Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
  • A61K31/05—Phenols
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/445—Non condensed piperidines, e.g. piperocaine
  • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
  • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• • 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
• • 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
  • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
• • 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 invention generally relates to new uses for TRPC3/6/7 channel activators.
• the present invention particularly relates to pharmaceutical compositions containing TRPC3/6/7 channel activators, specifically pharmaceutical compositions for preventing and/or treating heart failure or skeletal muscle failure, and
• the present invention relates to a method for screening substances for preventing and/or treating disorders.
• heart failure has been increasing around the world as society ages, and a situation that can be called a "heart failure pandemic" is progressing.
• the number of heart failure patients worldwide is on track to reach 30 million, and there are concerns that this number will continue to increase, leading to a shortage of hospital beds.
• heart failure is a pathological condition with a poor prognosis, with no complete recovery, and a gradual decline in heart function (chronic heart failure) through repeated periods of acute exacerbation (acute heart failure) and remission (chronic heart failure), eventually leading to treatment-resistant heart failure and death.
• chronic heart failure a pathological condition with a poor prognosis, with no complete recovery, and a gradual decline in heart function (chronic heart failure) through repeated periods of acute exacerbation (acute heart failure) and remission (chronic heart failure), eventually leading to treatment-resistant heart failure and death.
• acute heart failure acute heart failure
• remission chronic heart failure
• the pumping activity and morphological structure of the heart are regulated by various neurohumoral factors.
• the cells that make up the heart maintain cardiac homeostasis by sensing these neurohumoral factors on their cell membranes and transmitting appropriate signals within the cells.
• Cardiomyocyte hypertrophy and interstitial fibrosis that occur with the onset and progression of heart failure are caused by Ca 2+ signaling, especially Ca 2+
• the influx of Ca 2+ from outside the cell into the cell is deeply involved in the induction of expression of genes related to hypertrophy, and transient Ca 2+ influx is attracting attention as a molecular entity responsible for receptor-mediated Ca 2+ influx. It is a receptor potential canonical (TRPC) protein.
• TRPC receptor potential canonical
• TRPC1-TRPC7 There are seven isoforms (TRPC1-TRPC7) of animal TRPC channels, and among them, lipid (diacylglycerol)-operated TRPC3 and TRPC6 channels are important for cardiac remodeling, including the results of the present applicant. There have been many reports. In fact, it has been demonstrated at the animal level that compounds that inhibit TRPC3/C6 channel activity improve pathological cardiac hypertrophy and heart failure, suggesting their effectiveness as therapeutic targets (Non-patent Documents 1-7 ). Further, Patent Document 1 describes a benzisoxazole compound that modulates or inhibits the activity of TRPC3 channel or TRPC6 channel, and mentions cardiac hypertrophy as a TRPC3 and/or TRPC6-related disease. Substances that activate the channels are not described. In any case, the physiological role of TRPC3/6 channels in regulating cardiac function is not well understood.
• Kiyonaka S Kato K, Nishida M, Mio K, Numaga T, Sawaguchi Y, Yoshida T, Wakamori M, Mori E, Numata T, Ishii M, Takemoto H, Ojida A, Watanabe K, Uemura A, Kurose H, Morii T , Kobayashi T, Sato Y, Sato C, Hamachi I, Mori Y Selective and direct inhibition of TRPC3 channels underlies biological activities of a pyrazole compound. Proc. Natl. Acad. Sci. USA. 106: 5400-5405 (2009).
• therapeutic drugs to treat heart failure a condition with a poor prognosis
• TRPC6 gene-deficient mice to be vulnerable to heart failure, that is, the heart of TRPC6-deficient mice was unable to adapt to high blood pressure load, and activated TRPC3/6/7 channels.
• TRPC3/6/7 channels We attempted to apply substances that cause disease to pathological conditions.
• substances that activate TRPC3/6/7 channels have the same protective effect on heart failure even in different heart failure models and different mouse strains, and have completed the present invention.
• a pharmaceutical composition for preventing and/or treating heart failure or skeletal muscle failure containing a TRPC3/6/7 channel activator.
• the TRPC3/6/7 channel activator has the formula (1): [In the formula, A represents a carbon atom or a nitrogen atom, B represents a carbon atom or a nitrogen atom, R 1 represents an optionally substituted alkyl group, and when there is a plurality of them, they may be the same or different, m represents an integer from 0 to 5, R 2 is a halogen atom; a trihalogenomethyl group; an alkyl group; an acyloxy group; an alkoxycarbonyl group; an optionally substituted amide group; an optionally substituted piperidinylcarbonyl group; or an optionally substituted pyrrolidinyl Indicates a carbonyl group, and when there are multiple groups, they may be the same or different, n represents an integer
• the TRPC3/6/7 channel activator has the formula (1): [In the formula, A represents a carbon atom or a nitrogen atom, B represents a carbon atom or a nitrogen atom, R 1 represents a halogen atom, an alkyl group, an alkoxy group, an acyloxy group, an alkoxycarbonyl group, or an alkyl group substituted with a halogen atom, and when there is more than one, they may be the same or different; m represents an integer from 0 to 5, R 2 may be substituted with one or two groups selected from the group consisting of a halogen atom; a trihalogenomethyl group; an alkyl group; an acyloxy group; an alkoxycarbonyl group; an alkyl group, an alkoxycarbonyl group, and a halogen atom.
• a good amide group; a piperidinylcarbonyl group which may be substituted with one or more groups selected from the group consisting of an alkyl group, an alkoxycarbonyl group, and a halogen atom; or an alkyl group, an alkoxycarbonyl group, and a halogen atom Indicates a pyrrolidinylcarbonyl group which may be substituted with one or more groups selected from the group consisting of, and when there are multiple groups, they may be the same or different, n represents an integer from 0 to 5, p represents 0 or 1, and q represents 0 or 1.
• the TRPC3/6/7 channel activator is a compound represented by the formula (1), A represents a carbon atom or a nitrogen atom, B represents a carbon atom or a nitrogen atom, R 1 represents a fluorine atom, a chlorine atom, a bromine atom, a C1-C4 alkyl group, a C1-C4 alkoxy group, a C2-C5 acyloxy group, a C2-C5 alkoxycarbonyl group, or a trihalogenomethyl group, and when there is more than one, may be the same or different; m represents an integer from 0 to 4, R 2 is a fluorine atom; a chlorine atom; a bromine atom; a trifluoromethyl group; a trichloromethyl group; a tri
• the TRPC3/6/7 channel activator is a compound represented by the formula (1), A represents a nitrogen atom, B represents a carbon atom or a nitrogen atom, R 1 represents a fluorine atom, a chlorine atom, a C1-C4 alkyl group, a C1-C4 alkoxy group, a C2-C4 alkoxycarbonyl group, a trifluoromethyl group, a trichloromethyl group, or a tribromomethyl group, and when there is more than one, may be the same or different; m represents an integer from 0 to 3, R 2 is a fluorine atom; a chlorine atom; a trifluoromethyl group; a trichloromethyl group; a C1 to C4 alkyl group; a C2 to C4 acyloxy group; a C2 to C4 alkoxycarbonyl group; a C1 to C3 alkyl group and a C2 to C4 alkoxycarbonyl group
• the TRPC3/6/7 channel activator is a compound represented by the formula (1), A represents a nitrogen atom, B represents a carbon atom or a nitrogen atom, R 1 represents a chlorine atom, a C1-C3 alkyl group, a C1-C3 alkoxy group, a C2-C4 alkoxycarbonyl group, a trifluoromethyl group, or a trichloromethyl group, and when there is more than one, they may be the same or different, m represents an integer from 0 to 2, R 2 is selected from the group consisting of a fluorine atom; a chlorine atom; a trifluoromethyl group; a trichloromethyl group; a C1-C4 alkyl group; a C2-C4
• an amide group which may be substituted with one or two groups; or substituted with one or more groups selected from the group consisting of a C1-C3 alkyl group, a C2-C4 alkoxycarbonyl group, and a halogen atom; Indicates a piperidinyl carbonyl group that may be n represents an integer from 0 to 3; p indicates 1, q indicates 1, The pharmaceutical composition according to [5].
