WO2022210524A1 - Composé contenant de l'azote, composition comprenant ledit composé contenant de l'azote et marqueur pour la prédiction du degré d'une tumeur - Google Patents

Composé contenant de l'azote, composition comprenant ledit composé contenant de l'azote et marqueur pour la prédiction du degré d'une tumeur Download PDF

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WO2022210524A1
WO2022210524A1 PCT/JP2022/014962 JP2022014962W WO2022210524A1 WO 2022210524 A1 WO2022210524 A1 WO 2022210524A1 JP 2022014962 W JP2022014962 W JP 2022014962W WO 2022210524 A1 WO2022210524 A1 WO 2022210524A1
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mucolipin
nitrogen
containing compound
group
protein
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PCT/JP2022/014962
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Japanese (ja)
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美樹夫 林
幸樹 池田
亮一 岩田
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学校法人関西医科大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings

Definitions

  • compositions containing the nitrogen-containing compounds Disclosed herein are nitrogen-containing compounds, compositions containing the nitrogen-containing compounds, and predictive markers of tumor malignancy.
  • Non-Patent Document 1 a Non-Patent Document 1
  • An object of the present invention is to provide a compound that suppresses the proliferation of cancer stem cells.
  • Another object of the present invention is to provide a predictive marker for predicting the malignancy of a tumor.
  • Section 1 A step of detecting at least one selected from mucolipin protein and mucolipin mRNA present in tumor cells in a specimen collected from a subject, A method for detecting predictive markers for predicting tumor malignancy. Section 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 the malignancy of a tumor 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 the malignancy of a tumor, wherein the test reagent contains mucolipin protein. containing an antibody for detection, Said detection reagent. Section 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 the malignancy of a tumor, wherein the test reagent detects mucolipin mRNA.
  • Item 5 Detection for detecting mucolipin protein or mucolipin mRNA present in tumor cells in a specimen collected from a subject as a predictive marker for predicting tumor malignancy, comprising the detection reagent according to item 3. kit. Item 6.
  • a prediction device for predicting the malignancy of a tumor comprising a processing unit, wherein the processing unit determines the amount of mucolipin protein present in tumor cells in a sample collected from a subject, and mucolipin mRNA obtaining at least one selected measurement value, comparing the obtained measurement value with a reference value, and outputting a label indicating that the tumor is highly malignant when the measurement value exceeds the reference value; outputting a label indicating that the tumor is of low malignancy when the value is less than or equal to the reference value, and the subject has a malignant tumor; the prediction device.
  • the processing unit determines the amount of mucolipin protein present in tumor cells in a sample collected from a subject, and mucolipin mRNA obtaining at least one selected measurement value, comparing the obtained measurement value with a reference value, and outputting a label indicating that the tumor is highly malignant when the measurement value exceeds the reference value; outputting a label indicating that the tumor is of low malignancy when the value is less than or equal to the reference value
  • R 1 , R 2 , and R 3 are the same or different, a hydrogen atom, a C1-6 straight chain optionally having a halogen atom as a substituent, or a branched chain optionally having a halogen atom as a substituent It represents a chain alkyl group, 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.
  • R 4 represents a C1-6 branched or linear alkyl group optionally having a halogen atom as a substituent. ).
  • Item 8. Item 8. The nitrogen-containing compound according to Item 7, or a pharmaceutically acceptable salt thereof, wherein R 4 represents a C3-6 branched alkyl group.
  • Item 9. Item 9. The nitrogen-containing compound according to Item 7 or 8, or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 and R 3 are hydrogen atoms.
  • Item 10 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: .
  • 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 of Item 11, which is used to treat malignant tumors.
  • Item 13. The composition of paragraph 11, which is used to prevent recurrence of malignant tumors or to prevent metastasis of malignant tumors.
  • Item 14. Item 12. The composition according to Item 11, which is used for suppressing proliferation of tumor stem cells of malignant tumors.
  • Item 15. Item 15. The composition according to any one of Items 11 to 14, wherein the malignant tumor is glioblastoma or lung cancer.
  • a composition comprising: (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 optionally having a hydrogen atom or a halogen atom as a substituent. ); (In general formula (III), R 31 and R 32 are the same or different and represent a halogen atom.); (In general formula (IV), n represents an integer of 1-3.
  • 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 optionally having a hydrogen atom or a halogen atom as a substituent. );
  • 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 optionally having a hydrogen atom or a halogen atom as a substituent. ).
  • Item 17. A composition comprising the nitrogen-containing compound of Item 16, or a pharmaceutically acceptable salt thereof.
  • Item 18. 18.
  • Item 18. The composition according to Item 17, which is used for suppressing proliferation of tumor stem cells of malignant tumors.
  • Item 21. 21. The composition according to any one of Items 17 to 20, wherein the malignant tumor is glioblastoma or lung cancer.
  • cancer stem cells such as glioblastoma cancer stem cells and lung cancer stem cells.
  • the malignancy of a tumor can be predicted.
  • FIG. 2 shows a hardware configuration of a prediction device 10; 4 shows the flow of processing of the prediction program 104b.
  • FIG. 2 shows immunostaining images of mucolipin protein in histopathological specimens.
  • FIG. A shows the relationship between mucolipin MCOLN1 expression and the patient's Overall survival rate.
  • B shows the relationship between MCOLN1 expression of mucolipin and patient's Disease/Progression (D/P)-free survival rate.
  • C shows the relationship between the MCOLN2 expression of mucolipin and the patient's Overall survival rate.
  • D shows the relationship between MCOLN2 expression of mucolipin and patients' Disease/Progression (D/P)-free survival rate.
  • E shows the relationship between mucolipin MCOLN3 expression and patient Overall survival rats.
  • F shows the relationship between MCOLN3 expression of mucolipin and patients' Disease/Progression (D/P)-free survival rate.
  • A shows the cytostatic activity against glioblastoma cells when the respective compounds of KMU3, KMU5, KMU12, KMU13, KMU23, KMU7, and KMU9 were added to the medium.
  • B shows the relative growth rate (% of control) to control cells.
  • C indicates control
  • # indicates KMU5, KMU9, KMU12 (three growth curves are superimposed and thus indicated for convenience)
  • & indicates KMU7, KMU13, KMU23. (shown this way for convenience since the three growth curves are overlaid).
  • 3 indicates KMU3. It shows the concentration-dependent cytostatic activity of KMU3, SR33805, and temozolomide.
  • ⁇ indicates KMU3, ⁇ indicates SR33805, and ⁇ indicates temozolomide.
  • Fig. 2 shows changes in current passing through mucolipin in the presence of SR33805.
  • A shows the results of immunostaining with an anti-MCOLN3 antibody.
  • B shows the immunostaining results of an antibody absorption test in which the antigen peptide was reacted with an anti-MCOLN3 antibody.
  • C shows the cell proliferation rate of lung cancer stem cells in the presence of SR33805.
  • A shows a comparison of overall survival between the KMU3-administered group (K3) and the negative control group (Control).
  • B shows the overall survival of the lomerizine dihydrochloride administration group (Drug L3), the temozolomide administration group (Tmz), and the negative control group (Control).
  • C shows a comparison of overall survival between the KMU84 administration group (K84) and the negative control group (Control).
  • the present specification provides a nitrogen-containing compound that binds to mucolipin protein, or a pharmaceutically acceptable salt thereof, a method for producing the nitrogen-containing compound, and a mucolipin protein as a predictive marker for predicting tumor malignancy. , a method for detecting the predictive marker, an apparatus for detecting the predictive marker, a program for detecting the predictive marker, and the like are disclosed.
  • mucolipin proteins 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.
  • a tumor may be a malignant tumor or a benign tumor, preferably a malignant tumor.
