Therapeutic or prophylactic medicine for fragile x syndrome

Abstract

The present invention provides a therapeutic or prophylactic medicine for fragile X syndrome containing a compound represented by formula (I) (see the attached description for the symbols in formula (I)) or formula (II) (see the attached description for the symbols in formula (II)) or a salt thereof.

Description

Treatment or prevention of fragile X syndrome

[0001] The present invention relates to a therapeutic or preventive agent for fragile X syndrome. More specifically, the present invention relates to a therapeutic or preventive agent for fragile X syndrome, comprising dexrazoxane or an analog thereof, or crizotinib or an analog thereof.

[0002] Fragile X syndrome (FXS) is characterized by intellectual disability and affects approximately 1 in 3,600 males. Patients with FXS exhibit a broad range of autistic phenotypes, including intellectual, cognitive, and social impairments (e.g., Non-Patent Document 1). Physically, FXS patients are characterized by elongated faces, large ears, and large testes. After adolescence, various psychiatric symptoms often appear, with approximately 15% to 20% of male patients experiencing epilepsy. Other symptoms include joint hyperextensibility, flat feet, mitral valve prolapse, strabismus, otitis media, and eating disorders due to gastroesophageal reflux disease (GERD). These disorders result from the loss of the fragile X mental retardation protein (FMRP), encoded by the FMR1 (fragile X mental retardation 1) gene, during brain development. Currently, no cure exists for FXS.

[0003] FMRP is a neuronal RNA-binding protein that functions as a molecular brake on local protein synthesis during synaptic development, making it essential for maintaining normal synaptic plasticity (e.g., Non-Patent Document 2). It is known that mice with the FMR1 gene knocked out exhibit a prolonged "UP state" and increased neuronal firing rate (Non-Patent Document 1). Similarly, it is known that neuronal firing rate is increased in neurons derived from human pluripotent stem cells (iPS cells) (Non-Patent Document 3).

[0004] In FXS patients, the FMR1 gene contains repeated CGG codon sequences (usually consisting of over 200 repeats). Hypermethylation of these repeats and the CpG islands (promoter regions of the FMR1 gene) surrounding them, resulting in suppression of FMR1 gene expression, is thought to be central to the pathophysiology of FXS. It has also been reported that removal of these methylations restores FMR1 gene expression even in patients with expanded CGG codon repeats (Non-Patent Document 3).

[0005] Meanwhile, a new research concept called drug repositioning (DR) is being discussed as a way to break through the recent impasse in new drug development research. The idea is to discover new pharmacological effects from existing drugs whose safety and pharmacokinetics have already been confirmed in humans, and then to put them to practical use. Since a large amount of existing data can be used, development costs can be kept low, and there is also the added advantage of the existence of accumulated know-how and materials (such as related compounds).

[0006] Contractor A. et al., Neuron., 87(4):699-715 (2015)Darnell JC et al., Cell., 146(2):247-261 (2011)Liu XS et al., Cell., 172(5):979-992 (2018)

[0007] Therefore, an object of the present invention is to provide, through drug repositioning, a drug having therapeutic or preventive activity for FXS, and to accelerate the development of a practical pharmaceutical agent for the treatment or prevention of FXS.

[0008] The present inventors induced differentiation of neurons from iPS cells established from FXS patients, and then screened a known compound library, including drugs already on the market, for compounds that increase FMR1 gene expression using the survival of the neurons as an indicator. Through this screening, they successfully identified compounds that increase FMR1 gene expression. They also found that administering such compounds to cells increases FMR1 gene expression in a concentration-dependent manner. Based on these findings, the present inventors conducted further research and completed the present invention.

[0009] That is, the present invention is as follows: [1] A compound represented by the following formula (I):

[0010]

[0011] [In formula (I), R １ and R ２ are independently a hydrogen atom or a group selected from the group consisting of an aliphatic group having 1 to 6 carbon atoms and an alicyclic hydrocarbon group having 3 to 7 carbon atoms, in which each hydrogen atom may be substituted and which may have 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur, or R １ and R ２ may together form a crosslinking group having 2 to 6 carbon atoms] or the following formula (II):

[0012]

[0013] [In formula (II), Y represents a nitrogen atom or CZ [Z represents a hydrogen atom, a halogen atom, CN or an aliphatic group having 1 to 3 carbon atoms in which each hydrogen atom may be substituted], R １ and R ２and R independently represent a hydrogen atom or a group selected from the group consisting of an aliphatic group having 1 to 6 carbon atoms, an alicyclic hydrocarbon group having 3 to 7 carbon atoms, and a 5- to 10-membered monocyclic or bicyclic aromatic group, in which each hydrogen atom may be substituted and which may have 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or a salt thereof. [2] The therapeutic or preventive agent according to [1], wherein formula (I) is represented by the following formula (I-1):

[0014]