• ⁇ Screening method> [9] A method of screening for substances for preventing and/or treating heart failure or skeletal muscle failure, the screening method comprising selecting TRPC3/6/7 channel activators. [10] measuring the zinc concentration in cells in the presence of a test substance, and if the zinc concentration is increased compared to the zinc concentration in the absence of the test substance, the test substance.
• A represents a carbon atom or a nitrogen atom
• B represents a carbon atom or a nitrogen atom
• R 1 represents a halogen atom, an alkyl group, an alkoxy group, an acyloxy group, an alkoxycarbonyl group, or an alkyl group substituted with a halogen atom, and when there is more than one, they may be the same or different
• m represents an integer from 0 to 5
• n represents an integer from 2 to 5
• At least one of the plurality of R 2 is a halogen atom, an alkyl group, or a trifluoromethyl group; the remaining R 2 are 1) substituted with a single or multiple halogen atoms, and are selected from an alkyl group and an alkoxycarbonyl group.
• Figure 1 is a graph showing the increase in left ventricular contractility and heart rate due to intravenous administration of the vasodilator hydralazine (Hyd) in wild type (WT) and TRPC6 gene-deficient mice (TRPC6(-/-)). It is. ⁇ LVdP/dtmax: increase in left ventricular contractile force, ⁇ Heart Rate: increase in heart rate, AUC: area under the curve (total area under the curve for 10 minutes after administration). **; P ⁇ 0.01.
• Figure 2 shows the inhibitory effect of PPZ2 on the progression of heart failure in MLP-deficient mice.
• Figure 2a Graph showing left ventricular ejection fraction (EF).
• Figure 2b A graph showing fractional shortening (FS) of the left ventricle.
• Figure 3-1 shows the heart failure improving effect of PPZ2 in mice with pressure overload-induced heart failure.
• Figure 3-1a A graph showing left ventricular ejection fraction (EF).
• Figure 3-2 shows the heart failure ameliorating effect of PPZ2 in mice with pressure overload-induced heart failure.
• FIG. 1a A graph showing left ventricular ejection fraction (EF).
• Figure 3-1c A photograph of a WGA-stained image (left) and a graph
• Figure 3-2e Photograph showing Zn 2+ concentration (Zinpyr-1 staining) in myocardial tissue.
• FIG. 5 is a graph showing the dose dependence of enhancement of the baroreceptor reflex response (positive inotropic effect) in normal mice by administration of PPZ2 and the suppressive effect of the TRPC6 inhibitor BI749327.
• the vertical axis shows the hydralazine-induced increase in left ventricular contractile force ( ⁇ LVdP/dtmax) or heart rate ( ⁇ Heart Rate).
• the ⁇ value indicates the difference between the value before hydralazine (Hyd) administration and the maximum value 10 minutes after administration.
• FIG. 6 shows changes in intracellular Zn 2+ concentration caused by TRPC6 activators using TRPC6-expressing HEK293 cells.
• Figures 6a and 6b are graphs comparing the FluoZn-3 fluorescence intensity change and its increase rate (AUC; area under the curve) due to PPZ2 (30 ⁇ M) treatment.
• FIG. 7 shows the results of examining the myocardial contractility enhancing effect of TRPC6 activators using neonatal rat cardiomyocytes.
• Figure 7a Showing the effect of PPZ2 (30 ⁇ M) treatment on cardiomyocyte contraction induced by norepinephrine (NE) (0.1 ⁇ M) or the zinc chelator TPEN (N,N,N',N'-tetrakis(2-pyridylmethylethylenediamine).
• Figure 7b Graph showing the effect of GSK1702934A (1 ⁇ M) or hyperforin (1 ⁇ M) treatment on cardiomyocyte contraction induced by NE (1 ⁇ M).
• FIG. 8 is a graph showing that PPZ2 suppressed the increase in myopathy markers (left: LDH, right: CK) in muscular dystrophy model mice.
• FIG. 9 is a graph showing the effects of L687 and PPZ2 on Ca 2+ influx into HEK293 cells expressing human TRPC6 channels.
• FIG. 10 is a graph showing the effect of the compounds of the present invention on Ca 2+ influx into HEK293 cells expressing human TRPC6 channels.
• FIG. 11 is a graph showing the effect of the compounds of the present invention on Ca 2+ influx into HEK293 cells expressing human TRPC3 channels.
• Figure 12. Upper panel shows ⁇ -actinin staining images of neonatal rat ventricular myocytes.
• FIG. 14 is a graph showing the effect of L-0044 (1 ⁇ M) treatment on the contraction of cardiomyocytes stimulated by norepinephrine (NE) (0.1 ⁇ M).
• the present invention provides that a low-molecular compound that promotes activation of receptor-operated TRPC3/6/7 channels becomes an effective therapeutic agent for acute heart failure or acute exacerbation of chronic heart failure without having a hypotensive effect.
• This is a use invention showing the following. In a state of heart failure, the ability of the heart to pump blood is originally weakened, making it difficult for blood pressure to rise. When antihypertensive effects are added here, a state of chronic hypotension occurs, and a compensatory mechanism that attempts to raise blood pressure, ie, an increase in sympathetic nerve activity, comes into play.
• the present invention provides a pharmaceutical composition for preventing and/or treating heart failure or skeletal muscle failure, containing a TRPC3/6/7 channel activator, specifically a piperazine derivative (PPZ). provide.
• a TRPC3/6/7 channel activator specifically a piperazine derivative (PPZ).
• TRPC6 gene-deficient mice show a tendency to be vulnerable to heart failure, that is, the hearts of TRPC6-deficient mice are susceptible to increased oxidative stress and inflammation in response to hypertensive stress, and are unable to adapt to hypertensive stress. I paid attention to that.
• Norepinephrine released from sympathetic nerve terminals is a major neurotransmitter that enhances the pumping function of the heart, and plays an especially important role in compensatory cardiac hyperactivity (baroreceptive reflex) in response to sudden drops in blood pressure.
• Noradrenaline activates ⁇ -adrenergic receptors on the cardiac muscle cell membrane, causing contraction of the cardiac muscle cell and an increase in heart rate.
• TRPC6-deficient mice showed positive baroreflex responses (positive chronotropy (increased heart rate) and positive inotropy (increased contractile force)) induced by the antihypertensive drug hydralazine.
• the inotropic effect was significantly attenuated ( Figure 1).
• the baroreceptive reflex response is also markedly attenuated in heart failure, which is a cause of worsening prognosis, suggesting that TRPC6 channel activation may work to improve heart failure.