  • Tumors include both non-epithelial and epithelial malignancies. Specifically, malignant tumors of the central nervous system; respiratory system malignant tumors arising from the trachea, bronchi, lungs, etc.; nasopharynx, esophagus, stomach, duodenum, jejunum, ileum, cecum, appendix, ascending colon, transverse colon, S Gastrointestinal malignant tumors arising from the shaped colon, rectum, anus, etc.; liver cancer; pancreatic cancer; urological malignant tumors arising from the bladder, ureter, or kidney; breast cancer: prostate cancer; skin cancer; endocrine malignant tumors such as hypothalamus, pituitary gland, thyroid, parathyroid gland, adrenal gland; solid tumors such as malignant tumors arising from bone and soft tissue, and myelodysplastic syndrome, acute
  • Glioma is the most preferred brain tumor, and squamous cell carcinoma is the most preferred lung cancer.
  • Glioma includes glioblastoma, anaplastic oligodendroglioma, anaplastic astrocytoma, diffuse midline glioma, oligodendroglioma, diffuse astrocytoma, pilocytic astrocytoma, It may include subependymal giant cell viable cell tumor, pleomorphic xanthoastrocytoma, and the like.
  • Glioblastoma, anaplastic oligodendrogliomas, anaplastic astrocytomas, and diffuse midline gliomas are classified as high-grade gliomas, and are classified into oligodendrogliomas, diffuse astrocytomas, and pilocytic astrocytomas.
  • Cell tumors, subependymal giant cell viable cell tumors, etc. are classified as low-grade gliomas.
  • the degree of malignancy of brain tumors is generally expressed in 4 stages from grade 1 to 4 according to the type of tumor, with grades 3 and 4 considered to be highly malignant. The higher the grade, the lower the 2-year survival rate and 5-year survival rate.
  • Nitrogen-Containing Compound its Pharmaceutically Acceptable Salt, and Method for Producing Nitrogen-Containing Compound
  • Nitrogen-Containing Compound This embodiment relates to a nitrogen-containing compound.
  • the nitrogen-containing compound may bind to mucolipin protein.
  • said nitrogen-containing compound is capable of blocking the flow of ions through ion channels comprised by mucolipin proteins.
  • C in the descriptions of “C1-6”, “C1-4”, “C3-6”, etc. means 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
  • C3-6 indicates that the number of carbon atoms is 3 to 6. indicates that The nitrogen-containing compound according to this embodiment is represented by the following general formula (I).
  • m, n, and p are the same or different and represent an integer of 1-3.
  • 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 one.
  • R 1 , R 2 , and R 3 are the same or different, and are C1-6 linear or branched alkyl groups optionally having a hydrogen atom or a halogen atom as substituents; It represents a C1-6 linear or branched alkoxy group optionally having 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.
  • Examples of the C1-6 linear alkyl group for R 1 , R 2 and R 3 include methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group and n-hexyl group. I can give an example.
  • the C3-6 branched chain alkyl group includes isopropyl group, sec-butyl group, isobutyl group, tert-butyl group, isopentyl group, neopentyl group, 3-methylpentyl group, 3,3-dimethylbutyl group, or 3- An ethylbutyl group can be exemplified.
  • the C1-6 linear or branched alkyl group is preferably a C1-3 linear or branched alkyl group (that is, a methyl group, an ethyl group, an n-propyl group, or an isopropyl group), and more It is preferably a C1-3 linear alkyl group (that is, a methyl group, an ethyl group, or a 3-propyl group), more preferably a C1-2 alkyl group (that is, a methyl group, or an ethyl group). , and even more preferably a C1 alkyl group (ie, a methyl group).
  • a halogen atom as a substituent can be exemplified by a fluorine atom, a chlorine atom, a bromine atom, an iodine atom and the like.
  • the alkyl group in the C1-6 linear or branched alkoxy group optionally having a halogen atom as a substituent for R 1 , R 2 and R 3 is the above C1-6 linear alkyl group, and C3-6 branched chain alkyl groups.
  • a halogen atom as a substituent can be exemplified by a fluorine atom, a chlorine atom, a bromine atom, an iodine atom and the like.
  • Halogen atoms for R 1 , R 2 and R 3 can be exemplified by fluorine, chlorine, bromine and iodine atoms.
  • R 1 , R 2 and R 3 are preferably hydrogen atoms.
  • R 4 represents a C1-6 branched or linear alkyl group optionally having a halogen atom as a substituent.
  • the definition of the halogen atom and the branched or linear alkyl group at C1-6 are the same as the halogen atoms in R 1 , R 2 and R 3 .
  • R 4 is preferably a C3-6 branched alkyl group optionally having a halogen atom as a substituent (that is, isopropyl group, sec-butyl group, isobutyl group, tert-butyl group, isopentyl group, neopentyl group, 3-methylpentyl group, 3,3-dimethylbutyl group, and 3-ethylbutyl group), more preferably a C3-4 branched alkyl group optionally having a halogen atom as a substituent ( isopropyl group, sec-butyl group, isobutyl group, or tert-butyl group), more preferably unsubstituted C3-4 branched chain alkyl group (i.e., isopropyl group, sec- butyl group, isobutyl group, or tert-butyl group), and more preferably an unsubstituted C3 branched chain alkyl group (ie
  • the nitrogen-containing compound represented by general formula (I) is more preferably a nitrogen-containing compound represented by the following formula (1):
  • a nitrogen-containing compound represented by the following formula (11) is more preferable:
  • compositions are not particularly limited.
  • Such salts include, for example, inorganic acid addition salts such as hydrochlorides, hydrobromides, sulfates, nitrates; and acetates, tartrates, maleates, succinates, citrates, methanesulfonates. , malate, oxalate, benzenesulfonate and other organic acid addition salts.
  • These salts can be produced, for example, by treating the nitrogen-containing compound represented by the general formula (I) with an acid.
  • the nitrogen-containing compound represented by the general formula (I) is composed of the following general formula (IA) and (m, n, R 1 and R 2 are the same as above.) With the following general formula (IB) (p, R 3 and R 4 are the same as above.) can be produced by amide bonding under known conditions.
  • reaction between compound (IA) and compound (IB) is a method of reacting compound (IA) with the carboxylic acid of compound (IB) in a normal amide bond forming reaction.
  • amide bond formation reactions can be widely applied to the amide bond formation reaction.
  • mixed acid anhydride method i.e., compound (IB) is reacted with an alkylhalocarboxylic acid to form a mixed acid anhydride, which is reacted with compound (IA)
  • active ester method i.e., compound (IB) is an active ester such as p-nitrophenyl ester, N-hydroxysuccinimide ester, 1-hydroxybenzotriazole ester, or an active amide with benzoxazoline-2-thione, to which compound (IA) is added.
  • carbodiimide method i.e.
  • compound (IA) to compound (IB) with an activating agent such as dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (WSC), carbonyldiimidazole, etc.
  • an activating agent such as dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (WSC), carbonyldiimidazole, etc.
  • WSC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
  • COS carbonyldiimidazole
  • a method of reacting compound (IA) with an ester of and a lower alcohol under high pressure and high temperature a method of reacting compound (IA) with an acid halide of compound (IB), that is, a carboxylic acid halide, and the like.
  • the mixed acid anhydride used in the above mixed anhydride method (a) is obtained by a normal Schotten-Baumann reaction, which is usually isolated without being reacted with the compound (IA) of the general formula (I). A nitrogen-containing compound is produced.
  • the Schotten-Baumann reaction is carried out in the presence of a basic compound.
  • Examples of the basic compound that can be used include those commonly used in the Schotten-Baumann reaction, 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), 1,4-diazabicyclo”[2.2.2]octane (DABCO), etc.
