Compound or salt thereof and method for producing same, pharmaceutical composition containing compound or salt thereof and method for producing same

Abstract

Provided is a compound represented by formula (I) or a salt thereof. In formula (I), A represents a single bond or a substituted or unsubstituted divalent aromatic hydrocarbon group, L represents a substituted or unsubstituted linear or branched chain alkylene group having 1-13 carbon atoms, X represents a group represented by -(CH2CH2O)s-, -(CH2CH2CH2O)s-, -(CH(CH3)CH2O)s-, or -(CH2)tO-, s represents a number of 1-8, and t represents a number of 2-10.

Description

[Technical Field]

[0001] The present disclosure relates to a compound or a salt thereof and a method for producing the same, a pharmaceutical composition containing the compound or a salt thereof and a method for producing the same. This application claims priority based on Japanese Patent Application No. 2023-143583, filed on September 5, 2023, the contents of which are incorporated herein by reference. [Background technology]

[0002] Epigenetic modifications, such as methylation and acetylation of histone proteins, regulate gene expression and regulate various biological phenomena. Abnormalities in these modifications can cause cancer, neurological disorders, and other diseases. To combat these abnormalities and the diseases they cause, epigenetic modifications using small molecules have emerged as a promising therapeutic strategy.

[0003] Lysine demethylase 5 (KDM5) family proteins (KDM5A-D) are iron(II) / α-ketoglutarate-dependent oxidases that oxidatively remove methyl groups from tri- or dimethyllysine 4 of histone H3 (H3K4me3 / 2). In addition to its enzymatic activity, KDM5 acts as a scaffolding protein, recruiting several repressive transcription factors, such as histone deacetylase 1 (HDAC1) and HDAC2. In other words, KDM5 epigenetically regulates gene expression in cooperation with other proteins, rather than alone. Furthermore, KDM5 has been implicated in the development of various cancers, drug resistance, and neurodegenerative diseases such as Alzheimer's disease.

[0004] To date, several KDM5 inhibitors have been identified. Non-Patent Document 1 describes a compound (KDM5-C49) represented by the following formula as a KDM5 inhibitor. Non-Patent Document 2 describes a compound (CPI-455) represented by the following formula as a KDM5 inhibitor. Non-Patent Document 3 describes a compound (S1) represented by the following formula as a KDM5 inhibitor. Non-Patent Document 4 describes a compound (S2) represented by the following formula as a KDM5 inhibitor. Non-Patent Document 5 describes a compound (S3) represented by the following formula as a KDM5 inhibitor. Non-Patent Document 6 describes a compound (TK-129) represented by the following formula as a KDM5 inhibitor.

[0005] [ka]

[0006] However, these KDM5 inhibitors have not been able to produce significant pharmacological effects, such as anticancer activity, in cell and animal experiments. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] JohanssonC.,VelupillaiS.,TumberA.,SzykowskaA.,HookwayE.S.,NowakR.P.,Strain-DamerellC.,GileadiC.,PhilpottM.,Burgess-BrownN.,WuN.,KopecJ.,NuzziA.,S teuberH., EgnerU., BadockV., MunroS., LaThangueN.B., WestawayS., BrownJ., AthanasouN., PrinjhaR., BrennanP.E., OppermannU., Nat.Chem.Biol., 12, 539-545 (2016). [Non-patent document 2] Vinogradova M.,GehlingV.S.,GustafsonA.,AroraS.,TindellJ.A.,WilsonJ.,WilliamsonK.E.,GulerG.D.,Gan gourdeP.,ManieriW.,BusbyJ.,FlynnE.M.,LanF.,KimH.J.,OdateS.,CochranA.G.,LiuY.,WongchenkoM.,YangY. ,CheungT.K.,MaileT.M.,LauT.,CostaM.,HegdeG.W.,JacksonE.,PittyR.,ArnottD.,BaileyJ.,BellonS.,Cumm ingsR.T.,AlbrechtB.K.,HarmangeJ.J.,KieferJ.R.,TrojerP.,ClassonM.,Nat.Chem.Biol.12,531-538(2016).

Table 3

Fashion 4

Wood 5

Dude 6

[0008] The present disclosure has been made to solve the above-mentioned problems, and aims to provide a compound or a salt thereof that brings about a significant pharmacological effect such as an anticancer effect in cell and animal experiments. [Means for solving the problem]

[0009] The present inventors conducted extensive research to address the above-mentioned challenges. To identify compounds or salts thereof that exhibit more potent pharmacological activity, they focused on "proteolysis targeting chimeras (hereinafter also referred to as PROTACs)," which degrade target proteins and inhibit their overall function. PROTACs are small molecules composed of two ligands. Based on previously identified KDM5 inhibitors, novel KDM5PROTAC candidate compounds were designed and synthesized. Cellular assays revealed that the disclosed compounds or salts thereof exhibited significant neurite outgrowth-promoting activity in neuroblastoma Neuro-2a cells via the degradation of KDM5A. These results suggest that KDM5PROTACs are promising drug candidates for the treatment of neurological disorders. The present inventors discovered that the disclosed compounds or salts thereof can address the above challenges, leading to the completion of the present disclosure.

[0010] That is, the gist of the present disclosure lies in the following [1] to

[10] . [1] A compound represented by the following formula (I) or a salt thereof: [ka] In formula (I), A is a single bond. or replacement oris an unsubstituted divalent aromatic hydrocarbon group, L is a substituted or unsubstituted linear or branched alkylene group having 1 to 13 carbon atoms, and X is -(CH2CH2O) s -, -(CH2CH2CH2O) s -, -(CH(CH3)CH2O) s - or -(CH2) t It is a group represented by O—, in which s is a number from 1 to 8, and t is a number from 2 to 10. [2] In the formula (I), A is a single bond or a phenylene group, L is a linear alkylene group having 3 to 10 carbon atoms, and X is —(CH2CH2O) s -, -(CH2CH2CH2O) s -, -(CH(CH3)CH2O) s - or -(CH2) t The compound or salt thereof according to [1], wherein s is a group represented by O—, s is a number of 1 to 5, and t is a number of 2 to 10. [3] In the formula (I), A is a single bond, a p-phenylene group, or an m-phenylene group, L is a linear alkylene group having 3 to 10 carbon atoms, and X is —(CH2CH2O) s -, -(CH2CH2CH2O) s - or -(CH2) t The compound or salt thereof according to [1] or [2], wherein s is a group represented by O—, s is a number of 1 to 5, and t is a number of 2 to 10. [4] The compound or salt thereof according to any one of [1] to [3], wherein the compound is a compound represented by the following formula (I-1), a compound represented by the following formula (I-2), or a compound represented by the following formula (I-3): [ka] In formula (I-1), L 1 is a linear alkylene group having 3 to 10 carbon atoms, and X 1 Ha-(CH2CH2O) s -, s is a number of 1 to 5, and in formula (I-2), L 2 is a linear alkylene group having 3 to 10 carbon atoms, and X 2 Ha-(CH2CH2O) s -or-(CH2)t O—, s is a number of 1 to 5, and t is a number of 2 to 10; and in formula (I-3), L 3 is a linear alkylene group having 3 to 10 carbon atoms, and X 3 Ha-(CH2CH2O) s -or-(CH2) t It is a group represented by O—, in which s is a number of 1 to 5, and t is a number of 2 to 10. [5] The compound, In the above formula (I-1), L 1 is a linear alkylene group having 5 carbon atoms, and X 1 -(CH2CH2O) s -, and s is 3; In the above formula (I-1), L 1 is a linear alkylene group having 8 carbon atoms, and X 1 -(CH2CH2O) s -, and s is 3; In the above formula (I-2), L 2 is a linear alkylene group having 5 carbon atoms, and X 2 -(CH2CH2O) s -, and s is the number 3; In the above formula (I-2), L 2 is a linear alkylene group having 5 carbon atoms, and X 2 -(CH2) t a compound represented by O- and wherein t is the number 6; In the above formula (I-3), L 3 is a linear alkylene group having 5 carbon atoms, and X 3 -(CH2CH2O) s -, where s is the number 3, or In the above formula (I-3), L 3 is a linear alkylene group having 5 carbon atoms, and X 3 Ga-(CH2) t a compound represented by O- and wherein t is the number 6; The compound or salt thereof according to any one of [1] to [4], wherein: [6] A pharmaceutical composition comprising the compound or salt thereof according to any one of [1] to [5]. [7] The pharmaceutical composition according to [6], which is a KDM5 inhibitor and a KDM5 decomposer. [8] The pharmaceutical composition according to [6] or [7], which is an agent for preventing and / or treating a KDM5-related disease. [9] The compound or salt thereof according to any one of [1] to [5], for use in the prevention and / or treatment of a KDM5-related disease in which KDM5 inhibitory activity and KDM5 decomposition activity are effective.

[10] The pharmaceutical composition according to any one of [6] to [8], for use in the prevention and / or treatment of a KDM5-related disease in which the KDM5 inhibitory activity and KDM5 decomposition activity are effective. [ 11 Use of the compound or salt thereof according to any one of [1] to [5] and [9] in the manufacture of a pharmaceutical composition for the prevention and / or treatment of a KDM5-related disease in which KDM5 inhibitory activity and KDM5 decomposition activity are effective. [ 12

[0023] A preventive and / or therapeutic agent for a KDM5-associated disease, comprising the compound or salt thereof according to any one of [1] to [5] and [9] as an active ingredient. [Effects of the Invention]

