CUG repeat sequence binder

Abstract

This invention provides a novel binder for CUG repeat sequences. [Solution] The binder for the CUG repeat sequence contains compound A, which has a resonance unit of 10 RU or more as measured by surface plasmon resonance at a concentration of 25 nM and an RNA immobilization amount of (CUG)9 of 401 RU.

Description

Technical Field

[0001] The present invention relates to a binder for a novel CUG repeat sequence and the like.

Background Art

[0002] Repeat diseases (or triplet repeat diseases) are hereditary neurological diseases caused by abnormal elongation of a 3-base repeat (repeat) sequence on a gene. For example, myotonic dystrophy type 1 (DM1), which is a type of repeat disease, is a disease caused by abnormal elongation of the CTG repeat sequence present in myotonic dystrophy protein kinase (DMPK). Specifically, it is considered that the onset of DM1 is caused by a decrease in the function of a protein due to the binding and aggregation of RNA-binding proteins to RNA (CUG repeat RNA) generated by transcription from the abnormally elongated CTG repeat sequence.

[0003] So far, it has been reported that a molecule that dissociates an RNA-binding protein from an aggregate of CUG repeat RNA and an RNA-binding protein by binding to CUG repeat RNA is a molecular tool useful for the research of DM1 treatment (Non-Patent Documents 1 to 3).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0005] The object of the present invention is to provide a novel binder for CUG repeat sequences.

[0006] Another object of the present invention is to provide a novel therapeutic and / or preventive agent for recurrent disease. [Means for solving the problem]

[0007] As mentioned above, although molecules that may be useful in treating DM1 have been reported, there are no specific guidelines on which compounds will lead to the treatment of DM1, and molecular design of compounds had to be carried out through trial and error. The inventors, through diligent research to solve the above problems, discovered that the compound binds to CUG repeat sequences by exhibiting a finite value in a specific index (magnitude) determined by surface plasmon resonance (SPR) regarding binding affinity to CUG repeat sequences. They further found that this index is directly related to the improvement of splicing abnormalities caused by repeat diseases such as DM1, and that when the binding affinity is strong enough for the index to reach a specific value, it is effective in treating repeat diseases. They also found that such an index makes it easy to design molecules that can improve splicing abnormalities caused by repeat diseases such as DM1.

[0008] Meanwhile, the inventors also discovered novel compounds containing specific structural units. Furthermore, they found that these novel compounds performed well in light of the aforementioned specific indicators, and through further research, completed the present invention.

[0009] In other words, the present invention relates to the following inventions, etc. [1] A binder for CUG repeat sequences (a binder for CUG repeat sequences) containing compound A, in which the resonance units measured by surface plasmon resonance are 10 RU or more at a concentration of 25 nM and an RNA immobilization amount of (CUG)9 of 401 RU. [2] A therapeutic and / or prophylactic agent for repeat diseases, comprising compound A, wherein the resonance units measured by surface plasmon resonance are 10 RU or more at a concentration of 25 nM and an RNA immobilization amount of (CUG)9 of 401 RU. [3] The agent according to [1] or [2], wherein compound A has a skeleton capable of forming a complementary hydrogen bond with uracil. [4] Compound A is an agent according to any one of the following [1] to [3] having the structure of formula (1) below. [ka] (In the formula, Z represents an aromatic ring.) [5] Formula (1A) [ka] (In the formula, R 1 A compound having one or more constituent units of ). [6] In equation (1A), R 1 However, the compounds described in [5] are aromatic ring groups. [7] A drug described in any of the following [2]-[4], which is a disease caused by abnormal elongation of CTG repeats (CTG repeat disease). [8] A drug described in any of the following [2]-[4] and [7], in which the repeat disease is myotonic dystrophy type 1. [9] Formula (1A) [ka] (In the formula, R 1 A method of administering a compound having one or more constituent units of () to an animal (including a human).

[10] A method for treating and / or preventing recurrent disease, including the method of administration described in [9].

[11] A screening method for compound A (component A), in which the resonance unit measured by surface plasmon resonance is 10 RU or more at a concentration of 25 nM (CUG)9 RNA immobilization amount of 401 RU.

[12] The method according to

[11] , wherein compound A is a binder for a CUG repeat sequence [or a compound (component) for (effective for binding) a CUG repeat sequence].

[13] The method according to

[12] , wherein compound A is a therapeutic and / or preventive agent for recurrent disease [or a compound (component) for the treatment and / or prevention of recurrent disease (effective for the treatment and / or prevention of recurrent disease)].

[14] The use of compound A, which has a resonance unit of 10 RU or more as measured by surface plasmon resonance at a concentration of 25 nM and an RNA immobilization amount of (CUG)9 of 401 RU, as a binder for CUG repeat sequences (a binder to CUG repeat sequences).

[15] The use of compound A, which has a resonance unit of 10 RU or more as measured by surface plasmon resonance at a concentration of 25 nM and an RNA immobilization amount of (CUG)9 of 401 RU, as a therapeutic and / or prophylactic agent for repeat diseases.

[16] A method for binding compound A, which has a resonance unit of 10 RU or more as measured by surface plasmon resonance at a concentration of 25 nM and an RNA immobilization amount of (CUG)9 of 401 RU, to CUG repeat sequences by administering it to animals, including humans.

[17] A method for treating and / or preventing repeat diseases, comprising administering compound A, which has a resonance unit of 10 RU or more as measured by surface plasmon resonance at a concentration of 25 nM and an RNA immobilization amount of (CUG)9 of 401 RU, to an animal including a human. [Effects of the Invention]

[0010] According to the present invention, a novel binder for CUG repeat sequences can be provided. Such agents can improve splicing abnormalities caused by repeat diseases (e.g., DM1).