  • DBN 1,5-diazabicyclo[4 .3.0]nonene-5
  • DBU 1,8-diazabicyclo[5.4.0]undecene-7
  • DABCO 1,4-diazabicyclo”[2.2.2]octane
  • Organic bases carbonates such as sodium carbonate, potassium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, metal hydroxides such as sodium hydroxide, potassium hydroxide, calcium hydroxide, potassium hydride, sodium hydride, potassium, sodium , sodium amide, sodium methylate, sodium ethylate and other metal alcoholates.
  • the reaction is usually carried out at about -20 to 100°C, preferably about 0 to 50°C, and the reaction time is about 5 minutes to 10 hours, preferably about 5 minutes to 2 hours.
  • the reaction between the obtained mixed acid anhydride and compound (IA) is usually carried out at about ⁇ 20 to 150° C., preferably about 10 to 50° C., and the reaction time is about 5 minutes to 10 hours, preferably 5 minutes to 5 hours.
  • the mixed anhydride method is generally carried out in a solvent.
  • any solvent commonly used in the mixed acid anhydride method can be used as the solvent, and specific examples include halogenated hydrocarbons such as chloroform, dichloromethane, dichloroethane, carbon tetrachloride, benzene, toluene, and xylene.
  • halogenated hydrocarbons such as chloroform, dichloromethane, dichloroethane, carbon tetrachloride, benzene, toluene, and xylene.
  • aromatic hydrocarbons such as diethyl ether, diisopropyl ether, tetrahydrofuran, dimethoxyethane and other ethers, methyl acetate, ethyl acetate, isopropyl acetate and other esters, N,N-dimethylacetamide, N,N-dimethyl
  • aromatic hydrocarbons such as diethyl ether, diisopropyl ether, tetrahydrofuran, dimethoxyethane and other ethers, methyl acetate, ethyl acetate, isopropyl acetate and other esters, N,N-dimethylacetamide, N,N-dimethyl
  • aprotic polar solvents such as formamide, dimethylsulfoxide, hexamethylphosphoric acid triamide, and mixed solvents thereof.
  • alkyl halocarboxylic acids used in the mixed anhydride method include methyl chloroformate, methyl bromoformate, ethyl chloroformate, ethyl bromoformate, isobutyl chloroformate and the like.
  • the proportions of the carboxylic acid (IB), the alkylhalocarboxylic acid and the compound (IA) used in the method are usually equimolar. Each of them can be used in an equimolar to 1.5-fold molar amount range.
  • the condensation reaction in the presence of the activating agent, it is carried out in a suitable solvent in the presence or absence of a basic compound.
  • any of the solvents used in the method of reacting compound (IA) with carboxylic acid halide in other method (d) below can be used.
  • the amount of the activating agent used should be at least an equimolar amount, preferably an equimolar to 5-fold molar amount relative to compound (IA).
  • WSC is used as an activator, the addition of 1-hydroxybenzotriazole to the reaction system facilitates the reaction.
  • 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.
  • reaction is carried out in the presence of a basic compound in an appropriate solvent.
  • a wide range of known basic compounds can be used, and for example, any of the basic compounds used in the Schotten-Baumann reaction can be used.
  • solvents 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, methyl cellosolve, etc. Acetonitrile, pyridine, acetone, water and the like can be mentioned.
  • the ratio of the compound (IA) and the carboxylic acid halide to be used is not particularly limited and may be appropriately selected within a wide range. It is preferable to use about a molar amount.
  • 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.
  • the above amide bond forming reaction is performed by converting compound (IB) and compound (IA) into diphenylphosphinic chloride, phenyl-N-phenylphosphoramide chloride, diethylchlorophosphate, diethyl cyanophosphate, diphenylphosphate azide, bis (2-Oxo-3-oxazolidinyl)phosphinic chloride can also be carried out by a method of reacting in the presence of a condensing agent for a phosphorus compound.
  • the reaction is usually carried out at about -20 to 150°C, preferably about 0 to 100°C, in the presence of the solvent and basic compound used in the method of reacting compound (IA) with carboxylic acid halide.
  • the reaction is generally completed in about 5 minutes to 30 hours.
  • the condensing agent and compound (IB) are preferably used in at least equimolar amounts, preferably equimolar to 2-fold molar amounts, relative to compound (IA).
  • the compound produced by any of the above methods can be isolated and purified by known methods.
  • a nitrogen-containing compound different from the nitrogen-containing compound represented by the general formula (I) can bind to the mucolipin protein.
  • the nitrogen-containing compounds shown below are capable of blocking the flow of ions through ion channels composed of mucolipin proteins.
  • 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 : .
  • n represents 1 or 2.
  • n denotes 2.
  • R 21 , R 22 , R 23 and R 24 are the same or different, and are C1-3 straight or branched chain optionally having a hydrogen atom or a halogen atom as a substituent. Indicates an alkyl group.
  • Examples of C1-3 linear alkyl groups for R 21 , R 22 , R 23 and R 24 include methyl group, ethyl group and n-propyl group.
  • An isopropyl group can be exemplified as the C3 branched alkyl group. More preferably, R 21 , R 22 and R 24 are C1-3 linear alkyl groups and R 23 is a C3 branched alkyl group.
  • a halogen atom as a substituent can be exemplified by a fluorine atom, a chlorine atom, a bromine atom, an iodine atom and the like.
  • the nitrogen-containing compound represented by general formula (II) can be produced, isolated and purified by known methods.
  • the nitrogen-containing compound represented by general formula (II) is preferably a nitrogen-containing compound represented by the following formula (21): .
  • SR33805 is more preferred.
  • the pharmaceutically acceptable salt of the nitrogen-containing compound represented by general formula (II) above is not particularly limited.
  • Such salts include, for example, inorganic acid addition salts such as hydrochlorides, hydrobromides, sulfates, nitrates; and acetates, tartrates, maleates, succinates, citrates, methanesulfonates. , malate, oxalate, benzenesulfonate and other organic acid addition salts.
  • These salts can be produced, for example, by treating the nitrogen-containing compound represented by the general formula (II) with an acid.
  • the pharmaceutically acceptable salt of the nitrogen-containing compound represented by general formula (II) is preferably oxalate.
  • the oxalate salt of SR33805 is most preferred for this embodiment.
  • 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 : .
  • R 31 and R 32 are the same or different and represent a halogen atom.
  • a halogen atom can be exemplified by a fluorine atom, a chlorine atom, a bromine atom, an iodine atom and the like. Both halogen atoms are preferably fluorine atoms.
  • the nitrogen-containing compound represented by general formula (III) can be produced, isolated and purified by known methods.
  • the general formula (III) is preferably a nitrogen-containing compound represented by the following formula (31): .
  • the pharmaceutically acceptable salt of the nitrogen-containing compound represented by general formula (III) is not particularly limited.
  • Such salts include, for example, inorganic acid addition salts such as hydrochlorides, hydrobromides, sulfates, nitrates; and acetates, tartrates, maleates, succinates, citrates, methanesulfonates. , malate, oxalate, benzenesulfonate and other organic acid addition salts.
  • These salts can be produced, for example, by treating the nitrogen-containing compound represented by the general formula (III) with an acid.
  • the pharmaceutically acceptable salt of the nitrogen-containing compound represented by general formula (III) is preferably hydrochloride. Flunarizine dihydrochloride is most preferred for this embodiment.
  • 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 : .
  • n represents an integer of 1-3.
  • n denotes 2 or 3. More preferably, n represents 3.
  • R 41 and R 42 are the same or different and represent a halogen atom.
  • a halogen atom can be exemplified by a fluorine atom, a chlorine atom, a bromine atom, an iodine atom and the like. Both halogen atoms are preferably fluorine atoms.
  • R 43 represents a C1-3 linear or branched alkyl group optionally having a hydrogen atom or a halogen atom as a substituent.
  • the C1-3 linear alkyl group for R 43 can be exemplified by, for example, a methyl group, an ethyl group, or an n-propyl group.
  • An isopropyl group can be exemplified as the C3 branched alkyl group.