[0011] According to the present disclosure, it is possible to provide a compound or a salt thereof that exhibits a significant pharmacological effect such as an anti-cancer effect in cell and animal experiments. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the 1H-NMR spectrum of compound 14a. [Figure 2] FIG. 1 shows the 13C-NMR spectrum of compound 14a. [Figure 3] FIG. 1 shows the HPLC UV absorption spectrum of compound 14a. [Figure 4] FIG. 1 shows the 1H-NMR spectrum of compound 14b. [Figure 5] FIG. 1 shows the 13C-NMR spectrum of compound 14b. [Figure 6]FIG. 1 shows the HPLC UV absorption spectrum of compound 14b. [Figure 7] FIG. 1 shows the 1H-NMR spectrum of compound 20a. [Figure 8] FIG. 1 shows the 13C-NMR spectrum of compound 20a. [Figure 9] FIG. 1 shows the HPLC UV absorption spectrum of compound 20a. [Figure 10] FIG. 1 shows the 1H-NMR spectrum of compound 20b. [Figure 11] FIG. 1 shows the 13C-NMR spectrum of compound 20b. [Figure 12] FIG. 1 shows the HPLC UV absorption spectrum of compound 20b. [Figure 13] FIG. 1 shows the 1H-NMR spectrum of compound 23a. [Figure 14] FIG. 1 shows the 13C-NMR spectrum of compound 23a. [Figure 15] FIG. 1 shows the HPLC UV absorption spectrum of compound 23a. [Figure 16] FIG. 1 shows the 1H-NMR spectrum of compound 23b. [Figure 17] FIG. 1 shows the 13C-NMR spectrum of compound 23b. [Figure 18] FIG. 1 shows the HPLC UV absorption spectrum of compound 23b. [Figure 19] (A) Structures of KDM5 inhibitors 24 and vorinostat (25). (B) Effects of single treatment with compounds 24 and 25, and cotreatment with compounds 24 and 25, on N2a differentiation after 24 and 48 hours of treatment. [Figure 20] Representative images showing N2a cells treated for 24 and 48 hours with compounds 24 and 25 alone and with compounds 24 and 25 co-treated. Scale bar is 100 μm. [Figure 21](A) shows the design of the KDM5 PROTAC, (B) shows the effect of 0.2 μM 14a, 14b, 20a, 20b, 23a, and 23b on N2a differentiation after 48 hours of treatment, (C) shows the dose-dependent N2a cell neurite outgrowth activity of compounds 20b, 23b, and 26 after 48 hours of treatment, and (D) shows the time-dependent N2a cell neurite outgrowth activity of 20b and 23b at 0.02 μM. Bar graphs represent the mean ± SD from three independent experiments. [Figure 22] Representative images showing N2a cells treated with compounds 14a, 14b, 20a, 20b, 23a, and 23b for 48 hours. Scale bar: 100 μm. [Figure 23] Representative images showing N2a cells treated for 48 hours with compounds 20b, 23b, and 26. Scale bar is 100 μm. [Figure 24] Representative images showing N2a cells treated with compounds 20b and 23b for 24, 48, and 72 hours. Scale bar: 100 μm. [Figure 25] Figure 1 shows Western blot detection of KDM5A levels in N2a cells, (A) after 24 hours of treatment with compounds 20b and 23b, (B) after 24 hours of treatment with compound 23b and 24 hours of co-treatment with compounds 24 and 26, (C) after 24 hours of co-treatment with compound 20b and the proteasome inhibitor MG-132, and (D) after 24 hours of co-treatment with the proteasome inhibitor MG-132 and compound 23b. [Figure 26] FIG. 1 shows Western blot detection of H3K4me3 and H3K27Ac levels in N2a cells treated with compounds 20b and 23b for 24 hours. [Figure 27] FIG. 1 shows the KDM5A inhibitory activity and HDAC1 inhibitory activity of compounds 20b and 23b. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present disclosure will be described in further detail below. In addition to the compound represented by formula (I) and its salts, the present disclosure also includes N-oxides, solvates, isomers, mixtures of isomers, single crystal forms, mixtures of crystal forms, co-crystals, and isotopically labeled compounds thereof, which are hereinafter collectively referred to as "the present compounds." Unless otherwise specified, the term "alkyl group" includes linear, branched, and cyclic monovalent saturated hydrocarbon groups. The same applies to alkyl groups in alkoxy groups. Unless otherwise specified, the term "alkylene group" includes linear, branched and cyclic divalent saturated hydrocarbon groups. Unless otherwise specified, the term "alkenylene group" includes straight-chain, branched-chain and cyclic divalent unsaturated hydrocarbon groups. The "halogen atom" includes a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. The term "substituted or unsubstituted" includes both the replacement of a hydrogen atom (-H) with a monovalent substituent and the replacement of a methylene group (-CH-) or a methine group (>CH- or =CH-) with a divalent substituent. Examples of the "monovalent substituent" include an alkyl group, an alkoxy group, a halogen atom, a halogenated alkyl group, a hydroxy group, a carboxy group, an amino group, and a nitro group. The alkyl group as a substituent is preferably an alkyl group having 1 to 5 carbon atoms, and most preferably a methyl group, an ethyl group, a propyl group, an n-butyl group, or a tert-butyl group. The alkoxy group as a substituent is preferably an alkoxy group having 1 to 5 carbon atoms, more preferably a methoxy group, an ethoxy group, an n-propoxy group, an iso-propoxy group, an n-butoxy group, or a tert-butoxy group, and most preferably a methoxy group or an ethoxy group. As the halogen atom as a substituent, a fluorine atom or an iodine atom is preferred. Examples of halogenated alkyl groups as substituents include alkyl groups having 1 to 5 carbon atoms, such as methyl, ethyl, propyl, n-butyl, and tert-butyl groups, in which some or all of the hydrogen atoms have been substituted with the above-mentioned halogen atoms. The "divalent substituent" refers to -O-, -C(=O)-O-, -OC(=O)-, -C(=O)-, -OC(=O)-O-, -C(=O)-NH-, -C(=O)-NH-C(=O)-, -N=, -NH-, -NH-C(=NH)- (H may be substituted with a substituent such as a hydrocarbon group, an acyl group, or an alkoxyalkyl group), -S-, -S(=O)2-, -S(=O)2-O-, a group represented by the general formula -Y 21 -OY 22 -, -Y 21 -O-, -Y 21 -C(=O)-O-, -C(=O)-OY 21 -, -[Y 21 -C(=O)-O] m” -Y 22 -, -Y 21 -OC(=O)-Y 22 -or-Y 21 -S(=O)2-OY 22 -, wherein Y 21 and Y 22 are each independently a substituted or unsubstituted divalent hydrocarbon group, O is an oxygen atom, and m″ is an integer of 0 to 3.

[0014] ≪This compound≫ In the compound or salt thereof of the present disclosure, the compound is a compound represented by formula (I).

[0015] [ka]

[0016] In formula (I), A is a single bond. or replacement or is an unsubstituted divalent aromatic hydrocarbon group, L is a substituted or unsubstituted linear or branched alkylene group having 1 to 13 carbon atoms, and X is -(CH2CH2O) s -, -(CH2CH2CH2O) s -, -(CH(CH3)CH2O) s - or -(CH2) t It is a group represented by O—, in which s is a number from 1 to 8, and t is a number from 2 to 10.

[0017] As the A, a single bond, a phenylene group, a naphthylene group, and the like are preferred; a single bond, a 1,2-phenylene group, a 1,3-phenylene group (m-phenylene group), a 1,4-phenylene group (p-phenylene group), a 1,2-naphthylene group, a 1,4-naphthylene group, a 1,5-naphthylene group, a 2,3-naphthylene group, a 2,6-naphthylene group, and the like are more preferred; a single bond, a 1,3-phenylene group (m-phenylene group), and a 1,4-phenylene group (p-phenylene group) are even more preferred.

[0018] The L is preferably a linear alkylene group having 3 to 10 carbon atoms, more preferably a linear alkylene group having 4 to 9 carbon atoms, and even more preferably a linear alkylene group having 5 to 8 carbon atoms.

[0019] The X may be -(CH2CH2O) s -, -(CH2CH2CH2O) s -, -(CH(CH3)CH2O) s - or -(CH2) t A group represented by O- is preferred; -(CH2CH2O) s -, -(CH2CH2CH2O) s - or -(CH2) t A group represented by O- is more preferred, and -(CH2CH2O) s - or -(CH2) t A group represented by O- is more preferred. The aforementioned s is preferably a number of 1 to 5, more preferably 2 to 4, further preferably 2 or 3, and particularly preferably 3. The t is preferably a number from 2 to 10, more preferably from 3 to 8, further preferably from 4 to 7, and particularly preferably 6.

[0020] In the formula (I), A is a single bond or a phenylene group, L is a linear alkylene group having 3 to 10 carbon atoms, and X is —(CH2CH2O) s -, -(CH2CH2CH2O) s -, -(CH(CH3)CH2O) s - or -(CH2)t It is a group represented by O—, where s is a number of 1 to 5 and t is a number of 2 to 10. In the formula (I), A is a single bond, a p-phenylene group, or an m-phenylene group, L is a linear alkylene group having 3 to 10 carbon atoms, and X is —(CH2CH2O) s -, -(CH2CH2CH2O) s - or -(CH2) t It is more preferable that the group is represented by O—, s is a number of 1 to 5, and t is a number of 2 to 10.

[0021] The compound is more preferably a compound represented by the following formula (I-1), a compound represented by the following formula (I-2), or a compound represented by the following formula (I-3).

[0022] [ka]

[0023] In formula (I-1), L 1 is a linear alkylene group having 3 to 10 carbon atoms, and X 1 Ha-(CH2CH2O) s -, s is a number of 1 to 5, and in formula (I-2), L 2 is a linear alkylene group having 3 to 10 carbon atoms, and X 2 Ha-(CH2CH2O) s -or-(CH2) t O—, s is a number of 1 to 5, and t is a number of 2 to 10; and in formula (I-3), L 3 is a linear alkylene group having 3 to 10 carbon atoms, and X 3 Ha-(CH2CH2O) s -or-(CH2) t It is a group represented by O—, in which s is a number of 1 to 5, and t is a number of 2 to 10.

[0024] The compound is represented by the formula (I-1), 1 is a linear alkylene group having 5 carbon atoms, and X 1 -(CH2CH2O) s-, and s is 3; 1 is a linear alkylene group having 8 carbon atoms, and X 1 -(CH2CH2O) s -, and s is 3; 2 is a linear alkylene group having 5 carbon atoms, and X 2 -(CH2CH2O) s -, and s is the number 3; 2 is a linear alkylene group having 5 carbon atoms, and X 2 Ga-(CH2) t Compound (23a) is a group represented by O—, and t is the number 6; 3 is a linear alkylene group having 5 carbon atoms, and X 3 -(CH2CH2O) s -, and s is the number 3; and 3 is a linear alkylene group having 5 carbon atoms, and X 3 Ga-(CH2) t and compounds (23b) in which t is a group represented by O— and t is a number of 6.

[0025] Salts of the compounds include all pharmacologically acceptable salts. All pharmacologically acceptable salts are preferably low-toxicity, water-soluble salts. Examples of suitable salts include acid addition salts (e.g., inorganic acid salts [e.g., hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, nitrate, etc.], organic acid salts [e.g., acetate, trifluoroacetate, lactate, tartrate, oxalate, fumarate, maleate, benzoate, citrate, methanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, isethionate, glucuronate, gluconate, etc.], salts with acidic natural amino acids [e.g., aspartic acid, glutamic acid, etc.], etc.).

[0026] The present compounds may be converted into N-oxides and solvates. The N-oxide form refers to a compound in which the nitrogen atom is oxidized. The compound can be converted to the N-oxide form by known methods. The solvate refers to a solvated form with a pharmaceutically acceptable solvent such as water or ethanol, etc. The present compound can be converted into a solvate by a known method.

[0027] The compound may form a co-crystal with a suitable co-crystal former. The co-crystal is preferably a pharmaceutically acceptable co-crystal formed with a pharmaceutically acceptable co-crystal former. A co-crystal is generally defined as a crystal formed by two or more molecules through intermolecular interactions that are not ionic bonds. The co-crystal may be a complex of a neutral molecule and a salt. Co-crystals can be prepared according to known methods such as melt crystallization, recrystallization from a solvent, or physical grinding of the components.

