WO2022264971A1 - Procédé d'évaluation de la qualité du sperme - Google Patents

Procédé d'évaluation de la qualité du sperme Download PDF

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WO2022264971A1
WO2022264971A1 PCT/JP2022/023655 JP2022023655W WO2022264971A1 WO 2022264971 A1 WO2022264971 A1 WO 2022264971A1 JP 2022023655 W JP2022023655 W JP 2022023655W WO 2022264971 A1 WO2022264971 A1 WO 2022264971A1
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optionally substituted
group
compound
sperm
nitroxyl
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PCT/JP2022/023655
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English (en)
Japanese (ja)
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文紀 兵藤
政之 松尾
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国立大学法人東海国立大学機構
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Priority to JP2023529863A priority Critical patent/JPWO2022264971A1/ja
Publication of WO2022264971A1 publication Critical patent/WO2022264971A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/46—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with hetero atoms directly attached to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/92—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with a hetero atom directly attached to the ring nitrogen atom
    • C07D211/94—Oxygen atom, e.g. piperidine N-oxide
    • 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
    • C07D401/02—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 two hetero rings
    • C07D401/04—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 two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present disclosure relates to methods of evaluating sperm quality, compounds and kits therefor, and methods of testing or diagnosing male infertility.
  • Male sperm abnormalities are thought to be one of the causes of infertility, and in general, it is necessary to conduct tests to determine whether sperm are normal or not at a specialized medical institution.
  • the present disclosure provides a method of assessing sperm quality.
  • the present disclosure also provides methods of testing or diagnosing male infertility through such evaluation methods.
  • the present disclosure provides: (Item 1) A method of assessing sperm quality comprising: a step of mixing a population of spermatozoa with a test solution containing a nitrogen radical compound or an oxygen radical compound or a salt thereof to obtain a mixed solution; measuring a signal obtained from the mixture; and assessing sperm quality based on said signal.
  • (Item 1C) The method according to any one of the preceding items, wherein the nitrogen radical compound is 2,2-diphenyl-1-picrylhydrazyl or 2,2-di(alkyl-substituted phenyl)-1-picrylhydrazyl. .
  • (Item 2) A method of assessing sperm quality comprising: a step of mixing a population of spermatozoa with a test solution containing a nitroxyl radical compound or a salt thereof to obtain a mixed solution; measuring a signal obtained from the mixture; and assessing sperm quality based on said signal.
  • the nitroxyl radical compound is a 5- or 6-membered cyclic nitroxyl radical compound.
  • the nitroxyl radical compound has the following formula wherein Z is -C(R 6A )(R 6B )- or -N(R 6C )-, E is a bond, -C(R 10A )(R 10B )- or -N(R 10C )-; R 1 , R 2 , R 3 and R 4 are each independently hydrogen, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted cycloalkyl group, optionally substituted heterocycloalkyl group, is selected from the group consisting of an optionally substituted aryl group and an optionally substituted heteroaryl group, or R 1 and R 2 taken together are carbocyclic with the carbon atom to which
  • the nitroxyl radical compound has the following formula 5-membered cyclic nitroxyl radical compound represented by or the following formula wherein R 1 , R 2 , R 3 , R 4 , R 5A , R 5B , R 6A , R 6B , R 10A and R 10B are A method according to any one of the preceding items, each independently as described in any one of the preceding items.
  • the five-membered cyclic nitroxyl radical compound has the following formula wherein the 6-membered cyclic nitroxyl radical compound is represented by the following formula A method according to any one of the preceding items, represented by: (Item 6) A method according to any one of the preceding items, wherein R 1 , R 2 , R 3 and R 4 are each independently an optionally substituted alkyl group. (Item 6A) A method according to any one of the preceding items, wherein R 1 , R 2 , R 3 and R 4 are each independently an alkyl group. (Item 6B) A method according to any one of the preceding items, wherein R 1 , R 2 , R 3 and R 4 are methyl groups.
  • R 5A is a cyano group, an isothiocyano group, an optionally substituted alkyl group, an optionally substituted hydroxy group, an optionally substituted carboxyl group, an optionally substituted carbamoyl group or an optionally substituted A method according to any one of the preceding items, which is a good amino group.
  • the 5-membered cyclic nitroxyl radical compound is 3-(carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl free radical, 3-carbamoyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl free radical, 3-cyano-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl free radical, 3-[2-(2-iodoacetamido)acetamido]-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl free radical, 3-(2-isothiocyanatoethylcarbamoyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl free radical, 3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl free radical, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyloxy
  • the 6-membered cyclic nitroxyl radical compound is 4-acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-(2-chloroacetamido)-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-cyano-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-isothiocyanato-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-methacryloyloxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 2,2,6,6-tetramethyl-4-(propargyloxy)piperidine 1-oxyl free radical, 4-trimethylammonium-2,2,6,6-tetramethylpiperidine 1-oxyl free radical 4-oxo-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-carboxy-2,2, 6,6-tetramethylpiperidine 1-oxyl free radical, 4-hydroxy-2,
  • the nitroxyl compound is 3-[2-(2-iodoacetamido)acetamido]-2,2,5,5-tetramethyl-1-pyrrolidinyloxy free radical or 3-carbamoyl-2,2,5,5 -tetramethyl-1-pyrrolidinyloxy free radical.
  • the test solution further comprises a reducing agent.
  • the reducing agent comprises NADH or GSH.
  • the sperm quality comprises sperm motility.
  • (Item 12) A method according to any one of the preceding items, wherein the signal comprises a signal detected from the mixture by dynamic nuclear polarization (DNP) or electron spin resonance (ESR) methods.
  • DNP dynamic nuclear polarization
  • ESR electron spin resonance
  • (Item 13) A method according to any one of the preceding items, wherein the step of assessing sperm quality is performed by comparing the signal or a value derived therefrom with a reference value for assessing sperm quality.
  • the step of evaluating the quality of the sperm is performed by comparing the number and/or motility of the sperm calculated from the signal with a reference value for evaluating the quality of the sperm. or the method described in paragraph 1.
  • the step of evaluating the quality of the sperm comprises evaluating based on the mitochondrial metabolism or energy production of the sperm, wherein a decrease in the mitochondrial metabolism or the energy production is associated with a decrease in motility and/or function of the sperm.
  • a method according to any one of the preceding items, which means The step of evaluating the quality of the sperm comprises evaluating based on the mitochondrial metabolism or energy production of the sperm, wherein the mitochondrial metabolism or energy production value is observed in a normal semen test.
  • sperm motility and/or function is determined to be abnormal when compared to production and deviating from said normal values.
  • (Item 17A) A method for treating or preventing sperm or a sperm carrier comprising: If the evaluation by the method according to any one of items 1 to 17 determines that the sperm quality requires treatment or prevention, the treatment or prevention is given to the sperm or the sperm holder. A method comprising the step of applying.
  • (Item 18) A nitrogen radical compound or an oxygen radical compound or a salt thereof for evaluating sperm quality.
  • (Item 19) A nitroxyl radical compound or salt thereof for assessing sperm quality.
  • the nitroxyl radical compound has the following formula wherein Z is -C(R 6A )(R 6B )- or -N(R 6C )-, E is a bond, -C(R 10A )(R 10B )- or -N(R 10C )-; R 1 , R 2 , R 3 and R 4 are each independently an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted cycloalkyl group, optionally substituted heterocycloalkyl group, is selected from the group consisting of an optionally substituted aryl group and an optionally substituted heteroaryl group, or R 1 and R 2 taken together are carbocyclic with the carbon atom to which they are attached; or may form a heterocycle, R3 and R4 may together form a carbocyclic or heterocyclic ring with the carbon atom to which they are attached; R 1 and R 3 may together
  • the nitroxyl radical compound has the following formula 5-membered cyclic nitroxyl radical compound represented by or the following formula wherein R 1 , R 2 , R 3 , R 4 , R 5A , R 5B , R 6A , R 6B , R 10A and R 10B are A compound or a salt thereof according to any one of the above items, each independently as described in any one of the above items.
  • the five-membered cyclic nitroxyl radical compound has the following formula wherein the 6-membered cyclic nitroxyl radical compound is represented by the following formula
  • R 5A is a cyano group, an isothiocyano group, an optionally substituted alkyl group, an optionally substituted hydroxy group, an optionally substituted carboxyl group, an optionally substituted carbamoyl group or an optionally substituted
  • (Item 23A) The compound or salt thereof according to any one of the preceding items, wherein R 9 is bromoacetylamino or iodoacetylamino.
  • (Item 23B) The compound or salt thereof according to any one of the preceding items, wherein R 6A and R 10A are hydrogen.
  • (Item 25) 3-[2-(2-Iodoacetamido)acetamido]-2,2,5,5-tetramethyl-1-pyrrolidinyloxy free radical or a salt thereof for assessing sperm quality.
  • (Item 26) 3-carbamoyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxy free radical or salt thereof for assessing sperm quality.
  • a kit for assessing sperm quality comprising: A kit comprising a test solution containing a nitrogen radical compound or an oxygen radical compound or a salt thereof.
  • a kit for assessing sperm quality comprising: A kit containing a test solution containing a nitroxyl radical compound or a salt thereof.
  • nitroxyl radical compound is a 6-membered cyclic nitroxyl radical compound.
  • the test solution further contains a reducing agent.
  • Item 30 A kit according to any one of the preceding items, further comprising a DNP-MRI device.
  • the nitroxyl radical compound has the following formula wherein Z is -C(R 6A )(R 6B )- or -N(R 6C )-, E is a bond, -C(R 10A )(R 10B )- or -N(R 10C )-; R 1 , R 2 , R 3 and R 4 are each independently an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted cycloalkyl group, optionally substituted heterocycloalkyl group, is selected from the group consisting of an optionally substituted aryl group and an optionally substituted heteroaryl group, or R 1 and R 2 taken together are carbocyclic with the carbon atom to which they are attached; or may form a heterocycle, R3 and R4 may together form a carbocyclic or heterocyclic ring with the carbon atom to which they are attached; R 1 and R 3 may together
  • the nitroxyl radical compound has the following formula 5-membered cyclic nitroxyl radical compound represented by or the following formula wherein R 1 , R 2 , R 3 , R 4 , R 5A , R 5B , R 6A , R 6B , R 10A and R 10B are A kit according to any one of the preceding items, each independently as described in any one of the preceding items.
  • the five-membered cyclic nitroxyl radical compound has the following formula wherein the 6-membered cyclic nitroxyl radical compound is represented by the following formula
  • a kit according to any one of the preceding items, represented by (Item 32) The kit according to any one of the preceding items, wherein R 1 , R 2 , R 3 and R 4 are each independently an optionally substituted alkyl group.