  • a halogen atom as a substituent can be exemplified by a fluorine atom, a chlorine atom, a bromine atom, an iodine atom and the like.
  • the nitrogen-containing compound represented by general formula (IV) can be produced, isolated and purified by known methods.
  • the nitrogen-containing compound represented by general formula (IV) is preferably a nitrogen-containing compound represented by the following formula (41): .
  • the pharmaceutically acceptable salt of the nitrogen-containing compound represented by general formula (IV) above is not particularly limited.
  • Such salts include, for example, inorganic acid addition salts such as hydrochlorides, hydrobromides, sulfates, nitrates; and acetates, tartrates, maleates, succinates, citrates, methanesulfonates. , malate, oxalate, benzenesulfonate and other organic acid addition salts.
  • These salts can be produced, for example, by treating the nitrogen-containing compound represented by the general formula (IV) with an acid.
  • the pharmaceutically acceptable salt of the nitrogen-containing compound represented by general formula (IV) is preferably hydrochloride.
  • the dihydrochloride salt of lomerizine is most preferred for this embodiment.
  • 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 : .
  • n represents 1 or 2.
  • n denotes 2.
  • R 51 , R 52 and R 53 are the same or different and each a C1-3 linear or branched alkyl group optionally having a hydrogen atom or a halogen atom as a substituent. show.
  • Examples of the C1-3 linear alkyl group for R 51 , R 52 and R 53 include methyl group, ethyl group and n-propyl group.
  • An isopropyl group can be exemplified as the C3 branched alkyl group.
  • R 51 and R 52 are linear alkyl groups and R 53 is a branched alkyl group.
  • a halogen atom as a substituent can be exemplified by a fluorine atom, a chlorine atom, a bromine atom, an iodine atom and the like.
  • the nitrogen-containing compound represented by general formula (V) can be produced, isolated and purified by known methods.
  • the nitrogen-containing compound represented by the general formula (V) is preferably a nitrogen-containing compound represented by the following formula (51): .
  • the pharmaceutically acceptable salt of the nitrogen-containing compound represented by formula (V) above is not particularly limited.
  • Such salts include, for example, inorganic acid addition salts such as hydrochlorides, hydrobromides, sulfates, nitrates; and acetates, tartrates, maleates, succinates, citrates, methanesulfonates. , malate, oxalate, benzenesulfonate and other organic acid addition salts.
  • These salts can be produced, for example, by treating the nitrogen-containing compound represented by the general formula (V) with an acid.
  • a preferred pharmaceutically acceptable salt of the nitrogen-containing compound represented by formula (V) is hydrochloride.
  • composition This embodiment is the same as in 1. above. and 2.
  • a composition comprising a nitrogen-containing compound as described in .
  • the composition can be used to treat malignancies.
  • the composition can be used to prevent recurrence of malignant tumors.
  • the composition can be used to inhibit proliferation of tumor stem cells of malignant tumors.
  • the composition can prevent recurrence and metastasis of malignant tumors by suppressing proliferation of tumor stem cells of malignant tumors.
  • prevention includes preventing and/or delaying the onset or recurrence of tumors.
  • Treatment also includes shrinking and/or disappearing of developing tumors.
  • treatment may include treatment by administering the composition alone, as well as radiation therapy and surgical treatment in combination.
  • the maximum daily dosage of the composition is 0.001 to 10 mg per 1 kg of body weight in terms of the nitrogen-containing compound represented by general formula (I).
  • Administration of the composition may be carried out once a day at the above dosage, optionally divided into 2, 3, 4 or 5 times a day, preferably 2, 3, 4 or 5 times a day. Alternatively, it may be administered in 3 divided doses.
  • the composition can be prepared by combining the nitrogen-containing compound represented by the general formula (I) and a suitable carrier or additive for formulation.
  • a suitable carrier or additive for formulation various kinds commonly used for ordinary drugs depending on the dosage form of the composition, such as excipients, binders, disintegrants, lubricants, coloring agents, etc. agents, flavoring agents, flavoring agents, surfactants, and the like.
  • the dosage form is not particularly limited, but tablets, powders, granules, capsules (hard capsules and soft capsules) including), liquids, pills, suspensions, jelly formulations, and emulsions.
  • the above formulations or formulations are parenterally administered, injections, drops, suppositories, nasal drops, transpulmonary agents, and the like can be exemplified.
  • composition is an oral solid preparation such as tablets, powders, granules, pills, capsules, etc., carriers such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin , excipients such as crystalline cellulose, silicic acid, methylcellulose, glycerin, sodium alginate, gum arabic; simple syrup, grape sugar solution, starch solution, gelatin solution, polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, carboxymethylcellulose, shellac, methylcellulose , ethyl cellulose, water, ethanol, binders such as potassium phosphate; dried starch, sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, Disintegrants such as starch and lactose; Disintegration inhibitors such as star
  • Tablets here include oral tablets (plain tablets, sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double tablets, multi-layer tablets, etc.), chewable tablets (taken while chewing in the mouth). oral tablets (including lozenges that are dissolved in the oral cavity and then ingested), sublingual tablets, and buccal tablets.
  • composition is a solid preparation for oral administration of pills, excipients such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, kaolin, talc, etc.; gum arabic powder, tragacanth powder , a binder such as gelatin; a disintegrant such as laminaran and agar.
  • excipients such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, kaolin, talc, etc.
  • gum arabic powder, tragacanth powder a binder such as gelatin
  • a disintegrant such as laminaran and agar.
  • the capsule is prepared by mixing the active ingredient with the various carriers exemplified above and filling it into a hard capsule or a soft capsule. .
  • the composition when it is a liquid, it may be in a liquid form, and may be an aqueous or oily suspension, solution, syrup, elixir, or drink.
  • a liquid formulation is prepared according to a conventional method using a normal additive.
  • the container to be filled with the liquid agent is not limited as long as it can be sealed, and may be a glass container, an aluminum container, or a plastic container.
  • composition When the composition is an injection, diluents such as water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc.
  • diluents such as water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc.
  • pH adjusters such as sodium citrate, sodium acetate, sodium phosphate; buffers such as dipotassium phosphate, trisodium phosphate, sodium hydrogen phosphate, sodium citrate; sodium pyrosulfite, EDTA, thioglycolic acid, thio Stabilizers such as lactic acid; sugars such as mannitol, inositol, maltose, sucrose, lactose, and the like can be used as molding agents for freeze-drying. In this case, a sufficient amount of glucose or glycerin may be contained in the pharmaceutical preparation to adjust the isotonicity of the solution. Also good.
  • compositions By adding these carriers, subcutaneous, intramuscular and intravenous injections can be produced by conventional methods.
  • the composition when the composition is 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, and the like.
  • the detection method includes the step of 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 specimen 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 to predict tumor grade.
  • the detection of mucolipin protein or mucolipin mRNA in tumor cells is performed in vitro using specimens collected from the subject.
  • the specimen is not limited as long as it contains tumor cells.
  • Specimens may include body fluids, blood, which may contain tumor tissue or tumor cells.
  • the specimen can be collected, for example, by surgical excision or biopsy, or endoscopic excision or biopsy from a primary tumor or metastatic lesion.
  • Bodily fluids may include cerebrospinal fluid, ascites, pleural effusion, and the like.
  • Examples of blood include peripheral blood, venous blood, and arterial blood. Blood is preferably collected with an anticoagulant.
  • Whether the tissue is tumor tissue or normal tissue can be determined by macroscopic observation, microscopic observation, or the like. Alternatively, whether or not the tissue is a tumor tissue may be determined using the cell proliferation ability or expression of a tumor marker as an index.
  • tumor markers in the case of brain glioma, for example, deletion of chromosome 1p/19q, mutation of isocitrate dehydrogenase (IDH) 1 and 2, methylation of O(6)-MGMT promoter, defective expression of nuclear ATRX, CDKN2A homogeneous deletion can be mentioned.