[0011] The present invention may provide a novel therapeutic and / or preventive agent for recurrent disease. Because such agents can bind to CUG repeat sequences, they can treat and / or prevent diseases caused by CUG repeats (or diseases caused by abnormal elongation of CTG repeats, or CTG repeat diseases), which are transcripts of abnormally elongated CTG repeats. Furthermore, because such agents can bind to CUG repeat sequences, they can treat and / or prevent diseases caused by abnormal elongation of CAG repeats, which are complementary strands of CTG repeats (CAG repeat diseases).

[0012] The present invention may provide novel compounds.

[0013] One aspect of the present invention may provide a novel therapeutic and / or preventive agent for recurrent disease. [Brief explanation of the drawing]

[0014] [Figure 1] This shows the SPR measurement results in Example 1. [Figure 2] This shows the SPR measurement results in Example 2. [Figure 3] This figure shows normal splicing and splicing in DM1 patients for Atp2a1 (sarcoplasmic reticulum Ca2+ / ATPase gene). [Figure 4] These are the evaluation results for Example 3. [Figure 5] This is the evaluation result in Reference Example 1. [Figure 6] This figure shows normal splicing and splicing in DM1 patients in the Clcn1 (muscle-specific chloride channel gene). [Figure 7] These are the evaluation results for Example 4. [Figure 8] This is the evaluation result in Reference Example 2. [Modes for carrying out the invention]

[0015] [agent] The agent of the present invention comprises compound A, in which the resonance units (hereinafter sometimes simply referred to as "indicator A") measured by surface plasmon resonance at a concentration of 25 nM, an RNA immobilization amount of (CUG)9 of 401 RU, are 10 RU or more.

[0016] Surface plasmon resonance may be measured with a compound concentration of 25 nM and an RNA immobilization amount of 401 RU at (CUG)9, or it may be calculated using values ​​measured at other compound concentrations and / or RNA immobilization amounts. For example, if the resonance unit measured at compound concentration Y (nM) and RNA immobilization amount Z (RU) of (CUG)9 is W (RU), the resonance unit at compound concentration 25 nM and RNA immobilization amount 401 RU of (CUG)9 can be calculated using the formula W × (401 / Z) × (25 / Y).

[0017] Surface plasmon resonance can be measured, for example, by the method described later. For example, a BIAcore T200 (manufactured by GE Healthcare) can be used as a surface plasmon resonance measurement device. Furthermore, for surface plasmon resonance measurements, for example, a sensor tip coated with streptavidin can be used. Furthermore, as a measurement buffer for surface plasmon resonance measurements, for example, HEPES buffer (e.g., a sodium chloride solution of HEPES buffer, such as a 500 mM sodium chloride solution of 10 mM HEPES buffer) can be used.

[0018] Furthermore, among compounds where the above index A is 10RU or higher, compound A that does not show abrupt dissociation in the response curve of surface plasmon resonance, but rather dissociates after maintaining the bonded state to some extent, is more likely to exhibit good bonding properties with CUG repeats.

[0019] The agent of the present invention can function as a binder for CUG repeat sequences, a therapeutic and / or preventive agent for repeat disease, etc. The agent of the present invention may contain one or more compounds A.

[0020] (Compound A) Compound A typically has an index A of 10 RU or more, preferably 11 RU or more (for example, 12 RU or more, 13 RU or more, etc.). If the index A is 10 RU or higher, then Atp2a1 (sarcoplasmic reticulum Ca 2+ Because normal splicing is more likely to occur in genes such as the ATPase gene, it may be effective in treating and / or preventing repeat diseases. As will be discussed later, the skeleton, aromatic ring (and the type of substituents thereof), and number of these elements that can form hydrogen bonds complementary to uracil tend to be factors that increase the value of index A. Therefore, compounds having such skeletons can be suitably used not only as compound A, but also as a skeleton that constitutes part of compound A (or as a raw material or precursor, for example, JM608 described later).

[0021] Examples of compound A include those having hydrogen bonding groups (e.g., proton donors, proton acceptors, etc.). The hydrogen bonding group may be a hydrogen bond donor (or proton donor), a hydrogen bond acceptor (or proton acceptor), or the like. Compound A may have one or more hydrogen bonding groups. Furthermore, compound A may have one or more hydrogen bonding groups.

[0022] The hydrogen bond donor group (or proton donor) may be a group having a hydrogen atom bonded to a highly electronegative atom (for example, an atom with a Pauling electronegativity of 3 or higher, such as a nitrogen atom or an oxygen atom). Examples of hydrogen bond donor groups include -NH- groups, -NH2 groups, and -OH groups. In compound A, the number of hydrogen bond donor groups may be, for example, one or more (for example, two or more), preferably three or more.

[0023] The hydrogen bond acceptor (or proton acceptor) may be a group having an atom with a lone pair of electrons (e.g., a N atom, an O atom, etc.) (e.g., a carbonyl group, etc.). In compound A, the number of hydrogen bond acceptors may be, for example, one or more, preferably two or more.

[0024] Compound A is preferably one that has a skeleton capable of forming complementary hydrogen bonds with uracil. The number of hydrogen bonds may be, for example, one or more, preferably two or more, and more preferably three or more.

[0025] Compound A may be positively charged (cationic) when dissolved in water. Compound A may itself be basic. The basic group that compound A possesses may be, for example, an amino group. The number of basic groups is not particularly limited and may be one or more (e.g., 1 to 10, 1 to 7, 1 to 5, 1 to 3, etc.), two or more (e.g., 2 to 10, 2 to 7, 2 to 5, 2 to 3, etc.), etc.

[0026] Examples of compound A include those having an amide bond (-CONH-), those having an aromatic ring, and those having a structure of formula (1) below in which an amide bond and an aromatic ring are bonded.

[0027] Formula (1) [ka] (In the formula, Z represents an aromatic ring.)

[0028] In equation (1), Z may be any aromatic ring, a hydrocarbon aromatic ring, or a heterocycle. The number of elements in Z is not particularly limited and may be, for example, 3 to 12 (for example, 4 to 12). Furthermore, Z may be a monoring or a fused ring.