[0028] The present disclosure includes all geometric isomers arising from the structure of the compound, optical isomers based on asymmetric carbons, stereoisomers, tautomers, and other isomers and mixtures of isomers, and is not limited to the description of a convenient formula, and may be either one isomer or a mixture. Therefore, although optically active and racemic isomers may exist due to asymmetric carbon atoms in the molecule, the present disclosure is not limited to these and includes both. Furthermore, crystalline polymorphism may exist, but is similarly not limited thereto, and may be any single crystalline form or a mixture thereof, and may be a hydrate in addition to an anhydrous form, and all are within the scope of the present disclosure. The present disclosure also includes isotopically labeled compounds of the present compounds, which are identical to the present compounds except that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Isotopes that can be incorporated into the present compounds include, for example, isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, phosphorus, sulfur, iodine, and chlorine. 2 H, 3 H, 11 C. 14 C. 13 N, 15 O. 18F, 35 S, 123 I, and 125 Includes I etc. The present compounds and pharmaceutically acceptable derivatives (e.g., salts) thereof that contain the aforementioned isotopes and / or other isotopes are within the scope of the present disclosure. Isotopically labeled compounds of the present disclosure, e.g., 3 H and / or 14 Compounds into which radioactive isotopes such as C are incorporated are useful in drug and / or substrate tissue distribution assays. 3 H and 14 C are considered useful due to their ease of preparation and detection. 11 C and 18 F is considered useful in PET (positron emission tomography), and is an isotope 125 I is considered useful in SPECT (single photon emission computed tomography), all of which are useful in brain imaging. 2 Substitution with heavier isotopes, such as H, may confer certain therapeutic advantages, such as increased in vivo half-life or reduced dosage requirements due to greater metabolic stability, and therefore may be useful in certain circumstances. Isotopically labeled compounds of the present compounds can be uniformly prepared by following the procedures disclosed in the following schemes and / or examples, substituting readily available isotopically labeled reagents for non-isotopically labeled reagents.

[0029] A prodrug of the present compound means a compound that is converted into the present compound by reaction with an enzyme, gastric acid, etc. in vivo. When the present compound has an amino group, examples of the prodrug of the present compound include compounds in which the amino group has been acylated, alkylated, or phosphorylated (for example, the present compound in which the amino group has been converted to eicosanoyl, alanyl, pentylaminocarbonyl, (5-methyl-2-oxo-1,3-dioxolen-4-yl)methoxycarbonyl, tetrahydrofuranyl, pyrrolidylmethyl, pivaloyloxymethyl, acetoxymethyl, tert-butyl, etc.); when the present compound has a hydroxyl group, examples of the prodrug include compounds in which the hydroxyl group has been acylated, alkylated, phosphorylated, or converted to a borate (for example, the present compound in which the hydroxyl group has been converted to acetyl, palmitoyl, propanoyl, pivaloyl, succinyl, fumaryl, alanyl, dimethylaminomethylcarbonyl, etc.). Prodrugs of the present compound include those that are converted to the present compound under physiological conditions as disclosed in "Drug Development," Vol. 7, "Molecular Design," 1990, Hirokawa Publishing, pp. 163-198. Prodrugs of the present compound can be produced by methods known per se. Prodrugs of the present compound may form salts such as acid addition salts, or may form solvates with water or alcoholic solvents (ethanol, etc.), similar to the present compound.

[0030] <Method for producing the present compound> The present compound can be produced, for example, by the method shown below in Scheme I. In the production method described below, the starting compound may be a salt. Examples of salts include those described above as salts of the compound represented by general formula (I).

[0031] [ka]

[0032] In Scheme I, compounds 1, 6, 8, and 11 are commercially available or can be easily synthesized from commercially available products by known methods. In the chemical formula, A, L, and X are the same as A, L, and X described above in the description of the compound represented by formula (I). R 6 represents an alkyl group having 1 to 6 carbon atoms, and is preferably a methyl group, an ethyl group, or the like.

[0033] In step (a), compound 2 can be prepared by protecting the primary amino group of compound 1 with a Boc group (t-butoxycarbonyl group). Protection of a primary amino group with a Boc group is known. For example, it can be performed in a solvent such as methylene chloride in the presence of a Boc group-introducing agent such as (Boc)O and a base such as triethylamine at 0 to 80°C.

[0034] In step (b), compound 3 can be prepared by replacing the hydrogen atom on the nitrogen atom of compound 2 with a methyl group. The reaction of substituting a hydrogen atom on a nitrogen atom with a methyl group is known. For example, it can be carried out in a solvent such as N,N-dimethylformamide (hereinafter also referred to as DMF) in the presence of a methylating agent such as MeI and a base such as NaH at 0 to 80°C.

[0035] In step (c), compound 4 can be produced by a cross-coupling reaction between compound 3 and an organoboronic acid compound using a palladium catalyst. Palladium-catalyzed cross-coupling reactions are known. For example, they can be carried out in a solvent such as dimethyl sulfoxide (hereinafter also referred to as DMSO) in the presence of compound 3, an organoboronic acid compound such as bis(pinacolato)diboron, a palladium catalyst such as Pd(dppf)Cl, and a base such as KOAc at 40 to 190°C.

[0036] In step (d), compound 5 can be prepared by deprotecting the amino group of compound 4, which is protected with a Boc group. Deprotection of an amino group protected with a Boc group is known, and can be carried out, for example, in a solvent such as methylene chloride in the presence of an acid such as trifluoroacetic acid at 0 to 80°C.

[0037] In step (e), compound 7 can be prepared by reductive amination of the secondary amino group of compound 5 with organic aldehyde compound 6. Reductive amination is known and can be carried out, for example, in a solvent such as 1,2-dichloroethane in the presence of a reducing agent such as sodium triacetoxyborohydride at 0 to 80°C.

[0038] In step (f), compound 9 can be prepared by a cross-coupling reaction between compound 7 and organic halogen compound 8 using a palladium catalyst. Palladium-catalyzed cross-coupling reactions are known. For example, they can be carried out in a solvent such as 1,2-dimethoxyethane (hereinafter also referred to as DME) in the presence of Compound 7, Compound 8, a palladium catalyst such as Pd(dppf)Cl, and a base such as NaCO at 40 to 190°C.

[0039] In step (g), compound 10 can be prepared by hydrolyzing the ester of compound 9 to convert it into a carboxy group. The hydrolysis reaction for converting an ester to a carboxy group is known. For example, it can be carried out in a solvent such as tetrahydrofuran (hereinafter also referred to as THF), methanol, or water in the presence of a base such as sodium hydroxide at 0 to 80°C.

[0040] In step (h), compound 12 can be produced by an aryl ether synthesis reaction between compound 11 having a phenolic hydroxyl group and an alcohol having an amino group protected with a Boc group. The aryl ether synthesis reaction between an organic compound having a phenolic hydroxyl group and an alcohol is known. For example, it can be carried out in a solvent such as THF at 0 to 80°C in the presence of compound 11, the specific alcohol, a Mitsunobu reaction reagent such as di-2-methoxyethyl azodicarboxylate, and an organophosphorus compound such as triphenylphosphine.

[0041] Alternatively, in step (h), compound 12 can be prepared by the S reaction of compound 11 having a phenolic hydroxyl group with an alkyl halide having an amino group protected by a Boc group. N It can be produced by two reactions. Compound 11, which has a phenolic hydroxyl group, and alkyl halide N Reaction 2 is known. For example, it can be carried out in a solvent such as DMF in the presence of compound 11, the specific alkyl halide, a base such as KHCO3, and an iodinating agent such as KI at 0 to 150°C.

[0042] In step (i), compound 13 can be produced by deprotecting the amino group protected with a Boc group in compound 12 and then converting the amino group into a hydrochloride salt. Deprotection of an amino group protected with a Boc group is known, and can be carried out, for example, in a solvent such as methylene chloride in the presence of an acid such as trifluoroacetic acid at 0 to 80°C. The reaction of converting an amino group into a salt is known. For example, after deprotection, it can be carried out in ethyl acetate (hereinafter also referred to as AcOEt) in the presence of an acid such as hydrochloric acid at 0 to 80°C.

[0043] In step (j), compound 14 before conversion into the hydrochloride can be prepared by a condensation reaction between the carboxy group of compound 10 and the secondary amino group of compound 13. The condensation reaction between a carboxy group and an amino group is known. For example, it can be carried out in DMF at 0 to 80°C in the presence of a condensing agent such as Compound 10 or Compound 13, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (hereinafter also referred to as HATU), or a base such as diisopropylethylamine (hereinafter also referred to as i-PrNEt).

[0044] In step (k), the hydrochloride of compound 14 can be prepared by converting the tertiary amino group in compound 14 into a hydrochloride. The reaction of converting a tertiary amino group into a salt is known, and can be carried out, for example, in AcOEt in the presence of an acid such as hydrochloric acid at 0 to 80°C.

[0045] Additionally, the compound 6 can also be produced by the method described below, as shown in Scheme II below.

[0046] [ka]

[0047] In Scheme II, compounds 6a and 6b are commercially available, or can be easily synthesized from commercially available products by known methods. In the chemical formula, L is the same as L described above in the description of the compound represented by formula (I). A is a substituted or unsubstituted divalent aromatic hydrocarbon group; a phenylene group, a naphthylene group, and the like are preferred; a 1,2-phenylene group, a 1,3-phenylene group (m-phenylene group), a 1,4-phenylene group (p-phenylene group), a 1,2-naphthylene group, a 1,4-naphthylene group, a 1,5-naphthylene group, a 2,3-naphthylene group, and a 2,6-naphthylene group are more preferred; a 1,3-phenylene group (m-phenylene group) and a 1,4-phenylene group (p-phenylene group) are even more preferred. L' is a substituted or unsubstituted straight-chain or branched-chain alkenylene group having 2 to 13 carbon atoms, preferably a straight-chain alkenylene group having 3 to 10 carbon atoms, more preferably a straight-chain alkenylene group having 4 to 9 carbon atoms, and even more preferably a straight-chain alkenylene group having 5 to 8 carbon atoms. Hal represents a halogen atom, preferably a chlorine atom, a bromine atom, an iodine atom, or the like. R 6 represents an alkyl group having 1 to 6 carbon atoms, and is preferably a methyl group, an ethyl group, or the like.

[0048] In step (l), compound 6 can be prepared by a cross-coupling reaction of unsaturated alcohol 6a with aryl halide 6b using a palladium catalyst. Palladium-catalyzed cross-coupling reactions are known. For example, they can be carried out in a solvent such as DMF in the presence of Compound 6a, Compound 6b, a palladium catalyst such as Pd(OAc)2, a base such as LiOAc and tetrabutylammonium chloride (hereinafter also referred to as n-BuNCl), and a reaction promoter such as LiCl at 40 to 190°C.

[0049] <Application> Since the present compound has both KDM5 inhibitory activity and KDM5 degrading activity, it can be used as a KDM5 inhibitor and KDM5 degrader, and can be used as a drug for preventing and / or treating KDM5-related diseases in mammals, particularly humans.

[0050] The present compound can be used as a chemical probe for capturing a target protein of a physiologically active low-molecular-weight compound. That is, the present compound can be converted into an affinity chromatography probe, a photoaffinity probe, or the like by introducing a labeling group, a linker, or the like into a structural portion different from the portion essential for the activity of the compound, using the methods described in J. Mass Spectrum. Soc. Jpn. Vol. 51, No. 5, 2003, pp. 492-498 or WO2007 / 139149, for example. Examples of the labeling group, linker, etc. used in the chemical probe include groups shown in the group consisting of (1) to (5) below. (1) Protein labeling groups such as photoaffinity labeling groups (e.g., benzoyl group, benzophenone group, azide group, carbonylazide group, diaziridine group, enone group, diazo group, and nitro group, etc.) and chemical affinity groups (e.g., ketone groups in which the alpha carbon atom is substituted with a halogen atom, carbamoyl group, ester group, alkylthio group, α,β-unsaturated ketone, Michael acceptors such as ester, and oxirane group, etc.), (2) Cleavable linkers such as -SS-, -O-Si-O-, monosaccharides (glucose groups, galactose groups, etc.) or disaccharides (lactose, etc.), and oligopeptide linkers that can be cleaved by enzymatic reaction; (3) Fishing tag groups such as biotin and 3-(4,4-difluoro-5,7-dimethyl-4H-3a,4a-diaza-4-bora-s-indacen-3-yl)propionyl group; (4) 125 I, 32 P, 3 H, 14 Detectable markers such as radioactive labeling groups such as C; fluorescent labeling groups such as fluorescein, rhodamine, dansyl, umbelliferone, 7-nitrofurazanyl, and 3-(4,4-difluoro-5,7-dimethyl-4H-3a,4a-diaza-4-bora-s-indacen-3-yl)propionyl groups; chemiluminescent groups such as lumiferin and luminol; lanthanide metal ions, heavy metal ions such as radium ions, or the like. (5) Groups that can be bound to solid phase supports such as glass beads, glass beds, microtiter plates, agarose beads, agarose beds, polystyrene beads, polystyrene beds, nylon beads, nylon beds, etc. A probe prepared by introducing a labeling group selected from the group consisting of (1) to (5) into the present compound in accordance with the method described in the above document can be used as a chemical probe for identifying labeled proteins that are useful for searching for new drug discovery targets, etc.