• the present inventors focused on this relationship and found for the first time a relationship between activation of TRPC3/6/7 channels and prevention or treatment of heart failure or skeletal muscle failure.
• Test Examples 1-3 described below different experimental animals were used, and despite the fact that the phenotype often changes due to model differences, species differences, and strain differences, different heart failure models and different mouse strains were used. We were able to show that the same protective effect against heart failure exists (commonality/universality).
• TRPC3/6/7 channel activator refers to a group of channels that are directly activated by diacylglycerol (TRPC3/ 6/7), which activates the opening of diacylglycerol independently of diacylglycerol.
• TRPC3/6/7 channel activator means a group of substances that activate any or all of TRPC3, TRPC6, and TRPC7.
• the "TRPC3/6/7 channel activator” includes piperazine derivatives (PPZ), and specific examples thereof include Sawamura S, Hatano M, Takada Y, Hino K, Kawamura T, Tanikawa J, Nakagawa H, Hase H, Nakao A, Hirano M, Rotrattanadumrong R, Kiyonaka S, Mori MX, Nishida M, Hu Y, Inoue R, Nagata R, Mori Y. Screening of Transient Receptor Potential Canonical Channel Activators Identifies Novel Neurotrophic Piperazine Compounds. Examples include piperazine derivatives described in Mol Pharmacol. 89(3), 348-363 (2016). doi: 10.1124/mol.115.102863. For example, the formula: Examples include any of the compounds shown below.
• TRPC3/6/7 channel activator includes Xu X, Lozinskaya I, Costell M, Lin Z, Ball JA, Bernard R, Behm DJ, Marino JP, Schnackenberg (2013).
• TRPC3/6/7 channel activator includes cannabidiol, N. Qin et al., which is known to activate TRPV1 channel, TRPV2 channel, and TRPA1 channel. J. Neurosci. 28, 6231-6238 (2008)) is also included.
• TRPC3/6/7 channel activator includes compounds including the compounds described in WO2022/118966.
• the present invention provides a pharmaceutical composition for preventing and/or treating heart failure or skeletal muscle failure, which contains a TRPC3/6/7 channel activator;
• the TRPC3/6/7 channel activator has the formula (1): [In the formula, A represents a carbon atom or a nitrogen atom, B represents a carbon atom or a nitrogen atom, R 1 represents an optionally substituted alkyl group, and when there is a plurality of them, they may be the same or different, m represents an integer from 0 to 5, R 2 is a halogen atom; a trihalogenomethyl group; an alkyl group; an acyloxy group; an alkoxycarbonyl group; an optionally substituted amide group; an optionally substituted piperidinylcarbonyl group; or an optionally substituted pyrrolidinyl Indicates a carbonyl group, and when there are multiple groups, they may be the same or different, n represents an integer from 0 to 5, p
• the TRPC3/6/7 channel activator has the formula (1): [In the formula, A represents a carbon atom or a nitrogen atom, B represents a carbon atom or a nitrogen atom, R 1 represents a halogen atom, an alkyl group, an alkoxy group, an acyloxy group, an alkoxycarbonyl group, or an alkyl group substituted with a halogen atom, and when there is more than one, they may be the same or different; m represents an integer from 0 to 5, R 2 is one or two groups selected from the group consisting of a halogen atom; a trihalogenomethyl group; an alkyl group; an acyloxy group; an alkoxycarbonyl group; an alkyl group, an alkoxycarbonyl group, and a halogen atom, preferably a fluorine atom.
• the TRPC3/6/7 channel activator is a compound represented by the above formula (1), A represents a carbon atom or a nitrogen atom, B represents a carbon atom or a nitrogen atom, R 1 represents a fluorine atom, a chlorine atom, a bromine atom, a C1-C4 alkyl group, a C1-C4 alkoxy group, a C2-C5 acyloxy group, a C2-C5 alkoxycarbonyl group, or a trihalogenomethyl group, and when there is more than one, may be the same or different; m represents an integer from 0 to 4, R 2 is a fluorine atom; a chlorine atom; a bromine atom; a trifluoromethyl group; a trichloromethyl group; a tribromomethyl group; a C1 to C4 alkyl group; a C2 to C5 acyloxy group; a C2 to C5 alkoxycarbon
• the TRPC3/6/7 channel activator is a compound represented by the formula (1), A represents a nitrogen atom, B represents a carbon atom or a nitrogen atom, R 1 represents a fluorine atom, a chlorine atom, a C1-C4 alkyl group, a C1-C4 alkoxy group, a C2-C4 alkoxycarbonyl group, a trifluoromethyl group, a trichloromethyl group, or a tribromomethyl group, and when there is more than one, may be the same or different; m represents an integer from 0 to 3, R 2 is a fluorine atom; a chlorine atom; a trifluoromethyl group; a trichloromethyl group; a C1 to C4 alkyl group; a C2 to C4 acyloxy group; a C2 to C4 alkoxycarbonyl group; a C1 to C3 alkyl group and a C2 to C4 alkoxycarbonyl
• the TRPC3/6/7 channel activator is a compound represented by the formula (1), A represents a nitrogen atom, B represents a carbon atom or a nitrogen atom, R 1 represents a chlorine atom, a C1-C3 alkyl group, a C1-C3 alkoxy group, a C2-C4 alkoxycarbonyl group, a trifluoromethyl group, or a trichloromethyl group, and when there is more than one, they may be the same or different, m represents an integer from 0 to 2, R 2 is selected from the group consisting of a fluorine atom; a chlorine atom; a trifluoromethyl group; a trichloromethyl group; a C1-C4 alkyl group; a C2-C4 alkoxycarbonyl group; a C1-C3 alkyl group and a C2-C4 alkoxycarbonyl group.
• A represents a nitrogen atom
• B represents a carbon atom or a nitrogen atom
• amide group which may be substituted with one or two groups; or one or two selected from the group consisting of a C1-C3 alkyl group, a C2-C4 alkoxycarbonyl group, and a halogen atom, preferably a fluorine atom;
• A represents a carbon atom or a nitrogen atom
• B represents a carbon atom or a nitrogen atom.
• a and B are both carbon atoms
• A is a carbon atom and B is a nitrogen atom
• A is a nitrogen atom and B is a carbon atom
• a and B are both nitrogen atoms. That is, the ring containing A and B in formula (1) is a benzene ring, a pyridine ring, a pyridazine ring, a pyridine ring, or a pyridazine ring.
• both A and B are carbon atoms, A is a carbon atom and B is a nitrogen atom, or A is a nitrogen atom and B is a carbon atom; It is more preferable that both B are carbon atoms, or A is a nitrogen atom and B is a carbon atom.
• the carbon atom may have a substituent R 1 .
• R 1 is preferably a fluorine atom, a chlorine atom, a bromine atom, a C1-C4 alkyl group, a C1-C4 alkoxy group, a C2-C5 acyloxy group, a C2-C5 alkoxycarbonyl group, or a trihalogenomethyl group, and a fluorine atom, A chlorine atom, a C1-C4 alkyl group, a C1-C4 alkoxy group, a C2-C4 alkoxycarbonyl group, a trifluoromethyl group, a trichloromethyl group, or a tribromomethyl group are more preferable, and a chlorine atom, a C1-C3 alkyl group, a C1 -C3 alkoxy group, C2-C4 alkoxycarbonyl group, trifluoromethyl group, or trichloromethyl group is more preferred
• R 1 is located at the ortho position to the carbon atom directly bonding to the nitrogen atom constituting (NH) p among the carbon atoms constituting the benzene ring. Preferably, it is bonded to at least one of two carbon atoms.