[0015] [In formula (I-1), R １ and R ２ are independently a hydrogen atom or a group selected from the group consisting of an aliphatic group having 1 to 6 carbon atoms and an alicyclic hydrocarbon group having 3 to 7 carbon atoms, in which each hydrogen atom may be substituted and which may have 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur, or R １ and R ２ may form a crosslinking group having 2 to 6 carbon atoms.] [3-1] R in formula (I) or (I-1) １ and R ２ are independently a hydrogen atom or an aliphatic group having 1 to 6 carbon atoms in which each hydrogen atom may be substituted. [3-2] The therapeutic or prophylactic agent according to [1] or [2], １ and R ２ are independently a hydrogen atom or an aliphatic group having 1 to 3 carbon atoms in which each hydrogen atom may be substituted. [3-3] The therapeutic or prophylactic agent according to any one of [1] to [3-1], １ [3-4] The therapeutic or prophylactic agent according to any one of [1] to [3-2], wherein R in formula (I) or (I-1) is an aliphatic group having 1 to 3 carbon atoms. ２ [4] The therapeutic or prophylactic agent according to any one of [1] to [3-3], wherein R in formula (I) or (I-1) is a hydrogen atom. １ is a methyl group, and R ２is a hydrogen atom. [5-1] The therapeutic or prophylactic agent according to [1], wherein Y in formula (II) is CZ (Z represents a hydrogen atom, a halogen atom, or CN). [5-2] The therapeutic or prophylactic agent according to [1] or [5-1], wherein Y in formula (II) is CH. [6-1] The therapeutic or prophylactic agent according to [6-2], wherein R in formula (II) is CZ (Z represents a hydrogen atom, a halogen atom, or CN). １ is a 5- to 10-membered monocyclic or bicyclic aromatic group in which each hydrogen atom may be substituted and which may have 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. [6-2] The therapeutic or prophylactic agent according to any one of [1], [5-1], and [5-2], wherein R in formula (II) is １ is a 5- to 10-membered monocyclic aromatic group in which each hydrogen atom may be substituted and which may have 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. [6-3] The therapeutic or prophylactic agent according to any one of [5-1] to [6-2], in which the hydrogen atom is substituted with an alicyclic hydrocarbon group having 3 to 7 carbon atoms which may have 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. [6-4] The therapeutic or prophylactic agent according to any one of [6-1] to [6-3], in which at least one of the heteroatoms is a nitrogen atom. [6-5] The therapeutic or prophylactic agent according to any one of [6-1] to [6-4], in which all of the heteroatoms are nitrogen atoms. [7] The therapeutic or prophylactic agent according to any one of [1] and [5-1] to [6-5], in which formula (II) is the following formula (II-1):

[0016] [8] The following formula (I):

[0017]

[0018] [In formula (I), R １ and R ２are independently a hydrogen atom or a group selected from the group consisting of an aliphatic group having 1 to 6 carbon atoms and an alicyclic hydrocarbon group having 3 to 7 carbon atoms, in which each hydrogen atom may be substituted and which may have 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur, or R １ and R ２ may together form a crosslinking group having 2 to 6 carbon atoms] or the following formula (II):

[0019]

[0020] [In formula (II), Y represents a nitrogen atom or CZ [Z represents a hydrogen atom, a halogen atom, CN or an aliphatic group having 1 to 3 carbon atoms in which each hydrogen atom may be substituted], R １ and R ２ and independently represent a hydrogen atom or a group selected from the group consisting of an aliphatic group having 1 to 6 carbon atoms, an alicyclic hydrocarbon group having 3 to 7 carbon atoms, and a 5- to 10-membered monocyclic or bicyclic aromatic group, in which each hydrogen atom may be substituted and which may have 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or a salt thereof. [9] An agent for promoting expression of an FMR1 gene, comprising a compound represented by the following formula (I):

[0021]

[0022] [In formula (I), R １ and R ２ are independently a hydrogen atom or a group selected from the group consisting of an aliphatic group having 1 to 6 carbon atoms and an alicyclic hydrocarbon group having 3 to 7 carbon atoms, in which each hydrogen atom may be substituted and which may have 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur, or R １ and R ２ may together form a crosslinking group having 2 to 6 carbon atoms] or the following formula (II):

[0023]

[0024] [In formula (II), Y represents a nitrogen atom or CZ [Z represents a hydrogen atom, a halogen atom, CN or an aliphatic group having 1 to 3 carbon atoms in which each hydrogen atom may be substituted], R １ and R ２ and R independently represent a hydrogen atom or a group selected from the group consisting of an aliphatic group having 1 to 6 carbon atoms, an alicyclic hydrocarbon group having 3 to 7 carbon atoms, and a 5- to 10-membered monocyclic or bicyclic aromatic group, in which each hydrogen atom may be substituted and which may have 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or a salt thereof.

[10] A method for treating or preventing fragile X syndrome in a mammal, comprising administering an effective amount of a compound represented by the following formula (I):

[0025]

[0026] [In formula (I), R １ and R ２ are independently a hydrogen atom or a group selected from the group consisting of an aliphatic group having 1 to 6 carbon atoms and an alicyclic hydrocarbon group having 3 to 7 carbon atoms, in which each hydrogen atom may be substituted and which may have 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur, or R １ and R ２ may together form a crosslinking group having 2 to 6 carbon atoms] or the following formula (II):

[0027]

[0028] [In formula (II), Y represents a nitrogen atom or CZ [Z represents a hydrogen atom, a halogen atom, CN or an aliphatic group having 1 to 3 carbon atoms in which each hydrogen atom may be substituted], R １ and R ２and independently represent a hydrogen atom or a group selected from the group consisting of an aliphatic group having 1 to 6 carbon atoms, an alicyclic hydrocarbon group having 3 to 7 carbon atoms, and a 5- to 10-membered monocyclic or bicyclic aromatic group, in which each hydrogen atom may be substituted and which may have 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or a salt thereof.