[0051] Pharmaceutical Composition The pharmaceutical compositions of the present disclosure (hereinafter collectively referred to as the present pharmaceutical compositions) contain the present compounds. When the present compound is used for pharmaceutical purposes, the present compound can be used as a single agent, or can be used as a combination agent with other drugs added thereto, for the purposes of, for example, (1) supplementing and / or enhancing the effects of the present compound for the prevention, treatment, and / or amelioration of symptoms, (2) improving the pharmacokinetics and absorption, reducing the dosage of the present compound, and / or (3) alleviating the side effects of the present compound. The pharmaceutical composition of the present disclosure can contain the present compound in an amount of 0.000001 to 99.5% by weight, preferably 0.000001 to 90% by weight, based on the total mass of the pharmaceutical composition.

[0052] In combination with other drugs, the present compound and the other drug may be administered as a combined drug containing both ingredients in a single drug, or as separate preparations administered via the same or different administration routes. Separate preparations do not need to be administered simultaneously, and may be administered sequentially with a time lag. When administered sequentially, the order or method of administration is not particularly limited and can be adjusted appropriately to achieve the expected efficacy.

[0053] The dosage of other drugs used in combination with the present compound can be increased or decreased as appropriate depending on the clinical dosage or similar drugs. The ratio of the present compound to other drugs can be adjusted as appropriate, taking into account the subject's age, body weight, administration method, administration time, target disease, pathological condition, etc. Typically, 1 part by weight of the present compound can be combined with 0.01 to 100 parts by weight of other drugs. Multiple other drugs may be used. In addition to the above drugs, other drugs may also have a mechanism of action similar to that of the above drugs. These other drugs include not only known drugs but also drugs that will be discovered in the future.

[0054] The dosage of the compound varies depending on age, body weight, pathological condition, therapeutic effect, administration method, administration period, etc. The compound may be administered orally to an adult in a single dose of 0.1 mg to 300 mg once to several times a day, or may be administered parenterally to an adult in a single dose of 0.1 mg to 150 mg once to several times a day, or may be administered continuously intravenously over 1 to 24 hours a day.

[0055] The pharmaceutical composition containing the present compound can be formulated into various dosage forms. Examples of dosage forms include oral administration agents (e.g., tablets, capsules, granules, powders, oral liquids, syrups, oral jellies, etc.), oral preparations (e.g., oral tablets, oral sprays, oral semisolid preparations, oral gargles, etc.), injection preparations (e.g., injections, etc.), dialysis preparations (e.g., dialysis agents, etc.), inhalation agents (e.g., inhalation agents, etc.), ophthalmic preparations (e.g., eye drops, eye ointments, etc.), otorhinologic preparations (e.g., ear drops, etc.), nasal preparations (e.g., nasal drops, etc.), rectal preparations (e.g., suppositories, rectal semisolid preparations, enemas, etc.), vaginal preparations (e.g., vaginal tablets, vaginal suppositories, etc.), and dermatological preparations (e.g., solid topical preparations, liquid topical preparations, sprays, ointments, creams, gels, patches, etc.).

[0056] When producing oral solid preparations, tablets, granules, powders, capsules, etc. can be produced by adding excipients, binders, disintegrants, lubricants, colorants, etc. to the present compound as needed. Furthermore, tablets, granules, powders, capsules, etc. may be film-coated as needed. Examples of excipients include lactose, corn starch, and crystalline cellulose; examples of binders include hydroxypropyl cellulose and hydroxypropylmethyl cellulose; examples of disintegrants include carboxymethyl cellulose calcium and croscarmellose sodium; examples of lubricants include magnesium stearate and calcium stearate; examples of colorants include titanium oxide; and examples of film coating agents include hydroxypropyl cellulose, hydroxypropylmethyl cellulose, and methyl cellulose, but are not limited to these. These solid preparations such as tablets, capsules, granules, and powders can generally contain 0.001 to 99.5% by weight, preferably 0.001 to 90% by weight, of the present compound relative to the total mass of the solid preparation.

[0057] When preparing an injection (for intravenous administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, etc.), the compound may be added with a pH adjuster, buffer, suspending agent, solubilizing agent, antioxidant, preservative (antiseptic), isotonic agent, etc. as needed, and the injection may be prepared by a conventional method. Alternatively, the compound may be freeze-dried to prepare a freeze-dried preparation that can be dissolved immediately before use. Examples of pH adjusters and buffers include organic acids or inorganic acids and / or salts thereof; examples of suspending agents include methyl cellulose, polysorbate 80, sodium carboxymethyl cellulose, etc.; examples of solubilizing agents include polysorbate 80, polyoxyethylene sorbitan monolaurate, etc.; examples of antioxidants include α-tocopherol, etc.; examples of preservatives include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, etc.; and examples of isotonic agents include glucose, sodium chloride, mannitol, etc., but are not limited to these. These injections can usually contain 0.000001 to 99.5% by weight, preferably 0.000001 to 90% by weight, of the present compound relative to the total mass of the injection.

[0058] When preparing topical preparations, a base material is added to the present compound, and if necessary, the above-mentioned preservatives, stabilizers, pH adjusters, antioxidants, colorants, etc. are added, and then, for example, transdermal absorption preparations (ointments, patches, etc.), eye drops, nasal drops, suppositories, etc. can be prepared by conventional methods. The base raw material used can be, for example, various raw materials commonly used in pharmaceuticals, quasi-drugs, cosmetics, etc. Specific examples include animal and vegetable oils, mineral oils, ester oils, waxes, emulsifiers, higher alcohols, fatty acids, silicone oils, surfactants, phospholipids, alcohols, polyhydric alcohols, water-soluble polymers, clay minerals, purified water, and other raw materials. These topical preparations can usually contain 0.000001 to 99.5% by weight, preferably 0.000001 to 90% by weight, of the present compound relative to the total weight of the topical preparation. The dosage of the compound varies depending on the severity of symptoms, age, sex, body weight, dosage form, type of salt, specific type of disease, etc., but typically, for adults, the daily oral dosage is about 30 μg to 10 g, preferably 100 μg to 5 g, and more preferably 100 μg to 1 g, and the daily injection dosage is about 30 μg to 1 g, preferably 100 μg to 500 mg, and more preferably 100 μg to 300 mg, administered once or in divided doses.

[0059] <Application> Since the present compound and the present pharmaceutical composition have both KDM5 inhibitory activity and KDM5 degrading activity, they can be used as KDM5 inhibitors and KDM5 degraders, and can be used as agents for the prevention and / or treatment of KDM5-related diseases (in mammals, particularly humans).

[0060] Examples of such diseases include hyperproliferative disorders, cancer, stroke, diabetes, hepatomegaly, cardiovascular disease, multiple sclerosis, Huntington's disease, Alzheimer's disease, cystic fibrosis, viral diseases, autoimmune diseases, atherosclerosis, restenosis, psoriasis, rheumatoid arthritis, inflammatory bowel disease, asthma, allergic diseases, inflammation, neurological disorders, hormone-related diseases, conditions associated with organ transplants, immunodeficiency disorders, destructive bone disorders, proliferative disorders, infectious diseases, conditions associated with cell death, and thrombin-induced These conditions include platelet aggregation, liver disease, pathological immune conditions involving T-cell activity, central nervous system disorders, myeloproliferative disorders, Parkinson's disease, disease with Lewy bodies, frontotemporal lobar degeneration, mild cognitive impairment, dementia, cerebrovascular disease, schizophrenia, depression, anxiety disorders, bipolar disorder, autism spectrum disorder, attention deficit / hyperactivity disorder, learning disabilities, movement disorders, obsessive-compulsive disorder, personality disorders, sleep disorders, delirium, amyotrophic lateral sclerosis, developmental disorders, intellectual disabilities, post-traumatic stress disorder, and hepatitis.

[0061] In particular, the present compound and pharmaceutical composition are useful for the prevention and / or treatment of cancer, Huntington's disease, Alzheimer's disease, Parkinson's disease, Lewy body disease, frontotemporal lobar degeneration, mild cognitive impairment, dementia, cerebrovascular disease, schizophrenia, depression, anxiety disorder, bipolar disorder, autism spectrum disorder, attention-deficit / hyperactivity disorder, learning disability, movement disorder, obsessive-compulsive disorder, personality disorder, sleep disorder, delirium, amyotrophic lateral sclerosis, developmental disorder, intellectual disability, post-traumatic stress disorder, or hepatitis. The present compound or pharmaceutical composition is particularly suitable for the prevention and / or treatment of cancer and Alzheimer's disease.

[0062] When the pharmaceutical composition is used as a single agent or in combination with other drugs for the prevention and / or treatment of the above-mentioned diseases, the active ingredient, the compound, is typically formulated with pharmaceutically acceptable carriers such as various additives or solvents, and the resulting formulation is administered systemically or topically, orally, or parenterally. Here, "pharmaceutically acceptable carrier" refers to a substance other than the active ingredient commonly used in pharmaceutical formulations. Pharmaceutically acceptable carriers preferably exhibit no pharmacological activity at the dosage of the formulation, are harmless, and do not interfere with the therapeutic effect of the active ingredient. Pharmaceutically acceptable carriers may be used to enhance the usefulness of the active ingredient and formulation, facilitate formulation, stabilize quality, or improve usability. Specifically, substances listed in the "Dictionary of Pharmaceutical Additives" (2000, Yakuji Nipposha, IPEC JAPAN edition) can be appropriately selected as needed.