  • the tissue to be examined is It can be determined that there is a high possibility that it is a tumor tissue.
  • a tumor marker is used as an index, it is possible to determine that a tissue positive for tumor marker expression is highly likely to be a tumor tissue.
  • whether a cell is a tumor cell or a normal cell can be determined using the above tumor markers, cell proliferation ability, and the like. Tumor cells or tumor tissue taken from a subject are pretreated depending on the method of detection of mucolipin protein or mucolipin mRNA.
  • Methods for detecting mucolipin proteins as proteins 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), microarray, and RNA-Seq.
  • cells expressing mucolipin protein can be detected with a flow cytometer after the mucolipin protein is fluorescently labeled by immunostaining.
  • cells expressing mucolipin protein can be detected with a flow cytometer after fluorescently labeling mucolipin mRNA with in situ hybridization.
  • the tumor tissue embedded in the block may be only the tumor portion, or may include, for example, a normal portion.
  • the cells are smeared or collected on a slide glass and fixed with formalin, paraformaldehyde, ethanol, or the like.
  • the mucolipin protein is immunostained, a positive signal appears on the cell surface, that is, on the cell membrane.
  • mucolipin protein When mucolipin protein is detected as a protein by Western blotting or the like, tumor cells or tumor tissues are lysed with a predetermined lysis buffer as a pretreatment. The sample lysed with the lysis buffer is used as the test sample. When the mucolipin protein is detected as a protein by flow cytometry or the like, the sample is hemolyzed with a predetermined buffer as a pretreatment.
  • RNA or mRNA is extracted from tumor cells or tumor tissues as a pretreatment.
  • reverse transcription may be performed using the extracted total RNA or mRNA as a template to synthesize complementary DNA (cDNA).
  • Total RNA, mRNA, or cDNA is used as the test sample.
  • the primary antibody for detecting mucolipin protein by immunostaining, western blotting, or flow cytometry is not limited as long as it can detect mucolipin protein. Examples thereof include anti-MCOLN1 antibody (HPA031763; Atlas Antibodies), anti-MCOLN2 antibody (HPA019114; Atlas Antibodies), anti-MCOLN3 antibody (HPA018106; Atlas Antibodies) and the like.
  • a primary antibody bound to a mucolipin protein can be detected by reaction between an enzyme-labeled secondary antibody that binds to the primary antibody, the enzyme, and a substrate.
  • tissue sections may be treated with a proteolytic enzyme such as trypsin before immunostaining.
  • Methods for making probes for use in in situ hybridization are well known. Alternatively, commercially available probes may be used.
  • RNA-Seq the number of reads of mucolipin mRNA can be obtained using a next-generation sequencer (eg, manufactured by Illumina).
  • mucolipin protein When mucolipin protein is detected by immunostaining or in situ hybridization, human observation of the immunostained or in situ hybridization tissue specimen using a microscope, slide scanner, etc., reveals mucolipin protein. Presence or absence can be detected. It can be determined (determined) that mucolipin protein has been detected when immunostaining or in situ hybridization signals are confirmed in the tumor cells in the sample. If even one tumor cell having mucolipin protein is detected in the sample, it may be determined that "mucolipin protein is detected" or "positive expression of mucolipin protein".
  • tumor cells having intracellular mucolipin protein are 1% or more, preferably 5% or more, More preferably, it may be determined that "mucolipin protein is detected" or "expression of mucolipin protein is positive” when it is present at 10% or more.
  • mucolipin protein When detecting mucolipin protein by Western blotting, flow cytometry, RT-PCT, RNA-Seq, when mucolipin protein is detected in a sample extracted from tumor cells or tumor tissue in the specimen, "muco It may be determined that lipin protein is detected" or that mucolipin protein expression is positive.
  • mucolipin protein in a sample containing tumor cells or tumor tissue 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 tissue, When the amount of mucolipin protein is higher than the amount of mucolipin protein derived from normal cells or normal tissues, "mucolipin protein is detected” or "expression of mucolipin protein is positive” may be determined.
  • the number of cells expressing mucolipin protein in a specimen containing tumor cells or tumor tissue is compared with the number of cells expressing mucolipin protein in a specimen derived from normal cells or tissues, and tumor cells or tumor tissue is identified.
  • Mucolipin protein was detected when the number of cells expressing mucolipin protein in the sample containing the sample shows a higher value than the number of cells expressing mucolipin protein in the sample derived from normal cells or normal tissues Alternatively, it may be determined that "expression of mucolipin protein is positive".
  • the amount of mucolipin mRNA derived from a specimen containing tumor cells or tumor tissue is compared with the amount of mucolipin mRNA in a specimen derived from normal cells or normal tissue, and the amount of mucolipin mRNA in the specimen containing tumor cells or tumor tissue is determined. , when it shows a higher value than the amount of mucolipin mRNA in a sample derived from normal cells or normal tissues, even if it is determined that "mucolipin protein is detected" or "expression of mucolipin protein is positive” good.
  • mucolipin protein when the amount of mucolipin protein in a sample containing tumor cells or tumor tissue is similar to the amount of mucolipin protein in a sample derived from normal cells or tissue, the term "mucolipin protein has not been detected or "negative expression of mucolipin protein".
  • the number of cells expressing mucolipin protein in a sample containing tumor cells or tumor tissue is about the same as the number of cells expressing mucolipin protein in a sample derived from normal cells or normal tissue, "Mucolipin protein is not detected” or "expression of mucolipin protein is negative”.
  • “showing a high value” can be exemplified by a case showing a value that is 1.2 times or more, preferably 1.5 times or more, more preferably 2 times or more, and still more preferably 5 times or more. "Same degree” can be exemplified from 0.8 times to less than 1.2 times.
  • the amount of protein or RNA in each test sample was determined by measuring the amount of protein derived from housekeeping genes such as GAPDH, ⁇ 2-microglobulin, and ⁇ -actin. Or you may normalize by the amount of mRNA.
  • the amount of protein may be represented by mass or concentration, but may also be represented by the luminescence intensity of the substrate or the like.
  • the amount of mRNA may be the copy number or read number of mRNA, but may also be expressed by fluorescence intensity or the like.
  • reference values are determined in advance for the measured value of the amount of mucolipin protein, the measured value of the number of cells expressing the mucolipin protein, or the measured value of the amount of mucolipin mRNA, and the tumor cells or tumor tissue
  • Each measured value in the specimen containing is compared with the reference value corresponding to each measured value, and when the measured value is higher than the reference value, "mucolipin protein was detected” or "expression of mucolipin protein is positive.”
  • each measurement value in a specimen containing tumor cells or tumor tissue is compared with a reference value corresponding to each measurement value, and if the measurement value is lower than the reference value, "no mucolipin protein is detected” or It may be determined that "expression of mucolipin protein is negative".
  • the reference value is not limited as long as it is a value that can determine whether mucolipin protein, cells expressing mucolipin protein, or mucolipin mRNA is detected or positively expressed, and can be determined by a known method. can be done.
  • a value that can determine whether mucolipin protein, cells expressing mucolipin protein, or mucolipin mRNA is detected or positively expressed is ROC (receiver operating characteristic curve) curve, discriminant analysis method, modal method, It can also be determined by the Kittler method, the 3 ⁇ method, the p-tile method, or the like.
  • Examples of reference values include sensitivity, specificity, negative predictive value, positive predictive value, first quartile, and the like.
  • the method for detecting predictive markers may further comprise determining that the tumor is of low malignancy if mucolipin protein is not detected. Alternatively, the method may comprise determining that the tumor is aggressive if mucolipin protein is detected.
  • the method for detecting a predictive marker may include the step of presenting a label indicating that the tumor of the subject is of low malignancy when no mucolipin protein is detected in the subject.