• R 1 bonded to the carbon atom located at the ortho position include an alkoxy group or an alkoxycarbonyl group, preferably a C2-C5 alkoxycarbonyl group, more preferably a C2-C4 alkoxycarbonyl group, and a methoxycarbonyl group or an ethoxycarbonyl group.
• a carbonyl group is more preferred, and an ethoxycarbonyl group is particularly preferred.
• the ring to which R 1 is bonded is a benzene ring, and among the carbon atoms constituting the benzene ring, it is located at the meta position with respect to the carbon atom directly bonded to the nitrogen atom constituting (NH) p .
• a halogen atom is preferable, a chlorine atom or a fluorine atom is more preferable, and a fluorine atom is particularly preferable.
• R 1 is the carbon atom that directly bonds to the nitrogen atom that constitutes (NH) p among the carbon atoms that constitute the pyridine ring or pyridazine ring. It is preferable to bond to a carbon atom located between A and A.
• R 1 may be, for example, a halogen atom, an alkyl group, an alkoxy group, or an alkoxycarbonyl group, and may be a fluorine atom, a chlorine atom, a C1-C4 alkyl A C1-C4 alkoxy group or a C2-C5 alkoxycarbonyl group is preferred, a C1-C4 alkoxy group is more preferred, and a methoxy group or an ethoxy group is particularly preferred.
• m is preferably an integer of 0 to 4, more preferably an integer of 0 to 3, even more preferably 0, 1, or 2, even more preferably 0 or 1, and particularly preferably 1.
• R 2 When there is a plurality of R 2 s, they may be the same or different.
• R2 fluorine atom; chlorine atom; bromine atom; trifluoromethyl group; trichloromethyl group; tribromomethyl group; C1 to C4 alkyl group; C2 to C5 acyloxy group; C2 to C5 alkoxycarbonyl group; C1 to C3
• An optionally substituted pyrrolidinylcarbonyl group is preferred, and includes a fluorine atom; a chlorine atom; a trifluoromethyl group; a trichloro
• R 2 is one of the two carbon atoms located at the ortho position to the carbon atom directly bonded to the nitrogen atom forming the piperazine ring, among the carbon atoms forming the benzene ring to which R 2 is bonded. Or preferably, it is bonded to two carbon atoms, and more preferably to one ortho carbon atom. R 2 may be bonded to a carbon atom located at the para position. R 2 is (a) bonded to only one of the two carbon atoms in the ortho position, (b) one of the two carbon atoms in the ortho position and a carbon atom in the para position. and (c) preferably one of the two carbon atoms at the ortho position and one of the two carbon atoms at the meta position. R2 is (a) bonded to only one of the two carbon atoms in the ortho position, or (b) a carbon atom in the para position to one of the two ortho carbon atoms. More preferably, it is bonded to.
• R2 bonded to the carbon atom located at the ortho position is preferably a halogen atom, a trihalogenomethyl group, or a C1-C3 alkyl group, such as a fluorine atom, a chlorine atom, a bromine atom, a trifluoromethyl group, a methyl group, or an ethyl group.
• a group is more preferable, and a chlorine atom, a trifluoromethyl group, or a methyl group is particularly preferable.
• R2 bonded to the carbon atom located at the para position is substituted with one or two groups selected from the group consisting of, for example, a halogen atom, an alkyl group; an acyloxy group; an alkoxycarbonyl group; an alkyl group and an alkoxycarbonyl group.
• an amide group which may be substituted a piperidinylcarbonyl group which may be substituted with one or more groups selected from the group consisting of an alkyl group and an alkoxycarbonyl group; or a group consisting of an alkyl group and an alkoxycarbonyl group It may be a pyrrolidinylcarbonyl group which may be substituted with one or more groups selected from: chlorine atom; bromine atom; fluorine atom; C1-C3 alkyl group; C2-C4 acyloxy group; C2 -C4 alkoxycarbonyl group; amide group optionally substituted with one or two groups selected from the group consisting of C1-C3 alkyl group and C2-C4 alkoxycarbonyl group; C1-C3 alkyl group and C2-C4 a piperidinylcarbonyl group optionally substituted with one or more groups selected from the group consisting of alkoxycarbonyl groups; or selected from the group consisting of C1-C3
• amide group substituted with one or two groups piperidinylcarbonyl group; or piperidinyl optionally substituted with one or more groups selected from methoxycarbonyl group and ethoxycarbonyl group; More preferred is a carbonyl group.
• n is preferably an integer from 0 to 4, more preferably an integer from 0 to 3, even more preferably 0, 1, or 2, even more preferably 1 or 2, and particularly preferably 2.
• the "halogen atom” includes a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
• Preferable examples include fluorine atom, chlorine atom, and bromine atom, more preferably fluorine atom and chlorine atom.
• the "alkyl group” includes, for example, a C1-C8 alkyl group containing a linear, branched, or cyclic structure, preferably a C1-C6 alkyl group, more preferably a C1-C4 alkyl group. groups, particularly preferably C1-C3 alkyl groups.
• linear or branched alkyl groups include methyl group, ethyl group, 1-propyl group, 2-propyl group, 1-butyl group, 2-butyl group, isobutyl group, and tert-butyl group.
• alkyl groups containing a cyclic structure examples include cyclopropyl group, cyclopropylmethyl group, cyclobutyl group, cyclo Examples include butylmethyl group, cyclopentyl group, cyclopentylmethyl group, cyclohexyl group, cyclohexylmethyl group, cyclohexylethyl group, and the like.
• Preferred examples include methyl group, ethyl group, 2-propyl group, t-butyl group, and cyclopropyl group.
• the "alkyl group substituted with a halogen atom” includes, for example, the alkyl group substituted with a halogen atom and having a linear, branched, or cyclic structure.
• the number of substituents an alkyl group has is 1 to the maximum number that can be substituted, preferably 3 to the maximum number that can be substituted.
• there is a plurality of halogen atoms they may be the same or different, but are preferably the same.
• Examples include C1 to C4 alkyl groups substituted with the maximum number of substitutable halogen atoms, preferably C1 to C3 alkyl groups substituted with the maximum number of substitutable halogen atoms, more preferably substitutable A C1-C2 alkyl group substituted with the maximum number of halogen atoms, particularly preferably a trihalogenomethyl group.
• alkyl groups substituted with halogen atoms include perfluoro-n-butyl group, perfluoro-t-butyl group, perfluoro-n-propyl group, perfluoro-i-propyl group, pentylfluoroethyl group, Trifluoromethyl group, perchloro-n-butyl group, perchloro-t-butyl group, perchloro-n-propyl group, perchloro-i-propyl group, pentachloroethyl group, trichloromethyl group, perbromo-n-butyl group, perbromo -t-butyl group, perbromo-n-propyl group, perbromo-i-propyl group, pentabromoethyl group, tribromomethyl group, periodo-n-butyl group, periodo-t-butyl group, periodo-n-propyl group , periodo-i-propyl
• Preferred examples of the alkyl group substituted with a halogen atom include a pentylfluoroethyl group, a trifluoromethyl group, a pentachloroethyl group, a trichloromethyl group, a pentabromoethyl group, a tribromomethyl group, a pentayodoethyl group, and a triiodoethyl group.