[0063] <Method for producing pharmaceutical composition> A pharmaceutical composition containing the present compound can be produced by mixing the present compound with additives such as other drugs, carriers, excipients, etc., as necessary. [Example]

[0064] The present disclosure will be specifically explained below using examples, but the present disclosure is not limited to these examples. The chemical reagents and solvents used in this example were commercially available products of the highest purity available and were purchased from Sigma-Aldrich, Fujifilm Wako Chemicals, TCI Tokyo Chemical Industry, Nacalai Tesque, and Kanto Chemical. All air- and moisture-sensitive reactions were carried out using dry glassware under an argon (Ar) atmosphere. NMR spectra were obtained at 400 MHz ( 1 H), and the JEOLECS400 spectrometer operating at 700 MHz ( 1 H) or 175MHz ( 13 C) recorded on a Bruker AVANCE III 700 spectrometer operating at 1000 Hz. 1 HNMR and 13 C NMR chemical shift values ​​are the solvent peak or tetramethylsilane (TMS) (DMSO-d6: 1 HNMR 2.50, 13 Coupling constants are given in Hz and are reported as δ (ppm) relative to the CNMR (39.52). The purity of all test compounds was determined by HPLC using a Shimadzu UFLC (SPD-M20A UV detector, DGU-20A3R degassing unit, LC-20AD solvent delivery unit, CBM-20A system) and a COSMOSIL packed column (5C18-AR-II, 4.6 ID × 150 mm, Nacalai Tesque Inc.) at a flow rate of 1 mL / min with UV detection (λ = 254 nm). HPLC conditions: Eluent A: H2O containing 0.1% TFA; Eluent B: acetonitrile containing 0.1% TFA. Eluent B: 0–20 min, 10–90%; 20–30 min, 90%; 30–40 min, 90–10%. Positive and negative LRMS ion mass spectra were recorded on a Bruker HCT-Plus. High-resolution mass spectra (HRMS) were recorded on an LTQ Orbitrap XL (THERMO) or Shimadzu LCMS-IT-TOF mass spectrometer.

[0065] Abbreviation AcOEt: ethyl acetate; Boc2O = di-t-butyl dicarbonate; n-Bu4NCl: tetrabutylammonium chloride; DME: 1,2-dimethoxyethane; DMEAD = di-2-methoxyethyl azodicarboxylate DMF = N,N-dimethylformamide; DMSO = dimethyl sulfoxide; dppf = 1,1'-ferrocenebis(diphenylphosphine); HATU = 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; MeCN: acetonitrile; MeI: methyl iodide; MeOH: methanol; i-Pr2NEt: diisopropylethylamine; TFA = trifluoroacetic acid; THF = tetrahydrofuran.

[0066] (1) Compound synthesis Example 1: Synthesis of 7-{[4-(3-cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}-N-{2-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoindolin-4-yl]oxy}ethoxy}ethyl}heptanamide hydrochloride (14a-hydrochloride) Compound 14a was synthesized according to the following reaction scheme.

[0067] [ka]

[0068] Synthesis of 7-{[4-(3-cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}heptanoic acid (10a) Step 1: Synthesis of tert-butyl(4-bromophenethyl)carbamate (2) To a solution of 2-(4-bromophenyl)ethylamine (compound 1, 5.00 g, 25.0 mmol) and triethylamine (4.20 mL, 30.1 mmol) in CHCl (50 mL) was added BocO (5.72 g, 26.2 mmol) while cooling in an ice bath. The resulting mixture was stirred at room temperature for 5 h. The reaction was then quenched with 10% citric acid and extracted with AcOEt. The organic layer was separated, washed with brine, and dried over MgSO. After filtration, concentration under reduced pressure, and purification by flash column chromatography (silica gel, n-hexane / AcOEt = 9 / 1 to 4 / 1), compound 2 was obtained as a colorless solid (5.83 g, 78%). 1 HNMR(DMSO-d6,400MHz,δppm)7.46(2H,d,J=8.3Hz),7.15(2H,d,J=8.3Hz),6.86( 1H,t,J=5.2Hz),3.12(2H,td,J=6.8,6.8Hz),2.66(2H,t,J=7.0Hz),1.35(9H,s).

[0069] Step 2: Synthesis of tert-butyl(4-bromophenethyl)(methyl)carbamate (3) A solution of compound 2 (5.83 g, 19.4 mmol) in DMF (50 mL) was added to a suspension of 60% NaH (0.820 g, 20.5 mmol) in oil at 0° C. After 15 min, MeI (1.40 mL, 22.5 mmol) was added dropwise, and the resulting mixture was stirred at 0° C. for 5 h. The reaction mixture was poured into water and extracted with AcOEt. The organic layer was washed with brine and dried over MgSO. After filtration, concentration under reduced pressure, and purification by flash column chromatography (silica gel, n-hexane / AcOEt=9 / 1), compound 3 was obtained as a colorless oil (6.08 g, 99%). 1HNMR (DMSO-d6,400MHz,δppm)7.47(2H,d,J=8.1Hz),7.15(2H,d,J=8.1Hz),3.36(2H,t,J=6.5Hz),3.32(3H,s),2.72(2H,t,J=6.8Hz),1.25(9H,s).

[0070] Step 3: Synthesis of tert-butyl methyl [4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenethyl]carbamate (4) A solution of compound 3 (6.08 g, 19.3 mmol), bis(pinacolato)diboron (5.70 g, 22.4 mmol), KOAc (7.30 g, 74.4 mmol), and Pd(dppf)Cl (670 mg, 0.916 mmol) in DMSO (60 mL) was heated at 80 °C for 7 h. The reaction mixture was filtered, and the filtrate was extracted with AcOEt and washed with brine. The organic layer was separated and dried over MgSO. After filtration, concentration under reduced pressure, and purification by flash column chromatography (silica gel, n-hexane / AcOEt=4 / 1), compound 4 was obtained as a colorless solid (5.76 g, 82%). 1 HNMR(DMSO-d6,400MHz,δppm),7.59(2H,d,J=7.9Hz),7.21(2H,d,J=7.4Hz),3.36 (2H,t,J=7.2Hz),2.76(2H,t,J=7.2Hz),2.73(3H,s),1.36(9H,s),1.28(12H,s).

[0071] Step 4: Synthesis of N-methyl-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]ethan-1-amine (5) To a solution of compound 4 (5.76 g, 15.9 mmol) in CHCl (35 mL) was added trifluoroacetic acid (TFA) (16 mL) and cooled in an ice bath. The resulting mixture was stirred at 0 °C for 2 h. The solvent was removed under reduced pressure, and saturated aqueous NaHCO and AcOEt were added to the resulting residue, followed by separation. The organic layer was washed with brine, dried over MgSO, filtered, and the solvent was removed under reduced pressure. The residue was purified by flash chromatography (silica gel, n-hexane / AcOEt=4 / 1) to give compound 5 (3.50 g, 84%) as a colorless solid. 1 HNMR(DMSO-d6,400MHz,δppm),8.45(1H,brs),7.65(2H,d,J=8.1Hz),7.28(2H,d ,J=8.3Hz),3.15(2H,t,J=9.2Hz),2.94-2.89(2H,m),2.59(3H,s),1.28(12H,s).

[0072] Step 5: Synthesis of methyl 7-{methyl[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenethyl]amino}heptanoate (7a) A solution of methyl 7-oxoheptanoate (compound 6a, 401 mg, 2.53 mmol), compound 5 (632 mg, 2.42 mmol), and NaBH(OAc) (820 mg, 3.87 mmol) in ClCHCHCl (10 mL) was stirred at room temperature overnight. The reaction was quenched with water and extracted with AcOEt. The organic layer was separated, washed with brine, and dried over MgSO. After filtration, concentration under reduced pressure, and purification by flash column chromatography (silica gel, CHCl3 / AcOEt=4 / 1 to CHCl3 / MeOH=9 / 1), compound 7a was obtained as a colorless oil (253 mg, 26%). 1 HNMR(DMSO-d6,400MHz,δppm),7.57(2H,d,J=8.8Hz),7.22(2H,d,J=8.8Hz),3.57(3H,s),2.74-2.64(2H,m) ,2.35-2.23(4H,m),2.17(3H,brs),1.55-1.43(2H,m),1.41-1.32(2H,m),1.28(12H,s),1.25-1.14(6H,m).

[0073] Step 6: Synthesis of methyl 7-{[4-(3-cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}heptanoate (9a) A solution of compound 7a (147 mg, 0.364 mmol), compound 8 (78.3 mg, 0.331 mmol), NaCO (70.2 mg, 0.662 mmol), and Pd(dppf)Cl (24.2 mg, 0.0331 mmol) in DME / HO (1.5 mL / 0.5 mL) was heated at 110 °C for 3 h. The reaction mixture was filtered, and the filtrate was extracted with AcOEt and washed with brine. The organic layer was separated and dried over MgSO. After filtration, concentration under reduced pressure, and purification by flash column chromatography (silica gel, CHCl / AcOEt=4 / 1 to CHCl / MeOH=9 / 1), compound 9a was obtained as a colorless solid (23.5 mg, 15%). 1 HNMR(DMSO-d6,400MHz,δppm),8.03(1H,brs),7.39(2H,d,J=7.9Hz),7.32(2H,d,J=7.9Hz),3.57(3H,s),2.88-2.76(4H,m),2.60 -2.53(3H,m),2.38(3H,s),2.28(2H,t,J=7.2Hz),1.54-1.42(4H,m),1.30-1.23(4H,m),1.18(6H,d,J=8.2Hz);MS(ESI)m / z478(MH + ).

[0074] Step 7: Synthesis of 7-{[4-(3-cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}heptanoic acid (10a) To a solution of compound 9a (126 mg, 0.264 mmol) in MeOH (1.2 mL) was added an aqueous solution of NaOH (4 N, 0.200 mL, 0.791 mmol), and the mixture was stirred at room temperature overnight. 10% aqueous citric acid was added in portions, and the resulting mixture was stirred at room temperature. The insoluble material was collected by filtration and washed with water to give compound 10a (66.0 mg, 54%) as a colorless solid. 1 HNMR(DMSO-d6,400MHz,δppm),11.9(1H,brs),9.24(1H,brs),8.02(1H,s),7 .33(4H,d,J=9.6Hz),3.11-2.92(2H,m),2.90-2.78(2H,m),2.75-2.68(1H,m) ,2.52-2.50(2H,m),2.51(3H,s),2.21(2H,t,J=7.3Hz),1.68-1.60(2H,m),1. 54-1.48(2H,m),1.36-1.27(4H,m),1.23(6H,d,J=6.9Hz);MS(ESI)m / z464(MH + ).

[0075] Synthesis of 7-{[4-(3-cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}-N-{2-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoindolin-4-yl]oxy}ethoxy}ethyl}heptanamide hydrochloride (14a-hydrochloride) Step 1: Synthesis of {2-[2-(2-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoindolin-4-yl]oxy}ethoxy)ethoxy]ethyl}carbamate (12) To a solution of 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione (Compound 11, 300 mg, 1.09 mmol) and PPh3 (430 mg, 1.64 mmol) in THF (3 mL) was added 2-[2-(2-tert-butoxyaminoethoxy)ethoxy]ethanol (300 mg, 1.20 mmol) and DMEAD (383 mg, 1.64 mmol) at room temperature. The reaction mixture was stirred overnight at room temperature. The reaction was quenched with water and extracted with AcOEt. The organic layer was separated and dried over MgSO. After filtration, concentration under reduced pressure, and purification by flash column chromatography (silica gel, CHCl / AcOEt=4 / 1 to 1 / 1), compound 12 was obtained as a colorless oil (226 mg, 41%). 1HNMR(DMSO-d6,400MHz,δppm),11.10(1H,brs),7.85-7.78(1H,m),7.54(1H,d,J=7.8Hz) ,7.46(1H,d,J=7.4Hz),6.74(1H,brs),5.08(1H,dd,J=12.8,5.4Hz),4.35(2H,t,J=4.5H z),3.80(2H,t,J=4.5Hz),3.64(2H,t,J=4.7Hz),3.53-3.47(2H,m),3.40-3.36(2H,m),3 .08-3.01(2H,m),2.94-2.84(1H,m),2.62-2.51(2H,m),2.05-2.01(1H,m),1.36(9H,s).