  • the method for detecting a predictive marker may also include the step of presenting a label indicating that the tumor in the subject is highly malignant when the mucolipin protein is detected in the subject.
  • the detection method of the predictive marker is that when the amount of mucolipin protein in the sample, the number of cells expressing mucolipin protein, or the amount of mucolipin mRNA is below the reference value, the malignancy of the tumor in the subject is low. may include the step of presenting a label indicating the In addition, the detection method of the predictive marker is such that when the amount of mucolipin protein in the sample, the number of cells expressing mucolipin protein, or the amount of mucolipin mRNA exceeds a reference value, the degree of malignancy of the tumor possessed by the subject is A step of presenting a label indicating high may be included.
  • a label indicating that the tumor of the subject has a low degree of malignancy
  • a label indicating that the subject has a good prognosis or a high survival rate may be presented.
  • a label indicating that the subject has a highly malignant tumor a label indicating that the subject has a poor prognosis or a low survival rate may be presented.
  • Survival rate indicates Overall survival rate or Disease/Progression-free survival rate. Preferred is the 5-year survival rate.
  • Test Reagent 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-described tumor.
  • the test reagents may include mucolipin protein detection reagents and/or mucolipin mRNA detection reagents.
  • a mucolipin protein detection reagent comprises one or more antibodies (eg, primary antibodies) capable of binding to at least a portion of the mucolipin protein.
  • antibodies eg, primary antibodies
  • Any of polyclonal antibodies, monoclonal antibodies, and fragments thereof eg, Fab, F(ab'), F(ab)2, etc.
  • the immunoglobulin class and subclass are not particularly limited.
  • the antibody may be one screened from an antibody library, or may be a chimeric antibody, scFv, or the like.
  • the antibody does not necessarily have to be purified, and may be antibody-containing antiserum, ascitic fluid, an immunoglobulin fraction fractionated therefrom, or the like.
  • test reagents include stabilizers such as ⁇ -mercaptoethanol and DTT; protective agents such as albumin; surfactants such as polyoxyethylene (20) sorbitan monolaurate and polyoxyethylene (10) octylphenyl ether; At least one preservative such as sodium azide may be included.
  • Antibodies that bind to mucolipin proteins may be labeled with enzymes or fluorescent dyes. Antibodies that bind to mucolipin protein may be immobilized on microplates, magnetic beads, and the like.
  • the mucolipin protein detection reagent may be provided as a test kit that includes a test reagent and a package insert that describes how to use the reagent or describes the URL of a web page that describes how to use the reagent.
  • the test kit may contain a secondary antibody labeled with an enzyme or a fluorescent dye.
  • the test kit may contain a substrate that reacts with the enzyme.
  • the mucolipin mRNA detection reagent contains a nucleic acid that hybridizes with all or part of the mucolipin mRNA or the cDNA of the mucolipin mRNA.
  • the nucleic acid is preferably a detection nucleic acid (DNA or RNA) that functions as a primer and/or probe.
  • the length of the nucleic acid for detection is not particularly limited.
  • the sequence that hybridizes with mucolipin mRNA or cDNA of mucolipin mRNA is preferably 50 mer or less, more preferably 30 mer or less, and still more preferably , about 15 to 25 mer.
  • the primer may contain a sequence that does not hybridize to the cDNA of the mucolipin or mucolipintan mRNA.
  • the primer may be labeled with a fluorescent dye or the like.
  • RT-PCR can also use quantification probes that are degraded during the PCR reaction and are 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 about 5 to 20 mers containing a sequence that hybridizes with mucolipin mRNA or cDNA of mucolipin mRNA.
  • it is preferable that one end of the quantification probe is labeled with a fluorescent dye, and the other end of the quantification probe is labeled with a quencher of the fluorescent dye.
  • the sequence hybridizing with mucolipin mRNA or cDNA of mucolipin mRNA is preferably about 100 mer, more preferably about 60 mer, More preferably, it is about 20-30 mer.
  • the capture probe may contain a sequence that does not hybridize to the mucolipin mRNA or the cDNA of the mucolipin mRNA.
  • the capture probe is preferably immobilized on a chip.
  • the nucleic acid for detection when the nucleic acid for detection is a probe for in situ hybridization, the nucleic acid for detection may be an oligonucleotide having a sequence that hybridizes with mucolipin mRNA of about 15 to 100 mers, or a polynucleotide exceeding 100 mers. may be A polynucleotide may be DNA or RNA.
  • a probe for in situ hybridization can be bound with a labeling substance such as digoxigenin or fluorescent dye. Probes may contain sequences that do not hybridize to mucolipin mRNA.
  • the mucolipin mRNA detection reagent may be provided as a test kit that includes a test reagent and a package insert that describes how to use the reagent or describes the URL of a web page that describes how to use the reagent.
  • the test kit contains nucleic acid amplification reagents (including polymerase, buffer, dNTP, etc. even for heat-resistant DNA), reverse transcriptase, etc. You can stay. Nucleic acid amplification reagents may contain dyes such as SYBER GREEN (registered trademark) as necessary.
  • the test kit may contain a hybridization buffer, a washing buffer and the like.
  • the test kit may contain a protease such as proteinase K, a hybridization buffer, a washing buffer, and the like.
  • One embodiment of the present disclosure is a prediction system 1000 for predicting malignancy of a tumor (hereinafter abbreviated as "prediction system 1000") and a prediction device 10 for predicting malignancy of a tumor (hereinafter, “ abbreviated as “prediction device 10").
  • prediction system 1000 for predicting malignancy of a tumor
  • prediction device 10 for predicting malignancy of a tumor
  • "predicting the malignancy of a tumor” can be replaced with predicting the subject's prognosis and/or predicting the subject's survival rate.
  • FIG. 1 is a general view of a prediction system 1000, and as one aspect, the prediction system 1000 may include an analysis device 5a or an analysis device 5b in addition to the prediction device 10.
  • FIG. FIG. 2 shows a block diagram of the prediction device 10. As shown in FIG. Prediction device 10 may be connected to input unit 111 , output unit 112 , and storage medium 113 .
  • a processing unit 101 In the prediction device 10, a processing unit 101, a main storage unit 102, a ROM (read only memory) 103, an auxiliary storage unit 104, a communication interface (I/F) 105, an input interface (I/F) 106 and , an output interface (I/F) 107 and a media interface (I/F) 108 are connected to each other by a bus 109 so as to be capable of data communication.
  • the combination of the main memory unit 102 and the auxiliary memory unit 104 may be simply called a memory unit.
  • the storage unit stores measured values, reference values, and the like in a volatile or nonvolatile manner.
  • the processing unit 101 is the CPU of the prediction device 10 .
  • the processing unit 101 may cooperate with a GPU.
  • the prediction device 10 functions when the processing unit 101 executes the operation system 104a and the prediction program 104b stored in the auxiliary storage unit 104 and processes the acquired data.
  • the ROM 103 is composed of mask ROM, PROM, EPROM, EEPROM, etc., and stores computer programs executed by the processing unit 101 and data used for the programs.
  • 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 activated, and programs and settings related to hardware operations of the prediction device 10 .
  • the main storage unit 102 is composed of a RAM (random access memory) such as SRAM or DRAM.
  • the main storage unit 102 is used for reading computer programs stored in the ROM 103 and auxiliary storage unit 104 . Further, the main storage unit 102 is used as a working area when the processing unit 101 executes these computer programs.
  • the auxiliary storage unit 104 is composed of a hard disk, a semiconductor memory device such as a flash memory, an optical disc, or the like.
  • the auxiliary storage unit 104 stores an operating system 104a, a later-described prediction program 104b, 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.
  • the communication I/F 105 includes serial interfaces such as USB, IEEE1394 and RS-232C, parallel interfaces such as SCSI, IDE and IEEE1284, analog interfaces such as D/A converters and A/D converters, network interface controllers ( Network interface controller (NIC), etc.