[0029] When Z is a heterocycle, the heteroatoms are not particularly limited and include, for example, nitrogen atoms, oxygen atoms, sulfur atoms, etc., with nitrogen atoms, oxygen atoms, etc. being preferred. The heterocycle may have one or more heteroatoms. Furthermore, in a heterocycle, the number of heteroatoms is not particularly limited and may be one or more (for example, 1 to 5, 1 to 3, etc.). Furthermore, in a heterocycle, the bonding sites of heteroatoms are not particularly limited.

[0030] Furthermore, Z may have substituents. The substituents are not particularly limited and include, for example, alicyclic groups, aromatic ring groups, hydrocarbon groups [for example, alkyl groups (e.g., methyl group, ethyl group, etc.)]. 1-5 C(alkyl groups, etc.), alkenyl groups (e.g., ethyl group, propenyl group, etc.) 2-5 Alkenyl groups, etc., alkynyl groups (e.g., ethynyl group, propynyl group, etc.) 2-5 Examples of substituents include alkynyl groups, amino groups, and nitro groups, and those having a ring structure (e.g., alicyclic groups, aromatic ring groups) are preferred. Furthermore, if the substituent has a ring structure, the ring structure may be hydrocarbon-based or heterocyclic.

[0031] If the substituent at Z has a ring structure, the number of ring members is not particularly limited and may be, for example, 3 to 10 (e.g., 4 to 8).

[0032] The number of substituents in Z may be one or two or more. Furthermore, the substituents may be one or two or more types. The position of the substituents is not particularly limited, but it is preferable that they are bonded to a carbon atom.

[0033] Compound A having the structure of formula (1) is specifically the following: (1A) (wherein R 1 ' represents a substituent.' Examples include compounds having one or more constituent units of ').

[0034] Formula (1A) [Chemical formula]

[0035] In formula (1A), R 1 is not particularly limited, and examples thereof include the substituents of Z exemplified above. R 1 is preferably an aromatic ring group (e.g., a hydrocarbon-based aromatic ring group, a heteroaromatic ring group, etc.), an alkynyl group (e.g., a C 2-5 alkynyl group such as ethynyl group, propynyl group, etc.), an amino group, a nitro group, etc. Among them, in a repeat sequence (e.g., CUG repeat), a compound A bound to the repeat sequence and a base pair (e.g., G-C base pair in CUG repeat) can exhibit stacking interaction, or have a certain degree of rigidity and bulkiness. From the viewpoint that compound A is likely to be fixed to the repeat sequence (or RNA having a repeat sequence), an aromatic ring group is particularly preferred.

[0036] Typical examples of the heteroaromatic ring group include a pyridinyl group, a pyrimidinyl group, a pyrazinyl group, a pyrrolyl group, a furanyl group, etc. Typical examples of the hydrocarbon-based aromatic ring group include an aryl group (e.g., a phenyl group, a tolyl group, a xylyl group, a naphthyl group, etc.).

[0037] R 1 may further have a substituent (a). The number of the substituent (a) may be one or two or more. Also, the substituent (a) may be one type or two or more types. The position of the substituent (a) is not particularly limited, but it is preferably bonded to a carbon atom.

[0038] Examples of substituents (a) include substituents in Z as illustrated above, but from the viewpoint of having a certain degree of rigidity and bulkiness, compound A is more easily immobilized on the repeat sequence (or RNA having a repeat sequence), alicyclic heterocyclic groups (for example, groups derived from alicyclic amines) are preferred.

[0039] Typical substituents (a) include, for example, the following structures (groups).

[0040] [ka]

[0041] Also, representative R 1 Examples include the following structures (bases). In each of the following structures, X represents a substituent (a), and n represents 0 or an integer greater than or equal to 1. When n is 2 or greater, the substituents may be the same or different substituents. The upper limit of the number of substituents (a) n can be selected depending on the type of group to be substituted (aromatic ring group). For example, when the group to be substituted is pyridyl, the upper limit may be 4, and when the group to be substituted is furyl, it may be 3. n is typically 0 to 3, preferably 0 to 2, more preferably 0 or 1, and especially may be 1.

[0042] [ka]

[0043] The above R 1 Among these, the following structures (bases) are preferred.

[0044] [ka]

[0045] If compound A has the constituent units of formula (1A), the number of constituent units of formula (1A) in compound A may be one or more, but from the viewpoint of easily satisfying indicator A, it is preferable to have two or more (for example, 2 to 5 units), three or more units, etc.

[0046] If compound A has two or more constituent units of formula (1A), each constituent unit may be directly bonded or bonded via a linking group. The linking group is not particularly limited, but examples include heteroatom-containing groups [e.g., ether group (-O-), thioether group (-S-), carbonyl group (-CO-), thiocarbonyl group (-CS-), imino group (-NH-), amide group (-NCO-), carbamoyl group (-NCOO-), etc.], hydrocarbon groups [e.g., saturated or unsaturated hydrocarbon groups (e.g., alkylene or alkylidene groups (e.g., methylene group, ethylene group, trimethylene group, propylene group, tetramethylene group, etc.) C 1-10 Alkylene or alkylidene group), cycloalkylene or cycloalkylidene group (e.g., cyclopropylene group, cyclobutylene group, cyclohexylene group, etc.) 3-10 Cycloalkylene or cycloalkylidene group), alkenylene group (e.g., vinylene group, etc.) 2-10 Alkenylene group, etc., Arylene group (for example, phenylene group, etc.) 6-10 Examples include groups formed by combining (linking) two or more of these identical or different groups (arylene groups), etc. [for example, groups formed by combining a heteroatom-containing group (one or more heteroatom-containing groups) with a hydrocarbon group (one or more hydrocarbon groups) (for example, oxyalkylene groups, alkylenedioxy groups, iminoalkylene groups, etc.)]. The linking group may include a hydrogen bonding group (in particular, a group capable of forming a hydrogen bond with uracil).