  • R 5A is a cyano group, an isothiocyano group, an optionally substituted alkyl group, an optionally substituted hydroxy group, an optionally substituted carboxyl group, an optionally substituted carbamoyl group or an optionally substituted A kit according to any one of the preceding items, which is a good amino group.
  • (Item 35) A kit according to any one of the preceding items, wherein R9 is bromoacetylamino or iodoacetylamino.
  • (Item 35A) A kit according to any one of the preceding items, wherein R 6A and R 10A are hydrogen.
  • the nitroxyl compound is 3-[2-(2-iodoacetamido)acetamido]-2,2,5,5-tetramethyl-1-pyrrolidinyloxy free radical or 3-carbamoyl-2,2,5,5 -tetramethyl-1-pyrrolidinyloxy free radical.
  • the reducing agent comprises NADH or GSH.
  • a device for assessing sperm quality comprising: a signal measuring means for measuring a signal obtained from a mixed liquid in which a population of spermatozoa is mixed with a test liquid containing a nitrogen radical compound or an oxygen radical compound or a salt thereof; sperm quality evaluation means for evaluating sperm quality based on the signal.
  • (Item 41A) The compound or salt thereof according to any one of the above items, wherein the oxygen radical of the oxygen radical compound is a nitroxyl radical.
  • (Item 42) A device for assessing sperm quality, comprising: a signal measuring means for measuring a signal obtained from a mixed solution in which a population of spermatozoa and a test solution containing a nitroxyl radical compound are mixed; sperm quality evaluation means for evaluating sperm quality based on the signal.
  • the nitroxyl compound is a 5-membered cyclic nitroxyl compound.
  • nitroxyl compound is a 6-membered cyclic nitroxyl compound.
  • the nitroxyl radical compound has the following formula wherein Z is -C(R 6A )(R 6B )- or -N(R 6C )-, E is a bond, -C(R 10A )(R 10B )- or -N(R 10C )-; R 1 , R 2 , R 3 and R 4 are each independently an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted cycloalkyl group, optionally substituted heterocycloalkyl group, is selected from the group consisting of an optionally substituted aryl group and an optionally substituted heteroaryl group, or R 1 and R 2 taken together are carbocyclic with the carbon atom to which they are attached; or may
  • the nitroxyl radical compound has the following formula 5-membered cyclic nitroxyl radical compound represented by or the following formula wherein R 1 , R 2 , R 3 , R 4 , R 5A , R 5B , R 6A , R 6B , R 10A and R 10B are A device according to any one of the preceding items, each independently as described in any one of the preceding items.
  • the five-membered cyclic nitroxyl radical compound has the following formula wherein the 6-membered cyclic nitroxyl radical compound is represented by the following formula A device according to any one of the preceding items, represented by: (Item 44) A device according to any one of the preceding items, wherein R 1 , R 2 , R 3 and R 4 are each independently an optionally substituted alkyl group.
  • R 5A is a cyano group, an isothiocyano group, an optionally substituted alkyl group, an optionally substituted hydroxy group, an optionally substituted carboxyl group, an optionally substituted carbamoyl group or an optionally substituted A device according to any one of the preceding items, which is a good amino group.
  • (Item 45B) A device according to any one of the preceding items, wherein R 9 is bromoacetylamino or iodoacetylamino.
  • (Item 46) A device according to any one of the preceding items, wherein R 6A and R 10A are hydrogen.
  • the nitroxyl compound is 3-[2-(2-iodoacetamido)acetamido]-2,2,5,5-tetramethyl-1-pyrrolidinyloxy free radical or 3-carbamoyl-2,2,5,5 -tetramethyl-1-pyrrolidinyloxy free radical.
  • the test liquid further comprises a reducing agent.
  • (Item 49) A device according to any one of the preceding items, wherein the reducing agent comprises NADH or GSH.
  • the sperm quality comprises sperm motility.
  • the signal comprises a signal detected from the mixture by dynamic nuclear polarization (DNP) or electron spin resonance (ESR) methods.
  • the sperm quality assessment means compares the signal or a value derived therefrom with a reference value for assessing sperm quality.
  • the reference value includes a value indicating the quality of sperm obtained from sperm derived from a healthy subject.
  • reaction stopping means for performing a reaction stopping process for stopping the reaction between the test liquid and the sperm population on the mixed liquid.
  • the quenching treatment comprises addition of a surfactant or organic solvent, or heat treatment.
  • a method of testing or diagnosing male infertility in a subject comprising: a step of mixing a population of spermatozoa obtained from a subject with a test solution containing a nitrogen radical compound or an oxygen radical compound or a salt thereof to obtain a mixed solution; measuring a signal obtained from the mixture; and determining whether and/or the extent to which the subject is male infertile based on the signal.
  • a method of testing or diagnosing male infertility in a subject comprising: a step of mixing a population of spermatozoa obtained from a subject with a test solution containing a nitroxyl radical compound or a salt thereof to obtain a mixed solution; measuring a signal obtained from the mixture; and determining whether and/or the extent to which the subject is male infertile based on the signal.
  • a test/diagnostic kit for testing or diagnosing male infertility in a subject a test solution containing a nitrogen radical compound or an oxygen radical compound or a salt thereof; a container containing the test solution; If necessary, a reaction stopping solution for stopping the reaction between the test solution and the population of spermatozoa obtained from the subject; and instructions for use, the instructions comprising: A mixed solution is obtained by mixing a population of spermatozoa obtained from the subject and a test solution containing the nitrogen radical compound or the oxygen radical compound or a salt thereof, Measuring a signal obtained from the mixed solution, A kit directed to determining whether and/or the extent of male infertility in said subject based on said signal.
  • a test/diagnostic kit for testing or diagnosing male infertility in a subject a test solution containing a nitroxyl radical compound; a container containing the test solution; If necessary, a reaction stopping solution for stopping the reaction between the test solution and the population of spermatozoa obtained from the subject; and instructions for use, the instructions comprising: A mixed solution is obtained by mixing a population of spermatozoa obtained from the subject and a test solution containing the nitroxyl radical compound or a salt thereof, Measuring a signal obtained from the mixed solution, A kit directed to determining whether and/or the extent of male infertility in said subject based on said signal.
  • (Item B1) A composition comprising a nitrogen radical compound or an oxygen radical compound or a salt thereof for evaluating sperm quality.
  • (Item B2) The composition of item B1, further comprising at least one feature of any one or more of items 1-58A.
  • (Item C1) Use of a nitrogen radical compound or an oxygen radical compound or a salt thereof for manufacturing a pharmaceutical composition for evaluating sperm quality.
  • (Item C2) Use according to item C1, further comprising at least one feature according to any one or more of items 1-58A.
  • (Item D1) A composition comprising a nitroxyl radical compound or salt thereof for assessing sperm quality.
  • (Item D2) The composition of item D1, further comprising at least one feature of any one or more of items 1-58A.
  • (Item E1) Use of a nitroxyl radical compound or a salt thereof for manufacturing a pharmaceutical composition for evaluating sperm quality.
  • (Item E2) Use according to item E1, further comprising at least one feature according to any one or more of items 1-58A.
  • (Item F1) Use of a nitroxyl radical compound or salt thereof for assessing sperm quality.
  • (Item F2) Use according to item F1, further comprising at least one feature according to any one or more of items 1-58A.
  • (Item G1) A composition comprising a nitrogen radical compound or an oxygen radical compound or a salt thereof for testing or diagnosing male infertility in a subject.
  • (Item H1) Use of a nitrogen radical compound or an oxygen radical compound or a salt thereof for manufacturing a pharmaceutical composition for testing or diagnosing male infertility in a subject.
  • (Item H2) Use according to item H1, further comprising at least one feature according to any one or more of items 1-58A.
  • (Item I1) Use of a nitrogen or oxygen radical compound or a salt thereof for testing or diagnosing male infertility in a subject.
  • Item I2) Use according to item I1, further comprising at least one feature according to any one or more of items 1-58A.
  • (Item J1) A composition comprising a nitroxyl radical compound or a salt thereof for testing or diagnosing male infertility in a subject.
  • (Item J2) The composition of item J1, further comprising at least one feature of any one or more of items 1-58A.
  • (Item K1) Use of a nitroxyl radical compound or a salt thereof for manufacturing a pharmaceutical composition for testing or diagnosing male infertility in a subject.
  • (Item K2) Use according to item K1, further comprising at least one feature according to any one or more of items 1-58A.
  • (Item L1) Use of a nitroxyl radical compound or salt thereof to test or diagnose male infertility in a subject.
  • (Item L2) Use according to item L1, further comprising at least one feature according to any one or more of items 1-58A.
  • the quality of sperm can be evaluated by a simple method, and a simple sperm test can be easily performed at home or the like.
  • a simple sperm test can be easily performed at home or the like.
  • FIG. 1 is a graph showing reactivity of nitroxyl radical compounds to mitochondria and cytosol (including cytosol).
  • CMP is 3-carbamoyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (trade name: 3-carbamoyl-PROXYL).
  • homoge indicates the liver homogenate solution
  • home+kcn indicates the homogenate plus the liver-stopping drug potassium cyanide KCN
  • cytosol indicates the cytosol.
  • FIG. 2A is an image of sperm without and after heat treatment taken by a clinical sperm motility tester (CASA: computer-assisted sperm analysis). In FIG. 2A (left), sperm without heat treatment (37° C.) are active, while in FIG.
  • FIG. 2A (right), sperm after heat treatment (60° C.) have stopped motile.
  • FIG. 2B (left) shows a graph of sperm motility without and after heat treatment.
  • FIG. 2B (right) is a graph showing the difference in reactivity between sperm without and after heat treatment and the probe (nitroxyl radical compound).
  • FIG. 3A is an image of sperm without and after KCN addition taken by CASA. In FIG. 2A (left, no KCN addition), sperm motility was 92%, while in FIG. 2B (right, KCN addition), probe reaction rate was significantly reduced when KCN addition stopped mitochondrial function. .
  • FIG. 5 is a schematic diagram of a current sperm diagnostic system (top) and a diagnostic system according to the present disclosure (bottom).
  • group means a monovalent group unless otherwise specified. Examples of non-univalent groups include alkylene groups (divalent) and the like. In addition, in the following description of substituents and the like, the term “group” may be omitted.
  • substituents defined by “optionally substituted” or “substituted” is not particularly limited as long as it can be substituted, and 1 or Multiple. Also, unless otherwise indicated, the description of each substituent also applies when that substituent is part or a substituent of another substituent.
  • any portion of the group modified by "optionally substituted” or “substituted” in this specification may be substituted.
  • “optionally substituted arylalkyl” and “substituted arylalkyl” can be substituted on the aryl portion or substituted on the alkyl portion, and both the aryl and alkyl portions can be may be substituted.