  • the communication I/F 105 receives data from the analysis devices 5a and 5b or other external devices, and transmits information stored or generated by the prediction device 10 as necessary to the analysis devices 5a and 5b. 5b or transmit or display to the outside.
  • the communication I/F 105 may communicate with the analysis devices 5a, 5b or other external devices via a network.
  • the input I/F 106 includes serial interfaces such as USB, IEEE1394, and RS-232C, parallel interfaces such as SCSI, IDE, and IEEE1284, and analog interfaces such as a D/A converter and an A/D converter. be.
  • the input I/F 106 receives character input, click, voice input, and the like from the input unit 111 .
  • the accepted input content is stored in the main storage unit 102 or the auxiliary storage unit 104 .
  • the input unit 111 is composed of a touch panel, keyboard, mouse, pen tablet, microphone, etc., and performs character input or voice input to the prediction device 10 .
  • the input unit 111 may be connected from the outside of the prediction device 10 or integrated with the prediction device 10 .
  • the output I/F 107 is composed of an interface similar to the input I/F 106, for example.
  • 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 .
  • the output unit 112 is composed of, for example, a display, a printer, etc., and displays measurement results transmitted from the analysis devices 5a and 5b, various operation windows in the prediction device 10, analysis results, and the like.
  • the media I/F 108 reads, for example, application software stored in the storage medium 113 .
  • the read application software and the like are stored in the main storage unit 102 or the auxiliary storage 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 storage unit 104 to the storage medium 113 .
  • the storage medium 113 is composed of a flexible disk, CD-ROM, DVD-ROM, or the like.
  • a storage medium 113 is connected to the media I/F 108 by a flexible disk drive, CD-ROM drive, DVD-ROM drive, or the like.
  • the storage medium 113 may store application programs and the like for the computer to execute operations.
  • the processing unit 101 may acquire the prediction program 104b and various settings necessary for controlling the prediction device 10 via a network.
  • the application program is stored in an auxiliary storage unit of a server computer on the network, and the prediction device 10 accesses this server computer, downloads the computer program, and stores it in the ROM 103 or the auxiliary storage unit 104. It is possible.
  • the prediction device 10 may be a personal computer or the like.
  • the prediction system 1000 does not have to be installed in one place, and the prediction device 10 and the analysis devices 5a and 5b may be placed in different places and connected by a network. Further, the prediction device 10 may be a device that does not require an operator and does not include the input unit 111 and the output unit 112 .
  • the analysis device 5a is a device for measuring the amount or concentration of protein, and includes a sample storage space 51, a reaction section 52, and a detection section 53.
  • the cell lysate set in the sample storage area 51 is dispensed and incubated in a microplate on which an antigen-capturing antibody is immobilized, which is set in the reaction section 52 .
  • the detection antibody is dispensed into the microplate and incubated.
  • a substrate for detecting the detection antibody is dispensed into a microplate, the microplate is moved to the detection unit 53, and the signal generated by the reaction of the substrate is measured. .
  • another aspect of the analysis device 5a is a device for measuring the expression level of mRNA by microarray analysis. It is dispensed onto the chip, hybridized, washed, and then moved to the detection section 53 to detect the signal.
  • another aspect of the analysis device 5a is a device for measuring the expression level of mRNA by RT-PCR. Dispense into microtubes followed by quantitative PCR reagents into microtubes. While the PCR reaction is performed in the reaction section 52, the detection section 53 detects the signal in the tube.
  • the analysis device 5b is a device for measuring the expression level of mRNA by the RNA-Seq method, and includes a sequence analysis section 54a.
  • a sample subjected to RNA-Seq reaction is set in the sequence analysis unit 54, and the base sequence is analyzed in the sequence analysis unit 54a.
  • the analysis device 5b is a fully automatic western blotting device for measuring the amount of protein by western blotting, and includes a chemiluminescence signal detection section 54b.
  • a tumor cell or tumor tissue sample lysed with a lysis buffer is set at a predetermined position of an automatic western blotting device, subjected to SDS-PAGE, blotting to a membrane, antibody reaction, and chemiluminescence, and is sent to the chemiluminescence signal detection unit 54b. analysis and quantification of signal intensity.
  • the analysis 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 the cells equipped with a flow cell A detection unit 54c is provided.
  • the mucolipin protein present in the cells is fluorescently labeled by immunostaining, and the fluorescence intensity of the cells is measured in the cell detection section 54c.
  • 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 protein measurement value or the mRNA measurement value, and transmits it to the prediction device 10 as digital data.
  • the analyzer 5b A/D-converts the measured value of mRNA and transmits it to the prediction device 10 as digital data.
  • the prediction device 10 can acquire the measured value of protein or the measured value of mRNA as digital data that can be processed.
  • FIG. 3 shows an example of a flowchart of processing executed by the prediction program 104b.
  • the processing unit 101 of the prediction device 10 starts processing for predicting the degree of malignancy of the tumor when the operator inputs a processing start request from the input unit 111 .
  • step S11 the processing unit 101 acquires at least one selected from mucolipin protein amount and mucolipin mRNA present in tumor cells in the specimen collected from the subject, which is input by the operator through the input unit 111. .
  • at least one measurement value selected from mucolipin protein amount and mucolipin mRNA present in tumor cells in a specimen collected from a subject is obtained from the analyzer 5a or 5b.
  • the operator inputs from 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 may obtain this input data as measurements.
  • step S12 the processing unit 101 acquires the reference value corresponding to the measured value acquired in step S11 from the storage unit. The measured values are then compared with corresponding reference values.
  • step S13 when the acquired measurement value is less than the reference value, the processing unit 101 proceeds to step S14 (YES), determines that the malignancy of the tumor possessed by the subject is low, and outputs the determination result.
  • the indicated label is output to the output unit 112 (step S16).
  • step S13 when the acquired measurement value is equal to or greater than the reference value, the process proceeds to step S15 (NO), it is determined that the tumor of the subject is highly malignant, and a label indicating the determination result is is output to the output unit 112 (step S16).
  • the label may be a mark such as a cross, a circle, an exclamation point, or the like.
  • certain embodiments of the present invention relate to program products, such as storage media, storing 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 disc.
  • the storage format of the program in the storage medium is not limited as long as the prediction device 10 can read the program.
  • the storage in the storage medium is preferably non-volatile.
  • Cancer stem cell-like cells As cancer stem cell-like cells, cancer stem cell-like cells established from high-grade glioma (4 types: MD13, Me83, 1123, 30R) [Reference: Neuro Oncol 22(3): 333 44, 2020; Cancer Cell 24(3): 331-46, 2013] and lung squamous cell carcinoma-derived cancer stem cell-like cells (2 types: L1, L14). Metastatic brain tumor-derived cancer stem cell-like cells (2 types: B34, B67) were used.
  • ⁇ Method for establishing cancer stem cell-like cells The surgically resected cancer tissue (0.1-1 g) was minced with scissors. The minced tissue was transferred to a test tube containing 2 mL of a cell detachment solution (Accumax (TM); Nacalai Tesque), and shaken (20 times/min) for 5 minutes in a constant temperature bath at 37°C. 8 mL of cell culture medium was added, mixed, and centrifuged (40 xg, 5 minutes). The supernatant was discarded, 10 mL of cell culture medium was added, mixed, and cultured on an ultra-low adhesion surface dish (100 mm; Corning).
  • a cell detachment solution Acceler TM
  • Nacalai Tesque Nacalai Tesque
  • the cell culture medium was D-MEM/Ham's F-12 (Wako Pure Chemical Industries), NaHCO 3 (49 mM), glucose (26 mM), L-glutamine (3 mM), MACS NeuroBrew-21 (5 mL; Biotec), epidermal growth factor (EGF, 20 ng/mL; PeproTech), fibroblast growth factor (bFGF, 20 ng/mL; PeproTech), and penicillin (100 U/mL) and streptomycin (0.1 mg/mL). added was used.