[0047] The molecular weight of compound A is not particularly limited, but may be, for example, 200 or more, preferably 300 or more, and more preferably 400 or more.

[0048] Compound A having the constituent units of formula (1A) can be produced using known organic synthesis methods. Furthermore, if compound A has two or more constituent units of formula (1A), the method of linking each constituent unit is not particularly limited and can be done using known organic synthesis methods.

[0049] This invention also includes novel compounds. Examples of novel compounds include compounds in which Z is a heterofused ring in formula (1) above, and compounds having one or more constituent units of formula (1A) above. Among the compounds having the constituent unit of formula (1A) described above, a compound having one constituent unit of formula (1A) can serve as a raw material for a compound having two or more such constituent units. Furthermore, the compound in question does not need to satisfy the above indicator A requirement of 10 RU or more.

[0050] The method for measuring surface plasmon resonance is not particularly limited and may be measured by conventional methods.

[0051] Examples of surface plasmon resonance measurement devices include the BIAcore T200 (manufactured by GE Healthcare), as described in the examples below.

[0052] In surface plasmon resonance measurements, examples of sensor chips include streptavidin-coated sensor chips (SA chips).

[0053] In surface plasmon resonance measurements, examples of measurement buffers include HEPES buffer (for example, a sodium chloride solution of HEPES buffer, such as a 500 mM sodium chloride solution of 10 mM HEPES buffer).

[0054] In surface plasmon resonance measurements, examples of activation buffers include buffers of 50 mM NaOH and 1 M NaCl.

[0055] Examples of repeat diseases that the agent of the present invention is intended for include diseases caused by abnormal elongation of CTG repeats (CTG repeat diseases) [e.g., myotonic dystrophy type 1, Huntington disease-like 2, spinocerebellar degeneration type 8, Fuchs endothelial corneal dystrophy, etc.], and diseases caused by abnormal elongation of CAG repeats (CAG repeat diseases) [e.g., Huntington's disease, spinal and bulbar muscular atrophy, spinocerebellar degeneration type 2, etc.].

[0056] The target repeat disease may be one or more types.

[0057] The form of the agent of the present invention is not particularly limited, but may be, for example, an injectable preparation, eye drops, etc.

[0058] If the agent of the present invention is an injectable or ophthalmic preparation, the agent of the present invention may, in addition to compound A, optionally contain various commonly used additives (e.g., pH adjusters, buffers, stabilizers, isotonic agents, local anesthetics, etc.). Injectable or ophthalmic preparations can be prepared by adding various additives to compound A using conventional methods.

[0059] Examples of pH adjusters and buffering agents include sodium citrate, sodium acetate, sodium phosphate, and saline phosphate. Examples of stabilizers include sodium pyrosulfite, ethylenediaminetetraacetic acid (EDTA), thioglycolic acid, and thiolactic acid. Examples of local anesthetics include procaine hydrochloride and lidocaine hydrochloride. Examples of isotonic agents include sodium chloride and glucose.

[0060] Additives may be used individually or in combination of two or more types.

[0061] In the agent of the present invention, the content of compound A is not particularly limited and may be set as appropriate depending on the dose range, the number of doses, etc.

[0062] The method of administering the agent of the present invention is not particularly limited, but examples include intravenous administration, intramuscular administration, subcutaneous administration, intrathecal administration, nasal administration, etc.

[0063] The dosage range of the agent of the present invention is not particularly limited and may be set as appropriate depending on the form of administration, method of administration, type of disease, and the characteristics of the subject (body weight, age, condition, and whether or not other pharmaceuticals are being used, etc.).

[0064] The present invention is not limited to the embodiments described above, and various modifications are possible. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the present invention. [Examples]

[0065] The present invention will now be described in more detail based on examples, but the present invention is not limited to such examples.

[0066] (Synthesis Example 1) Synthesis of compounds JM608 and JM642 Compounds JM608 and JM642, with the structures shown below, were synthesized using the following synthetic scheme.

[0067] [ka]

[0068] [ka]

[0069] [ka]

[0070] In this synthesis example, commercially available reagents were used without purification. HPLC was performed using a Gilson 811C Dynamic Mixer (measurement wavelength 254 nm, Cosmosil 5C). 18 Measurements were performed using an MS-II column (150 × 20 mm) and two solvent systems (0.1% AcOH / H2O and MeCN). 1 1H NMR and 13 ¹³C NMR spectra were measured using ECS400 (JEOL), ECA600 (JEOL), and AvanceIII700 (BRUKER). ESI mass spectrometry was performed using a JEOL AccuTOF-T100N mass spectrometer.

[0071] The synthesis methods for each compound in the above synthesis scheme are shown below.

[0072] Synthesis of 5-bromo-1,3-dichloroisoquinoline (compound 2) (step a above) 1,3-Dichloroisoquinoline (compound 1) (2.0 g, 10.1 mmol) and N-bromosuccinimide (2.3 g, 12.9 mmol) were mixed in super-dehydrated acetonitrile (50 mL), and sulfuric acid (2 mL) was added dropwise. The mixture was stirred at room temperature for 3 days. The resulting solid was filtered and washed with hexane. The resulting white solid was dried to obtain compound 2 (1.2 g, 44% yield).

[0073] The measurement results for compound 2 are as follows: 1 H NMR (600MHz, CDCl3): δ=8.30(d,J=8.2Hz,1H),8.03(d,J=7.6Hz,2H),7.53(m,1H). 13 C NMR (150MHz, CDCl3): δ=151.6,144.9,138.4,136.0,129.0,126.9,126.5,121.1,119.5. HRMS(ESI)m / z: Calculated value [C9H4 79 Br 35 Cl2N+Na] + ,297.8796; measured value 297.8798.