[11] A compound represented by the following formula (I):

[0029]

[0030] [In formula (I), R １ and R ２ are independently a hydrogen atom or a group selected from the group consisting of an aliphatic group having 1 to 6 carbon atoms and an alicyclic hydrocarbon group having 3 to 7 carbon atoms, in which each hydrogen atom may be substituted and which may have 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur, or R １ and R ２ may together form a crosslinking group having 2 to 6 carbon atoms] or the following formula (II):

[0031]

[0032] [In formula (II), Y represents a nitrogen atom or CZ [Z represents a hydrogen atom, a halogen atom, CN or an aliphatic group having 1 to 3 carbon atoms in which each hydrogen atom may be substituted], R １ and R ２ are independently a hydrogen atom or a group selected from the group consisting of an aliphatic group having 1 to 6 carbon atoms, an alicyclic hydrocarbon group having 3 to 7 carbon atoms, and a 5- to 10-membered monocyclic or bicyclic aromatic group, in which each hydrogen atom may be substituted and which may have 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or a salt thereof.

[0033] The present invention makes it possible to treat or prevent FXS. In particular, because the present invention uses an existing drug whose safety has been confirmed as an active ingredient, it is expected that the development period for clinical application can be shortened.

[0034] This figure shows the results of measuring the methylation of the promoter region of the FMR-1 gene in iPS cells derived from healthy subjects (HC) and FXS patients, as well as in neurons derived from these cells. The promoter region of the FMR-1 gene is highly methylated in FXS patient iPS cells and neurons derived from FXS patient iPS cells. The CpG positions in the figure are, from left to right, 30, 42, 44, 54, 65, 81, 97, 99, 103, 107, 112, 114, 116, 127, 132, 140, 144, 157, 159, 165, 167, and 169. This figure shows the results of measuring the expression of FMRP in iPS cells derived from healthy subjects (HC) and FXS patients, as well as in neurons derived from these cells. FMRP expression is not observed in iPS cells derived from FXS patient iPS cells or neurons derived from FXS patient iPS cells. This figure shows the results of measuring the number of synaptic puncta and neuronal activity in neurons generated from iPS cells derived from healthy subjects (HC) and FXS patients. Neurons generated from FXS patient iPS cells show an increase in the number of synapses stained with Synapsin-1 antibodies. Error bars: standard error. This figure shows raster plots of neuronal activity detection in neurons generated from iPS cells derived from healthy subjects (HC) and FXS patients. Neurons generated from FXS patient iPS cells show increased neuronal activity as assessed using a microelectrode array (MEA). This figure shows a schematic diagram and results of compound screening of approximately 600 compounds to increase FMR-1 expression. Two compounds selected from the hit compounds show an increase in FMRP expression.

[0035] As shown in the Examples below, the present inventors have discovered that administering dexrazoxane hydrochloride or crizotinib to cells increases the expression level of the FMR1 gene in the cells. Since increasing the expression level of the FMR1 gene in vivo can improve the symptoms of fragile X syndrome (FXS), dexrazoxane or its analogs and crizotinib or its analogs can be used to treat or prevent FXS. Therefore, the present invention provides a therapeutic or preventive agent for FXS (hereinafter sometimes referred to as the "medicine of the present invention") comprising dexrazoxane or its analog or crizotinib or its analog (hereinafter sometimes referred to as the "compound of the present invention"). Furthermore, unless otherwise specified, the therapeutic or preventive agent (or method) for FXS also encompasses a pharmaceutical agent (or method) capable of both treating and preventing the disease.

[0036] As used herein, the term "therapeutic drug" encompasses not only drugs intended to cure FXS, but also drugs intended to inhibit the progression of FXS, alleviate symptoms (e.g., improvement to minimal manifestations (MM) that do not interfere with daily life or work), or alleviate sequelae. For example, because FXS is a disease that progresses over a long period of time (usually over years), early initiation of treatment can prevent the progression of symptoms. Furthermore, as used herein, the term "prophylactic drug" encompasses not only drugs intended to reduce the risk of developing FXS in subjects who have not yet developed FXS, but also drugs intended to reduce the risk of FXS recurrence in subjects who have developed FXS. For example, the onset of FXS can be prevented by administering the drug of the present invention to patients with a genetic background potentially predisposed to FXS before the onset of FXS symptoms. The same applies to "therapeutic method" and "prophylactic method."

[0037] The pharmaceutical composition of the present invention can be administered orally or parenterally as an appropriate pharmaceutical composition containing the active ingredient, a compound of the present invention, either alone or mixed with a pharmacologically acceptable carrier, excipient, diluent, etc. Furthermore, the pharmaceutical composition of the present invention can be administered to mammals (e.g., humans, rats, mice, guinea pigs, rabbits, sheep, horses, pigs, cattle, dogs, cats, and monkeys). Therefore, a method for treating or preventing FXS in a mammal is also provided, which comprises administering an effective amount of a compound of the present invention to the mammal.

