Sterol derivative having innate immune regulatory activity

Abstract

To provide a novel compound associated with regulation of a STING pathway activity, or to provide a novel use for a specific sterol derivative.SOLUTION: In some embodiments, provided is a compound represented by the general formula (I) or (II), a pharmaceutically acceptable salt thereof, or a prodrug thereof. In some embodiments, provided is a STING pathway modulator or an agent for treating a STING-related disease, comprising a compound represented by the general formula (I) or (II), a pharmaceutically acceptable salt thereof, or a prodrug thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a sterol derivative having a natural immune regulating effect. [Background technology]

[0002] Defense mechanisms against viruses and pathogens include the adaptive immune system, which remembers and prepares for pathogens that have invaded the body once, and the innate immune system, which recognizes molecules characteristic of pathogens and initiates a response to eliminate the pathogen. The STING (Stimulator of interferon genes) pathway is one of the innate immune systems, and is known to detect DNA derived from viruses in the cytoplasm and initiate a defense response through the production and secretion of interferon. In the STING pathway, when cytoplasmic DNA binds to cGAS (cyclic GMP-AMP synthase), the enzyme activity of cGAS is activated, and the second messenger cyclic dinucleotide cGAMP is synthesized. cGAMP binds to STING, a membrane protein of the endoplasmic reticulum, and causes a conformational change, leading to transport to the Golgi apparatus, palmitoylation of STING, and oligomerization. On the STING oligomer, the kinase TBK1 phosphorylates itself, STING, and the transcription factor IRF3. Phosphorylated IRF3 becomes active and activates the transcription of antiviral cytokines such as interferon, resulting in antiviral and inflammatory responses.

[0003] Activation of the STING pathway plays an important role in the immune response to cancer cells. It is expected that STING pathway activation may be used as a cancer treatment, either alone or in combination with existing cancer immunotherapy, and compounds that activate the STING pathway have been explored. Derivatives based on cGAMP-like nucleic acids with improved stability, etc., and small molecule STING agonists without nucleic acid structure are known, and many compounds are currently undergoing clinical trials.

[0004] On the other hand, excessive activation of the STING pathway is not only the direct cause of STING-associated vasculitis in infancy (SAVI), a genetic disease, but also the activation of the STING pathway is known to exacerbate inflammatory and autoimmune diseases classified as type I interferonopathies, such as systemic lupus erythematosus. Therefore, compounds that suppress activation of the STING pathway have the potential to be used as therapeutic agents for inflammatory and autoimmune diseases, including those mentioned above.

[0005] The search for compounds that inhibit the STING pathway has lagged behind the search for activators, and no clinical trials have been conducted (Non-Patent Document 1). Non-Patent Document 2 describes a compound that competitively binds to the cGAMP binding site of STING, but although its activity at the protein level is high at the nM order, its activity at the cellular level is low. Non-Patent Document 3 also reports a compound that is said to act on the cGAMP binding site. In addition, nitrated unsaturated fatty acids have been reported as endogenous compounds that covalently bind to cysteine, which is the palmitoylation site necessary for STING activation (Non-Patent Document 4), and other synthetic compounds have also been reported (Non-Patent Document 5).

[0006] In addition, compounds that have the same structure as STING agonists have been reported as compounds that are thought to act competitively at the cGAMP binding site (Patent Document 1). Compounds that bind covalently to the palmitoylation site have also been reported (Non-Patent Document 6).

[0007] In addition, Non-Patent Document 7 describes 25-hydroxycholesterol (25HC) as a compound that has an inhibitory effect on the activation of the STING pathway. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. WO2019 / 269270 [Non-patent literature]

[0009]

Outline Building1

Outdoor Tool2

Outdoor Tools3

Outdoor Tools 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Summary of the Invention

Problems to be Solved by the Invention

[0010] An object of the present invention is to provide a new compound involved in regulating the activity of the STING pathway, or to provide a new use of a specific sterol derivative. [Means for solving the problem]

[0011] In order to solve the above problems, the inventors conducted intensive research and discovered that sterol derivatives having a specific structure have the effect of regulating the activity of the STING pathway, thereby completing the present invention.