[0076] Step 2: Synthesis of 4-(2-(2-aminoethoxy)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione hydrochloride (13-hydrochloride) To a solution of compound 12 (226 mg, 0.448 mmol) in CHCl (2 mL) was added trifluoroacetic acid (TFA) (0.34 mL) while cooling in an ice bath. After stirring at 0 °C for 3 h, the reaction mixture was concentrated under reduced pressure. 4N hydrochloric acid in AcOEt was added to the residue, and the solvent was evaporated under reduced pressure to give compound 13-hydrochloride (239 mg) as a crude product, which was used in the next reaction without further purification.

[0077] Step 3: Synthesis of 7-{[4-(3-cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}-N-{2-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoindolin-4-yl]oxy}ethoxy}ethyl}heptanamide (14a) To a solution of compound 10a (19.9 mg, 0.0429 mmol), i-PrNEt (0.0180 mL, 0.104 mmol), and crude compound 13 hydrochloride (19.0 mg) in DMF (0.2 mL) was added HATU (20.0 mg, 0.0526 mmol) at room temperature. The reaction mixture was stirred overnight at room temperature. Water was added to the reaction mixture, followed by extraction twice with AcOEt. The combined organic layers were washed with brine, dried over MgSO, filtered, and concentrated. The filtrate was concentrated and purified by reverse-phase flash chromatography (MeCN / 0.1% TFA) to give compound 14a, which was converted to the hydrochloride salt in the next step.

[0078] Step 4: Synthesis of 7-{[4-(3-cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}-N-{2-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoindolin-4-yl]oxy}ethoxy}ethyl}heptanamide hydrochloride (14a-hydrochloride) Compound 14a was treated with 4N hydrochloric acid in AcOEt, and the solvent was evaporated under reduced pressure to give compound 14a-hydrochloride (5.8 mg, 15% from compound 10a) as a colorless solid. 1HNMR(DMSO-d6,700MHz,δppm),δ13.44(1H,s),11.13(1H,s),8.41(1H,s),7.88(1H,brs),7.81(1H,t,J=8.4Hz),7.54(1H,d,J=8.6Hz),7.50-7.48(4H,m),7.47(1H,d,J=7.0Hz),5.09(1H,dd,J=12.9,5.6Hz),4.34(2H,t,J=4.3Hz),3.81-3.80(2H,m),3.66-3.64(2H,m),3.53-3.52(2H,m),3.41-3.39(2H,m),3.32-3.25(1H,m),3.19-2.99(6H,m),2.91-2.80(1H,m),2.82(3H,s),2.60-2.58(2H,m),2.55-2.49(2H,m),2.08-2.06(2H,m),2.04-2.00(1H,m),1.71-1.63(2H,m),1.51-1.46(2H,m),1.31-1.25(4H,m),1.23(6H,d,J=7.0Hz,6H); 13 CNMR(DMSO-d6,175MHz,δppm),172.22,171.48,169.37,166.20,164.69,155.18,154.40,147.27,144.73,142.85,138.43,136.42,132.62,130.91,128.34,128.25,119.38,115.65,114.81,113.38,112.16,73.37,69.48,68.99,68.56,68.23,68.06,54.83,54.27,48.11,37.80,34.47,30.33,28.60,27.75,27.53,25.20,24.35,22.47,21.37,20.47,19.34;HRMScalcdforC 45 H 55 N8O9 + 851.4092,found851.4077;HPLCR t 12.27min,97.84%purity.

[0079] Example 2: Synthesis of 10-{[4-(3-cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}-N-{2-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoindolin-4-yl]oxy}ethoxy}ethyl}decanamide hydrochloride (14b-hydrochloride) Compound 14b-hydrochloride was synthesized using a procedure similar to that described for the synthesis of compound 10a and compound 14a-hydrochloride, except that methyl 10-oxodecanoate (6b) was used instead of methyl 7-oxoheptanoate (6a); it was a yellow solid (5.9 mg, 16% from compound 10b). 1 HNMR(DMSO-d6,700MHz,δppm),13.43(1H,s),11.14(1H,s),8.42(1H,s),7.84-7.81(2H,m),7.54(1H,d,J=8.6Hz),7.50(4H,s),7. 47(1H,d,J=7.3Hz),5.10(1H,dd,J=12.9,5.2Hz),4.35(2H,t,J=4.3Hz),3.81(2H,t,J=4.5Hz),3.65(2H,t,J=4.7Hz),3.53(2H,t, J=4.7Hz),3.50-3.35(2H,m),3.32-3.29(1H,m),3.22-3.04(6H,m),2.94-2.86(1H,m),2.85(3H,s),2.61-2.57(2H,m),2.55-2.48 (2H,m),2.06-2.02(1H,m),2.04(2H,t,J=7.0Hz),1.72-1.60(2H,m),1.48-1.44(2H,m),1.29-1.23(10H,m),1.23(6H,d,J=7.0Hz); 13CNMR(DMSO-d6,175MHz,δppm),172.21,171.54,169.35,166.19,164.67,157.35,157.16,155.18,154.39,147. 23,144.72,142.84,138.27,136.41,132.62,130.94,128.34,128.25,119.36,115.64,114.80,113.36,112.16, 73.36,69.47,68.99,68.56,68.22,68.06,54.87,54.42,52.88,48.10,41.15,37.79,34.63,30.32,28.64,28. 11,28.07,28.02,27.88,27.75,25.35,24.60,22.67,21.36,20.45,19.34,17.43,16.09,11.84;HRMScalcdforC 48 H 61 N8O9 + 893.4562,found893.4545;HPLCR t 13.26min,95.59%purity.

[0080] Example 3: Synthesis of 4-(6-{[4-(3-cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}hexyl)-N-{2-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoindolin-4-yl]oxy}ethoxy)ethoxy]ethyl}benzamide (20a) Compound 20a was synthesized according to the following reaction scheme.

[0081] [ka]

[0082] Step 1: Synthesis of ethyl 4-(6-oxohexyl)benzoate (16a) A solution of compound 15a (2.42 g, 8.77 mmol), 5-hexen-1-ol (1.05 g, 10.5 mmol), LiOAc (1.44 g, 21.8 mmol), LiCl (371 mg, 8.75 mmol), tetrabutylammonium chloride (1.22 g, 4.39 mmol), and Pd(OAc) (195 mg, 0.869 mmol) in DMF (10 mL) was heated at 70 °C for 6.5 h. The reaction mixture was filtered, and the filtrate was extracted with AcOEt and washed with brine. The organic layer was separated and dried over MgSO. After filtration, concentration under reduced pressure, and purification by flash column chromatography (silica gel, n-hexane / AcOEt=4 / 1), compound 16a was obtained as a colorless oil (1.96 g, 90%). 1 HNMR(DMSO-d6,400MHz,δppm),9.65(1H,t,J=1.5Hz),7.87(2H,d,J=8.4Hz),7.34(2H,d,J=8.4Hz),4.29(2H,q,J=7.1 Hz),2.64(2H,t,J=7.6Hz),2.41(2H,td,J=7.0,2.0Hz),1.63-1.51(4H,m),1.33-1.26(2H,m),1.31(3H,t,J=7.2Hz).

[0083] Step 2-4: Synthesis of 4-(6-{[4-(3-cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}hexyl)benzoic acid (19a) Compound 19a was synthesized from compounds 8 and 16a using a procedure similar to that described for the synthesis of compound 10a (steps 5-7); yellow solid (25.0 mg, 7% from compound 16a). 1HNMR(DMSO-d6,400MHz,δppm),12.8(1H,brs),8.01(1H,brs),7.86(2H,d,J=7.8Hz),7.33-7.31(6H,m),3.33-3.24(1H,m),3.06 -2.93(4H,m),2.80(3H,brs),2.68-2.64(4H,m),1.67-1.56(4H,m),1.37-1.29(4H,m),1.24(6H,d,J=9.8Hz);MS(ESI)m / z540(MH + ).

[0084] Step 5: Synthesis of 4-(6-{[4-(3-cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}hexyl)-N-{2-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoindolin-4-yl]oxy}ethoxy)ethoxy]ethyl}benzamide (20a) Compound 20a was synthesized from compound 13-hydrochloride and compound 19a in a similar manner to that described for the synthesis of 14a-hydrochloride (step 3); a yellow solid (9.1 mg, 21%). 1 HNMR(DMSO-d6,700MHz,δppm),11.14(1H,s),8.44(1H,t,J=5.6Hz),8.04(1H,s),7.82-7.76(3H,m),7.52(1H,d,J =7.0Hz),7.47(1H,d,J=7.0Hz),7.32-7.26(6H,m),5.10(1H,dd,J=12.9,5.2Hz),4.33-4.23(2H,m),3.84-3.80(2 H,m),3.66(2H,t,J=4.7Hz),3.57-3.53(4H,m),3.43-3.30(6H,m),2.92-2.87(2H,m),2.74(1Ht,J=6.5Hz),2.65- 2.55(5H,m),2.54-2.48(2H,m),2.04-2.00(1H,m),1.63-1.46(4H,m),1.35-1.28(4H,m),1.30(6H,d,J=18.1Hz); 13CNMR(DMSO-d6,175MHz,δppm),172.70,169.85,166.69,166.00,165.16,156.73,155.67,145.54 ,143.81,136.87,133.10,131.75,128.00,127.94,127.16,127.07,126.61,124.80,119.83,116 .14,115.26,110.56,73.97,69.95,69.55,68.81,68.68,68.54,59.65,48.59,34.72,34.27,30. 85,30.82,30.47,30.28,29.11,28.72,28.22,21.96,21.86,20.66,20.44,13.87;HRMScalcdforC 51 H 59 N8O9 + 927.4405,found927.4390;HPLCR t 13.73min,95.89%purity.

[0085] Example 4: Synthesis of 4-(6-{[4-(3-cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}hexyl)-N-{2-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoindolin-4-yl]oxy}ethoxy)ethoxy]ethyl}benzamide hydrochloride (20b-hydrochloride) Compound 20b-hydrochloride was synthesized using procedures similar to those described for the synthesis of compound 20a (steps 1-5) and compound 14a-hydrochloride (step 4), except that ethyl 3-(6-oxohexyl)benzoate (16b) was used instead of ethyl 4-(6-oxohexyl)benzoate (16a); yellow solid (16.6 mg, 17% from compound 8). Compound 16b was synthesized using procedures similar to those described for the synthesis of compound 16a (step 1), except that compound 15b was used instead of compound 15a. 1HNMR(DMSO-d6,700MHz,δppm),13.44(1H,s),11.15(1H,d,J=5.2Hz,1H),8.51(1H,t,J=5.6Hz),8.43(1H,s),7.82(2H,t,J=8.0Hz),7.74-7.67(2H,m),7.53-7.47(6H,m),7.39-7.36(2H,m),5.10(1H,dd,J=12.9,5.2Hz),4.33(2H,t,J=4.5Hz),3.81(2H,t,J=4.5Hz),3.67(2H,t,J=4.7Hz),3.58(2H,t,J=4.7Hz),3.53(2H,t,J=4.7Hz),3.52-3.37(6H,m),3.35-3.27(1H,m),3.22-3.00(4H,m),2.92-2.87(1H,m),2.86(3H,s),2.65-2.59(4H,m),2.55-2.48(2H,m),2.04-2.01(1H,m),1.70-1.66(2H,m),1.66-1.63(2H,m),1.40-1.32(4H,m),1.25(6H,d,J=7.0Hz); 13 CNMR(DMSO-d6,175MHz,δppm),172.71,171.93,169.86,166.69,166.20,165.17,155.66,154.89,147.74,145.22,143.34,142.15,138.82,136.88,134.24,133.11,131.42,130.98,128.83,128.74,128.04,127.05,124.40,119.84,116.14,115.28,113.86,112.66,73.86,69.96,69.53,68.78,68.69,68.54,55.33,54.83,48.59,34.77,30.82,30.49,29.12,28.25,28.00,25.71,23.07,22.02,21.85,20.96,19.84;HRMScalcdforC 51 H 59 N8O9 + 927.4405,found927.4388;HPLCR t 13.80min,97.80%purity.