• a methyl group is mentioned, a trifluoromethyl group, a trichloromethyl group, and a tribromomethyl group are more preferable, and a trifluoromethyl group and a trichloromethyl group are even more preferable.
• the "alkoxy group” includes, for example, a C1-C8 alkoxy group containing a linear, branched, or cyclic structure, preferably a C1-C6 alkoxy group, more preferably a C1-C4 alkoxy group. groups, particularly preferably C1-C3 alkoxy groups.
• linear or branched alkoxy groups include methoxy group, ethoxy group, 1-propoxy group, 2-propoxy group, 1-butoxy group, 2-butoxy group, isobutoxy group, and tert-butoxy group.
• alkoxy groups containing a cyclic structure include cyclopropoxy group, cyclopropylmethoxy group, cyclobutyroxy group, cyclobutylmethoxy group, cyclopentyloxy group, cyclopentylmethoxy group, cyclohexyloxy group, cyclohexylmethoxy group, cyclohexylethoxy group, etc.
• Preferred examples include methoxy group, ethoxy group, 2-propoxy group, tert-butoxy group, and cyclopropoxy group.
• the "acyloxy group” includes, for example, a C2-C9 acyloxy group containing a linear, branched, or cyclic structure, preferably a C2-C7 acyloxy group, more preferably a C2-C5 acyloxy group. groups, particularly preferably C2-C4 acyloxy groups.
• the linear or branched acyloxy group includes a methoxycarbonyl group, an ethoxycarbonyl group, a 1-propoxycarbonyl group, a 2-propoxycarbonyl group, a 1-butoxycarbonyl group, a 2-butoxycarbonyl group, Examples include isobutoxycarbonyl group, tert-butoxycarbonyl group, n-pentyloxycarbonyl group, neopentyloxycarbonyl group, n-hexyloxycarbonyl group, isohexyloxycarbonyl group, and 3-methylpentyloxycarbonyl group.
• Acyloxy groups containing a cyclic structure include cyclopropoxycarbonyl group, cyclopropylmethoxycarbonyl group, cyclobutyroxycarbonyl group, cyclobutylmethoxycarbonyl group, cyclopentyloxycarbonyl group, cyclopentylmethoxycarbonyl group, cyclohexyloxycarbonyl group, cyclohexylmethoxy Examples include carbonyl group, cyclohexylethoxycarbonyl group, and the like. Preferred examples include methoxycarbonyl group, ethoxycarbonyl group, 2-propoxycarbonyl group, tert-butoxycarbonyl group, and cyclopropoxycarbonyl group.
• the "alkoxycarbonyl group” includes a carbonyl group bonded to the alkoxy group, such as a carbonyl group bonded to a C1 to C8 alkoxy group, which has a linear, branched, or cyclic structure. and is preferably a C2-C7 alkoxycarbonyl group, more preferably a C2-C5 alkoxycarbonyl group, particularly preferably a C2-C4 alkoxycarbonyl group.
• linear or branched alkoxy groups constituting the alkoxycarbonyl group include methoxy group, ethoxy group, 1-propoxy group, 2-propoxy group, 1-butoxy group, 2-butoxy group, isobutoxy group, tert- Examples include butoxy group, n-pentyloxy group, neopentyloxy group, n-hexyloxy group, isohexyloxy group, and 3-methylpentyloxy group.
• alkoxy groups containing a cyclic structure constituting an alkoxycarbonyl group include a cyclopropoxy group, a cyclopropylmethoxy group, a cyclobutyroxy group, a cyclobutylmethoxy group, a cyclopentyloxy group, a cyclopentylmethoxy group, a cyclohexyloxy group, and a cyclohexylmethoxy group. , cyclohexylethoxy group, and the like.
• Preferred examples of the alkoxy group constituting the alkoxycarbonyl group include methoxy group, ethoxy group, 2-propoxy group, tert-butoxy group, and cyclopropoxy group.
• an amide group which may be substituted with one or two groups selected from the group consisting of an alkyl group and an alkoxycarbonyl group means an amide group, a mono- or di-alkyl amide group, a mono- or di- It includes an alkoxycarbonylamide group, an alkyl group, and an amide group di-substituted with an alkoxycarbonyl group, preferably an amide group or a dialkylamide group, and more preferably a dialkylamide group.
• the substituents may be the same or different.
• Examples of the monoalkyl amide group include amide groups mono-substituted with the above-mentioned alkyl groups, preferably amide groups mono-substituted with C1-C4 alkyl groups, more preferably amide groups mono-substituted with C1-C3 alkyl groups. Among these groups, particularly preferred are methylamide and ethylamide groups.
• Examples of dialkylamide groups include amide groups di-substituted with the above-mentioned alkyl groups, preferably amide groups di-substituted with C1-C4 alkyl groups, more preferably amide groups di-substituted with C1-C3 alkyl groups.
• dimethylamide group diethylamide group, and ethylmethylamide group.
• the monoalkoxycarbonylamide group include an amide group mono-substituted with the above-mentioned alkoxycarbonyl group, preferably an amide group mono-substituted with a C2-C5 alkoxycarbonyl group, more preferably a mono-substituted amide group with a C2-C4 alkoxycarbonyl group.
• Substituted amide groups particularly preferred are methoxycarbonylamide and ethoxycarbonylamide groups.
• the dialkoxycarbonylamide group includes an amide group di-substituted with the alkoxycarbonyl group described above, preferably an amide group di-substituted with a C2-C5 alkoxycarbonyl group, more preferably a di-substituted amide group with a C2-C4 alkoxycarbonyl group.
• Substituted amide groups particularly preferred are di(methoxycarbonyl)amide and di(ethoxycarbonyl)amide groups.
• the amide group substituted with an alkyl group and an alkoxycarbonyl group includes an amide group di-substituted with the alkyl group and the alkoxycarbonyl group, preferably a di-substituted amide group with a C1-C4 alkyl group and a C2-C5 alkoxycarbonyl group.
• a substituted amide group more preferably a di-substituted amide group with a C1 to C3 alkyl group and a C2 to C4 alkoxycarbonyl group, particularly preferably a methylmethoxycarbonylamide group, a methylethoxycarbonylamide group, an ethylmethoxycarbonylamide group, Examples include ethyl ethoxycarbonylamide group.
• the number of substituents that the piperidinylcarbonyl group has is The number is 0 to 5, preferably 0 to 4, more preferably 0 to 3, even more preferably 0 to 2, particularly preferably 0 or 1.
• the piperidinylcarbonyl group has a substituent at least one substituent is attached to the carbon atom at the 3-position of the piperidine ring (specifically, the 3-position when the nitrogen atom constituting the piperidine ring is the 1-position). Preferably, they are bonded.
• substituents include the above-mentioned alkyl group and the above-mentioned alkoxycarbonyl group, preferably a C1-C4 alkyl group, a C2-C5 alkoxycarbonyl group, more preferably a C1-C3 alkyl group, a C2-C4 alkoxycarbonyl group. Particularly preferred are a methyl group, an ethyl group, a methoxycarbonyl group, and an ethoxycarbonyl group.