  • the substituents in the case of "optionally substituted” may be one or more identical or different substituents selected from Substituent Group I below.
  • the types of atoms in the substituents involved in bonding are not particularly limited by the types of substituents, it is preferred that the compound formed by substitution exists stably.
  • the atom to which the substituent is attached is an oxygen atom, a nitrogen atom, or a sulfur atom
  • the attachment point in the substituent below is generally limited to a carbon atom and selected from them.
  • Substituent Group I includes halogen, hydroxy, oxo, carboxy, amino, imino, hydroxyamino, hydroxyimino, formyl, formyloxy, carbamoyl, sulfamoyl, sulfanyl, sulfino, sulfo, thioformyl, thiocarboxy, dithiocarboxy, thiocarbamoyl, cyano, nitro, nitroso, azide, hydrazino, ureido, amidino, amidinoamino, unsubstituted or substituted alkyl, unsubstituted or substituted alkenyl, unsubstituted or substituted alkynyl, unsubstituted or substituted aryl, unsubstituted or substituted cycloalkyl, unsubstituted or substituted heteroaryl, unsubstituted or substituted heterocycloalkyl,
  • C 1-6 means having 1 to 6 carbon atoms.
  • C 1-4 means that the number of carbon atoms is 1 to 4
  • C 1-3 means that the number of carbon atoms is 1 to 3. means.
  • the description with a carbon number limitation is only a preferred numerical range, and the present disclosure is intended to include groups having substituents with a carbon number other than the specified carbon number within the scope of the present disclosure. .
  • heteroatom refers to atoms other than carbon atoms and hydrogen atoms, such as oxygen atoms, nitrogen atoms, sulfur atoms, and the like.
  • a group containing a heteroatom is a hetero group (e.g., a heteroaryl group (meaning that an aryl group contains at least a heteroatom)) or a hetero group (e.g., a heterocyclic group (a ring group ( It means that the carbocyclic group) contains at least one heteroatom.)) and the like.
  • nitrogen radical compound means a compound in which one or more nitrogen atoms among the atoms constituting the compound have unpaired electrons.
  • stable nitrogen radical compounds include, but are not limited to, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the like.
  • DPPH 2,2-diphenyl-1-picrylhydrazyl
  • nitrogen radical compound it is sufficient that "one or more nitrogen atoms among the atoms constituting the compound have unpaired electrons", and other atoms ( For example, oxygen) may or may not have an unpaired electron.
  • oxygen radical compound means a compound in which one or more oxygen atoms among atoms constituting the compound have unpaired electrons.
  • stable oxygen radical compounds include the galvinoxyl free radical (aka 2,6-di-tert-butyl- ⁇ -(3,5-di-tert-butyl-4-oxo-2,5-cyclohexadiene-1 -ylidene)-p-tolyloxy, free radical), and nitroxyl radical compounds.
  • oxygen radical compound when the term “oxygen radical compound” is used, it is sufficient that "one or more oxygen atoms among the atoms constituting the compound have unpaired electrons", and other atoms ( nitrogen) may or may not have an unpaired electron.
  • nitroxyl radical compound means a compound containing a nitroxyl radical group, and the nitroxyl radical group (>N—O.) forms a nitroxide group in which an oxygen atom and a nitrogen atom are bonded. It is a group characterized in that the oxygen atom having an unpaired electron.
  • the nitroxyl radical compound of the present disclosure is not particularly limited as long as it has a nitroxyl radical group and can exhibit the functions of the present disclosure.
  • a "cyclic nitroxyl radical compound” means that the nitrogen atom of the nitroxyl radical is a ring (e.g., non-aromatic or aromatic carbocycle, non-aromatic or It is a compound that is one of the constituent atoms.
  • cyclic nitroxyl radical compounds include five-membered cyclic nitroxyl radical compounds (eg, 2,2,5,5-tetramethyl-1-pyrrolidinyloxy and derivatives thereof such as 3-(carboxy) -2,2,5,5-tetramethyl-1-pyrrolidinyloxy), and 6-membered cyclic nitroxyl radical compounds (e.g., TEMPO (2,2,6,6-tetramethylpiperidine 1-oxyl), AZADO (2-azaadamantane-N-oxyl), Me-AZADO (1-methyl-2-azaadamantane-N-oxyl), nor-AZADO (9-azanoradamantane-N-oxyl)), It is not limited to these.
  • TEMPO 2,2,6,6-tetramethylpiperidine 1-oxyl
  • AZADO 2-azaadamantane-N-oxyl
  • Me-AZADO 1-methyl-2-azaadamantane-N-oxyl
  • halogen-free anions include carbonate, hydrogen carbonate, nitrate, sulfate, phosphate, acetate, propionate, butyrate, formate, maleate, fumarate, tartrate, citrate, stearate, succinate, ethylsuccinate, malonate, lactobionate, gluconate, glucoheptonate, benzoate, methanesulfonate, benzenesulfonic acid, paratoluenesulfonate (tosylate ion), lauryl sulfate ion, malate ion, ascorbate ion, mandelate ion, saccharinate ion, xinafoate ion, pamoate ion, cinnamate ion, adipate ion, cysteine ion, N-acetylcysteine ion , ionic acid ion, nicotinate i
  • radical compounds are highly reactive chemical species, and many of them are unstable and disappear after a certain amount of life due to interactions with surrounding substances.
  • nitroxyl radical compounds the electron-withdrawing property of the nitrogen atom stabilizes the unpaired electrons on the oxygen.
  • This nitroxy radical is P-type, that is, the type that releases electrons from the nitroxy radical and becomes an oxoammonium cation. It is divided into types in which type reactions are stably performed.
  • P-type can be used as a positive electrode active material, N, and a negative electrode active material.
  • an alkyl group refers to an atomic group obtained by removing one hydrogen atom from an aliphatic saturated hydrocarbon.
  • the alkyl group is, for example, a saturated aliphatic hydrocarbon group such as methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, sec-butyl group, tert-butyl group, and has a substituent. It may or may not have
  • the substituent is not particularly limited, and examples thereof include an alkoxy group, an aryl group, and a heteroaryl group. These substituents may further have a substituent.
  • the number of carbon atoms in the alkyl group is not particularly limited, but from the viewpoint of availability and cost, It can be 8, 1-6, 1-5, 1-4, 1-3, 1-2, or 1.
  • the alkenyl group refers to, for example, a vinyl group, an allyl group, an unsaturated aliphatic hydrocarbon group containing a double bond such as a butadienyl group, and may or may not have a substituent. good.
  • the number of carbon atoms in the alkenyl group is not particularly limited, but 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to It can be 4, 2-3, or 2.
  • the alkoxy group refers to a functional group in which one of the ether bonds is substituted with an aliphatic hydrocarbon group, such as a methoxy group, an ethoxy group, a propoxy group, and the aliphatic hydrocarbon group has a substituent. may or may not have.
  • the number of carbon atoms in the alkoxy group is not particularly limited, but is 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-5, 1- It can be 4, 1-3, 1-2, or 1.
  • the aryl group refers to, for example, an aromatic hydrocarbon group such as a phenyl group, a naphthyl group, a biphenyl group, an anthracenyl group, a phenanthryl group, a terphenyl group, and a pyrenyl group. It does not have to be.
  • the number of carbon atoms in the aryl group is not particularly limited, but is preferably in the range of 6-18.
  • the arylalkyl group is an alkyl group having an aromatic hydrocarbon as a substituent, and specific examples include a phenylmethyl group (benzyl group), ⁇ , ⁇ -dimethylbenzyl group, 1- phenylethyl group, 2-phenylethyl group, 1-phenylpropyl group, 2-phenylpropyl group, 3-phenylpropyl group, naphthalen-1-ylmethyl group, naphthalen-2-ylmethyl group, naphthalen-1-ylethyl group, naphthalene -2-ylethyl group, anthracen-1-ylmethyl group, anthracen-2-ylmethyl group and anthracen-9-ylmethyl group.
  • the number of carbon atoms in the arylalkyl group is not particularly limited, but is preferably in the range of 7-19.
  • the heteroaryl group refers to an aromatic group having one or more atoms other than carbon in the ring, such as furanyl, thiophenyl, benzofuranyl, dibenzofuranyl, pyridyl, quinolinyl, etc. It may or may not have a substituent.
  • the number of carbon atoms in the heteroaryl group is not particularly limited, but is preferably in the range of 3-18.
  • Halogen as used herein means an atom selected from fluorine, chlorine, bromine and iodine.
  • a hydroxy group refers to an atomic group —OH in which one oxygen atom and one hydrogen atom are bonded.
  • a "cyano group” is a -CN monovalent group.
  • an amino group refers to a monovalent group represented by —NH 2 or a group in which H is substituted with another substituent.
  • Substituents for substitution include, for example, aryl groups, heteroaryl groups, linear alkyl groups, branched alkyl groups, and the like.
  • aryl group and the heteroaryl group a phenyl group, a naphthyl group, a pyridyl group and a quinolinyl group are preferable. These substituents may be further substituted.
  • the number of carbon atoms is not particularly limited, but is preferably 2 or more and 50 or less, more preferably 6 or more and 40 or less, and particularly preferably 6 or more and 30 or less.
  • an alkylamino group refers to a functional group in which an aliphatic hydrocarbon group such as a methyl group, an ethyl group, or an n-propyl group is bonded to an amino group.
  • the alkylamino group may or may not have a substituent.
  • the number of carbon atoms in the alkylamino group is not particularly limited, it is preferably in the range of 2 to 20.
  • a dialkylamino group as used herein refers to a functional group in which two aliphatic hydrocarbon groups such as a dimethyl group and a diethyl group are bonded to an amino group.
  • the two aliphatic hydrocarbon groups may be the same or different.
  • a dialkylamino group may or may not have a substituent. Although the number of carbon atoms in the dialkylamino group is not particularly limited, it is preferably in the range of 2 to 20.
  • the arylamino group refers to, for example, phenylamino, p-tolylamino, 2-naphthylamino groups, and the like.
  • a diarylamino group refers to a substituent such as a diphenylamino group or a dinaphthylamino group, and the aromatic ring in these substituents may be substituted with an alkyl group, an alkoxy group, or the like.
  • an alkylcarbonyloxy group refers to a functional group such as an acetoxy group in which one side of an ether bond is substituted with an alkylcarbonyl group.
  • An alkylcarbonyloxy group may or may not have a substituent.
  • the number of carbon atoms in the alkylcarbonyloxy group is not particularly limited, it is preferably in the range of 2 to 20.
  • an arylcarbonyloxy group refers to a functional group such as a benzoyloxy group in which one side of an ether bond is substituted with an arylcarbonyl group.
  • An arylcarbonyloxy group may or may not have a substituent.