[0086] Example 5: Synthesis of 4-(6-{[4-(3-cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}hexyl)-N-(9-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoindolin-4-yl]oxy}nonyl)benzamide hydrochloride (23a-hydrochloride) Compound 23a-hydrochloride was synthesized according to the following reaction scheme.

[0087] [ka]

[0088] Step 1: Synthesis of tert-butyl (6-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl]oxy}hexyl)carbamate (21) A solution of compound 11 (274 mg, 1.00 mmol), tert-butyl(6-bromohexyl)carbamate (280 mg, 1.00 mmol), KHCO (150 mg, 1.50 mmol), and KI (17.0 mg, 0.102 mmol) in DMF (3 mL) was heated at 80 °C for 14 h. After cooling to room temperature, water and AcOEt were added and the organic layer was separated. The organic layer was washed with brine and dried over MgSO. It was filtered, concentrated under reduced pressure, and purified by flash column chromatography (CHCl / AcOEt = 10 / 1 → 1 / 1) to give compound 21 as a colorless oil (373 mg, 79%). 1 HNMR(DMSO-d6,400MHz,δppm),11.08(1H,brs),7.82-7.78(1H,m),7.51(1H, d,J=8.2Hz),7.44(1H,d,J=7.6Hz),6.75(1H,brs),5.07(1H,dd,J=12.8,5.4H z),4.20(2H,t,J=6.5Hz),2.93-2.90(1H,m),2.93-2.83(2H,m),2.61-2.53( 2H,m),2.04-2.00(1H,m),1.78-1.71(2H,m),1.49-1.26(6H,m),1.36(9H,s).

[0089] Step 2: Synthesis of 4-[(6-aminohexyl)oxy]-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione hydrochloride (22-hydrochloride) Following the same procedure as described for the synthesis of compound 14a-hydrochloride (step 2), compound 22-hydrochloride was synthesized as a crude product (404 mg), which was used directly in the next reaction without further purification.

[0090] Step 3: Synthesis of 4-(6-{[4-(3-cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}hexyl)-N-(6-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoindolin-4-yl]oxy}hexyl)benzamide hydrochloride (23a-hydrochloride) Compound 23a-hydrochloride was synthesized from compound 19a and compound 22-hydrochloride using a procedure similar to that described for the synthesis of compound 14a-hydrochloride (steps 3 and 4); yellow solid (26.0 mg, 52%). 1HNMR(DMSO-d6,700MHz,δppm),13.43(1H,s),11.12(1H,s),8.42(1H,s),8.41-8.38(1H,m),7.81(1H,dd,J=7.0,7.0Hz),7.76(2H,d,J=8.2Hz),7.52(1H,d,J=8.6Hz),7.50-7.48(4H,m),7.44(1H,d,J=6.9Hz),7.27(2H,d,J=8.2Hz),5.08(1H,dd,J=12.9,5.6Hz),4.21(2H,t,J=6.5Hz),3.32-3.28(1H,m),3.27-3.24(2H,m),3.20-2.99(4H,m),2.91-2.86(1H,m),2.83(3H,s),2.65-2.61(2H,m),2.60-2.56(2H,m),2.53-2.47(2H,m),2.03-2.00(1H,m),1.79-1.75(2H,m),1.73-1.65(2H,m),1.65-1.57(2H,m),1.57-1.52(2H,m),1.52-1.46(2H,m),1.41-1.36(2H,m),1.36-1.31(4H,m),1.24(6H,d,J=7.0Hz); 13 CNMR(DMSO-d6,175MHz,δppm),172.20,169.37,166.23,165.28,164.70,155.35,154.38,147.23,144.76,144.71,142.83,136.42,132.60,131.60,130.92,128.32,128.23,127.47,126.55,126.08,119.12,115.54,114.51,113.35,112.14,73.35,68.08,54.82,54.35,48.06,34.14,30.31,29.83,28.62,28.50,27.74,27.48,25.53,25.19,24.45,22.58,21.35,20.45,19.33;HRMScalcdforC 51 H 59 N8O7 + 895.4507,found895.4490;HPLCR t 15.16min,95.44%purity.

[0091] Example 6: Synthesis of 3-(6-{[4-(3-cyano-6-isopropyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)phenethyl](methyl)amino}hexyl)-N-(6-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoindolin-4-yl]oxy}hexyl)benzamide hydrochloride (23b-hydrochloride) Compound 23b-hydrochloride was synthesized using a procedure similar to that described for the synthesis of compound 23a-hydrochloride (steps 1-3); yellow solid (10.4 mg, 11%). 1 HNMR(DMSO-d6,700MHz,δppm),13.43(1H,s),11.12(1H,s),8.47-8.41(2H,m),7.80(1H,dd,J=8.0,8.0Hz),7.71-7.64(2H,m),7.52-7. 43(6H,m),7.36-7.34(2H,m),5.08(1H,dd,J=12.9,5.6Hz),4.20(2H,t,J=6.5Hz),3.33-3.30(1H,m),3.28-3.24(2H,m),3.17-3.00(4H ,m),2.90-2.85(1H,m),2.82(3H,s),2.65-2.61(2H,m),2.61-2.56(2H,m),2.54-2.47(2H,m),2.04-2.00(1H,m),1.79-1.75(2H,m),1. 73-1.66(2H,m),1.65-1.59(2H,m),1.57-1.52(2H,m),1.52-1.47(2H,m),1.41-1.37(2H,m),1.37-1.31(4H,m),1.23(6H,d,J=7.0Hz); 13CNMR(DMSO-d6,175MHz,δppm),172.20,169.37,166.22,165.51,164.71,155.35,141.62,13 6.42,134.06,132.60,130.31,128.30,128.20,127.49,126.50,123.87,119.12,115.54,114 .52,73.35,68.08,54.80,54.28,48.06,34.28,30.31,30.00,28.59,28.47,27.74,27.50,2 5.55,25.21,24.45,23.62,22.52,21.52,21.45,21.35,20.45,19.34,13.36;HRMScalcdforC 51 H 59 N8O7 + 895.4507,found895.4494;HPLCR t 15.26min,95.66%purity.

[0092] (2) Neurite outgrowth assay The mouse Neuro-2a (N2a) cell line was obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank. N2a cells were grown at 1 × 10 in Dulbecco's modified Eagle's medium (DMEM) containing high glucose, 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. 4 cells / mL at 37°C in a humidified atmosphere of 5% CO2. For the neurite outgrowth assay, the medium was changed to DMEM supplemented with 2% FBS. After incubation with the test compounds, the cell morphology was observed using a microscope (Olympus CKX41). Differentiated cells were defined as cells with at least one neurite with a length greater than or equal to twice the diameter of the cell body. Results are expressed as the percentage of differentiated cells among counted cells. These experiments were performed in triplicate.

[0093] (3) Western blotting N2a cells (5×10 5Cells (2 mL / dish) were treated with the indicated concentrations of test compounds in cell culture medium supplemented with 10% FBS for 24 hours. Cells were harvested and extracted with SDS sampling buffer. The protein concentration of the lysates was measured using a BCA protein assay. Equal amounts of protein from each lysate were separated on a 5%-20% SDS-polyacrylamide gel, and bands were transferred to PVDF membranes (Millipore, #IPVH00010 for β-actin detection, #ISEQ10100 for H3, H3K4me3, and H3K27Ac detection). After blocking with TBS-T containing 5% nonfat milk, the transblotted membranes were probed with primary antibodies. The primary antibodies used were: rabbit monoclonal KDM5A antibody (CST #3876, 1:1000 dilution); mouse monoclonal β-actin antibody (SantaCruz, #sc-47778, 1:2000 dilution); rabbit polyclonal H3K4me3 antibody (Abcam, #ab8580, 1:5000 dilution); rabbit polyclonal H3K27Ac antibody (Abcam, #ab4729, 1:1000 dilution); or rabbit polyclonal histone H3 antibody (Abcam, #ab1791, 1:200000 dilution). The probed membranes were washed three times with TBS-T and incubated with ECL rabbit IgG HRP-conjugated whole antibody (GE Healthcare LifeScience, #NA934, 1:2500 dilution) or ECL mouse IgG HRP-conjugated whole antibody (GE Healthcare LifeScience, #NA931, 1:2500 dilution), and washed three times with TBS-T again. Immunoblots were visualized by enhanced chemiluminescence using chemiluminescent HRP substrate (Millipore, #P90718).

[0094] (4) Results and Discussion Before investigating KDM5 PROTACs, we tested compound 24 (Figure 19(A)) in a neurite outgrowth assay, which is commonly used for early screening of neurodegenerative disease drugs, because compounds that inhibit KDM5A have been suggested as potential therapeutic candidates for neurodegenerative diseases, including Alzheimer's disease. As shown in Figure 19(B), incubation of mouse neuroblastoma N2a cells with 0.2 μM compound 24 for 24 or 48 hours failed to significantly induce neurite outgrowth. This indicates that conventional KDM5 inhibitors, which inhibit only the catalytic function of KDM5A, do not exhibit strong neurite outgrowth activity. Based on the assumption that the overall function of KDM5A, including the scaffold that interacts with HDACs 1 and 2 to regulate histone deacetylation, is important for neurite outgrowth, we examined the neurite outgrowth-promoting activity of a KDM5 inhibitor in combination with the HDAC inhibitor vorinostat (25) (Figure 19(A)). Notably, this combination significantly induced neurite outgrowth compared with compounds 24 or 25 alone (Figure 19(B)). This indicates that the dual approach of inducing both histone methylation and acetylation by inhibiting KDM5A and HDAC, respectively, is an effective means to promote neurite outgrowth. These results suggest that TPD (target protein degradation)-mediated degradation of KDM5A can potently induce neurite outgrowth in N2a cells by disrupting both the catalytic and scaffolding functions of KDM5A. This idea prompted the synthesis of KDM5 PROTAC candidates that exhibit potent neurite outgrowth activity.

[0095] Figure 19(A) shows the structures of the KDM5 inhibitors 24 and vorinostat (25), and Figure 19(B) shows the effects of compounds 24 and 25 alone and co-treatment with compounds 24 and 25 on N2a differentiation after 24 and 48 hours of treatment. Bar graphs represent mean values ​​± SD from three independent experiments. p values ​​were determined using Tukey's multiple comparison test; *p<0.05. Representative images showing N2a cells treated with compounds 24 and 25 alone and co-treatment with compounds 24 and 25 are shown in Figure 20.