• the number of substituents that the pyrrolidinylcarbonyl group has is The number is 0 to 4, preferably 0 to 3, more preferably 0 to 2, particularly preferably 0 or 1. When there are multiple substituents, they may be the same or different.
• the pyrrolidinylcarbonyl group has a substituent at least one substituent is attached to the carbon atom at the 3-position of the pyrrolidine ring (specifically, the 3-position when the nitrogen atom constituting the pyrrolidine ring is the 1-position). Preferably, they are bonded.
• substituents include the above-mentioned alkyl group and the above-mentioned alkoxycarbonyl group, preferably a C1-C4 alkyl group, a C2-C5 alkoxycarbonyl group, more preferably a C1-C3 alkyl group, a C2-C4 alkoxycarbonyl group. Particularly preferred are a methyl group, an ethyl group, a methoxycarbonyl group, and an ethoxycarbonyl group.
• prevention means preventing the onset of heart failure or skeletal muscle failure in advance.
• treatment means: (1) Delaying the onset of heart failure or skeletal muscle failure; (2) Slowing down or stopping the progression, exacerbation, or deterioration of symptoms of heart failure or skeletal muscle failure; (3) Heart failure or (4) means a method or process aimed at curing heart failure or skeletal muscle failure. Treatment may be administered before the onset of the disease or condition as a prophylactic measure, or alternatively, treatment can be administered after the onset of the disease.
• heart failure refers to an abnormality in heart function in which the heart does not pump blood at the rate required by metabolic tissues.
• Heart failure includes a wide range of disease states such as congestive heart failure, myocardial infarction, tachycardia, familial hypertrophic cardiomyopathy, ischemic heart disease, idiopathic dilated cardiomyopathy, and myocarditis.
• Heart failure can result from many causes including, but not limited to, ischemic, congenital, rheumatic, viral, toxic or idiopathic forms.
• Chronic cardiac hypertrophy a component of heart failure, is a serious disease state that is a precursor to congestive heart failure and cardiac arrest.
• the present invention also encompasses pharmaceutical compositions for preventing and/or treating chronic cardiac hypertrophy.
• skeletal muscle insufficiency refers to a state in which muscles do not have the ability to perform their work, and symptoms appear as a decrease in muscle strength, a decrease in endurance, or both. This includes muscular dystrophy.
• a pharmaceutical composition usually means a drug for preventing or treating a disease, or for testing or diagnosing a disease.
• the TRPC3/6/7 channel activator of the present invention does not lower blood pressure and exhibits efficacy against acute exacerbation.
• TRPC3/6/7 channel inhibitors are not known to have any effect on acute exacerbation. Therefore, the present invention provides a treatment method in which the TRPC3/6/7 channel activator of the present invention is administered and then the TRPC3/6/7 channel inhibitor is administered in the early acute stage of heart failure or skeletal muscle failure. , is considered ideal.
• the pharmaceutical composition of the present invention can be formulated by methods known to those skilled in the art.
• the pharmaceutical composition of the present invention can be administered either parenterally or orally.
• the composition can be, for example, in the form of an injection, a nasal administration, a pulmonary administration, or a transdermal administration.
• parenteral administration the composition can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.
• the composition may be a tablet, capsule, pill, granule, powder, syrup, or the like.
• the administration method can be selected as appropriate depending on the age and symptoms of the patient.
• the dosage of the pharmaceutical composition containing the polypeptide can be set, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight per dose. Alternatively, the dosage may be, for example, 0.001 to 100000 mg per patient, although the present invention is not necessarily limited to these values.
• the dosage and method of administration vary depending on the patient's weight, age, symptoms, etc., and those skilled in the art can take these conditions into account and set an appropriate dosage and method of administration.
• Another aspect of the present invention is a method for treating or preventing heart failure or skeletal muscle failure, which comprises administering a TRPC3/6/7 channel activator to a subject in need of such treatment. , preferably administering a compound of the invention to such a subject.
• the present invention relates to TRPC3/6/7 channel activators for treating or preventing heart failure or skeletal muscle failure.
• the present invention relates to the use of a TRPC3/6/7 channel activator for the manufacture of a medicament for treating or preventing heart failure or skeletal muscle failure.
• formula (1) [In the formula, A represents a carbon atom or a nitrogen atom, B represents a carbon atom or a nitrogen atom, R 1 represents a halogen atom, an alkyl group, an alkoxy group, an acyloxy group, an alkoxycarbonyl group, or an alkyl group substituted with a halogen atom, and when there is more than one, they may be the same or different; m represents an integer from 0 to 5, n represents an integer from 2 to 5, At least one of the plurality of R 2 is a halogen atom, an alkyl group, or a trifluoromethyl group; the remaining R 2 are 1) substituted with one or more halogen atoms, and are selected from an alkyl group and an alkoxycarbonyl group.
• the present invention provides a method of screening for substances for preventing and/or treating heart failure or skeletal muscle failure, the screening method selecting for TRPC3/6/7 channel activators.
• the screening method of the present invention includes a step of measuring zinc concentration in cells in the presence of a test substance, and a step in which the zinc concentration increases compared to the zinc concentration in the absence of the test substance.
• a method for screening a candidate substance for a TRPC3/6/7 channel activating substance comprising the step of determining that the test substance is a candidate substance that causes activation of a TRPC3/6/7 channel.
• the cells are not limited as long as they are cultured cells. For example, when using cultured mouse or rat cardiomyocytes, it is preferable to isolate them from the heart and perform primary culture.
• HEK293 cells are used as a commercially available cell line, and activity can be observed by overexpressing TRPC3/6/7 channels.
• the cells are preferably HEK293 cells, more preferably cultured mouse or rat cardiomyocytes.
• test substance is a substance that can be evaluated to determine whether it is a candidate substance for a TRPC3/6/7 channel activator, and is not particularly limited. Examples include compounds, proteins, peptides, nucleic acids, lipids, carbohydrates, glycolipids, glycoproteins, metals, and the like. The method of administering the test substance is also not particularly limited.
• Reference example 1 Compensatory cardiac hyperactivity in response to a transient decrease in blood pressure due to intravenous administration of the vasodilator hydralazine (Hyd) Wild type (WT) and TRPC6 gene-deficient mice (TRPC6(-/-) obtained from the NIEHS Institute in the United States) (Dietrich A, Mederos YSM, Gollasch M, Gross V, Storch U, Dubrovska G, et al. (2005). Increased vascular smooth muscle contractility in TRPC6-/- mice. Molecular and cellular biology 25(16): 6980-6989.
• FIG. 1 shows that compared to wild type (WT) mice, the increase in left ventricular contractile force ( ⁇ LVdP/dtmax) is significantly attenuated in TRPC6 gene-deficient mice, while there is no difference in the increase in heart rate ( ⁇ Heart Rate). It is shown that.
• TRPC6-deficient mice showed a positive change in hydralazine-induced baroreflex responses (positive chronotropy (increase in heart rate) and positive inotropy (increase in contractile force)) compared to wild-type mice. This means that the force action is significantly weakened.
• TRPC3/6/7 channel activator PPZ2, perforin, and GSK1702934A used in the test example are represented by the following formula.