[0012] That is, the present invention relates to the following. [1] A compound represented by the following general formula (I) or (II), a pharmacologically acceptable salt thereof, or a prodrug thereof: [ka] (In the formula, R 1 -OR 4 , and -NHR 5 selected from the group consisting of; [ka] teeth, [ka] selected from the group consisting of; R 2 ' is CH 2 and NH; R 2 " is selected from CH; R 3 teeth, [ka] selected from the group consisting of; R 4 -H, and -C(=O)R 6 selected from the group consisting of; R 5 is selected from the group consisting of -H, -C(=O)R 7 , and -S(=O) 2 R 7 ; R 6 is

Chem.

Chem.

Chem.

Chem.

Chem.

Chemical formula

Chemical formula

[10] The agent according to any one of [7] to [9], R 8 But -CH 3 Selected from; R 22 is selected from -H; [ka] but, [ka] selected from the group consisting of; [ka] but, [ka] selected from the group consisting of; [ka] but, [ka] selected from the group consisting of; [ka] but, [ka] An agent selected from:

[11] The agent according to any one of [7] to

[10] , X, [ka] JPEG2025079637000047.jpg48170.

[12] A STING pathway regulator or a STING-related disease treatment agent, comprising any one of the following compounds, a pharmacologically acceptable salt thereof, or a prodrug thereof: [ka] JPEG2025079637000049.jpg226170JPEG2025079637000050.jpg196170

[13] The agent described in any of [7] to

[12] , wherein the regulation of the STING pathway or the treatment of a STING-related disease is achieved by partial activation of the STING pathway.

[14] The agent according to any one of [7] to

[13] , wherein the STING-associated disease is STING-associated vasculitis, COPA (Coatomer protein alpha) syndrome, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, Aicardi-Goutières syndrome, systemic lupus erythematosus, Parkinson's disease, or Huntington's disease.

[0013] Alternatively, the present invention relates to the following: <1> A method for regulating STING pathway activity or treating a STING-associated disease, comprising administering to a subject a compound represented by general formula (I) or (II), a pharmacologically acceptable salt thereof, or a prodrug thereof. <2> A compound represented by general formula (I) or (II), a pharmacologically acceptable salt thereof, or a prodrug thereof, or a pharmaceutical composition comprising the same, for use in a method for modulating STING pathway activity or in treating a STING-associated disease. <3> Use of a compound represented by general formula (I) or (II), a pharmacologically acceptable salt thereof, or a prodrug thereof for the manufacture of a STING pathway regulator or an agent for treating a STING-related disease. Effect of the Invention

[0014] According to the present invention, there are provided sterol derivatives having a regulating effect on the STING pathway, as well as STING pathway regulators and STING-related disease therapeutic agents each containing the sterol derivatives. [Brief description of the drawings]

[0015] [Figure 1] Figure 1 shows the results of using OG828 (C1) and ELN025 (C29) as an example of evaluating the concentration dependency of the STING pathway regulating effect of sterol derivatives on mouse STING-expressing cells. H151: STING antagonist. [Diagram 2] Figure 2 shows the results of Western blotting to evaluate the effects of sterol derivatives (OGK4005 (C26), OGK4006 (C27), OGK4007 (C28)) on STING agonist-dependent phosphorylation of STING, TBK1, and IRF3. DMXAA: vadimezan (mouse STING agonist), 25-HC: 25-hydroxycholesterol. [Diagram 3] 3 is a graph showing the results of evaluating the STING pathway regulating effect of the sterol derivative ELN025 (C29) in cells expressing human STING SAVI mutant. SN-011, H-151: STING antagonist. [Figure 4] Figure 4 is a graph showing the results of evaluating the STING pathway regulatory effect of the sterol derivative ELN025 (C29) alone or in the presence of an agonist in mouse STING-expressing cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention will now be described. Note that the features of the present invention described below can be combined in any combination.

[0017] [Compound] In one embodiment, the present invention relates to a compound represented by the following general formula (I) or (II), a pharmacologically acceptable salt thereof, or a prodrug thereof. In another embodiment, the present invention relates to a compound represented by the following general formula (I) or (II), or a pharmacologically acceptable salt thereof. Hereinafter, when referring to the compound of the present invention, it refers to one or more of the compound represented by the general formula (I) and the compound represented by the general formula (II).

[0018] [ka]

[0019] In the formula, a bond represented by a dotted line may not exist (the same applies below). The dotted line means that [ka] Refers to, [ka] This does not refer to the above.

[0020] In the present invention, [ka] , respectively. [ka] It is sometimes written as:

[0021] In another aspect, the present invention relates to a compound represented by the general formula (I), a pharmacologically acceptable salt thereof, or a prodrug thereof. In yet another aspect, the present invention relates to a compound represented by the general formula (I), or a pharmacologically acceptable salt thereof.