[0074] Synthesis of tert-butyl(3-((5-bromo-3-chloroisoquinoline-1-yl)aminopropyl)carbamate (compound 3) (step b above) Compound 2 (1.0 g, 3.6 mmol) was dissolved in 1,4-dioxane (8 mL), then diisopropylamine (1 mL) and N-Boc-1,3-propanediamine (3 mL) were added, and the resulting mixture was refluxed overnight. The resulting solution was neutralized with aqueous NH4Cl solution and then extracted with dichloromethane. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The resulting product was purified by silica gel column chromatography (using 1% methanol / CHCl3) to obtain compound 3 as a white solid (1.5 g, yield 54%).

[0075] The measurement results for compound 3 are as follows: 1 H NMR (600MHz, CDCl3): δ=7.87(d,J=8.2Hz,1H),7.83(d,J=7.6Hz,1H),7.27(t,J=8.2Hz,1H),7.21(s,1H),6.79(s ,1H),5.05(t,J=5.8Hz,1H),3.70(q,J=6.0Hz,2H),3.25(q,J=5.5Hz,2H),1.79(quin,J=5.8Hz,2H),1.48(s,9H). 13 C NMR (150MHz CDCl3): δ=157.3,155.9,146.5,138.1,134.4,126.0,121.9,121.1,118.1,106.9,79.8,37.7,37.2,29.9,28.6. HRMS(ESI)m / z: Calculated value [C 17 H 29 79 Br 35 ClN3O2+Na] + ,436.0398; measured value 436.0398.

[0076] tert-butyl 4-(4-(1-((3-((tert-butoxycarbonyl)amino)propyl)amino)-3-chloroisoquinoline-5-yl)pyridine-2-yl)piperazine-1-carboxylate (compound 4) (steps c and d above) Compound 3 (400 mg, 0.96 mmol), 1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-2-yl)piperazine (Compound 8) (335 mg, 1.16 mmol), potassium carbonate (400 mg, 2.89 mmol), and Pd(PPh3)4 (111 mg, 96 μmol) were mixed with a mixed solvent (1,4-dioxane (12 mL) and water (1 mL)) and stirred at 80°C for 14 hours under an argon atmosphere. After the resulting reaction mixture was cooled to room temperature, Boc2O (1.5 mL) was added and the mixture was stirred for 1 hour. The resulting reaction mixture was neutralized with an aqueous solution of NH4Cl. The organic layer was dried over anhydrous MgSO4 and then concentrated under reduced pressure. The obtained product was purified by silica gel column chromatography (30% ethyl acetate / hexane) to obtain compound 4 as a pale yellow solid (493 mg, yield 86%).

[0077] The measurement results for compound 4 are as follows: 1 H NMR (600MHz, CDCl3): δ=8.27(d,J=2.4Hz,11H),7.93(t,J=4.7Hz,1H),7.48(d,J=4.8Hz,2H),6.85(s,1H),6.71(q,J=2.1Hz,1H),6.68(t,J=5.8Hz) ,1H),6.65(s,1H),5.14(t,J=6.2Hz,1H),3.72(q,J=6.0Hz,2H),3.58(t, J=4.8Hz,8H),3.26(q,J=6.0Hz,2H),1.79(q,J=5.5Hz,2H),1.48(s,18H). 13 C NMR(150MHz,CDCl3):δ=159.6,157.2,155.9,155.0,149.3,148.1,145.5,137.3,136.7,130.7,125.3,122.4,117.0,115.2,108.1,105.5, 80.1,79.6,45.2,44.0,42.9,37.6,37.2,30.0, 28.6,28.5. HRMS(ESI)m / z: Calculated value [C 30 H 42 35 ClN6O4+H] +,597.2951; measured value 597.2955.

[0078] Synthesis of tert-butyl 4-(4-(3-(((3-((tert-butoxycarbonyl)amino)propoxy)carbonyl)amino)-1-((3((tert-butoxycarbonyl)amino)propyl)amino)isoquinoline-5-yl)pyridine-2-yl)piperazine-1-carboxylate (compound 5) (step e above) A mixture of compound 4 (144 mg, 241 μmol), tert-butyl(3-(carbamoyloxy)propyl)carbamate (compound 9) (158 mg, 722 μmol), cesium carbonate (235 mg, 722 μmol), XPhos Pd G3 (20 mg, 24 μmol), and super-dehydrated 1,4-dioxane (9 mL) was refluxed under an argon atmosphere for 15 hours. The resulting reaction mixture was cooled to room temperature, diluted with ethyl acetate, filtered through a silica short plug, and concentrated under vacuum. The resulting product was purified by silica gel column chromatography (40% ethyl acetate / hexane) to obtain compound 5 as a pale yellow solid (73.2 mg, yield 39%).

[0079] The measurement results for compound 5 are as follows: 1 H NMR (600MHz, CDCl3): δ=8.28(d,J=5.6Hz,1H),7.84(d,J=8.2Hz,1H),7.50(s,1H),7.46(d ,J=7.3Hz,1H),7.34(t,J=7.7Hz,1H),7.12(s,1H),6.78(d,J=4.7Hz,2H),6.34(s,1H),5.1 0(s,1H),4.84(s,1H),4.16(t,J=6.0Hz,2H),3.65(q,J=6.2Hz,2H),3.58(d,J=26.2Hz,8H) ,3.26(q,J=5.7Hz,2H),3.18(s,2H),1.80(m,2H),1.78(m,2H),1.48(s,18H),1.42(s,9H). 13C NMR(150MHz,CDCl3):δ=159.5,157.0,156.1,155.2,155.0,153.1,149.9,148.0,145.4,137.4,137.1,130.7,123.4,122 .2,116.1,115.2,108.6,92.6,80.0,79.6,79.3,62.7,45.2,44.1,42.9,37.6,37.5,37.3,29.9,29.5,28.6,28.6,28.6. HRMS(ESI)m / z: Calculated value [C 40 H 58 N8O8+H] + ,779.4450; measured value 779.4443.