• Test example 1 TRPC6 channel activation in MLP-deficient mice
• Muscle LIM protein (MLP)-deficient mouse a dilated cardiomyopathy model mouse (Knoll R, Hoshijima M, Hoffman HM, Person V, Lorenzen-Schmidt I, Bang ML, Hayashi T, Shiga N, Yasukawa H, Schaper W, McKenna W, Yokoyama M, Schork NJ, Omens JH, McCulloch AD, Kimura A, Gregorio CC, Poller W, Schaper J, Schultheiss HP and Chien KR
• the cardiac mechanical stretch sensor machinery involves a Z disc complex that is defective in a sub set of human dilated cardiomyopathy. Cell 111 (2002b), pp. 943-55.) develops heart failure 5 weeks after birth.
• Test example 2 TRPC6 channel activation in pressure overload-induced heart failure model mice
• Pressure overload-induced heart failure model mice Male mice of the 129Sv strain (Kitajima N, et al. TRPC3 positively regulates reactive oxygen species driving maladaptive cardiac remodeling. Sci Rep 6, 37001 (2016))
• TAC transverse aortic constriction
• PPZ2 phosphodiesterase 5A regulates cardiac adrenergic stimulation by NOS3-dependent mechanism.
• Figure 3-2f TRPC6 deficiency
• Fig. 3 shows that PPZ2-treated mice had higher levels of cancer compared to mice treated with vehicle (50% (v/v) diethyl sulfoxide (DMSO)/50% (v/v) polyethylene glycol 300) (control). , showing a significant improvement in heart failure after TAC.
• vehicle 50% (v/v) diethyl sulfoxide (DMSO)/50% (v/v) polyethylene glycol 300
• Test example 3 TRPC6 channel activation in post-myocardial infarction heart failure model mice (Nishimura A, et al. Hypoxia-induced interaction of filamin with Drp1 causes mitochondrial hyperfission-associated myocardial senescence. Sci Signal 11, (2016).)
• the left anterior descending coronary artery was ligated to induce myocardial infarction (MI).
• MI myocardial infarction
• an osmotic pump containing PPZ2 was implanted, and subsequent changes in cardiac function were analyzed.
• MI cardiac function gradually decreased over time in control mice, whereas in PPZ-treated mice, the rate of increase in EF and FS increased significantly for 3 to 4 weeks after administration ( Figure 4 ).
• Test example 4 Effect on baroreceptive reflex response by inducing a transient decrease in blood pressure Hydralazine (0.5 mg/kg, 1.2 mL/h) was administered to mice that had been intraperitoneally administered PPZ2 30 minutes prior to inducing a transient decrease in blood pressure.
• TRPC6 channel inhibitor BI749327 (30 mg/kg) (In vivo selective inhibition of TRPC6 by antagonist BI 749327 ameliorates fibrosis and dysfunction in cardiac and renal disease, https://doi.org/10.1073/pnas.1815354116) was combined with PPZ2 ( 2.5 mg/kg) was administered orally 5 hours before administration.
• the obtained results are shown in Figure 5.
• the vertical axis shows the hydralazine-induced increase in left ventricular contractile force ( ⁇ LVdP/dtmax) or heart rate ( ⁇ Heart Rate).
• Administration of PPZ2 increases hydralazine-induced ⁇ LVdP/dtmax in a PPZ2 concentration-dependent manner.
• PPZ2 administration did not change the hydralazine-induced ⁇ Heart Rate.
• the hydralazine-induced increase in ⁇ LVdP/dtmax due to administration of PPZ2 was suppressed by administration of BI749327, a TRPC6 channel inhibitor. From the above, it was found that PPZ2 enhances the positive inotropic effect of the heart caused by the baroreceptive reflex in a concentration-dependent manner through activation of the TRPC6 channel.
• Test example 5 Zn 2+ concentration enhancement effect of TRPC6 channel activator GSK1702934A TRPC6 channel activator other than PPZ2 GSK1702934A (In vivo selective inhibition of TRPC6 by antagonist BI 749327 ameliorates fibrosis and dysfunction in cardiac and renal disease, https://doi.org /10.1073/pnas.1815354116) was investigated to enhance Zn 2+ concentration.
• TRPC6 WT
• Zn 2+ permeability-defective mutant KYD
• Test example 6 Effect of TRPC6 channel activator GSK1702934A and hyperforin on enhancing myocardial contractility TRPC6 channel activator other than PPZ2 GSK1702934A and hyperforin (GSK1702934A and M085 directly activate TRPC6 via a mechanism of stimulating the extracellular cavity formed by the pore helix and transmembrane helix S6, https://doi.org/10.1016/j.jbc.2021.101125) to investigate the effect of enhancing myocardial contractility.
• Neonatal rat cardiomyocytes were treated with a TRPC6 activator for 30 minutes to examine whether norepinephrine (NE) (1 ⁇ M) increased the contractile force of cardiomyocytes.
• NE norepinephrine
• FIG. PPZ2 enhanced NE-induced myocardial contraction.
• the myocardial contraction enhancing effect of PPZ2 was attenuated by treatment with TPEN, a Zn 2+ chelator. Therefore, it was revealed that PPZ2 enhances NE-induced myocardial contraction in a Zn 2+ -dependent manner (Fig. 7a). It was found that GSK1702934A and hyperforin also showed effects similar to PPZ2 ( Figure 7b).
• Test example 7 TRPC6 channel activation in muscular dystrophy model mice
• Muscular dystrophy model mice C57BL/10-mdx mice (6 weeks old, purchased from CLEA Japan) were implanted with an osmotic pump containing PPZ2 (2.5 mg/kg) intraperitoneally for 4 weeks. Continuous administration was performed.
• PPZ2 blood levels, which are markers of myopathy, were significantly reduced compared to mice treated with vehicle (50% (v/v) diethyl sulfoxide (DMSO)/50% (v/v) polyethylene glycol 300) (control). It was found that LDH (lactate dehydrogenase) and CK (creatinine kinase) were significantly inhibited (Figure 8).
• Preparation example 7 (3-chloro-4-(piperazin-1-yl)phenyl)(4,4-difluoropiperidin-1-yl)methanone dihydrochloride 1 of 4-(2-chloro-4-(4,4-difluoropiperidine-1-carbonyl)phenyl)piperazine-1-carboxylic acid tert-butyl ester (1933 mg, 4.35 mmol) prepared in Preparation Example 6 A 4 N HCl/1,4-dioxane solution (10 mL) was added to a solution of ,4-dioxane (5 mL), and the mixture was stirred at room temperature overnight.
• Example 1 2-(4-(2-chloro-4-(4,4-difluoropiperidine-1-carbonyl)phenyl)piperazin-1-yl)-N-(2-ethoxyphenyl)acetamide (3-chloro-4-(piperazin-1-yl)phenyl)(4,4-difluoropiperidin-1-yl)methanone dihydrochloride (55 mg, 0.13 mmol) prepared in Preparation Example 7, Preparation Example 1 A solution of 2-bromo-N-(2-ethoxyphenyl)acetamide (52 mg, 0.2 mmol) prepared in The mixture was heated and stirred for 10 hours.
• Example 2 2-(4-(2-chloro-4-(4,4-difluoropiperidine-1-carbonyl)phenyl)piperazin-1-yl)-N-(2-(trifluoromethoxy)phenyl)acetamide (3-chloro-4-(piperazin-1-yl)phenyl)(4,4-difluoropiperidin-1-yl)methanone dihydrochloride (41.7 mg, 0.1 mmol) prepared in Preparation Example 7, Preparation Example 2 A solution of 2-bromo-N-(2-(trifluoromethoxy)phenyl)acetamide (29.8 mg, 0.1 mmol) and DBU (60 ⁇ L, 0.4 mmol) in DMF (1 mL) was added under a nitrogen atmosphere.