[0022] In one embodiment, R 1 -OR 4 , and -NHR 5In another embodiment, R 1 -OR 4 is selected from.

[0023] In one embodiment, [ka] teeth, [ka] In another embodiment, the compound is selected from the group consisting of: [ka] teeth, [ka] In yet another embodiment, the compound is selected from the group consisting of: [ka] teeth, [ka] is selected from.

[0024] In one embodiment, R 2 ' is CH 2 and NH. In another embodiment, R 2 ' is CH 2 In one embodiment, R 2 " is selected from CH.

[0025] In one embodiment, R 3 teeth, [ka] In another embodiment, R 3 teeth, [ka] is selected from.

[0026] In one embodiment, R 4 -H, and -C(=O)R 6 In another embodiment, R 4 is selected from -H.

[0027] In one embodiment, R 5 -H, -C(=O)R 7 , and -S(=O) 2 R 7 In another embodiment, R 5 is selected from -H.

[0028] In one embodiment, R 6 teeth, [ka] is selected from.

[0029] In one embodiment, W 1 , W 2 , and W 3 are each independently 8 , and N.R. 8 Here, "independently" means, for example, that 1 , W 2 , and W 3 may be selected from different ones (the same applies hereinafter in the present invention). In another embodiment, W 1 , and W 2 are each independently 8 Selected from W 3 is NR 8 In yet another embodiment, W 1 , and W 2 is CH 2 Selected from W 3 is NR 8 is selected from.

[0030] In one embodiment, R7 -C 1-6 In another embodiment, R 7 -C 1-5 Alkyl, and -C 1-4 In yet another embodiment, R 7 -C 1-3 alkyl. 7 The alkyl group in R may be linear or branched, linear or cyclic, but is preferably linear. 7 Non-limiting examples of alkyl include propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, and the like. 7 is -CH 2 CH 3 , and -CH 3 In yet another embodiment, R 7 is -CH 3 is selected from.

[0031] In one embodiment, R 8 -H, and -C 1-6 In another embodiment, R 8 -C 1-6 In yet another embodiment, R 8 -C 1-5 Alkyl, and -C 1-4 In yet another embodiment, R 8 -C 1-3 alkyl. 8 The alkyl group in R may be linear or branched, linear or cyclic, but is preferably linear. 8 Non-limiting examples of alkyl include propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, and the like. 8 is -CH2 CH 3 In yet another embodiment, R 8 is selected from -CH.

[0032] In some embodiments, X is [ka] In another embodiment, X is selected from the group consisting of: [ka] is selected from the group consisting of:

[0033] In yet another embodiment, X is [ka] JPEG2025079637000068.jpg143170 (any combination of one or more of the above Xs may be selected).

[0034] In one embodiment, R 9 , R 12 , and R 14 are each independently -C 1-6 In another embodiment, R 9 , R 12 , and R 14 are each independently -C 1-5 In yet another embodiment, R is selected from alkyl or absent. 9 , R 12 , and R 14 are each independently -C 1-4 In yet another embodiment, R is selected from alkyl or absent. 9 , R 12 , and R 14 are each independently -C 1-3 alkyl or absent. 9 , R 12 , and R14 Each alkyl may be independently linear or branched, linear or cyclic, but is preferably linear. 9 , R 12 , and R 14 Non-limiting examples of alkyl include propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, and the like. 9 , R 12 , and R 14 are each independently -CH 2 CH 3 and -CH, or absent.

[0035] In the present invention, there are cases where a substituent or the like is "not present." For example, [ka] In R 9 If does not exist, [ka] (In the present invention, the same applies hereinafter when "does not exist" is mentioned.)