[0038] As used herein, FXS refers to a disease caused by the expansion of CGG codon repeats (also referred to as "CGG repeats") in the 5' untranslated region within the first exon of the FMR1 gene, and in typical FXS patients, the FRM1 gene has more than 200 CGG repeats. Hypermethylation of these repeats and the CpG islands nearby, which results in suppression of FMR1 gene expression, is thought to be central to the pathophysiology of FXS.

[0039] The compounds of the present invention can also be used as FMR1 gene expression promoters. Therefore, in another aspect of the present invention, an FMR1 gene expression promoter (hereinafter, sometimes referred to as the "expression promoter of the present invention") comprising a compound of the present invention is provided. As used herein, the expression promoter of the present invention includes any agent that promotes FMR1 gene expression at the cellular or organismal level. Furthermore, unless otherwise specified, the term "gene expression" is used herein to mean at least "production of a functional protein encoded by FMR1 mRNA," but also includes "production of FMR1 mRNA." Therefore, promotion of FMR1 gene expression may include not only an increase in the amount of functional protein encoded by the gene in cells, but also an increase in the amount of mRNA transcribed from the gene in cells, as a result of administration of a compound of the present invention.

[0040] The expression promoter of the present invention is prepared as a general pharmaceutical composition or pharmaceutical preparation, or as a cosmetic or food product, and administered orally or parenterally. The expression promoter of the present invention can also be used as a reagent. The expression promoter of the present invention can be administered to subjects, including humans (e.g., mammals, mammalian cells, tissues, organs, etc.). Therefore, a method for promoting expression of the FMR1 gene in a subject is also provided, which comprises administering a compound of the present invention to the subject.

[0041] Examples of dexrazoxane or its analogues used in the present invention include the compound represented by the following formula (I) or a salt thereof:

[0042]

[0043] [In formula (I), R １ and R ２ are independently a hydrogen atom or a group selected from the group consisting of an aliphatic group having 1 to 6 carbon atoms and an alicyclic hydrocarbon group having 3 to 7 carbon atoms, in which each hydrogen atom may be substituted and which may have 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur, or R １ and R ２ may form a crosslinking group having 2 to 6 carbon atoms.

[0044] In one embodiment, the above formula (I) includes a compound represented by the following formula (I-1) or a salt thereof.

[0045]

[0046] [In formula (I-1), R １ and R ２ is the same as that in formula (I).

[0047] In the compound represented by the above formula (I) or (I-1) or a salt thereof, R １ and R ２ are each independently a hydrogen atom or an aliphatic group having 1 to 6 carbon atoms in which each hydrogen atom may be substituted. １ and R ２ At least one of (preferably R １), each hydrogen atom of which may be substituted (preferably unsubstituted) is more preferably an aliphatic group (preferably an alkyl group) having 1 to 3 carbon atoms (preferably 1). ２ It is also preferred that R is a hydrogen atom. １ is an aliphatic group (preferably an alkyl group) having 1 to 3 carbon atoms (preferably 1), and R ２ is a hydrogen atom, or a salt thereof.

[0048] Specific examples of dexrazoxane or its analogues include a compound represented by the following formula (I-1-a) (i.e., dexrazoxane; 4-[(2S)-2-(3,5-dioxopiperazin-1-yl)propyl]piperazine-2,6-dione) (in the above formula (I-1), R １ is a methyl group, and R ２ is a hydrogen atom). Specific examples of salts of the compounds include salts represented by the following formula (I-1-b) (i.e., dexrazoxane hydrochloride; 4-[(2S)-2-(3,5-dioxopiperazin-1-yl)propyl]piperazine-2,6-dione; hydrochloride).

[0049]

[0050]

[0051] Examples of crizotinib or its analogues used in the present invention include a compound represented by the following formula (II) or a salt thereof:

[0052]

[0053] [In formula (II), Y represents a nitrogen atom or CZ [Z represents a hydrogen atom, a halogen atom, CN or an aliphatic group having 1 to 3 carbon atoms in which each hydrogen atom may be substituted], R １ and R ２are independently a hydrogen atom or a group selected from the group consisting of an aliphatic group having 1 to 6 carbon atoms, an alicyclic hydrocarbon group having 3 to 7 carbon atoms, and a 5- to 10-membered monocyclic or bicyclic aromatic group, in which each hydrogen atom may be substituted and which may have 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0054] The above formula (II) can also be shown as the following formula (II-a):

[0055]

[0056] [In formula (II-a), R １ , R ２ The definitions of Y are the same as those in formula (II).

[0057] In one embodiment, the compound or salt thereof of the formula (II) in which Y is CZ (Z represents a hydrogen atom, a halogen atom, or CN) is mentioned. Among these, the compound or salt thereof in which Z represents a hydrogen atom is preferred.

[0058] In another embodiment, R １ Examples of compounds or salts thereof include compounds in which each hydrogen atom may be substituted and which are 5- to 10-membered (preferably 5-membered) monocyclic or bicyclic aromatic groups (preferably monocyclic aromatic groups) which may have 1 to 4 (preferably 2) heteroatoms independently selected from nitrogen, oxygen, and sulfur. Furthermore, the substitution of hydrogen atoms with an alicyclic hydrocarbon group (preferably 6) having 3 to 7 carbon atoms (preferably a cycloalkyl group) which may have 1 to 4 (preferably 1) heteroatoms independently selected from nitrogen, oxygen, and sulfur (preferably a nitrogen atom) is preferred. １ When contains heteroatoms, it is preferred that at least one (and preferably all) of the heteroatoms is a nitrogen atom.