  • the number of carbon atoms in the arylcarbonyloxy group is not particularly limited, it is preferably in the range of 6 or more and 40 or less.
  • an alkylcarbonylamino group refers to a functional group in which an alkylcarbonyl group such as an acetyl group is bonded to an amino group.
  • An alkylcarbonylamino group may or may not have a substituent.
  • the number of carbon atoms in the alkylcarbonylamino group is not particularly limited, it is preferably in the range of 2 to 20.
  • an arylcarbonylamino group represents a carbonylamino group substituted with a 5- to 10-membered monocyclic or bicyclic carboaryl group such as benzoylamino or naphthoylamino.
  • cycloalkyl means a non-aromatic saturated hydrocarbon ring group, which has a partially bridged structure, a partially spirolated structure, and one or two or more carbonyl structures. Also includes those with “C 3-20 cycloalkyl” means monocyclic or bicyclic cycloalkyl having 3 to 20 carbon atoms. “C 3-6 cycloalkyl” means monocyclic cycloalkyl having 3 to 6 carbon atoms. Specific examples of C 3-6 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.
  • C 3-10 cycloalkyl means monocyclic or bicyclic cycloalkyl having 3 to 10 carbon atoms. Specific examples of C 3-10 cycloalkyl include C 3-6 cycloalkyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, bicyclo[4.1.0]heptyl, bicyclo[3.3.0]octyl, bicyclo Examples include, but are not limited to, [4.2.0]octyl, bicyclo[4.3.0]nonyl, decahydronaphthyl, and the like.
  • a cycloalkyl group as a substituent or part thereof may be fused with an aryl and/or heteroaryl ring.
  • a cyclohexyl group may be fused with a benzene ring to form a 1,2,3,4-tetrahydronaphthalenyl group, where any of the possible carbon atoms on the cyclohexane ring are Or it binds to a group or an atom thereof close to the mother skeleton.
  • Cycloalkyl groups are 1,2,3,4-tetrahydronaphthalen-1-yl, 1,2,3,4-tetrahydronaphthalen-2-yl, indan-1-yl, indan-2-yl, 5,6 , 7,8-tetrahydroquinolin-5-yl, 5,6,7,8-tetrahydroquinolin-6-yl.
  • heterocycloalkyl is composed of 3 or more atoms, including the same or different 1 or 2 or more heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur atoms. means a non-aromatic saturated or partially unsaturated heterocyclic ring, including those with partially bridged structures and those that are partially spiroified. Heterocycloalkyls can have structures in which a non-aromatic heterocyclic ring is fused with an aryl ring and/or a heteroaryl ring.
  • R acyl is hydrogen, optionally substituted alkyl, optionally substituted alkenyl , optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted heterocycloalkyl.
  • Specific examples of acyl include, but are not limited to, formyl, and the illustrated groups of alkylcarbonyl, arylcarbonyl, and heteroarylcarbonyl, and the like.
  • haloacyl include, but are not limited to, fluoroacetyl, chloroacetyl, bromoacetyl, iodoacetyl and the like.
  • alkoxy is a monovalent radical of -O-alkyl.
  • Preferred examples of alkoxy include C 1-6 alkoxy (ie C 1-6 alkyl-O-), C 1-4 alkoxy (ie C 1-4 alkyl-O-) and the like.
  • C 1-4 alkoxy examples include methoxy (CH 3 O—), ethoxy (CH 3 CH 2 O—), n-propoxy (CH 3 (CH 2 ) 2 O—), isopropoxy ((CH 3 ) 2 CHO-), n-butoxy (CH 3 (CH 2 ) 3 O-), isobutoxy ((CH 3 ) 2 CHCH 2 O-), tert-butoxy ((CH 3 ) 3 CO-), sec-butoxy (CH 3 CH 2 CH(CH 3 )O—) and the like.
  • C 1-6 alkoxy examples include C 1-4 alkoxy, n-pentyloxy (CH 3 (CH 2 ) 4 O-), isopentyloxy ((CH 3 ) 2 CHCH 2 CH 2 O-), neopentyloxy ((CH 3 ) 3 CCH 2 O—), tert-pentyloxy (CH 3 CH 2 C(CH 3 ) 2 O—), 1,2-dimethylpropoxy (CH 3 CH(CH 3 )CH( CH 3 )O—) and the like, but are not limited thereto.
  • haloalkyl refers to a monovalent halogenated alkyl group in which one or more hydrogens on the alkyl group have been replaced with a halogen.
  • perhaloalkyl also means a haloalkyl in which all hydrogens on the alkyl group have been replaced with halogens.
  • perfluoroethyl is --CF 2 CF 3
  • perchloro-n-propyl is --CCl 2 CCl 2 CCl 3 .
  • haloalkyl include C 1-6 haloalkyl, C 1-4 haloalkyl, C 1-3 haloalkyl and the like.
  • C 1-3 alkyl include fluoromethyl, chloromethyl, bromomethyl, difluoromethyl, dichloromethyl, dibromomethyl, trifluoromethyl, trichloromethyl, tribromomethyl, fluorochloromethyl, difluorochloromethyl, fluorodichloromethyl , fluoroethyl, chloroethyl, bromoethyl, trifluoroethyl, trichloroethyl, tribromoethyl, perfluoroethyl, perchloroethyl, perbromoethyl, perfluoropropyl, perchloropropyl, perbromopropyl, perfluoroisopropyl, perchloroisopropyl, perbromo Examples include, but are not limited to, isopropyl and the like.
  • C 1-4 alkyl include, but are not limited to, C 1-3 haloalkyl, perfluorobutyl, perchlorobutyl, perbromobutyl, perfluoroisobutyl, perfluoro-t-butyl and the like.
  • C 1-6 alkyl include C 1-4 haloalkyl, perfluoro-n-pentyl, perfluoroisopentyl, perfluoroneopentyl, perfluorotert-pentyl, perfluoro-1,2-dimethylpropyl, etc. It is not limited to these.
  • haloalkoxy and haloalkyloxy are —O-haloalkyl monovalent radicals in which one or more hydrogens on the alkyl group have been replaced with halogen.
  • perhaloalkoxy means a haloalkoxy in which all hydrogens on the alkyl group have been replaced with halogens.
  • perfluoroethoxy is -OCF 2 CF 3
  • perchloro-n-propoxy is -OCCl 2 CCl 2 CCl 3 .
  • Preferred examples of haloalkoxy include C 1-6 haloalkoxy, C 1-4 haloalkoxy, C 1-3 haloalkoxy and the like.
  • C 1-3 alkoxy include fluoromethoxy, chloromethoxy, bromomethoxy, difluoromethoxy, dichloromethoxy, dibromomethoxy, trifluoromethoxy, trichloromethoxy, tribromomethoxy, fluorochloromethoxy, difluorochloromethoxy, fluorodichloro Methoxy, fluoroethoxy, chloroethoxy, bromoethoxy, trifluoroethoxy, trichloroethoxy, tribromoethoxy, perfluoroethoxy, perchloroethoxy, perbromoethoxy, perfluoropropoxy, perchloropropoxy, perbromopropoxy, perfluoroisopropoxy, perchloro Examples include, but are not limited to, isopropoxy, perbromoisopropoxy, and the like.
  • C 1-4 alkoxy examples include, but are not limited to, C 1-3 haloalkoxy, perfluorobutoxy, perchlorobutoxy, perbromobutoxy, perfluoroisobutoxy, perfluoro-t-butoxy and the like.
  • C 1-6 alkoxy examples include C 1-4 haloalkoxy, perfluoro-n-pentyloxy, perfluoroisopentyloxy, perfluoroneopentyloxy, perfluorotert-pentyloxy, perfluoro-1,2-dimethylpropoxy and the like. include, but are not limited to:
  • alkylsulfonyl means a sulfonyl group substituted with the above “alkyl”.
  • C 1-6 alkylsulfonyl is preferably “C 1-4 alkylsulfonyl”. Specific examples of “C 1-6 alkylsulfonyl” include, but are not limited to, methylsulfonyl, propionylsulfonyl, butyrylsulfonyl and the like.
  • Protecting group refers to a group of atoms that, when attached to a reactive functional group in a molecule, masks, reduces or prevents the reactivity of the functional group.
  • the compounds of the present disclosure may be substituted with protecting groups as appropriate or necessary at any position such as any of R 1 to R 4 or substituents thereof or other substituents thereof, and the protecting groups are Compounds containing are also within the scope of this disclosure.
  • protecting groups can be selectively removed during the synthetic process, if desired. Examples of protecting groups are in Greene and Wuts, Protective Groups in Organic Chemistry, 5th Edition, 2014, John Wiley & Sons, NY and Harrison et al., Compendium of Synthetic Organic Methods, vols.
  • nitrogen protecting groups include formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl (“CBZ”), tert-butoxycarbonyl (“Boc”), trimethylsilyl (“TMS”), 2-trimethylsilylethanesulfonyl. (“TES”), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (“FMOC”), nitro-veratryloxycarbonyl (“NVOC”), etc. not.
  • hydroxyl protecting groups are those in which the hydroxyl group is acylated (esterified) or alkylated, such as benzyl and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers such as TMS, triethylsilyl, t-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS)), alkyldiarylsilyl ethers (eg t-butyldiphenylsilyl (TBDPS)), triarylsilyl ethers (eg triphenylsilyl), glycol Ethers (eg, ethylene glycol ether, propylene glycol ether, etc.), and allyl ethers include, but are not limited to.
  • the amino group possessed by the compound of the present disclosure may be protected with a nitrogen protecting group.
  • An amino group in the substituents listed in the substituent group may be further protected with a nitrogen protecting group, and the protected substituent may be used as the substituent.
  • a hydroxy group possessed by the compound of the present disclosure (e.g., a hydroxy group as a substituent, a hydroxy group in the substituent possessed by the compound, a hydroxy group in the above substituent groups, etc.) is also protected with a hydroxy-protecting group.
  • a hydroxy group in a substituent listed in a substituent group may be further protected with a hydroxyl protecting group (such as a silyl ether) described herein, and the protected substituent may be may be used.
  • the nitroxyl radical compound has the formula wherein Z is -C(R 6A )(R 6B )- or -N(R 6C )-, E is a bond, -C(R 10A )(R 10B )- or -N(R 10C )-; R 1 , R 2 , R 3 and R 4 are each independently an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted cycloalkyl group, optionally substituted heterocycloalkyl group, is selected from the group consisting of an optionally substituted aryl group and an optionally substituted heteroaryl group, or R 1 and R 2 taken together are carbocyclic with the carbon atom to which they are attached; or may form a heterocycle, R3 and R4 may together form a carbocyclic or heterocyclic ring with the carbon atom to which
  • the nitroxyl radical compound has the formula 5-membered cyclic nitroxyl radical compound represented by or the following formula wherein R 1 , R 2 , R 3 , R 4 , R 5A , R 5B , R 6A , R 6B , R 10A , and R 10B are , as defined herein (eg, Section 1).