[0036] In one embodiment, R 10 , R 11 , R 13 , and R 15 are each independently -H, -C 1-6 Alkyl, -R 16 C.C.H. [ka] In addition, R in the formula is selected from the group consisting of 17 , R 18 , R 19 , R 20 , R 21In another embodiment, R 10 , R 11 , R 13 , and R 15 are each independently -H, -C 1-5 Alkyl, -C 1-4 Alkyl, -C 1-3 Alkyl, -CH 2 CH 3 , -CH 3 , -R 16 C.C.H. [ka] With the proviso that in certain embodiments, R 1 is OH, [ka] but, [ka] and R 2 ' is CH 2 (i) R 10 and R 11 All of the above are -CH 3 (ii) R 10 and R 11 All of the above are -CH 2 CH 3 and (iii) R 10 and R 11 One of them is -H and the other is -CH 3 It is not possible to be R 10 , R 11 , R 13 , and R 15 The alkyl, alkenyl, and alkynyl in R may each independently be a straight chain or a branched chain, and may be a chain or a cyclic group, but is preferably a straight chain. 10 , R 11 , R 13 , and R15 Non-limiting examples of alkyl include propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, and the like. 10 , R 11 , R 13 , and R 15 Non-limiting examples of alkenyl include allyl, prenyl, pentenyl, and the like. 10 , R 11 , R 13 , and R 15 Non-limiting examples of alkynyl include propargyl, butynyl, pentynyl, hexynyl and the like.

[0037] In one embodiment, [ka] teeth, [ka] is selected from the group consisting of:

[0038] In one embodiment, [ka] teeth, [ka] is selected from the group consisting of:

[0039] In one embodiment, [ka] teeth, [ka] is selected from the group consisting of:

[0040] In one embodiment, [ka] is selected from

Chem.

[0041] In one aspect, the present invention relates to any of the following compounds, their pharmaceutically acceptable salts, or their prodrugs (it is possible to select by combining one or more arbitrary compounds from the following).

Chem.

[0042] In one aspect, Y 1 and Y 2 and Y 3 are each independently selected from the group consisting of CHR 22 and NR 22 . In another aspect, Y 1 and Y 2 and Y 3 are each independently selected from CHR 22 .

[0043] In one aspect, Z 1 and Z 2 and Z 3 are each independently selected from the group consisting of CH and N. In another aspect, Z 1 is selected from CH, and Z 2 and Z 3 are each independently selected from the group consisting of CH and N.

[0044] In one aspect, R 16 is selected from -C 1-6 alkyl or does not exist. In another aspect, R 16 is -C 1-5is selected from alkyl or does not exist. In yet another aspect, R 16 is, -C 1-4 is selected from alkyl or does not exist. In yet another aspect, R 16 is, -C 1-3 is selected from alkyl or does not exist. Note that the alkyl of R 16 may be linear or branched, and may be chain or cyclic, but is preferably linear. Non-limiting examples of the alkyl of R 16 include propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, etc. In yet another aspect, R 16 is, -CH 2 CH 3 , and -CH, or does not exist.

[0045] In one aspect, R 17 , and R 18 are each independently, -NH 2 , -COOH, -C(=O)NH 2 , -NHC(=O)CH 3 , -NHCOOC(CH 3 ) 3 , -NHS(=O) 2 CH 3 , -OH, -C 1-3 alkyl, -CCH, -CCSi(CH 3 ) 3 , -NHC(=O)CH 2 CH 2 CCH, -NHC(=O)CH 2 CH 2 CH 2 CH 3 , -N 3 , -F, -Cl, -Br, -I, -CN, and -CF 3 , or does not exist. In another aspect, R 17 , and R 18 are each independently, -NH 2 , -NHC(=O)CH 3, -NHCOOC(CH 3 ) 3 , -CH 3 , -CH 2 CH 3 , -CCH, -CCSi(CH 3 ) 3 , -NHC(=O)CH 2 CH 2 CCH, -NHC(=O)CH 2 CH 2 CH 2 CH 3 , -N 3 , -CN, and -CF 3 or absent.

[0046] In one embodiment, Z 4 , NH, NCH 3 , O, and S.

[0047] In one embodiment, R 19 -F, -Cl, -Br, -I, -CH 3 , -COOR 23 , and -C(=O)NR 24 R 25 or absent.

[0048] In one embodiment, R 20 , and R 21 are each independently -H, -NH 2 , -NHC(=O)CH 3 , -NHCOOC(CH 3 ) 3 , -NHC(=O)CH 2 CH 2 CH 2 CH 3 , and -NHC(=O)CH 2 CH 2 In another embodiment, R is selected from the group consisting of CCH. 20 , and R 21 are each independently -H, -NH 2 , -NHC(=O)CH 3 , -NHCOOC(CH3 ) 3 , -NHC(=O)CH 2 CH 2 CH 2 CH 3 , and -NHC(=O)CH 2 CH 2 In yet another embodiment, R is selected from the group consisting of CCH. 20 , and R 21 are each independently -H, -NHCOOC(CH 3 ) 3 , -NHC(=O)CH 2 CH 2 CH 2 CH 3 , and -NHC(=O)CH 2 CH 2 CCH or absent.