[0059] Specific examples of crizotinib or its analogs include the compound represented by the following formula (II-1) (i.e., crizotinib; 3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-2-amine). The above formula (II-1) can also be represented by the following formula (II-1-a):

[0060]

[0061]

[0062] As used herein, the term "aliphatic group" refers to a straight or branched hydrocarbon chain that is completely saturated or contains one or more unsaturated bonds. Examples of aliphatic groups include straight or branched alkyl groups (e.g., methyl, ethyl, propyl, isopropyl, butyl, 2-butyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, 3-pentyl, 3-methylbutyl, hexyl, and 3-hexyl groups), alkenyl groups (e.g., vinyl, allyl, 2-propynyl, 2-butenyl, and 3-methyl-2-butenyl groups), and the like. Examples of such groups include alkynyl groups (e.g., ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 4-methyl-2-pentynyl, and the like). Furthermore, the term "alicyclic hydrocarbon group" refers to a monocyclic or bicyclic hydrocarbon group that is completely saturated or contains one or more unsaturated bonds and does not belong to the category of aromatic hydrocarbon groups, or an aliphatic group having such a hydrocarbon group. Examples of alicyclic hydrocarbon groups include cycloalkyl groups (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl groups), cycloalkenyl groups (e.g., cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, and cycloheptenyl groups), (cycloalkyl)alkyl groups, (cycloalkenyl)alkyl groups, and (cycloalkyl)alkenyl groups.

[0063] Examples of the monocyclic or bicyclic aromatic group include an aryl group and a heteroaryl group. Examples of the aryl group include aryl groups having 10 or less carbon atoms, such as a phenyl group and a naphthyl group. Examples of heteroaryl groups include 5- to 6-membered monocyclic groups containing 1 or 2 nitrogen atoms, 5- to 6-membered monocyclic groups containing 1 or 2 nitrogen atoms and 1 oxygen atom or 1 sulfur atom, 5-membered monocyclic groups containing 1 oxygen atom or 1 sulfur atom, and bicyclic groups containing 1 to 4 nitrogen atoms in which a 6-membered ring is fused with a 5- or 6-membered ring. Specific examples include a 2-pyridyl group, a 3-pyridyl group, a 4-pyridyl group, a 2-thienyl group, a 3-thienyl group, a 3-oxadiazolyl group, a 1-imidazolyl group, a 2-imidazolyl group, a 2-thiazolyl group, a 3-isothiazolyl group, a 2-oxazolyl group, a 3-isoxazolyl group, a 2-furyl group, a 3-furyl group, a 3-pyrrolyl group, an 8-quinolyl group, a 2-quinazolinyl group, and an 8-purinyl group.

[0064] Examples of halogen atoms include fluorine, chlorine, bromine, and iodine.

[0065] As used herein, the phrase "each hydrogen atom may be substituted (or has been substituted)" means that at least one hydrogen atom of the group may be substituted (or has been substituted) with another atom or group. In other words, a group in which each hydrogen atom may be substituted (or has been substituted) can be rephrased as a group that may have a substituent (or has a substituent). In this specification, examples of the substituent include a halogen atom, a cyano group, a benzyloxy group, a trifluoromethyl group, a hydroxyl group, a lower alkoxy group, a lower alkanoyloxy group, an amino group, a mono-lower alkylamino group, a di-lower alkylamino group, a carbamoyl group, a lower alkylaminocarbonyl group, a di-lower alkylaminocarbonyl group, a lower alkoxycarbonylamino group, a carboxyl group, a lower alkoxycarbonyl group, a lower alkylthio group, a lower alkylsulfinyl group, a lower alkylsulfonyl group, a lower alkanoylamino group, a lower alkylsulfonamido group, a phthalimido group, a heteroaryl group, an aryl having a substituent (substituted aryl), a heteroaryl having a substituent (substituted heteroaryl), a saturated heterocyclic group, or a group of the formula: -NR ７ R ８ (R ７ and R ８ each independently represents a hydrogen atom, a lower alkoxy group, a lower alkyl group, a substituted lower alkyl group, a cycloalkyl group, or an aralkyl group, or R ７ and R ８are bonded to each other and, together with the nitrogen atom to which they are bonded, represent a saturated cyclic amino group having 4 to 8 carbon atoms constituting the ring. Examples of heteroaryl groups include the same heteroaryl groups as described above. Examples of saturated heterocyclic groups include 5- to 8-membered ring groups having one nitrogen atom, such as 1-piperidinyl and 1-pyrrolidinyl, 6- to 8-membered ring groups having two nitrogen atoms, and 6- to 8-membered ring groups having one nitrogen atom and one oxygen atom. Examples of substituted alkyl groups include alkyl groups having 1 to 6 carbon atoms substituted with a cycloalkyl group or a substituted cycloalkyl, and aralkyl groups or substituted aralkyl groups. Examples of aralkyl groups and substituted aralkyl groups include alkyl groups having 1 to 6 carbon atoms substituted with the above-mentioned aryl group or substituted aryl group, such as benzyl, 1-phenylethyl, 2-phenylethyl, and 2-naphthylmethyl. Unless otherwise specified, in this specification, unless there is a statement that "each hydrogen atom may be substituted," this means an unsubstituted group. In addition, in this specification, the term "lower" means having 5 or less carbon atoms (preferably 3 or less).