  • the 5-membered cyclic nitroxyl radical compound has the formula wherein said 6-membered cyclic nitroxyl radical compound is represented by the following formula wherein R 1 , R 2 , R 3 , R 4 , R 5A , R 6A , and R 10A are as defined herein (eg, item 1).
  • the nitroxyl radical compounds of the present disclosure are molecular be.
  • Such nitroxyl radical compounds include TEMPO (2,2,6,6-tetramethylpiperidinooxyl) moieties or PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moieties is a molecule having
  • the oxidizing agent is not particularly limited as long as it is an oxidizing agent that oxidizes nitroxyl radicals, and known oxidizing agents can be used. Specific examples of such oxidizing agents include hypochlorite, iodobenzene diacetate, oxygen and the like.
  • hypochlorites include sodium hypochlorite, potassium hypochlorite, calcium hypochlorite (bleaching powder), and the like.
  • the hypochlorite may be in a solid state or in the form of an aqueous solution, and even a hydrate can be suitably used.
  • these hypochlorites may be used singly or in combination with a plurality of hypochlorites.
  • R 5A is a cyano group, an isothiocyano group, an optionally substituted alkyl group, an optionally substituted hydroxy group, an optionally substituted carboxyl group, an optionally substituted carbamoyl group or an optionally substituted amino group.
  • R9 is bromoacetylamino or iodoacetylamino.
  • R 6A and R 10A are hydrogen.
  • the nitroxyl radical compound has the formula wherein Q is any n-valent group, n is an integer greater than or equal to 1, and RF is any one hydrogen atom in the nitroxyl radical compound of Formula I. is the resulting monovalent group.
  • Q is any n-valent group
  • n is an integer greater than or equal to 1
  • RF is any one hydrogen atom in the nitroxyl radical compound of Formula I. is the resulting monovalent group.
  • multiple RF may be the same or different from each other, and the points of attachment to Q may also be the same or different.
  • Q can also be a polymer chain, examples of which include ethylene glycol poly(meth)acrylic acid, poly(meth)acrylic acid esters, poly(meth)acrylic acid amides, polyacrylonitrile, polyamides, polyesters, polystyrenes, polyurethanes, etc. include, but are not limited to.
  • R F attached to Q include: include, but are not limited to.
  • At least two of R 1 , R 2 , R 3 and R 4 are each independently an optionally substituted alkyl group. In one embodiment, at least two of R 1 , R 2 , R 3 and R 4 are each independently alkyl groups. In one embodiment, at least two of R 1 , R 2 , R 3 and R 4 are methyl groups.
  • At least three of R 1 , R 2 , R 3 and R 4 are each independently optionally substituted alkyl groups. In one embodiment, at least three of R 1 , R 2 , R 3 and R 4 are each independently alkyl groups. In one embodiment, at least three of R 1 , R 2 , R 3 and R 4 are methyl groups.
  • R 1 , R 2 , R 3 and R 4 are each independently an optionally substituted alkyl group. In one embodiment, R 1 , R 2 , R 3 and R 4 are each independently alkyl groups. In one embodiment, R 1 , R 2 , R 3 and R 4 are methyl groups.
  • R 1 and R 2 together may form a carbocyclic or heterocyclic ring with the carbon atom to which they are attached.
  • R3 and R4 together may form a carbocyclic or heterocyclic ring with the carbon atom to which they are attached.
  • R 1 and R 3 may together form a heterocycle (eg, a pyrrolidine or piperidine ring) containing the nitrogen of the nitroxyl group.
  • a heterocycle eg, a pyrrolidine or piperidine ring
  • R5A is hydrogen, cyano group, isothiocyano group, optionally substituted alkyl group, optionally substituted hydroxy group, optionally substituted carboxyl group, optionally substituted carbamoyl or an optionally substituted amino group
  • R 5B is hydrogen or an optionally substituted alkyl group, or R 5A and R 5B together are an oxo group, or a substituted It is a benzoylmethylidene group which may be substituted.
  • R5A is hydrogen, cyano group, isothiocyano group, haloacylaminoalkyl group, amino group, dialkylamino group, trialkylammonium group, acylamino group, haloacylamino group, maleimido group, acyloxy group, alkyl an oxy group, an alkynyloxy group, a phosphonooxy group, an alkylsulfonyloxy group, -COOH, a carbamoyl group, a substituted carbamoyl group, -OH, or an acyl group, and R 5B is hydrogen or an alkyl group, or R 5A and R5B together are an oxo group or a benzoylmethylidene group.
  • R5A is hydrogen, amino group, dimethylamino group, trimethylammonium group, acetylamino group, chloroacetylamino group, bromoacetylamino group, haloacetylamino group, haloacetylaminoacetylamino group, cyano group , isothiocyano group, methacryloxy group, benzoyloxy group, 3,5-di-tert-butyl-4-hydroxybenzoyloxy group, methoxy group, propargyloxy group, phosphonooxy group, methylsulfonyloxy group, -COOH group, carbamoyl group , an isothiocyanatoalkylcarbamoyl group, —OH, a haloacetylaminomethyl group, a maleimido group, or an acetyl group, and R 5B is hydrogen or a methyl group, or R 5A and R 5B together are It
  • R 9 is preferably bromoacetylamino or iodoacetylamino.
  • nitrogen radical compounds and oxygen radical compounds are 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl free radical, 3-(aminomethyl)-PROXYL free radical 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl free radical, 16-DOXYL-stearic acid free radical, 16-DOXYL-methyl stearate free radical, 4-acetoxy-2,2,6,6-tetramethylpiperidine-1-oxyl free radical 2-(4-methoxy-4-oxobutyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxy free radicals (aka Methyl 5-DOXYL-stearate, free radicals), 4′,4′-dimethylspiro(5 ⁇ -cholestane-3,2′-oxazolidin)-3′-yloxy free radical (aka 3 ⁇ -DOXY
  • the 5-membered cyclic nitroxyl radical compound may have R 1 , R 2 , R 3 and R 4 as methyl groups, and in another embodiment R 5A as a cyano group, even if substituted It may be an alkyl group, an optionally substituted carboxyl group, an optionally substituted carbamoyl group, or an optionally substituted amino group.
  • R 9 is preferably bromoacetamide or iodoacetamide.
  • Z is -N(R 6C )-
  • the compounds of formula I are AZADO (2-azaadamantane-N-oxyl), Me-AZADO ( 1-methyl-2-azaadamantane-N-oxyl), or nor-AZADO (9-azanoradamantane-N-oxyl).
  • a "pharmaceutically acceptable salt” is a disclosed compound in which a parent compound, such as a compound of the disclosure, is modified by converting an existing acid or base moiety to its salt form. (eg, reacting the free base group with a suitable organic acid).
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; not something.
  • representative acid addition salts include acetate, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzenesulfonic acid, benzoate, bisulfate, boric acid.
  • alkali or alkaline earth metal salts examples include sodium, lithium, potassium, calcium, magnesium, etc., and non-toxic ammonium, quaternary ammonium, and amine cations, examples of which include ammonium , tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.
  • Pharmaceutically acceptable salts of the present disclosure include conventional non-toxic salts of the parent compounds formed, for example, from non-toxic inorganic or organic acids.
  • Pharmaceutically acceptable salts of the disclosure can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods.
  • such salts are formed by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or in a mixture of the two. can be prepared.
  • non-aqueous media like ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. See below for a list of suitable salts. Remington's Pharmaceutical Sciences, 17th ed . , Mack Publishing Company, Easton, Pa.; , 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P.M. H. Stahl and C.I. G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al. , Journal of Pharmaceutical Science, 66, 1-19 (1977); the contents of each of these are hereby incorporated by reference in their entirety.
  • solvate means a compound of the present disclosure that has molecules of a suitable solvent incorporated in the crystal lattice.
  • a suitable solvent is physiologically tolerable at the dosage administered.
  • solvates may be prepared by crystallization, recrystallization, or precipitation from solutions containing organic solvents, water, or mixtures thereof.
  • Suitable solvents are ethanol, water (eg monohydrate, dihydrate and trihydrate), N-methylpyrrolidone (NMP), dimethylsulfoxide (DMSO), N,N'-dimethylformamide (DMF), N,N′-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H )-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate and the like.
  • NMP N-methylpyrrolidone
  • DMSO dimethylsulfoxide
  • DMF N,N'-dimethylformamide
  • DMAC N,N′-dimethylacetamide
  • DMEU 1,3-dimethyl-2-imidazolidinone
  • DMPU 1,3-dimethyl
  • the term "subject” or “subject (person)” refers to an entity that is the target of diagnosis, detection, treatment, etc. of the present invention (e.g., organisms such as humans or cells taken from organisms, blood, serum, etc.). etc.). When an organism is intended, it is referred to as a subject.
  • specimen refers to any substance obtained from a subject or the like, and includes, for example, cells, serum, and the like.
  • samples refers to any substance obtained from a subject or the like, and includes, for example, cells, serum, and the like.
  • a person skilled in the art can appropriately select a preferable specimen or sample based on the description of this specification.
  • agent As used herein, “agent”, “agent” or “factor” (both equivalent to the English equivalent of agent) are used broadly and interchangeably to describe any agent capable of achieving its intended purpose. It may be matter or other elements (eg, energy such as light, radiation, heat, electricity, etc.).
  • Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (including DNA such as cDNA and genomic DNA, RNA such as mRNA), poly Saccharides, oligosaccharides, lipids, organic small molecules (e.g., hormones, ligands, signaling substances, organic small molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (e.g., small molecule ligands, etc.), etc.) , including but not limited to these complex molecules.
  • proteins proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (including DNA such as cDNA and genomic DNA, RNA such as mRNA), poly Saccharides, oligosaccharides, lipids, organic small
  • diagnosis refers to the identification of various parameters associated with a disease, disorder, condition (e.g., mitochondrial-associated disease), etc. in a subject to determine the current or future status of such disease, disorder, condition. means to judge.
  • conditions within the body can be investigated, and such information can be used to determine the disease, disorder, condition, treatment or prophylactic formulation to be administered in a subject.
  • various parameters such as method can be selected.
  • diagnosis in a narrow sense means diagnosing the current situation, but in a broader sense it includes “early diagnosis”, “predictive diagnosis”, “pre-diagnosis” and the like.
  • the diagnostic method of the present invention is industrially useful because, in principle, it is possible to use what comes out of the body, and it can be performed without the hands of medical professionals such as doctors.
  • "predictive diagnosis, preliminary diagnosis or diagnosis” is sometimes referred to as "assisting”.