[0049] In one embodiment, R 22 , R 23 , R 24 , and R 25 are each independently -H, and -C 1-6 In another embodiment, R 22 , R 23 , R 24 , and R 25 are each independently -H, -C 1-5 Alkyl, and -C 1-4 In yet another embodiment, R 22 , R 23 , R 24 , and R 25 are each independently -H, and -C 1-3 alkyl. 22 , R 23 , R 24 , and R 25 Each alkyl may be independently linear or branched, linear or cyclic, but is preferably linear. 22 , R 23 , R 24 , and R25 Non-limiting examples of alkyl include propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, and the like. 22 , R 23 , R 24 , and R 25 are each independently -H, -CH 2 CH 3 , and -CH 3 In yet another embodiment, R 22 , R 23 , R 24 , and R 25 are each independently -H, and -CH 3 is selected from the group consisting of:

[0050] The "pharmacologically acceptable salt" of a compound can be an acidic or basic salt. Examples of basic salts include, but are not limited to, alkali metal salts such as sodium and potassium; alkaline earth metal salts such as calcium and magnesium; ammonium salts; and nitrogen-containing organic base salts such as trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenethylamine, 1-ephenamine, and N,N'-dibenzylethylenediamine. Examples of acid salts include, but are not limited to, mineral acid salts such as hydrochloride, hydrobromide, nitric acid, and sulfuric acid; organic carboxylates such as formic acid, acetic acid, citric acid, oxalic acid, fumaric acid, maleic acid, succinic acid, malic acid, tartaric acid, aspartic acid, trichloroacetic acid, and trifluoroacetic acid; and sulfonates such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid, and naphthalenesulfonic acid.

[0051] The compound of the present invention, its pharmacologically acceptable salt, or their prodrugs may have any crystal structure, and may further be a solvate. A solvate refers to a compound containing solvent molecules in a crystal at a certain stoichiometric ratio. The compound of the present invention, its pharmacologically acceptable salt, or their prodrugs may contain any solvent molecule (water, alcohol, etc.), and may be, for example, a hydrate containing water molecules. In addition, the number of solvent molecules in the solvate is not particularly limited, and for example, the solvate may be a dihydrate containing two water molecules. In addition, the solvate also includes an anhydride (anhydrous product) obtained by removing water molecules from a hydrate.

[0052] A prodrug is a derivative that is converted into the compound of the present invention or a pharmacologically acceptable salt thereof in vivo. Selection and preparation of a derivative suitable for a prodrug can be performed, for example, by referring to "Design of Prodrugs" (Elseveier, 1985) by Bundgard.

[0053] In some embodiments, the compound of the present invention, its pharmacologically acceptable salt, or a prodrug thereof may have any functional group in its structure protected. A functional group is protected when a protecting group is bonded to the functional group, thereby reducing the reactivity of the functional group. A commonly used protecting group can be arbitrarily selected by referring to, for example, Greene's Protective Groups in Organic Synthesis 5th Edition (John Wiley & Sons, 2014). When the functional group is an amino group, the protecting group may be, for example, a tert-butoxycarbonyl group (Boc group), a benzyloxycarbonyl group (Cbz group), a 9-fluorenylmethyloxycarbonyl group (Fmoc group), a 2,2,2-trichloroethoxycarbonyl group (Troc group), an allyloxycarbonyl group (Alloc group), a phthaloyl group (Pht group), a p-toluenesulfonyl group (Ts group), a 2-nitrobenzenesulfonyl group (Ns group), an allyl group, etc. When the functional group is a hydroxy group, the protecting group may be, for example, a C group such as a methyl group, a tBu group, etc. 1-6 The protecting group may be an alkyl group, an aralkyl group such as a benzyl group (Bn group) or a p-methoxybenzyl group (PMB group), an acyl group such as an acetyl group (Ac group) or a benzoyl group (Bz group), or a silyl group such as a trimethylsilyl group (TMS group), a triethylsilyl group (TES group), a tert-butyldimethylsilyl group (TBS group), a mesyl group (Ms group), or a tert-butyldiphenylsilyl group (TBDPS group). The protecting group is preferably one which is easily deprotected, but is not particularly limited.

[0054] [Manufacturing scheme] The compound of the present invention can be produced by combining methods known to those skilled in the art, for example, according to the scheme shown below.