[0066] Dexrazoxane or its analogs, or crizotinib or its analogs, can be commercially available or produced by known methods for each compound. Dexrazoxane or its analogs can be produced, for example, by methods described in U.S. Patent No. 3,941,790, International Publication No. WO 01 / 19358, or the literature cited therein. Dexrazoxane is also sold by Kissei Pharmaceutical Co., Ltd. and other companies as a drug that suppresses tissue damage caused by extravasation (EV) of anthracycline anticancer drugs. Crizotinib or its analogs can be produced, for example, by methods described in U.S. Patent No. 7,858,643, or the literature cited therein. Crizotinib is also sold by Pfizer as an anti-cancer drug (trade name: Xalkori (registered trademark)).

[0067] Dexrazoxane or an analog thereof, or crizotinib or an analog thereof, encompasses not only the free form but also pharmacologically acceptable salts thereof. While pharmacologically acceptable salts vary depending on the type of compound, examples include inorganic base salts such as alkali metal salts (sodium salt, potassium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, etc.), aluminum salts, and ammonium salts; base addition salts such as organic base salts such as trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine; inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate; and organic acid salts such as citrate, oxalate, acetate, formate, propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, and paratoluenesulfonate.

[0068] When dexrazoxane or an analog thereof, or crizotinib or an analog thereof, has isomers such as optical isomers, stereoisomers, positional isomers, or rotational isomers, either isomer or mixture thereof is encompassed by the compounds of the present invention. For example, when dexrazoxane or an analog thereof, or crizotinib or an analog thereof, has optical isomers, optical isomers resolved from the racemate are also encompassed by the compounds of the present invention. These isomers can be obtained individually by known synthesis methods, separation methods (e.g., concentration, solvent extraction, column chromatography, recrystallization, etc.), optical resolution methods (e.g., fractional recrystallization, chiral column method, diastereomer method, etc.), etc.

[0069] Dexrazoxane or an analog thereof, or crizotinib or an analog thereof may be in the form of a crystal, and both a single crystal form and a mixture of crystal forms are encompassed in the compounds of the present invention. The crystals can be produced by crystallization using a crystallization method known per se.

[0070] Dexrazoxane or an analog thereof, or crizotinib or an analog thereof may be a solvate (e.g., a hydrate, etc.) or a non-solvate (e.g., a non-hydrate, etc.), and both are encompassed in the compounds of the present invention.

[0071] Also, isotopes (e.g., 3 H, 14 C, 35 S, 125 Compounds labeled with , for example, I, are also encompassed by the compounds of the present invention.

[0072] Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions, etc. On the other hand, compositions for parenteral administration include, for example, injections, suppositories, etc., and injections may include dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, and drip injections. These preparations contain excipients (e.g., sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, alpha starch, and dextrin; cellulose derivatives such as crystalline cellulose; organic excipients such as gum arabic; dextran; and pullulan; and inorganic excipients such as silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminometasilicate; phosphates such as calcium hydrogen phosphate; carbonates such as calcium carbonate; and sulfates such as calcium sulfate), lubricants (e.g., metal stearates such as stearic acid, calcium stearate, and magnesium stearate; talc; colloidal silica; waxes such as beeswax and Gay's wax; boric acid; adipic acid; sulfates such as sodium sulfate; glycol; fumaric acid; sodium benzoate; DL-leucine; and sodium lauryl sulfate). , lauryl sulfates such as magnesium lauryl sulfate; silicic acids such as silicic anhydride and silicic acid hydrate; and the above-mentioned starch derivatives), binders (e.g., hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, and compounds similar to the above-mentioned excipients), disintegrants (e.g., cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium, and internally crosslinked sodium carboxymethyl cellulose; chemically modified starch / celluloses such as carboxymethyl starch, carboxymethyl starch sodium, and crosslinked polyvinylpyrrolidone), emulsifiers (e.g., colloidal clays such as bentonite and Veegum; metal hydroxides such as magnesium hydroxide and aluminum hydroxide; anionic surfactants such as sodium lauryl sulfate and calcium stearate;The compositions are produced by well-known methods using additives such as cationic surfactants such as benzalkonium chloride; and nonionic surfactants such as polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, and sucrose fatty acid esters), stabilizers (paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosal; dehydroacetic acid; and sorbic acid), flavoring agents (for example, commonly used sweeteners, acidulants, fragrances, etc.), diluents, etc.;

[0073] The dosage of the compound of the present invention, which is the active ingredient of the medicament or expression promoter of the present invention, can vary depending on various conditions such as the type of compound, the symptoms, age, body weight, and drug tolerance of the subject. However, for oral administration, the lower limit is 0.1 mg (preferably 0.5 mg) and the upper limit is 1000 mg (preferably 500 mg), and for parenteral administration, the lower limit is 0.01 mg (preferably 0.05 mg) and the upper limit is 100 mg (preferably 50 mg), and can be administered 1 to 6 times per day to an adult. The dosage may be increased or decreased depending on the symptoms. In particular, when the compound of the present invention is already marketed as a pharmaceutical for diseases other than those mentioned above, the dosage can be selected appropriately for each compound within the range of confirmed safety.

