity against glioblastoma cells (MD13), cancer stem cell-like cells derived from metastatic brain tumors (B34), and cancer stem cell-like cells derived from lung squamous cell carcinoma (L1). As a result, the nitrogen-containing compound represented by the following formula (1) was found to have the highest cell proliferation inhibitory activity.

[0160] [ka]

[0161] The compound represented by formula (1) is a known compound represented by the following formula (1A) (6-(pyrrolidin-3-yloxy)pyridin-3-amine) [ka] and a known compound (6-isopropoxypicolinic acid) represented by the following formula (1B): [ka] and were produced by amide bond formation.

[0162] The synthesis and purity of the nitrogen-containing compound represented by formula (1) were confirmed by liquid chromatography mass spectrometry (LCMS) under the conditions shown in Figure 7. The LCMS results are shown in Figure 8. LCMS showed a single peak, indicating that a single compound was produced from the compound represented by formula (1A) and the compound represented by formula (1B). Hereinafter, the nitrogen-containing compound represented by formula (1) may be referred to as "KMU3."

[0163] In addition, we commissioned Pistan Co., Ltd. (Kanagawa Prefecture) to analyze the 1H-NMR spectrum of KMU3. The results were as follows:

[0164] 1 H NMR (DMSO-d6, δ in ppm) 10.16-10.06 (m, 5H) 8.59(s, 1H), 8.18(dd, 1H, J=9.0, 2.5 Hz),7.89-7.52 (m, 2H), 7.00-6.83 (m2, 1H) 5.72-5.49(m, 2H)3.31(br, 4H), 2.15(br, 2H), 1.21(d, 6H. J=5.7Hz)

[0165] Figure 9 shows the chemical formulas of six nitrogen-containing compounds (KMU5, KMU12, KMU13, KMU23, KMU7, and KMU9) ​​that were structurally similar to KMU3 among the novel nitrogen-containing compounds identified in this screening study. Figure 10 shows the cytostatic activity of KMU3, KMU5, KMU12, KMU13, KMU23, KMU7, and KMU9 against glioblastoma cells when added to the culture medium at a final concentration of 1 μM (Figure 10A) and the relative growth rate (% of control) compared to control cells (cells not treated with the test drug) (Figure 10B). In Figure 10, "C" indicates the control; "#" indicates KMU5, KMU9, and KMU12 (these are shown for convenience because the growth curves of the three compounds overlap); and "&" indicates KMU7, KMU13, and KMU23 (these are shown for convenience because the growth curves of the three compounds overlap). "3" indicates KMU3. Compared with KMU5, KMU12, KMU13, KMU23, KMU7, and KMU9, KMU3 had higher cell growth inhibitory activity.

[0166] Therefore, it was demonstrated that nitrogen-containing compounds having the structure represented by the above general formula (I) are important in inhibiting the proliferation of glioblastoma cells.

[0167] Figure 11 shows the concentration-dependent cell growth inhibitory activity of KMU3, SR33805, and temozolomide. Black circles represent KMU3, white circles represent SR33805, and crosses represent temozolomide. MD13 cells were used as glioblastoma cells, and each drug was added to the culture medium at concentrations of 10 nM, 100 nM, 1 μM, and 10 μM. Cell viability was measured four days later. KMU3 demonstrated cell growth inhibitory activity comparable to that of SR33805.

[0168] Next, to verify that SR33805 actually inhibits the function of mucolipin, we observed changes in the current through mucolipin in the presence of SR33805. The results are shown in Figure 12. The current through mucolipin decreased in a concentration-dependent manner by varying the final concentration of SR33805 added (0 nM (control), 1 nM, 10 nM, and 100 nM). This indicates that SR33805 and similar compounds with mucolipin docking activity function as mucolipin blockers.

[0169] IV. Expression of mucolipin in lung cancer stem cells and the inhibitory effect of SR33805 on cell proliferation Mucolipin expression in lung cancer stem cells (L1) derived from squamous cell carcinoma was confirmed by immunostaining. The results are shown in Figure 13. Figure 13A shows the results of immunostaining using an anti-MCOLN3 antibody, and Figure 13B shows the results of an antibody absorption test in which an antigen peptide was reacted with the anti-MCOLN3 antibody. A positive signal was detected in Figure 13A. Furthermore, no signal was detected in Figure 13B, suggesting that the positive signal in Figure 13A is specific to mucolipin. Similar results were obtained with immunostaining using an anti-MCOLN1 antibody. These results indicate that mucolipin is also expressed in lung cancer stem cells. Next, the cell proliferation rate of lung cancer stem cells was observed in the presence of SR33805. The results are shown in Figure 13C. When SR33805 was added to the cells at final concentrations of 0 μM (control), 1 μM, or 10 μM, the proliferation of lung cancer stem cells was also inhibited in a concentration-dependent manner.