• Example 3 2-(4-(2-chloro-4-(4,4-difluoropiperidine-1-carbonyl)phenyl)piperazin-1-yl)-N-(2-(2,2,2-trifluoroethoxy) phenyl)acetamide (3-chloro-4-(piperazin-1-yl)phenyl)(4,4-difluoropiperidin-1-yl)methanone dihydrochloride (41.7 mg, 0.1 mmol) prepared in Preparation Example 7, Preparation Example 3 2-bromo-N-(2-(2,2,2-trifluoroethoxy)phenyl)acetamide (31.2 mg, 0.1 mmol) and DBU (54 ⁇ L, 0.4 mmol) in DMF (1 mL).
• the solution was heated and stirred at 80° C. for 10 hours under a nitrogen atmosphere.
• the reaction solution was returned to room temperature, a saturated aqueous ammonium chloride solution was added, and extraction was performed with ethyl acetate.
• the organic layer was washed with a saturated aqueous sodium bicarbonate solution and then with saturated brine, dried over sodium sulfate, and concentrated.
• the residue was purified by medium pressure preparative silica gel column chromatography (hexane/ethyl acetate) to obtain crystals of the desired product (14.8 mg). Yield 26%.
• Test example 8 Influx of Ca 2+ into HEK293 cells that transiently express TRPC channels
• TRPC3 or TRPC6 genes were introduced into HEK293 cells (human embryonic kidney-derived cells) by lipofection. TRPC6 channel was expressed in the cell membrane. That is, by introducing TRPC6 plasmid DNA (pCI-neo (Promega)) into HEK293 cells using a lipofection agent and culturing them at 37°C for 24 to 48 hours, HEK293 cells with TRPC6 channels expressed in their cell membranes were produced. (TRPC6-expressing HEK293 cells) were generated.
• Non-Patent Document 1 S. Sawamura et al., Mol. Pharmacol., 89, 348-363 (2016)
• TRPC3 HEK293 cells in which the TRPC3 channel was expressed in the cell membrane (TRPC3-expressing HEK293 cells) were produced in the same manner.
• Ca2 + -containing solution (2mM CaCl2 , 132mM NaCl, 4mM KCl, 1mM MgCl2, 5mM glucose.5mM HEPES (pH7.4)
• Ca2 + -free solution (132mM NaCl, 4mM KCl , 1mM MgCl2 , 5mM glucose, 5mM HEPES (pH7.4)).
• Test example 8-1 Effect of Example Compounds on Ca 2+ Influx into HEK293 Cells Expressing TRPC6 Channel Changes in calcium ion concentration of the channel were measured according to the measurement method described in Test Example 8.
• PPZ2, L687, L-0042 to L-0046 (compounds shown below) are used as test compounds, and the amount added is 1 to 30 ⁇ M. Additionally, 0.1% DMSO was used instead of the test compound as a control.
• the fluorescence intensity (Ratio) of Fura-2 By measuring the fluorescence intensity (Ratio) of Fura-2 over time, the increase in Ca 2+ in HEK293 cells expressing TRPC6 channel (from the maximum Ratio value measured after addition of the test compound to the initial value (before addition of the test compound)) was determined. ⁇ Ratio value) was calculated.
• Test compounds that caused a greater Ca 2+ increase than the DMSO control were designated as substances that activate TRPC6 channels.
• L687 and PPZ2 are shown in FIG. 9, and the results for the compounds of the invention are shown in FIG. 10.
• PPZ2 is known to activate TRPC6 channels and cause Ca 2+ to flow into cells, and in this study, addition of PPZ2 resulted in a concentration-dependent increase in the ⁇ Ratio value.
• Similar compounds L687 ( Figure 9) and L-0042 to L-0046 ( Figure 10) also increased intracellular Ca 2+ concentration in a concentration-dependent manner, indicating that these compounds activate TRPC6 channels. was confirmed. In particular, the strongest TRPC6-dependent increase in intracellular Ca 2+ concentration was observed in L-0044 (Example 1 compound).
• Test example 8-2 Effect of example compounds on Ca 2+ influx into HEK293 cells expressing TRPC3 channels TRPC3-expressing HEK293 cells (TRPC3/HEK293) prepared by the method described in Test Example 81 were used, and L-0042 was used as the test compound. ⁇ L-0046 (1-30 ⁇ M) was used. The amount of Ca 2+ increase ( ⁇ Ratio) into each cell was determined in the same manner as Test Example 8-1. The results obtained are shown in FIG. In HEK293 cells expressing human TRPC3 channels, compounds L-0042 to L-0046 of the present invention increased intracellular Ca 2+ concentration in a concentration-dependent manner, indicating that these compounds also activate TRPC3 channels. This was confirmed. In particular, the strongest TRPC3-dependent increase in intracellular Ca 2+ concentration was observed in L-0043 (Example 2 compound).
• Test example 9 Suppressive effect of L-0044 on in vitro cardiac hypertrophic response
• Neonatal rat cardiomyocytes were preincubated with or without 3 ⁇ M L-0044 (Example 1 compound) for 1 hour.
• Cells were then stimulated with 100 nM endothelin-1 (ET-1) for 24 hours.
• ET-1 endothelin-1
• FIG. 12 shows that ET-1 administration induces cardiomyocyte hypertrophy
• L-0044 pretreatment reduces ET-1-mediated cardiac hypertrophy. This revealed that although L-0044 alone tends to cause weak myocardial hypertrophy, treatment with 3 ⁇ M L-0044 suppresses pathological cardiac hypertrophy caused by endothelin-1 stimulation.
• Test example 10 Effect of Example Compounds on Zn 2+ Influx into HEK293 Cells Expressing TRPC6 Channel Using TRPC6-expressing HEK293 cells (HEK293 expressing TRPC6-mCherry) or control HEK293 cells, L-0043, L- 0044, L-0046 (10 ⁇ M) or PPZ2 (30 ⁇ M) were used. In the same manner as Test Example 5, the amount of increase in Zn 2+ into each cell was determined. The obtained results are shown in FIG. 13. Figure 13 shows that L-0043 and L-0044 potently enhance Zn 2+ influx through TRPC6 at lower concentrations than PPZ2.
• Test example 11 Neonatal rat cardiomyocytes were preincubated with L-0044 (1 ⁇ M) for 30 min. The cells were placed in a microscope equipped with a high-speed camera (60 fps) and videos of cell movement were taken. Cells were electrically stimulated for 3 minutes (5 V, 1 Hz, pulse width 10 ms) and then stimulated with norepinephrine (NE) (0.1 ⁇ M) to evaluate changes in cell movement. Cardiomyocyte contractility was analyzed using the ImageJ plug-in MUSCLEMOTION (A Versatile Open Software Tool to Quantify Cardiomyocyte and Cardiac Muscle Contraction In Vitro and In Vivo, https://doi.org/10.1161/CIRCRESAHA.117.312067). The obtained results are shown in FIG. 14. Figure 14 shows that L-0044 pretreatment enhances the contractile force of cardiac myocytes induced by NE stimulation.

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