[0074] The medicament or expression-promoting agent of the present invention can also be used in combination with therapeutic or preventive agents for FXS (including those currently undergoing clinical trials) (e.g., metabotropic glutamate receptor antagonists, GABA receptor modulators, minocycline, selective serotonin reuptake inhibitors, lovastatin, metformin, cannabidiol, etc.). When used as a concomitant drug, such a concomitant drug can be formulated together with the compound of the present invention and administered as a single formulation, or can be formulated separately from the compound of the present invention and administered simultaneously or at different times via the same or a different route as the medicament or expression-promoting agent of the present invention. The dosage of these concomitant drugs may be the amount normally used when the drug is administered alone, or may be reduced from the amount normally used.

[0075] When the expression promoter of the present invention is administered to cells, the administration can typically be carried out by culturing the cells in a culture medium containing the expression promoter of the present invention. Such culturing can be carried out under conditions commonly used in the art, as long as the conditions are such that the expression of the FMR1 gene can be promoted.

[0076] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0077] <Materials and Methods> Generation of iPS cells and cell culture. Human iPS cells were generated from peripheral blood mononuclear cells (PBMCs) or human skin fibroblasts derived from FXS patients (FX1, FX2, and FX3) with an expanded CGG repeat in the 5'UTR of the FMR1 gene and healthy controls (HC1 and HC2) without the expanded repeat, using lentiviral or episomal vectors carrying OCT3 / 4, Sox2, Klf4, L-Myc, Lin28, dominant-negative p53, or OCT3 / 4, Sox2, Klf4, L-Myc, Lin28, or p53-shRNA, according to previously published methods (Table 1). Cultures were maintained in feeder-free medium.

[0078]

[0079] Neuronal cell induction: Neurons were generated using the method described in WO 2014 / 148646. Briefly, a construct for Neurogenin 2 expression was introduced into iPS cells using a PiggyBac vector under tetracycline induction, and stable iPS cell lines were generated. Doxycycline was added to the iPS cells, and they were cultured in neural differentiation medium for 7 days to generate neurons.

[0080] Measurement of CpG island methylation in the FMR1 gene in iPS cells and neurons. Bisulfite sequencing was performed. Bisulfite treatment converts C to U, but not methylated C. Methylated CpG islands were identified by sequencing the target promoter region of bisulfite-treated DNA. As a result, FX patient-derived iPS cells and neurons derived from FX patient iPS cells showed nearly 100% methylation, whereas healthy donor-derived iPS cells and neurons derived from healthy donor iPS cells showed less than 20% methylation. This suggests that the FMR1 promoter region was methylated due to the expansion of CGG repeats.

[0081] Western blotting. Neurons (Day 10) generated from iPS cells were lysed in RIPA buffer and subjected to SDS-PAGE. The proteins were then transferred to a hydrophobic membrane, and FMRP protein was detected using an FMRP antibody.

[0082] Measurement of synaptic puncta numbers. Neurons generated from iPS cells were immunostained using Synapsin-1 and βIII-tubulin antibodies. Images were captured using an InCell6000, and the number of Synapsin-1-positive puncta was quantified.

[0083] Measurement of neuronal activity Neurons generated from iPS cells were cultured on an MEA electrode dish for one month, and neuronal activity was detected. The detected neuronal activity is shown as a raster plot.

[0084] High-throughput screening An overview of the screening method is shown in Figure 5. Neurons generated from iPS cells of FX patients were cultured in 96-well plates, and approximately 600 compounds were added on Day 10. RNA was extracted on Day 12, and qPCR was performed.

[0085] Statistical Analysis. All data are presented as mean ± standard error. Group comparisons were performed using one- or two-way ANOVA followed by post-hoc testing with Scheffe's multiple comparison test. p<0.05 was considered statistically significant.

[0086] Measurement of FMR1 gene expression levels Neurons generated from FX patient iPS cells were cultured in 24-well plates, and compounds were added on day 10. Protein was collected on day 12, and FMRP was quantified by ELISA.

[0087] Example 1: Analysis of FMRP expression levels in cells with expanded CGG repeats in the FMR1 gene. The level of methylation and FMRP levels in the CpG islands of the FMR1 gene were measured in iPS cells and neurons. The results are shown in Figures 1 and 2. Figures 1 and 2 show that in iPS cells and neurons with expanded CGG repeats, methylation was observed in approximately 100% of the CpG islands, and FMRP was not detected. Therefore, we decided to screen for substances that promote FMR1 gene expression.

[0088] Example 2: Phenotypic analysis of neurons with expanded CGG repeats in the FMR1 gene To analyze the phenotype of neurons with expanded CGG repeats in the FMR1 gene, the number of synaptic puncta and neuronal activity were measured. The results are shown in Figures 2 and 3. Figures 2 and 3 demonstrate that neurons with expanded CGG repeats in the FMR1 gene had a higher number of synaptic puncta and higher neuronal activity than controls. Thus, neurons with expanded CGG repeats in the FMR1 gene reflect the pathophysiology of FXS patients.