[0170] This indicates that mucolipin blockers also exert a cell proliferation-inhibitory effect on lung cancer stem cells.

[0171] V. Comparison of the effects of test drugs in glioblastoma model mice We generated glioblastoma model mice by transplanting cancer stem cells into immunodeficient mice. Immunodeficient mice (BALB / cAJcl-nu / nu, 6-8 weeks old, female) were anesthetized with a triple-dose combination (Domitor 0.75 mg / kg, midazolam 4 mg / kg, and betolfar 5 mg / kg) to minimize pain. Using a stereotaxic instrument (SR-6M-HT; Narishige Scientific Instruments Research Institute), the head was held and the forehead skin was incised. Cancer stem-like cells (MD13, 100,000 cells) established from a high-grade glioma were injected over 5 minutes, 2 mm rightward and 1 mm anterior to bregma, at a depth of 3 mm. After the injection, the wound was closed with 5-0 nylon. Starting on day 7 after transplantation, the test drugs KMU3, temozolomide (Temodar®, MSD), lomerizine dihydrochloride (Alomone Labs), and KMU84 (expressed by the formula below) were orally administered for 5 consecutive days using a probe (FTP-20-38-50; Instech Laboratories). Efficacy was evaluated by overall survival (Kaplan-Meier curve). Statistical analysis was performed using the log-rank test. [ka] KMU3 and KMU84 were diluted in 0.5 w / v% methylcellulose 400 solution (Fujifilm Wako Pure Chemical Industries) to 5 mg / kg body weight and administered once daily. This dose was the optimal dose for KMU3. Lomerizine dihydrochloride was adjusted to 3 mg / kg body weight and administered once daily. Temozolomide was adjusted to 5 mg / kg body weight and administered once daily. Dimethyl sulfoxide (Nacalai Tesque), the solvent for the test drug, was administered as a negative control.

[0172] The results are shown in Figure 14. Panel A shows a comparison of overall survival between the KMU3-administered group and the negative control group. The KMU3-administered group (K3) had a significantly longer overall survival than the negative control group (Control) (p = 0.014). Panel B shows overall survival between the lomerizine dihydrochloride-administered group, the temozolomide-administered group, and the negative control group. The lomerizine dihydrochloride-administered group (Drug L3) had a significantly longer overall survival than the negative control group (Control) (p = 0.006). The temozolomide-administered group (Tmz) had a significantly longer overall survival than the negative control group (Control) (p = 0.008). Panel C shows a comparison of overall survival between the KMU84-administered group (K84) and the negative control group (Control). No difference was observed between the two groups. These results suggest that KMU3 and lomerizine dihydrochloride are effective in treating tumors.

Claims

1. A nitrogen-containing compound represented by the following general formula (I), or a pharmaceutically acceptable salt thereof: 【Chemistry 1】 (where, m, n, and p are the same or different and each represents an integer of 1 to 3. R １ , R ２ , and R ３ are the same or different and represent a hydrogen atom, a C1-6 linear or branched alkyl group which may have a halogen atom as a substituent, a C1-6 linear or branched alkoxy group which may have a halogen atom as a substituent, a halogen atom, an amino group, a hydroxyl group, a nitro group, a carboxy group or a cyano group. R ４ represents a C1-6 branched or linear alkyl group which may have a halogen atom as a substituent.

2. R ４ The nitrogen-containing compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein represents a C3-6 branched alkyl group.

3. R １ , R ２ , and R ３ The nitrogen-containing compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein is a hydrogen atom.

4. The nitrogen-containing compound according to claim 3, wherein the nitrogen-containing compound represented by general formula (I) is represented by the following formula (1), or a pharmaceutically acceptable salt thereof: 【Chemistry 2】 。

5. A composition comprising the nitrogen-containing compound of any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof.

6. The composition of claim 5 used to treat malignant tumors.

7. The composition according to claim 5, which is used to prevent the recurrence of a malignant tumor or to prevent the metastasis of a malignant tumor.

8. The composition according to claim 5, which is used to inhibit the proliferation of tumor stem cells of malignant tumors.

9. The composition according to any one of claims 5 to 8, wherein the malignant tumor is glioblastoma or lung cancer.

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