  • detection agent assay agent
  • test agent assay agent
  • diagnostic agent broadly refers to any agent capable of diagnosing a target condition (for example, a disease such as a mitochondrial-related disease).
  • an “agent” or agent, detection agent, etc. that "specifically” interacts (or binds) to a biological agent, such as an intracellular component
  • a biological agent e.g., mitochondrial-associated components
  • the biological agent e.g., mitochondrial-associated components
  • the affinity for other unrelated components e.g., non-mitochondrial components, e.g., non-mitochondrial cytosolic components, etc.
  • label refers to an entity (for example, substance, energy, electromagnetic waves, etc.) that distinguishes a target molecule or substance from others. Examples of such labeling methods include RI (radioisotope) method, fluorescence method, biotin method, chemiluminescence method and the like. Reagents, compositions of the present disclosure can be labeled as such.
  • in vivo refers to the inside of a living organism. In certain contexts, “in vivo” refers to a location where a substance of interest is to be placed.
  • in vitro refers to a state in which a part of a living body is "outside the body” (for example, into a test tube) or released for various research purposes. It is a term in contrast to in vivo.
  • contacting means arranging a plurality of substances such that interaction or binding between the substances occurs. This can be accomplished by bringing the (eg, compounds of the present disclosure) into physical proximity, either directly or indirectly, to the sample containing the mitochondrial associated target.
  • the compounds of this disclosure can exist in many buffers, salts, solutions, and the like. Contacting includes, for example, placing a compound in a beaker, microtiter plate, cell culture flask, microarray (eg, gene chip), or the like.
  • any sample may be used as long as it is considered to contain components related to the mitochondrial redox system, and for example, serum can be used. Serum can be obtained by conventional methods.
  • kits refers to a unit in which parts to be provided (for example, test drugs, diagnostic drugs, drugs, labels, instructions, etc.) are provided, usually divided into two or more compartments.
  • This kit form is preferred when the purpose is to provide a composition that should not be provided in a mixed form for reasons such as stability, and is preferably used in a mixed form immediately before use.
  • kits preferably include the parts provided (e.g., instructions or instructions describing how to use the reagents, diagnostic agents, agents, or how the reagents should be handled).
  • the kit When the kit is used as a reagent kit in the present specification, the kit usually includes instructions describing how to use test agents, diagnostic agents, drugs, and the like.
  • the term "instructions" refers to instructions for physicians or other users on how to use the present invention.
  • the instructions include words instructing the detection method of the present invention, how to use the diagnostic agent, or the administration of a medicine or the like.
  • the instructions may include words that instruct oral administration or esophageal administration (for example, by injection) as the administration site.
  • This instruction is prepared in accordance with the format prescribed by the regulatory authority of the country in which the invention is implemented (for example, the Ministry of Health, Labor and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, etc.), and is approved by the regulatory authority.
  • FDA Food and Drug Administration
  • package inserts which are usually provided in paper form, but are not limited to that, for example, in the form of electronic media (e.g., homepages provided on the Internet, e-mail). can also be provided.
  • the nitroxyl radical compound of the present disclosure can be prepared by the production method described below or by known literatures and methods, such as Molecules 2021, 26, 677, NATURE COMMUNICATIONS 6:6070, Chem.Pharm.Bull. 61(12) 1197. -1213 (2013), Can. J. Chem. 52, 3381 (1974), Current Organic Chemistry, 2014, 18, 459-474, WO 2008/093881, which are incorporated herein by reference in their entirety. It can be produced by the methods described or with reference to them, but is not limited to these. These production methods can be appropriately improved based on the knowledge of those skilled in organic synthetic chemistry.
  • the object compound can be obtained by protecting the site other than the reaction site as necessary and deprotecting after the reaction is completed or after a series of reactions.
  • Protective groups used in these processes include literature (Peter G. M. Wuts, "Greene's Protective Groups in Organic Synthesis", 5th Ed., John Wiley & Sons, Inc., Hoboken, New Jersey (2014)), etc.
  • the usual protecting groups described in can be used.
  • introduction and removal of a protecting group can be carried out by methods commonly used in organic synthetic chemistry (eg, methods described in the above literature) or methods based thereon.
  • the starting materials and intermediates in the production method below can be purchased as commercial products, or can be obtained by synthesizing from known compounds according to methods described in known literature or known methods. Moreover, these starting materials and intermediates may be salts thereof as long as they do not interfere with the reaction.
  • the inert solvent in the following production method means a solvent that does not react with raw materials, reagents, bases, acids, catalysts, ligands, etc. used in the reaction (hereinafter sometimes referred to as "raw materials used in the reaction, etc.”). means.
  • raw materials used in the reaction etc.
  • the solvent used in each step reacts with the raw materials used in the reaction, it can be used as an inert solvent as long as the desired reaction proceeds to obtain the desired compound.
  • Synthetic scheme 1 (1) Route 1 Synthesis of Compound XII and Compound XIV wherein R 1 , R 2 , R 3 , R 4 , R 6A , R 6B , R 10A and R 10B are as defined herein.
  • an oxidizing agent is used, which can be anything as long as it can oxidize the secondary nitrogen of the piperidine or pyrrolidine ring to a nitroxyl radical.
  • a reducing agent is used, which can be any reagent as long as it can reduce a carbonyl group to a hydroxy group.
  • Synthetic scheme 2 (1) Route 2 Synthesis of compound XX wherein R 1 , R 2 , R 3 , R 4 , R 6A , R 6B , R 10A and R 10B are as defined herein.
  • Nu — is any nucleophile, including but not limited to OH, OR, NH 2 , NHR.
  • a reducing agent is used, which can be any reagent as long as it is capable of saturating (eg, hydrogenating, etc.) the double bonds.
  • the intermediates and target compounds in the above production method can be purified by purification methods commonly used in synthetic organic chemistry (e.g., neutralization, filtration, extraction, washing, drying, concentration, recrystallization, various chromatography, etc.). It can be isolated and purified. Further, each intermediate can be subjected to the next reaction without particular purification.
  • purification methods commonly used in synthetic organic chemistry e.g., neutralization, filtration, extraction, washing, drying, concentration, recrystallization, various chromatography, etc.
  • An optically active form of the compound of the present disclosure can be produced by using an optically active starting material or intermediate, or by optically resolving the intermediate or final product racemate.
  • Examples of the optical resolution method include, but are not limited to, a separation method using an optically active column, a separation method such as a fractional crystallization method, and the like.
  • Diastereomers of the compounds of the present disclosure can be prepared by separation methods such as, but not limited to, column chromatography and fractional crystallization.
  • a salt of the nitroxyl radical compound of the present disclosure can be produced from a nitroxyl radical by a method known to those skilled in the art.
  • oxoammonium salts can be produced by reacting a nitroxyl radical compound with nitrogen dioxide.
  • sperm quality indicates the characteristics and attributes of sperm, and means, for example, an index that indicates the function of sperm.
  • sperm quality refers to any molecular biological or biochemical factors such as sperm motility, fertility, sperm viability, mitochondrial activity, normality of sperm morphology, normality of sperm constituent proteins, etc. Indices that can chemically assess cells are included.
  • sperm motility is an index that indicates the motility of sperm and includes movement speed, movement time, kinetic energy, and the like.
  • VSL linear progression velocity, ⁇ m/sec
  • VAP cell pathway average velocity, ⁇ m/sec
  • VCL forward curve velocity, ⁇ m/sec
  • ALH head amplitude, ⁇ m
  • STR linear linearity, VSL/VAP
  • LIN linearity, VSL/VCL
  • BCF head frequency, Hz
  • a method for evaluating sperm quality comprising the step of mixing a sperm population and a test solution containing a nitroxyl radical compound to obtain a mixed solution; A method is provided comprising measuring a signal obtained from the mixture and assessing sperm quality based on the signal.
  • the method of the present disclosure is specifically implemented as follows. That is, a bottle or tube containing a nitroxyl radical and a reducing agent is prepared, and semen is added thereto and mixed. After reacting a population of spermatozoa with a test solution containing a nitroxyl radical compound, reaction termination treatment is performed as necessary.
  • the reaction time between the sperm population and the test solution containing the nitroxyl radical compound may be any time, for example, from immediately after mixing to 120 minutes, preferably from about 1 to about 60 minutes.
  • the signal obtained by the DNP-MRI/electron spin resonance method can be read from this mixed solution, the rate of change from the signal value before the reaction can be calculated, and the sperm quality can be evaluated.
  • Any animal-derived sperm can be used as the sperm to be subjected to the method of the present disclosure.
  • animals include, but are not limited to, humans, cows, pigs, goats, horses, sheep, dogs, cats, rabbits, mice, hamsters, rats, and chickens.
  • Any sperm derived from the testis, epididymis, sperm ejaculate, stem cells, testicular stem cells, iPS cells, cultured cells, or the like can be used as the sperm.
  • the sperm may be refrigerated or cryopreserved. Fresh semen and frozen semen are preferable, and the sperm reflects the quality immediately before being subjected to artificial insemination, in vitro fertilization, microinsemination, or the like.
  • human-derived sperm can be ejaculated sperm, and when obtaining non-human sperm, for example, if the sperm is testis-derived, a method of collecting the testis and aspirating the sperm, etc. If the sperm is derived from the epididymis, the epididymis is collected and the sperm is aspirated or scraped out. If the sperm is derived from the ejaculated semen, it is collected after ejaculation into the female body, electrical stimulation, or an artificial vagina. and the like can be used. Stem cells, testicular stem cells, iPS cells, and cultured cells can also be collected by performing cell culture. The obtained sperm may be suspended in seminal plasma immediately after obtaining, or may be further diluted or washed with an aqueous solution or the like.
  • the population of spermatozoa of the present disclosure can contain multiple spermatozoa.
  • the sperm is obtained as described above, it is usually collected not as a single sperm, but as a group of sperm.
  • the sperm population is selected from the sperm population obtained as described above, depending on the purpose or quality to be evaluated, for example, a population excluding dead sperm, It is also possible to select a population or the like from which spermatozoa whose constituent protein quality is outside a certain range are excluded.
  • Sperm motility can be mentioned as sperm quality evaluated by the method of the present disclosure.
  • the test solution of the present disclosure contains a nitroxyl radical compound, and any solution can be used as long as it allows quality evaluation of individual sperm in a sperm population.
  • nitroxyl radical compounds as a result of comprehensively examining the reactivity with mitochondria and cytoplasm for 37 kinds of nitroxyl radical compounds (functional contrast agents) shown in Table 1, it was found that nitroxyl radical compounds with a five-membered ring structure are mitochondria. It has been found to tend to detect metabolism well.