[0089] Example 3: Screening of candidate substances We used neurons with expanded CGG repeats in the FMR1 gene to screen for existing drugs that increase the expression level of the FMR1 gene. The screening results are shown in Figure 5. From the hit compounds, the following two existing drugs were selected. The expression level of the FMR1 gene is shown as a relative expression level, with the protein expression level after vehicle (DMSO) administration set to 1.

[0090] (1) Dexrazoxane hydrochloride Relative expression level of FMR1 gene: 368.2509661

[0091] (2) Crizotinib Relative expression level of FMR1 gene: 727.3654052

[0092] Example 4: Verification of the concentration-dependent FMR1 gene expression promoting effect of dexrazoxane hydrochloride or crizotinib Next, we verified whether dexrazoxane hydrochloride or crizotinib can promote FMR1 gene expression in a concentration-dependent manner. The results are shown in Figure 6. Figure 6 demonstrates that both dexrazoxane hydrochloride and crizotinib can promote FMR1 gene expression in a concentration-dependent manner.

[0093] These results demonstrate that dexrazoxane hydrochloride and crizotinib can promote FMR1 gene expression in a concentration-dependent manner, strongly suggesting that these compounds may be effective in treating or preventing fragile X syndrome.

[0094] Dexrazoxane or an analog thereof and crizotinib or an analog thereof are useful for treating or preventing fragile X syndrome. In particular, drugs that are already on the market as pharmaceuticals for other diseases have accumulated clinical and non-clinical data on safety, etc., and libraries of related compounds already exist, so it may be possible to develop pharmaceuticals that can treat or prevent fragile X syndrome quickly and at low cost.

[0095] This application is based on patent application No. 2022-045557 filed in Japan (filing date: March 22, 2022), the contents of which are incorporated in their entirety herein.

Claims

1. The following formula (II): 【Chemistry 1】 [In formula (II), Y represents a nitrogen atom or CZ (where Z represents a hydrogen atom, a halogen atom, CN, or an aliphatic group having 1 to 3 carbon atoms, in which case each hydrogen atom may be substituted), R1 and R2 independently represent a group selected from the group consisting of a hydrogen atom, or an aliphatic group having 1 to 6 carbon atoms, an alicyclic hydrocarbon group having 3 to 7 carbon atoms, and a monocyclic or bicyclic aromatic group having 5 to 10 members, where each hydrogen atom may be substituted, and which may have 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Or the following formula (I): 【Chemistry 2】 [In formula (I), R １ and R ２ R is a group that is independently selected from the group consisting of a hydrogen atom, or an aliphatic group having 1 to 6 carbon atoms and an alicyclic hydrocarbon group having 3 to 7 carbon atoms, where each hydrogen atom may be substituted, and which may have 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or R １ and R ２ However, this indicates that both may form a crosslinking group having 2 to 6 carbon atoms. A therapeutic or prophylactic agent for fragile X syndrome comprising the compound shown or a salt thereof.

2. The therapeutic or prophylactic agent according to claim 1, wherein Y in formula (II) is CZ [where Z represents a hydrogen atom, a halogen atom, or CN].

3. The therapeutic or prophylactic agent according to claim 1, wherein R1 of formula (II) is a 5-10 member monocyclic or bicyclic aromatic group in which each hydrogen atom may be substituted and which may have 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

4. The therapeutic or prophylactic agent according to any one of claims 1 to 3, wherein formula (II) is formula (II-1) shown below. 【Transformation 3】

5. R in equation (I) １ and R ２ The therapeutic or prophylactic agent according to claim 1, wherein each hydrogen atom is independently a hydrogen atom or an aliphatic group having 1 to 3 carbon atoms, in which case each hydrogen atom may be substituted.

6. A therapeutic or prophylactic agent according to claim 1 or 5, wherein formula (I) is represented by the following formula (I-1). 【Chemistry 4】 [In formula (I-1), R １ and R ２ This independently represents a hydrogen atom or an aliphatic group having 1 to 3 carbon atoms, in which case each hydrogen atom may be substituted.

7. R of formula (I-1) １ is a methyl group, and R ２ is a hydrogen atom, the therapeutic or prophylactic agent according to claim 6.

8. The following formula (II): 【Transformation 5】 [In formula (II), Y represents a nitrogen atom or CZ (where Z represents a hydrogen atom, a halogen atom, CN, or an aliphatic group having 1 to 3 carbon atoms, in which case each hydrogen atom may be substituted), R1 and R2 independently represent a group selected from the group consisting of a hydrogen atom, or an aliphatic group having 1 to 6 carbon atoms, an alicyclic hydrocarbon group having 3 to 7 carbon atoms, and a monocyclic or bicyclic aromatic group having 5 to 10 members, where each hydrogen atom may be substituted, and which may have 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Or the following formula (I): 【Transformation 6】 [In formula (I), R １ and R ２ This independently represents a group selected from the group consisting of a hydrogen atom, or an aliphatic group having 1 to 6 carbon atoms and an alicyclic hydrocarbon group having 3 to 7 carbon atoms, where each hydrogen atom may be substituted, and which may have 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or R １ and R ２ However, this indicates that both may form a crosslinking group having 2 to 6 carbon atoms. An FMR1 gene expression promoter comprising a compound represented by or a salt thereof.

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