[0080] Synthesis of 3-aminopropyl(1-((3-aminopropyl)amino)-5-(2-(piperazine-1-yl)pyridine-4-yl)isoquinoline-3-yl)carbamate (compound JM608) (step g above)

[0081] [ka]

[0082] To a solution of compound 5 (8.5 mg, 10.8 μmol) in CHCl3 (1 mL), ethyl acetate containing 4 M HCl (2 mL) was added, and the mixture was stirred at room temperature for 1 hour. The solvent was evaporated until dry, and compound JM608 was obtained as a yellow solid (5.7 mg, 90% yield). The product was further purified by HPLC.

[0083] The measurement results for JM608 are as follows: 1 H NMR (600MHz, D2O): δ=8.21(d,J=5.5Hz,1H),7.96(d,J=8.2Hz,1H),7.54(d,J=7. 6Hz,1H),7.44(t,J=7.9Hz,1H),7.06(s,1H),6.94(s,1H),6.92(d,J=5.5Hz,1H), 4.20(t,J=5.8Hz,2H),3.73(t,J=5.2Hz,4H),3.67(t,J=6.5Hz,2H),3.31(t,J=5 .2Hz,4H),3.09(t,J=7.2Hz,2H),3.05(t,J=7.2Hz,2H),1.92(m,2H),1.91(m,2H) 13 C NMR(150MHz,D2O):δ=158.9,156.1,154.9,150.8,147.0,144.5,135.9,135.9,131.2,1 24.2,122.9,116.6,115.6,109.9,92.9,62.7,43.4,43.1,37.3,36.9,36.9,27.1,26.3. HRMS(ESI)m / z: Calculated value [C 25 H 34 N8O2 + 2H] 2+ ,240.1475; measured value 240.1477.

[0084] Synthesis of tert-butyl 4-(4-(3-(((3-(((benzyloxy)carbonyl)amino)propoxy)carbonyl)amino)-1-((3((tert-butoxycarbonyl)amino)propyl)amino)isoquinoline-5-yl)pyridine-2-yl)piperazine-1-carboxylate (compound 6) (step f above) A mixture of compound 4 (500 mg, 837 μmol), benzyl (3-(carbamoyloxy)propyl)carbamate (compound 10) (317 mg, 1.26 mmol), cesium carbonate (818 mg, 2.51 mmol), XPhos Pd G3 (71 mg, 84 μmol), and super-dehydrated 1,4-dioxane (35 mL) was refluxed under an argon atmosphere for 15 hours. The resulting reaction mixture was cooled to room temperature, diluted with ethyl acetate, filtered through a silica short plug, and concentrated under vacuum. The resulting product was purified by silica gel column chromatography (20-60% ethyl acetate / hexane) to obtain compound 6 as a pale yellow solid (356 mg, yield 52%).

[0085] The measurement results for compound 6 are as follows: 1HNMR(600MHz,CDCl3):δ=8.28(d,J=5.5Hz,1H),7.84(d,J=7.6Hz,1H),7.51(s,1H),7.47(d,J=6.9Hz, 1H),7.32-7.36(5H),7.30(t,J=4.1Hz,1H),7.10(s,1H),6.78(d,J=4.1Hz,2H),6.33(s,1H),5.18(s, 1H),5.08(s,2H),4.18(t,J=5.8Hz,2H),3.65(q,J=5.5Hz,2H),3.61(d,J=5.6Hz,4H),3.56(d,J=5.5H z,4H),3.26(d,J=5.5Hz,4H),1.84(t,J=6.2Hz,2H),1.78(t,J=5.5Hz,2H),1.48(s,9H),1.48(s,9H). 13 C NMR(150MHz,CDCl3):δ=159.51,156.97,156.59,155.22,155.05,153.14, 149.81,148.06,145.40,137.40,137.10,136.69,130.75,128.61,128.22, 128.19,123.45,122.15,116.09,115.13,108.61,92.67,79.99,79.67,66. 75,62.65,45.31,43.98,42.89,37.95,37.66,37.37,29.91,29.49,28.57. HRMS(ESI)m / z: Calculated value [C 43 H 56 N8O8+H] + ,813.4294; measured value 813.4298.

[0086] Synthesis of tert-butyl 4-(4-(3-(((3-aminopropoxy)carbonyl)amino)-1-((3((tert-butoxycarbonyl)amino)propyl)amino)isoquinoline-5-yl)pyridine-2-yl)piperazine-1-carboxylate (compound 7) (step h above) Compound 6 (316 mg, 389 μmol) was dissolved in methanol (170 mL), and then palladium-carbon (Pd / C) (10% by mass) (60 mg) was added. The resulting mixture was stirred at room temperature under hydrogen gas for 24 hours. After filtering out the Pd / C using a short-padded Celite column, the solvent was concentrated. The resulting product was eluted with 2% MeOH / CHCl3 using amino-coated silica gel to obtain compound 7 as a pale yellow solid (219 mg, yield 74%).

[0087] The measurement results of Compound 7 are summarized below. 1 H NMR (600MHz, CDCl3: δ=8.28(d,J=4.8Hz,1H),7.83(d,J=8.2Hz,1H),7.52(s,1H),7.47(d,J=7. 6Hz,1H),7.35(t,J=7.9Hz,1H),7.04(s,1H),6.79(d,J=5.5Hz,2H),6.28(s,1H),5.10(s,1H), 4.21(t,J=6.2Hz,2H),3.66(q,J=6.2Hz,2H),3.61(d,J=5.9Hz,4H),3.57(d,J=5.9Hz,4H),3.2 6(q,J=5.7Hz,2H),2.78(t,J=6.2Hz,2H),1.80(m,2H),1.79(m,2H),1.49(s,9H),1.48(s,9H). 13 C NMR (176MHz, CDCl3): δ=159.58,156.95,155.20,155.03,153.19,149.85,148.04,145.46,137.46,137.13,130.78,123.43,1 22.10,116.04,115.19,108.57,92.71,79.99,79.69,63.03,45.29,44.11,42.91,38.96,37.63,37.32,32.94,29.95,28.59. HRMS(ESI)m / z: Calculation value [C] 35 H 50 N8O6+H] + ,679.3926; measured value 679.3930.