  • nitroxyl radical compounds include 3-carbamoyl-2,2,5,5-tetramethyl-1-pyrrolidine-N-oxyl (carboxyl-PROXYL), 3- Carboxy-2,2,5,5-tetramethyl-1-pyrrolinyloxy, 2,2,6,6-tetramethyl-4-piperidinol-N-oxyl (Tempol), N-d16-triacetoneamine-N -oxyl and triacetoneamine-N-oxyl, methoxycarbonyl-PROXYL, carboxy-PROXYL, etc.
  • nitroxyl radical compound can be mentioned, preferably a five-membered cyclic nitroxyl radical compound can be used, more preferably in the method of the present disclosure
  • the nitroxyl radical compound used is 3-[2-(2-iodoacetamido)acetamido]-proxyl or carbamoylproxyl.
  • the test solution of the present disclosure can further contain a reducing agent.
  • a reducing agent Any reducing agent may be used as long as it reacts with sperm mitochondria together with a nitroxyl radical compound and is metabolized.
  • the reducing agent of the present disclosure include, but are not limited to, NADH, GSH, and the like.
  • the test liquid of the present disclosure is typically an aqueous solution. If the sperm is derived from the testis, epididymis, ejaculate, stem cells, testicular stem cells, iPS cells, cultured cells, etc., it is also possible to mix a nitroxyl radical compound with the liquid itself collected with the sperm and use it as a test solution. can.
  • the test solution can also include buffering agents, egg yolk components, glycerin, and the like.
  • the pH of the test solution of the present disclosure may be any pH as long as the quality of sperm can be evaluated, and is usually about 6.0 to about 9.0. Preferably it is about 6.8 to 7.5.
  • the component concentration in the test solution of the present disclosure is determined so that the pH of the aqueous solution falls within the above range.
  • the test solution of the present disclosure can contain any buffer as long as it achieves the above desired pH.
  • any buffering agent having a buffering action near neutrality can be selected. liquid, citrate buffer, acetate buffer, carbonate buffer and the like.
  • acids or bases can be used to achieve the desired pH.
  • tris(hydroxymethyl)aminomethane, citrate, or phosphate buffers are used.
  • the test solution of the present disclosure can also contain any sugar or energy source as an energy source for sperm.
  • sugars and energy sources include glucose, xylose, fructose, mannose, galactose, sucrose, lactose, maltose, trehalose, dextrin, ATP, ADP, succinic acid and NADH.
  • the test solution of the present disclosure can contain any antibiotic for the purpose of preventing bacterial growth.
  • antibiotics include penicillin, streptomycin, gentamicin, dibekacin and the like.
  • sterilization may be performed to prevent bacterial growth.
  • any mixing method can be used, for example, manual pipette, automatic pipette, dispenser etc. can be used.
  • the temperature at which the population of sperm and the test solution of the present disclosure are mixed may be any temperature as long as the quality of the sperm can be evaluated.
  • it can be mixed at about 15 to 45° C., preferably about 25 to about 45° C., which is a temperature near the body temperature of the animal from which the sperm is derived, more preferably about 28 to about 42° C., particularly preferably about It can be from 30°C to about 38°C.
  • the reaction in the method of the present disclosure, can be stopped as necessary after mixing the population of spermatozoa with the test solution containing the nitroxyl radical compound.
  • the reaction termination treatment is not particularly limited as long as it can dissolve cells or terminate mitochondrial function. can contain.
  • DNP-MRI or electron spin is performed from the mixture.
  • the signal obtained by the resonance (ESR) method can be read.
  • ESR resonance
  • DNP is an abbreviation for Dynamic Nuclear Polarization, and unless otherwise defined, a method of increasing the polarization of nuclear spins by transferring the energy of excited electron spins to nuclear spins. That's what I mean.
  • MRI refers to Magnetic Resonance Imaging without hyperpolarization, such as dynamic nuclear polarization (DNP).
  • the nitroxyl radical compound functions as a contrast agent (also referred to as a probe) for DNP, and free radical concentration data can be obtained.
  • a contrast agent also referred to as a probe
  • free radical concentration data can be obtained.
  • the free radical concentration can be imaged quantitatively, so information on the free radical consumption rate, which serves as an index for evaluating sperm quality, can be obtained more accurately.
  • the percentage change from the pre-reaction signal value can be calculated to assess sperm quality.
  • the signal obtained from the mixture can be measured, and the sperm quality can be evaluated based on the signal. Since the method of the present disclosure is an evaluation based on the mitochondrial metabolism (energy production) of sperm, in one embodiment, the energy production of mitochondria is predicted from the signal obtained from the mixed solution, thereby improving sperm motility, function, etc. Quality can be evaluated. For example, the signal obtained from the mixed solution or its value may be compared with a reference value for evaluating the quality of sperm. good too. In this case, the sperm function can be evaluated by comparison with the reaction rate of normal sperm (including sperm motility and mitochondrial metabolic capacity that reflect mitochondrial function). For example, when the response rate is at least about 5% lower, preferably at least about 10% lower, and more preferably about 20% lower than the reaction rate of normal sperm, it can be evaluated as having decreased function.
  • the reference values for evaluating the quality of sperm are, for example, semen volume of 1.5 ml or more, sperm concentration of 15 million / ml or more, total sperm count of 39 million or more, motility of 40% or more, normal morphology rate of 4% or more. (malformation rate less than 96%), total motile sperm count (total sperm count x motility rate) 15,600,000 or more, and the like can also be used.
  • the number and/or motility of sperm calculated from the signal obtained from the mixture can be compared with a reference value for evaluating the quality of sperm.
  • Sperm count can also be used as a correction parameter if necessary.
  • a method for testing or diagnosing male infertility can be provided by evaluating sperm quality as described above. That is, the present disclosure provides a method for testing or diagnosing male infertility in a subject, comprising the step of mixing a population of spermatozoa obtained from the subject with a test solution containing a nitroxyl radical compound to obtain a mixed solution; A method is provided comprising measuring a signal obtained from the mixture and determining whether and/or the degree of male infertility in the subject based on the signal.
  • the degree of normality and/or abnormality of sperm function through evaluating sperm quality.
  • the degree is preferably evaluated in stages, more preferably in 3 to 6 stages. can.
  • kits for evaluating sperm quality including a test solution containing a nitroxyl radical compound.
  • a test/diagnosis kit for testing or diagnosing male infertility in a subject comprising a test solution containing a nitroxyl radical compound, a container containing the test solution, and optionally the test a reaction stopping solution for stopping the reaction between the solution and the sperm population obtained from the subject; and instructions describing a method of use, wherein the instructions include the sperm population obtained from the subject
  • a test solution containing a nitroxyl radical compound is mixed to obtain a mixed solution, a signal obtained from the mixed solution is measured, and based on the signal, whether or not the subject is male infertile and/or the degree thereof Kits are provided that are instructed to determine the
  • the use of the method and kit of the present disclosure enables stepwise evaluation of sperm quality and whether or not a subject is male infertile.
  • semen is collected using the kit at home, the collected semen is sent to a testing company for testing, the diagnostic results are fed back, and if the sperm function declines If it is evaluated that the patient is in good health, it may be possible to set up a system that promotes consultation with a urologist.
  • a small DNP-MRI device in the kit it is possible to easily obtain test results at home.
  • by installing a high-precision DNP-MRI apparatus in a urology department, etc. it is possible to perform high-precision diagnosis using the method and apparatus of the present disclosure in addition to conventional microscopic examination.
  • an apparatus for evaluating the quality of sperm wherein a signal for measuring a signal obtained from a mixed liquid in which a population of sperm and a test liquid containing a nitroxyl radical compound are mixed.
  • An apparatus comprising measuring means and sperm quality evaluating means for evaluating sperm quality based on the signal.
  • the signal measurement means performs a step of measuring a signal obtained from a mixed solution in which the population of spermatozoa and the test solution containing the nitroxyl radical compound are mixed in the above-described method of the present disclosure.
  • the signal measuring means is a component for measuring the value of the individual sperm quality index of the sperm population mixed with the test liquid.
  • the signal measuring means can be provided with various devices and devices that are provided in a normal DNP-MRI apparatus.
  • the sperm quality evaluation means performs the step of evaluating sperm quality in the method of the present disclosure described above.
  • the sperm quality evaluation means is a constituent part for evaluating sperm quality based on the signal obtained by the signal measurement means. For this purpose, various arithmetic processes are performed on data stored in a storage unit or the like. Arithmetic processing is performed by the CPU included in the device of the present disclosure.
  • This CPU includes functional modules for controlling the signal measuring means, the sperm quality evaluating means, and the like of the device of the present disclosure, and can perform various controls. Each of these units may be composed of an independent integrated circuit, microprocessor, firmware, or the like.
  • the drug and composition of the present disclosure can be provided as follows.
  • formulations of the pharmaceutical compositions described herein can be prepared by any method known or later developed in the field of pharmacy. Generally, such methods of preparation include the step of bringing into association the active ingredient with the excipients and/or one or more other accessory ingredients.
  • a pharmaceutical composition according to the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as multiple single unit doses.
  • unit dose refers to discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of active ingredient will generally be the dose of active ingredient used in a subject or subject and/or a fraction (e.g., half or one-third of such dose) to meet the specific needs of such dose. be equivalent to.
  • compositions according to the present disclosure will vary depending on the identity, size and/or condition of the subject being treated. and will also vary depending on the route by which the composition is administered.
  • the composition can consist of from about 0.1% to about 99% (w/w) active ingredient.
  • the composition contains about 0.1% to about 100% (eg, about 0.5 to about 50%, about 1 to about 30%, about 5 to about 80%, at least about 80% (w/w ))).
  • the pharmaceutically acceptable excipient is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% pure. you can In some embodiments, the excipient is approved for use in human and veterinary medicine. In some embodiments, the excipient is approved by the US Food and Drug Administration. In some embodiments, the excipients are of pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (USP), European Pharmacopoeia (EP), British Pharmacopoeia, Japanese Pharmacopoeia and/or International Pharmacopoeia.
  • USP United States Pharmacopoeia
  • EP European Pharmacopoeia
  • British Pharmacopoeia Japanese Pharmacopoeia and/or International Pharmacopoeia.
  • excipients include any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, as long as they are suitable for the particular dosage form desired; Including, but not limited to, surfactants, isotonic agents, thickening or emulsifying agents, preservatives and the like.
  • Various excipients for preparing pharmaceutical compositions and techniques for preparing the compositions are known in the art (Remington: The Science and Practice of Pharmacy, 21st Edition, AR Gennaro , See Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety).
  • Any conventional excipient medium may interact with a substance or derivative thereof by producing any undesired biological effect or by adversely interacting with other component(s) of the pharmaceutical composition. Unless incompatible, use of conventional excipient vehicles is contemplated within the scope of the present disclosure.
  • diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium lactose phosphate, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol. , sodium chloride, dried starch, corn starch, powdered sugar, etc., and/or combinations thereof.