  • a patch-clamp method was used to measure current passing through mucolipin.
  • a patch electrode was prepared from a glass tube (G-1.5, Narishige Scientific Instruments Laboratory) using a micropipette preparation device (PP-83, Narishige Scientific Instruments Laboratory).
  • a patch electrode with an electrical resistance of 3-7 M ⁇ when filled with the electrode internal solution was used.
  • Electrode internal solution is 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), glycol ether diamine tetraacetic acid (10 mM), ethylenediaminetetraacetic acid disodium salt (0.5 mM), and adenosine triphosphate disodium salt (2 mM) adjusted to pH 7.4 with CsOH.
  • the cover glass was attached to the chamber on the inverted microscope (Olympus).
  • the experimental perfusate used was NaCl (150 mM) and HEPES (5 mM) adjusted to pH 7.4 with NaOH. Experiments were performed at 23-30°C.
  • An EPC 800 patch clamp amplifier (HEKA Elektronik) was used for current measurements.
  • a data acquisition device Digidata 1322A; Axon Instruments
  • software clampex 9; Axon Instruments
  • Mucolipin Expression and Survival Rate Mucolipin protein was expressed in the histopathological specimens in 4 of the 5 cases of glioblastoma, which is a high-grade glioma (Fig. 4). This suggests that mucolipin expression is characteristic of glioblastoma.
  • survival curves are shown in FIG. In FIG. 5, A and B show the relationship between mucolipin MCOLN1 expression and patient survival, C and D show the relationship between mucolipin MCOLN2 expression and patient survival, and E and F show the relationship between mucolipin MCOLN3 expression and patient survival.
  • Figure 2 shows the relationship between expression and patient survival.
  • A, C, and E indicate the Overall survival rate
  • B, D, and F indicate the Disease/Progression (D/P)-free survival rate.
  • FIG. 6 shows the cytostatic activity and blood-brain barrier permeability of each compound.
  • Cells used were glioblastoma cells (MD13, 30R), metastatic brain tumor-derived cancer stem-like cells (B34), and lung squamous cell carcinoma-derived cancer stem-like cells (L1).
  • SR33805 which had the strongest cytostatic activity, and lomerizine and flunarizine, which are calcium channel blockers known to have blood-brain barrier permeability, were selected.
  • Mucolipin protein (MCOLN1; NG_015806, PDB: 5WJ9) was searched for drug-binding regions using the Molsite method (Fukunishi Y and Nakamura H, Protein Sci, 2011, PMID: 21064162). After that, the 3D structures of lomerizine (PubChem ID: 122125), flunarizine (PubChem ID: 941361), and SR33805 (PubChem ID: 129426) were acquired from PubChem and used with myPresto software (https://www.mypresto5.jp).
  • the compound represented by the formula (1) is a known compound represented by the following formula (1A) (6-(pyrrolidin-3-yloxy)pyridin-3-amine) and a known compound represented by the following formula (1B) (6-isopropoxypicolinic acid) was generated by an amide bond.
  • FIG. 8 shows the results of LCMS.
  • 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).
  • the nitrogen-containing compound represented by formula (1) may be referred to as "KMU3".
  • FIG. 10 shows the cell growth inhibitory activity against glioblastoma cells when the nitrogen-containing compounds of KMU3, KMU5, KMU12, KMU13, KMU23, KMU7, and KMU9 were added to the medium at a final concentration of 1 ⁇ M (FIG. 10A). , relative growth rate (% of control) to control cells (cells not treated with test drug) (shown in FIG. 10B).
  • FIG. 10A shows the cell growth inhibitory activity against glioblastoma cells when the nitrogen-containing compounds of KMU3, KMU5, KMU12, KMU13, KMU23, KMU7, and KMU9 were added to the medium at a final concentration of 1 ⁇ M.
  • the nitrogen-containing compound having the structure represented by the above general formula (I) is important for suppressing growth of glioblastoma cells.
  • Figure 11 shows the concentration-dependent cytostatic activity of KMU3, SR33805, and temozolomide.
  • Filled circles indicate KMU3, open circles indicate SR33805, and crosses indicate temozolomide.
  • MD13 cells as glioblastoma cells
  • each drug was added to the medium at 10 nM, 100 nM, 1 ⁇ M, and 10 ⁇ M, and the viability of the cells was measured 4 days later.
  • KMU3 showed a cell growth inhibitory activity comparable to that of SR33805.
  • FIG. 13A shows the results of immunostaining with an anti-MCOLN3 antibody
  • FIG. 13B shows the results of an antibody absorption test in which an antigen peptide was reacted with an anti-MCOLN3 antibody.
  • a positive signal was detected in FIG. 13A.
  • the positive signal in FIG. 13A was considered to be a signal specific to mucolipin.
  • Immunostaining with an anti-MCOLN1 antibody also gave similar results.
  • the mucolipin blocker also exerts a cytostatic effect on lung cancer stem cells.
  • Cancer stem-like cells (MD13, 100,000 cells) established from a high-grade glioma were injected over 5 minutes at a depth of 3 mm, 2 mm to the right and 1 mm anterior to bregma. After the injection was completed, the wound was closed with 5-0 nylon. From the 7th day after transplantation, using a sonde (FTP-20-38-50; Instech Laboratories), the test drugs, KMU3, temozolomide [Temodar (registered trademark), MSD Corporation], Lomerizine dihydrochloride (Alomone Labs), KMU84 represented by the following formula was orally administered for 5 consecutive days, and efficacy was evaluated by overall survival (Kaplan-Meier curve).
  • KMU3 and KMU84 were adjusted to 0.5 w/v% methylcellulose 400 solution (Fuji Film Wako Pure Chemical Industries, Ltd.) so as to be 5 mg per 1 kg of body weight, and administered once a day. This dose is the optimal dose of 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) used as the solvent for the test drug was administered to the negative controls to which the test drug was not administered.
  • FIG. A shows the comparison of the overall survival between the KMU3 administration group and the negative control group.
  • B shows the overall survival of the Lomerizine dihydrochloride-administered group, the temozolomide-administered group, and the negative control group.

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Abstract

La présente invention aborde le problème de la fourniture d'un composé contenant de l'azote efficace pour inhiber la prolifération des cellules souches cancéreuses.　La solution selon l'invention concerne un composé contenant de l'azote représenté par la formule générale (I) ou un sel pharmaceutiquement acceptable de celui-ci (dans la formule : m, n et p sont identiques ou différents et représentent un nombre entier de 1 à 3 ; R1, R2 et R3 sont identiques ou différents et représentent un atome d'hydrogène, un groupe alkyle linéaire ou ramifié en C1-6 ayant éventuellement un atome d'halogène en tant que substituant, un groupe alcoxy linéaire ou ramifié en C1-6, un atome d'halogène, un groupe amino, un groupe hydroxyle, un groupe nitro, un groupe carboxy ou un groupe cyano ; et R4 représente un groupe alkyle linéaire ou ramifié en C1-6 ayant éventuellement un atome d'halogène en tant que substituant).
PCT/JP2022/014962 2021-03-29 2022-03-28 Composé contenant de l'azote, composition comprenant ledit composé contenant de l'azote et marqueur pour la prédiction du degré d'une tumeur WO2022210524A1 (fr)

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WO2024075736A1 (fr) * 2022-10-05 2024-04-11 学校法人関西医科大学 Composé azoté, composition comprenant ledit composé azoté et marqueur pour la prédiction du degré d'une tumeur

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024075736A1 (fr) * 2022-10-05 2024-04-11 学校法人関西医科大学 Composé azoté, composition comprenant ledit composé azoté et marqueur pour la prédiction du degré d'une tumeur

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