[0088] Synthesis of di-tert-butyl 4,4'-((((10-(tert-butoxycarbonyl)-7,13-dioxo-2,18-dioxa-6,10,14-triazanonadecanedioyl)bis(azandiyl))bis(1-((3-((tert-butoxycarbonyl)amino)propyl)amino)isoquinoline-3,5-diyl))bis(pyridine-4,2-diyl))bis(piperazine-1-carboxylate) (compound Boc-JM642) (step i above) Compound 7 (50 mg, 73.7 μmol) and bis(perfluorophenyl) 3,3'-((tert-butoxycarbonyl)azandiyl)dipropionate (Compound 11) (19 mg, 32.5 μmol) were dissolved in CHCl3 (1 mL), and triethylamine (47 μL, 338 μmol) was added. The mixture was stirred at 50°C for 24 hours. The resulting reaction mixture was cooled to room temperature, neutralized with aqueous NH4Cl solution, and extracted with chloroform. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The resulting product was eluted by 1% MeOH / CHCl3 using silica gel column chromatography to obtain compound Boc-JM642 as a pale yellow solid (51.9 mg, yield 89%).

[0089] The measurement results for compound Boc-JM642 are as follows: 1 H NMR (600MHz, CDCl3): δ=8.25(d,J=5.5Hz,2H),7.84(d,J=8.2Hz,2H),7.50(s,2H),7.44(d,J=6.9Hz,2H), 7.39(s,2H),7.31(t,J=7.6Hz,2H),6.77(d,J=5.5Hz,4H),6.40(s,2H),5.16(t,J=6.2Hz,2H),4.11(d,J= 5.5Hz,4H),3.63(m,4H),3.61(m,8H),3.57(m,8H),3.49(m,4H),3.27(d,J=4.8Hz,4H),3.22(d,J=6.2Hz, 4H),2.42(s,4H),1.80(t,J=6.2Hz,4H),1.74(t,J=5.5Hz,4H),1.46(s,18H),1.45(s,18H),1.40(s,9H). 13C NMR(150MHz,CDCl3):δ=159.47,156.97,155.86,155.21,155.03,153.16,149.83,147.96,14 5.50,137.24,137.01,130.69,123.35,122.21,116.06,115.13,108.61,92.57,80.30,80.00, 79.56,62.86,45.33,45.14,42.85,37.71,37.42,36.46,36.07,29.81,28.94,28.53,28.48. HRMS(ESI)m / z: Calculated value [C 81 H 115 N 17 O 16 +2H] 2+ 791.9427; measured value 791.9434.

[0090] Synthesis of ((3,3'-Azandiylbis(propanoyl))bis(Azandiyl))bis(propan-3,1-diyl)bis((1-((3-aminopropyl)amino)-5-(2-(piperazine-1-yl)pyridine-4-yl)isoquinoline-3-yl)carbamate) (compound JM642)

[0091] [ka]

[0092] To a chloroform solution (2 mL) of compound Boc-JM642 (51.9 mg, 32.8 μmol), ethyl acetate containing 4 M HCl (4 mL) was added, and the mixture was stirred at room temperature for 1 hour. The solvent was evaporated until dry, and compound JM642 was obtained as a yellow solid (31.9 mg, 90% yield). The product was further purified by HPLC.

[0093] The measurement results for compound JM642 are as follows: 1H NMR(600MHz,D2O):δ=7.88(d,J=4.8Hz,2H),7.49(d,J=8.2Hz,2H),7.09(d,J=6.9Hz,2H),6.97( t,J=7.6Hz,2H),6.74(s,2H),6.52(d,J=4.8Hz,2H),6.37(s,2H),3.88(t,J=5.8Hz,4H),3.50(t ,J=5.8Hz,4H),3.40(t,J=4.8Hz,8H),3.16(t,J=4.9Hz,8H),3.10(m,4H),3.06(t,J=5.1Hz,4H) ,2.84(t,J=5.1Hz,4H),2.80(t,J=6.9Hz,4H),2.38(t,J=5.5Hz,4H),1.86(m,4H),1.62(s,4H). 13 C NMR(176MHz,D2O):δ=172.86,158.28,155.60,154.72,150.29,146.64,144.38,135.39,134.98,131.10,123.63 ,122.62,116.32,115.24,109.67,91.98,62.88,43.51,43.03,42.97,37.04,36.67,35.94,32.01,27.96,27.17. HRMS(ESI)m / z: Calculated value [C 57 H 76 N 16 [06+2H] 2+ ,541.8816; measured value 541.8821.

[0094] (Surface plasmon resonance (SPR) measurement) A streptavidin-coated sensor chip (SAchip, manufactured by GE Healthcare) is used in HBS-EP + After washing with buffer (10 mM HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, and 0.05% v / v Surfactant P20) for 6 minutes, the cells were activated by continuously injecting 30 μL of activation buffer (50 mM NaOH and 1 M NaCl) for 1 minute three times in succession. 5'-biotinylated r(CUG)9 (manufactured by Thermo Fisher Scientific Inc.) was diluted to 0.1 μM with HEPES buffer (10 mM HEPES and 500 mM NaCl), and then flowed through the sensor tip until the binding amount reached approximately 400 RU. The amount of r(CUG)9 immobilized on the surface of the sensor tip was 401 RU. Surface plasmon resonance was measured using the BIAcore T200 SPR system (manufactured by GE Healthcare). Furthermore, the following sequence was used for r(CUG)9.

[0095] [Table 1]

[0096] (Example 1) To perform the single-cycle kinetics (SCK) method, HBS-EP is applied to the sensor chip surface at 25°C. + After preparing the mixture by running the buffer at 30 μL / min for 120 seconds, compound JM608 was added using HBS-EP to achieve concentrations of 0.063 μM, 0.125 μM, 0.25 μM, 0.50 μM, and 1.0 μM. + The solution was dissolved in buffer, and the resulting solution was sequentially injected onto the sensor surface at a rate of 30 μL / min per cycle for 60 seconds at a time. The obtained SPR response curves were analyzed using BIAcore T200 evaluation software (version 2.0). The results are shown in Figure 1.