[0096] To design the KDM5 PROTAC, compound 24 was selected as a KDM5 ligand. Simulations of compound 24 bound to KDM5A suggested that the hexyl chain faces the protein surface. Based on these simulations, a linker was introduced to the end of the hexyl group. Because the linker structure often influences degradation activity, we tested various linkers, including an amide-bonded polyethylene glycol linker (14) and benzene ring-containing linkers (20 and 23) (Figure 21(A)). Furthermore, we selected thalidomide (26), the most widely used compound in PROTAC research, as the E3 ligand. Next, we synthesized compounds 14a, 14b, 20a, 20b, 23a, and 23b as novel KDM5 PROTAC candidates (Figure 21(A)) (see the chemical formula above) and screened the synthesized compounds in a neurite outgrowth activity assay. As shown in Figure 21(B), treatment of N2a cells with compounds 14a, 14b, 20a, 20b, 23a, and 23b promoted neurite outgrowth. Compound 14b exhibited stronger neurite outgrowth-promoting activity than compound 14a. This indicates that the introduction of a spacer between the hexyl group of the KDM5 inhibitor and the amide group of the linker is important for neurite outgrowth-promoting activity. Furthermore, replacing the spacer with a benzene ring is expected to contribute to the rigidity of the linker orientation, but the effect was maintained or even increased. Importantly, the activity of meta-substituted compounds 20b and 23b was superior to that of para-substituted compounds 20a and 23a. Furthermore, the activity of the carbon-linked compound 23b was slightly superior to that of the polyethylene-linked compound 20b. Preliminary structure-activity relationship studies revealed that compounds with linkers containing a meta-substituted benzene ring exhibited potent neurite outgrowth-promoting activity. As a result, compounds 20b and 23b exhibited significant neurite outgrowth-promoting activity. Compounds 20b and 23b were also tested at different concentrations and incubation times (Figures 21(C) and 21(D)). Both compounds promoted neurite outgrowth in the range of 0.02-2 μM (Figure 21(C)). When applied at 0.02 μM, they increased the number of neurite cells in a time-dependent manner (Figure 21(D)). Furthermore, the effect of the E3 ligand, compound 26, on neurite outgrowth activity was examined (Figure 21(C)). Importantly, compound 26 did not exhibit as strong neurite outgrowth activity as compounds 20b and 23b. These results suggest that PROTAC compound-mediated degradation of KDM5 leads to strong neurite outgrowth.

[0097] Figure 21(A) shows the design of the KDM5 PROTAC. Figure 21(B) shows the effect of 0.2 μM compounds 14a, 14b, 20a, 20b, 23a, and 23b on N2a differentiation after 48 hours of treatment. Figure 21(C) shows the dose-dependent N2a cell neurite outgrowth activity of compounds 20b, 23b, and 26 after 48 hours of treatment. Figure 21(D) shows the time-dependent N2a cell neurite outgrowth activity of compounds 20b and 23b at 0.02 μM. Bar graphs represent the mean ± SD from three independent experiments. Representative images showing N2a cells treated with test compounds are shown in Figures 22-24.

[0098] Next, the KDM5A degradation activity of compounds 20b and 23b was examined by Western blot analysis, confirming their function as KDM5 PROTACs. Treatment of N2a cells with compounds 20b and 23b reduced KDM5A levels in a dose-dependent manner (Figure 25(A)). Combination of compounds 20b and 23b with their parent compounds, compounds 24 and 26, did not reduce KDM5A levels (Figure 25(B)). Furthermore, the reduction of KDM5A levels by compounds 20b and 23b was blocked by the proteasome inhibitor MG-132 (Figures 25(C) and 25(D)). These results suggest that compounds 20b and 23b function as PROTACs to reduce KDM5A levels in N2a cells.

[0099] Figure 25 shows Western blot detection of KDM5A levels in N2a cells, where Figure 25(A) shows the results after 24 hours of treatment with compounds 20b and 23b, Figure 25(B) shows the results after 24 hours of treatment with compound 23b and 24 hours of co-treatment with compounds 24 and 26, Figure 25(C) shows the results after 24 hours of co-treatment with compound 20b and the proteasome inhibitor MG-132, and Figure 25(D) shows the results after 24 hours of co-treatment with the proteasome inhibitor MG-132 and compound 23b.

[0100] Finally, we investigated the effects of PROTAC compounds 20b and 23b on histone methylation and acetylation. Because H3K4me3 is a substrate of KDM5s, we analyzed the H3K4 methylation levels in N2a cells treated with the KDM5 PROTAC. As shown in Figure 26, treatment with 20b and 23b resulted in a dose-dependent accumulation of H3K4me3 levels. This suggests that 20b and 23b inhibit the catalytic function of KDM5s in N2a cells. We then investigated whether compounds 20b and 23b, through their interaction with HDACs 1 and 2, affect the levels of acetylated lysine 27 of histone H3 (H3K27Ac), which is a direct substrate of HDACs 1 and 2 and an indirect substrate of KDM5s. As expected, 20b and 23b also increased H3K27Ac levels (Figure 26). In addition, compounds 20b and 23b potently inhibited KDM5A in in vitro assays without affecting HDAC1 (Figure 27). Here, when evaluating their inhibitory activity against HDAC1, the HDAC inhibitor vorinostat (25) was used as a positive control, demonstrating 99% inhibition at 1 μM. These results indicate that degradation of KDM5A by 20b and 23b inhibited both the catalytic function of KDM5s, which regulates H3K4me3, and the scaffolding function of KDM5s, which interferes with the deacetylation activity of HDAC1 and 2, in N2a cells.

[0101] In addition, FIG. 26 shows Western blot detection of H3K4me3 and H3K27Ac levels in N2a cells treated with compounds 20b and 23b for 24 hours.

[0102] (5) Conclusion Although inhibition of KDM5s has been reported to be effective in treating neurodegenerative diseases, in this study, the conventional KDM5 inhibitor 24 did not induce strong neurite outgrowth in N2a cells. On the other hand, the combination of compound 24 with the HDAC inhibitor vorinostat (25) significantly induced neurite outgrowth in N2a cells, suggesting that inhibition of both KDM5 catalytic activity and HDAC scaffolding binding influences neurite outgrowth. Therefore, we focused on KDM5 PROTACs that can inhibit both of these functions and synthesized compounds 14a, 14b, 20a, 20b, 23a, and 23b as KDM5 PROTAC candidates. Among these, compounds 20b and 23b significantly promoted neurite outgrowth in N2a cells. Furthermore, biological assays indicated that treatment of N2a cells with compounds 20b and 23b resulted in the degradation of KDM5A via PROTAC-mediated proteasomal degradation. This degradation affected the overall function of KDM5, in contrast to conventional KDM5 inhibitors, and therefore we believe that KDM5PROTACs hold promise as therapeutic agents for neurological diseases. [Industrial Applicability]

[0103] This compound exhibits remarkable pharmacological effects, including anti-cancer activity, in cell and animal experiments.

Claims

1. A compound represented by the following formula (I) or a salt thereof: 【Chemical 1】 In formula (I), A is a single bond or a substituted or unsubstituted divalent aromatic hydrocarbon group, L is a substituted or unsubstituted linear or branched alkylene group having 1 to 13 carbon atoms, and X is -(CH ２ CH ２ O) ｓ -, -(CH ２ CH ２ CH ２ O) ｓ -, -(CH(CH ３ ) CH ２ O) ｓ - or -(CH ２ ) ｔ It is a group represented by O—, in which s is a number from 1 to 8, and t is a number from 2 to 10.

2. In the formula (I), A is a single bond or a phenylene group, L is a linear alkylene group having 3 to 10 carbon atoms, and X is —(CH ２ CH ２ O) ｓ -, -(CH ２ CH ２ CH ２ O) ｓ -, -(CH(CH ３ ) CH ２ O) ｓ - or -(CH ２ ) ｔ 2. The compound or salt thereof according to claim 1, wherein s is a group represented by O—, s is a number from 1 to 5, and t is a number from 2 to 10.

3. In the formula (I), A is a single bond, a p-phenylene group, or an m-phenylene group, L is a linear alkylene group having 3 to 10 carbon atoms, and X is —(CH ２ CH ２ O) ｓ -, -(CH ２ CH ２ CH ２ O) ｓ - or -(CH ２ ) ｔ 2. The compound or salt thereof according to claim 1, wherein s is a group represented by O—, s is a number from 1 to 5, and t is a number from 2 to 10.

4. The compound or salt thereof according to claim 1, wherein the compound is a compound represented by the following formula (I-1), a compound represented by the following formula (I-2), or a compound represented by the following formula (I-3): 【Chemistry 2】 In formula (I-1), L １ is a linear alkylene group having 3 to 10 carbon atoms, and X １ Ha-(CH ２ CH ２ O) ｓ -, s is a number of 1 to 5, and in formula (I-2), L ２ is a linear alkylene group having 3 to 10 carbon atoms, and X ２ Ha-(CH ２ CH ２ O) ｓ - or - (CH ２ ) ｔ O—, s is a number from 1 to 5, and t is a number from 2 to 10; and in formula (I-3), L ３ is a linear alkylene group having 3 to 10 carbon atoms, and X ３ Ha-(CH ２ CH ２ O) ｓ - or - (CH ２ ) ｔ It is a group represented by O—, in which s is a number from 1 to 5, and t is a number from 2 to 10.

5. The compound is In the formula (I-1), L １ is a linear alkylene group having 5 carbon atoms, and X １ Ga-(CH ２ CH ２ O) ｓ -, and s is 3; In the formula (I-1), L １ is a linear alkylene group having 8 carbon atoms, and X １ Ga-(CH ２ CH ２ O) ｓ -, and s is 3; In the formula (I-2), L ２ is a linear alkylene group having 5 carbon atoms, and X ２ Ga-(CH ２ CH ２ O) ｓ -, and s is the number 3; In the formula (I-2), L ２ is a linear alkylene group having 5 carbon atoms, and X ２ Ga-(CH ２ ) ｔ a compound in which t is a group represented by O— and t is the number 6; In the formula (I-3), L ３ is a linear alkylene group having 5 carbon atoms, and X ３ Ga-(CH ２ CH ２ O) ｓ -, where s is the number 3, or In the formula (I-3), L ３ is a linear alkylene group having 5 carbon atoms, and X ３ Ga-(CH ２ ) ｔ a compound in which t is a group represented by O— and t is the number 6; The compound or salt thereof according to claim 4, wherein:

6. A pharmaceutical composition comprising the compound or salt thereof according to any one of claims 1 to 5.

7. The pharmaceutical composition according to claim 6, which is a KDM5 inhibitor and a KDM5 degrader.

8. The pharmaceutical composition according to claim 6, which is an agent for preventing and / or treating a KDM5-related disease.

9. 6. The compound or salt thereof according to any one of claims 1 to 5, for use in the prevention and / or treatment of a KDM5-related disease in which KDM5 inhibitory activity and KDM5 decomposition activity are effective.

10. The pharmaceutical composition according to claim 6, for use in the prevention and / or treatment of KDM5-related diseases in which KDM5 inhibitory activity and KDM5 decomposition activity are effective.

11. Use of the compound or salt thereof according to any one of claims 1 to 5 in the manufacture of a pharmaceutical composition for the prevention and / or treatment of a KDM5-related disease in which KDM5 inhibitory activity and KDM5 decomposition activity are effective.

12. A preventive and / or therapeutic agent for a KDM5-related disease, comprising the compound or salt thereof according to any one of claims 1 to 5 as an active ingredient.

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