  • formulations may include at least one inactive ingredient.
  • inactive ingredient refers to one or more inert agents included in the formulation.
  • all or part of the inactive ingredients that can be used in the formulations of the present invention are approved by the Ministry of Health, Labor and Welfare of Japan or by PMDA as its reviewing agency, the United States Food and Drug Administration (FDA), etc. , or not approved.
  • the detection, testing or diagnostic agents of the present invention have other substances (e.g., labels, etc.) attached to moieties (e.g., compounds of the present disclosure, etc.) that enable detection, testing, or diagnosis.
  • moieties e.g., compounds of the present disclosure, etc.
  • complexes or complex molecules means any construct comprising two or more moieties.
  • the other portion may be any other substance (eg, low-molecular compounds, sugars, lipids, nucleic acids, other carbohydrates, etc.).
  • two or more moieties that constitute a complex may be covalently bonded or otherwise bonded (e.g., hydrogen bond, ionic bond, hydrophobic interaction, van der Waals force, etc.).
  • complex includes molecules formed by linking multiple types of molecules such as polypeptides, polynucleotides, lipids, sugars, and small molecules.
  • kits for detection, testing and/or diagnosis for performing methods for detection, testing and/or diagnosis according to the present disclosure comprises a detection agent, test agent and/or diagnostic agent of the disclosure or a compound of the disclosure.
  • the embodiment can use any of the embodiments described herein alone or in combination.
  • the means used are mass spectrometer, nuclear magnetic resonance spectrometer, X-ray spectrometer, SPR, chromatography (e.g. HPLC, thin layer chromatography, gas chromatography), immunological chemical means (e.g. Western blotting, EIA (enzyme immunoassay), RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay)), biochemical means (e.g.
  • pI electrophoresis Southern blotting, two-dimensional electrophoresis), electrophoresis equipment, chemical analysis equipment, fluorescence two-dimensional differential electrophoresis (2DE-DIGE), isotope labeling method (ICAT), tandem affinity purification method (TAP method), physical means, laser microdissection and these is selected from the group consisting of combinations of
  • a standard curve can be drawn and quantified based thereon.
  • Quantification means that may be used in such quantification include determination means that compares the standard curve and the measurement results to determine whether the values obtained with the agents of the present disclosure are within normal values.
  • determination means can be realized using a computer.
  • Evaluation of sperm quality in the present disclosure is based on sperm mitochondrial metabolism or energy production, and the mitochondrial metabolism or energy production value is observed in a normal semen test mitochondrial metabolism or energy production and if it deviates from the normal value, it is determined that there is an abnormality in sperm motility and/or function.
  • abnormalities are determined by observing the speed and direction of sperm migration and the normal morphology rate (malformation rate) under a microscope. Since it is an evaluation based on the basics, if the energy production decreases, the motility rate and function can also decrease.
  • the normal value of the semen test is generally specified using a microscope, and can be separately referred to when evaluating with the method of the present disclosure.
  • the World Health Organization (WHO) semen test normal values are as follows. (2010 latest version) ⁇ Semen volume 1.5ml or more ⁇ Sperm concentration 15 million/ml or more ⁇ Total sperm count 39 million or more ⁇ Motility rate 40% or more ⁇ Total motile sperm count (total sperm count x motility rate) 15.6 million or more
  • a reference value for normal spermatozoa for mitochondrial metabolism or energy production measured in the present disclosure is created from spermatozoa having the above normal values, and diagnosis can be made based on this reference value.
  • a liver homogenate solution, cytosol was prepared.
  • a homogenate solution, a cytosol, and a mixture of a mitochondrial blocking drug (KCN) in the homogenate were prepared, and each nitroxyl radical (probe) was added and allowed to react for 5 minutes.
  • the radical intensity after the reaction was measured by electron spin resonance ESR.
  • Nitroxyl radicals with a 5-membered ring structure tended to detect mitochondrial metabolism well overall. It was also found that the nitroxyl radical (functional contrast agent) at No. 22 is the most metabolized nitroxyl radical (functional contrast agent) by mitochondria.
  • Example 2 Measurement of sperm motility
  • bovine sperm motility measured using a sperm motility analyzer (CASA) and It was compared with the data obtained by adding a xyl radical compound and measuring the radical change by ESR.
  • the left micrograph of FIG. 2A shows the activity state of bovine spermatozoa at 37°C
  • the right micrograph shows the activity state of bovine spermatozoa at 60°C.
  • signals shown in pink and blue represent active sperm
  • yellow represents non-motile sperm.
  • the left graph in FIG. 2B is a bar graph of the sperm motility obtained from this micrograph. As can be seen from this bar graph, it was found that the heat treatment at 60° C. resulted in almost zero sperm motility.
  • the graph on the right of Figure 2B is the result of adding nitroxyl radicals to bovine sperm and measuring radical changes with ESR. Radical signals obtained by ESR measurement were plotted with time on the horizontal axis and radical concentration (logarithmic display) on the vertical axis. The slope of this graph indicates the metabolic rate. As can be seen from this graph, nitroxyl radicals are metabolized without heat treatment, whereas the reaction rate (metabolism) of nitroxyl radicals stops with heat treatment. From this, it was found that sperm activity can be evaluated by using nitroxyl radicals.
  • Example 3 Effect of mitochondrial function inhibitor on sperm motility It was confirmed whether sperm motility can be evaluated by the method using the nitroxyl radical compound of the present disclosure when a mitochondrial function inhibitor (KCN) is added to bovine sperm to reduce motility.
  • KCN mitochondrial function inhibitor
  • the micrograph on the left of Fig. 3A is the result of measuring the motility of normal bovine sperm using CASA, which showed a motility of 92%.
  • mitochondrial function inhibitor KCN
  • KCN mitochondrial function inhibitor
  • the graph in Fig. 3B is the result of adding nitroxyl radicals to bovine sperm and measuring radical changes with ESR.
  • nitroxyl radicals probes
  • bovine spermatozoa and nitroxyl radicals were mixed
  • nitroxyl radicals were metabolized when KCN was not added. It has been found that stopping mitochondrial function with KCN stops the metabolism of nitroxyl radicals.
  • Example 4 Relationship between sperm quality or number and nitroxyl radical metabolic rate
  • sperm quality or number and nitroxyl radical (probe) metabolic rate was investigated.
  • the graph on the left side of FIG. 4 shows the results of preparing spermatozoa having different motility rates and reacting them with nitroxyl radicals.
  • Sperm with different motility were prepared by measuring the motility of sperm at 0, 2, and 4 hours after thawing with CASA, and reactions with nitroxyl radicals were observed at each time. From this graph, it was found that the metabolic rate of nitroxyl radicals also changed in proportion to the motility of sperm.
  • the graph on the right side of FIG. 4 is a graph showing the relationship between sperm count and nitroxyl radical metabolism.
  • Thawed bovine sperm was diluted 2-fold, 4-fold, and 8-fold with a buffer solution to prepare solutions with different numbers of sperm cells, which were then mixed with nitroxyl radicals to confirm reactivity. From this graph, it was found that diluting the bovine sperm to reduce the number decreased the metabolic rate of nitroxyl radicals proportionally.
  • Example 5 Diagnostic system
  • a test kit containing a test solution containing a nitroxyl radical compound is sent to a person's home or the like.
  • the test subject himself/herself mixes the sperm into the tube containing the test drug and leaves it for a certain period of time.
  • the tube is mixed with a reaction terminating agent to stop the reaction.
  • the tube containing the mixed sperm solution is returned to the testing institution, and the nitroxyl radical metabolism is measured by ESR or DNP to test or diagnose male infertility in the subject.
  • This disclosure is useful when performing a simple sperm test easily at home.

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Abstract

La présente invention concerne un procédé d'évaluation de la qualité du sperme.　La présente invention concerne un procédé d'évaluation de la qualité du sperme, ledit procédé comprenant les étapes suivantes : obtention d'une solution mixte par mélange d'une population de sperme et d'un liquide d'inspection contenant un composé radical nitroxyle ; mesure d'un signal obtenu à partir du liquide mélangé ; et évaluation de la qualité du sperme sur la base du signal.
PCT/JP2022/023655 2021-06-14 2022-06-13 Procédé d'évaluation de la qualité du sperme WO2022264971A1 (fr)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
JPH1054812A (ja) * 1987-06-23 1998-02-24 Hafslund Nycomed Innov Ab 磁気共鳴像形成のための造影剤
WO2016194073A1 (fr) * 2015-05-29 2016-12-08 株式会社日立製作所 Procédé de mesure de résonance de spin électronique et son dispositif
WO2017217340A1 (fr) * 2016-06-13 2017-12-21 国立大学法人九州大学 Procédé d'acquisition d'informations de vitesse de consommation de radicaux libres et procédé de détermination de nash

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JPH1054812A (ja) * 1987-06-23 1998-02-24 Hafslund Nycomed Innov Ab 磁気共鳴像形成のための造影剤
WO2016194073A1 (fr) * 2015-05-29 2016-12-08 株式会社日立製作所 Procédé de mesure de résonance de spin électronique et son dispositif
WO2017217340A1 (fr) * 2016-06-13 2017-12-21 国立大学法人九州大学 Procédé d'acquisition d'informations de vitesse de consommation de radicaux libres et procédé de détermination de nash

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DIKALOV SERGEY I., DIKALOVA ANNA E., MOROZOV DENIS A., KIRILYUK IGOR A.: "Cellular accumulation and antioxidant activity of acetoxymethoxycarbonyl pyrrolidine nitroxides", FREE RADICAL RESEARCH, TAYLOR & FRANCIS, GB, vol. 52, no. 3, 4 March 2018 (2018-03-04), GB , pages 339 - 350, XP093015683, ISSN: 1071-5762, DOI: 10.1080/10715762.2017.1390744 *
IANNONE, A. ; BINI, A. ; SWARTZ, H.M. ; TOMASI, A. ; VANNINI, V.: "Metabolism in rat liver microsomes of the nitroxide spin probe tempol", BIOCHEMICAL PHARMACOLOGY, ELSEVIER, US, vol. 38, no. 16, 15 August 1989 (1989-08-15), US , pages 2581 - 2586, XP025514154, ISSN: 0006-2952, DOI: 10.1016/0006-2952(89)90541-8 *
PHADKE RATNA S., SRIVASTAVA SUDHA, GOVIL GIRJESH: "Magnetic resonance methods for studying intact spermatozoa", JOURNAL OF BIOSCIENCES, SPRINGER INDIA, NEW DELHI, vol. 15, no. 3, 1 September 1990 (1990-09-01), New Delhi, pages 125 - 134, XP093015679, ISSN: 0250-5991, DOI: 10.1007/BF02703872 *

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