[0097] (Example 2) SPR measurements were performed in the same manner as in Example 1, except that compound JM642 was used instead of compound JM608, and compound JM642 was sequentially injected at concentrations of 6 nM, 13 nM, 25 nM, 50 nM, and 100 nM. The results are shown in Figure 2.

[0098] Furthermore, Table 2 shows the resonance units for each compound at a concentration of 25 nM for Examples 1 and 2. For Example 1, the measured value (1.9 RU) at a compound concentration of 63 nM was used, and the resonance unit at a compound concentration of 25 nM was calculated using the formula 1.9 RU × (25 nM / 63 nM).

[0099] [Table 2]

[0100] As shown in Figures 1-2 and Table 2, binding to the CUG repeat sequence was confirmed in Examples 1 and 2.

[0101] (Evaluation of improvement in splicing abnormalities in DM1 mouse models) (Example 3) Atp2a1 (Sarcoplasmic Reticulum Ca 2+ Evaluation of the ATPase gene. Homozygous HSALR transgenic mice (Science 2000, 289, 1769–1772) of strain 20b (FVB inbred background), sex and age-matched (less than 3 months old), were administered JM642 intraperitoneally daily for 5 days at the specified dose (10 mg / kg / day or 20 mg / kg / day). After treatment, the rectus femoris (quadriceps femoris) muscle was collected for splicing analysis. Total RNA extraction, cDNA synthesis, and polymerase chain reaction (PCR) were performed from the tissue according to the method described in Ann. Clin. Transl. Neurol. 2016, 3, 42–54. PCR products were separated by agarose gel electrophoresis, and the gels were stained with GelRed (Biotium). The gels were imaged using a Typhoon laser fluoroimager (GE Healthcare), and gel bands were quantified using ImageQuant (GE Healthcare). Furthermore, total RNA extraction, cDNA synthesis, and PCR were performed on mice under the same conditions as above, except that JM642 was not administered, as well as on normal mice, and the PCR products were subjected to electrophoresis in the same manner as above. The results are shown in Figure 4.

[0102] As shown in Figure 3, in ATP2a1, under normal splicing conditions, exon 22 is incorporated (remains) (Wild Type in Figure 3), but in DM1 patients, exon 22 is not incorporated. In contrast, as shown in Figure 4, when the compound JM642 was administered to the DM1 mouse model, the retention rate of Exon 22 (ex22) in Atp2a1 was high.

[0103] (Reference example 1) The evaluation was carried out in the same manner as in Example 3, except that the following compound (DDAP), synthesized according to the method described in Non-Patent Document 3 above, was used instead of compound JM642. The results are shown in Figure 5.

[0104] [ka]

[0105] As shown in Figures 4 and 5, for example, from the remaining Exon 22 percentage at a compound dose of 20 mg / kg / day, Example 3 (remaining percentage 70%) showed greater improvement in DM1 splicing abnormalities compared to Reference Example 1 (remaining percentage 30%).

[0106] (Example 4) Evaluation of Clcn1 (muscle-specific chloride channel gene) The evaluation was performed in the same manner as in Example 3, except that Clcn1 was used instead of Atp2a1. The results are shown in Figure 7.

[0107] As shown in Figure 6, in Clcn1, Exon 7a is not incorporated under normal splicing conditions (Wild Type), but it is incorporated in DM1 patients. In contrast, as shown in Figure 7, when the compound JM642 was administered to the DM1 mouse model, the skipping rate of Exon 7a (ex7a) in Clcn1 was high.

[0108] (Reference example 2) The evaluation was carried out in the same manner as in Example 4, except that compound DDAP, synthesized according to the method described in Non-Patent Document 3 above, was used instead of compound JM642. The results are shown in Figure 8.

[0109] As shown in Figures 7 and 8, for example, from the Exon 7a skip rate at a compound dose of 20 mg / kg / day, Example 4 (skip rate 70%) showed better improvement in DM1 splicing abnormalities compared to Reference Example 2 (skip rate 60%). [Industrial applicability]

[0110] According to the present invention, it is possible to provide agents useful as treatments and / or preventive agents for recurrent diseases.

Claims

1. Concentration 25nM, (CUG) ９ A binder for CUG repeat sequences, comprising compound A, in which the resonance units measured by surface plasmon resonance are 10 RU or more at an RNA immobilization amount of 401 RU.

2. Concentration 25nM, (CUG) ９ A therapeutic and / or prophylactic agent for repeat diseases, comprising compound A, wherein the resonance units measured by surface plasmon resonance are 10 RU or more at an RNA immobilization amount of 401 RU.

3. The agent according to claim 1 or 2, wherein compound A has a skeleton capable of forming a hydrogen bond complementary to uracil.

4. The agent according to any one of claims 1 to 3, wherein compound A has the structure of the following formula (1). 【Chemistry 1】 (In the formula, Z represents an aromatic ring.)

5. Formula (1A) 【Chemistry 2】 (In the formula, R １ A compound having one or more constituent units of ).

6. In equation (1A), R １ However, the compound according to claim 5 is an aromatic ring group.

7. The agent according to any one of claims 2 to 4, wherein the repeat disease is a disease caused by abnormal elongation of CTG repeats.

8. The agent according to any one of claims 2 to 4 and 7, wherein the repeat disease is myotonic dystrophy type 1.

9. Concentration 25nM, (CUG) ９ A screening method for compound A, in which the resonance units measured by surface plasmon resonance are 10 RU or more at an RNA immobilization level of 401 RU.

10. The method according to claim 9, wherein compound A is a binder for CUG repeat sequences, a therapeutic agent for repeat disease, and / or a preventive agent for repeat disease.

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