[0055] (Scheme 1) [ka]

[0056] In the formula, R9 can be selected as described above in the [Compound] section. For example, R 9 Ga-CH 2 CH 2 -, the starting compound for this reaction is 3β-hydroxy-Δ5-cholenic acid, which is commercially available. In the formula, a is, for example, R 10 , or R 11 may be selected from. The solvent used in this reaction is not particularly limited as long as it does not affect the reaction, and may be, for example, halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, aromatic hydrocarbons, etc., and may be preferably tetrahydrofuran (THF). The amount of the solvent is not particularly limited, and may be, for example, 1 to 1000 times (v / w) the amount of the starting compound. This reaction can be carried out in the presence of a suitable base. Non-limiting examples of suitable bases include triethylamine, N,N-diisopropylethylamine, 4-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene, N,N-dimethylaminopyridine, and the like. Preferably, the base is triethylamine (Et 3 The amount of the base used is not particularly limited, and may be, for example, 0.1 to 10 times the molar amount of the starting compound. This reaction can be carried out in the presence of a suitable condensing agent. Non-limiting examples of suitable condensing agents include isobutyl chloroformate, dicyclohexylcarbodiimide, diisopropylcarbodiimide, p-toluenesulfonyl chloride, 1-ethyl-3-(N,N-dimethylaminopropyl)carbodiimide hydrochloride, carbonyldiimidazole, ethyl chloroformate, isobutyl chloroformate, 2,3,6-trichlorobenzoic acid chloride, 2-methyl-6-nitrobenzoic anhydride, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and the like. Preferably, the condensing agent is isobutyl chloroformate. The amount of the condensing agent used is not particularly limited, and may be, for example, 0.1 to 10 times the molar amount of the starting compound. This reaction may be carried out in a state where the hydroxyl group of the sterol ring is protected. Non-limiting examples of protecting groups for protecting the hydroxyl group include TBS group, Ms group, C 1-6 Examples of the protecting group include an alkyl group, a Bn group, a PMB group, an Ac group, a Bz group, a tri-TMS group, a TES group, and a TBDPS group, and preferably, the protecting group may be a TBS group. The method of protecting the hydroxyl group of the sterol ring with a TBS group is not particularly limited, and may be, for example, a method of reacting the above starting compound with t-butyldimethylchlorosilane and imidazole in a solvent, etc. The reaction temperature for this reaction may be, for example, −30° C. to 100° C., and the reaction time may be, for example, 5 minutes to 48 hours.

[0057] (Scheme 2) [ka]

[0058] In the formula, R 12 , and R 13 can be selected as described above in the [Compound] section. For example, R 12 Ga-CH 2 CH 2 In the case of -, the starting compound is 3β-hydroxy-Δ5-cholenic acid, which is commercially available. The solvent used in this reaction is not particularly limited as long as it does not affect the reaction, and may be, for example, halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, aromatic hydrocarbons, etc., and may be preferably dimethylformamide (DMF). The amount of the solvent is not particularly limited, and may be, for example, 1 to 1000 times (v / w) the amount of the starting compound. This reaction can be carried out in the presence of a suitable acid trapping agent. Non-limiting examples of suitable acid trapping agents include inorganic bases such as potassium carbonate and sodium hydroxide, epoxy compounds such as propylene oxide and allyl glycidyl ether, and organic tertiary amines such as pyridine, triethylamine, and N,N-dimethylaniline. The acid trapping agent may be potassium carbonate. The amount of the base used is not particularly limited, and may be, for example, 0.1 to 10 times the molar amount of the starting compound. The reaction temperature for this reaction may be, for example, −30° C. to 100° C., and the reaction time may be, for example, 5 minutes to 72 hours.

[0059] (Scheme 3) [ka]

[0060] In the formula, R 9 , R 10 , and R 11 can be selected as described above in the [Compound] section. For example, R 9 Ga-CH 2 CH 2 -, the starting compound for this reaction is 3β-hydroxy-Δ5-cholenic acid, which is commercially available. The solvent used in this reaction is not particularly limited as long as it does not affect the reaction, and may be, for example, halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, aromatic hydrocarbons, etc., and may be preferably dimethylformamide (DMF). The amount of the solvent is not particularly limited, and may be, for example, 1 to 1000 times (v / w) the amount of the starting compound. This reaction can be carried out in the presence of a suitable base. Non-limiting examples of suitable bases include organic bases such as N-diisopropylethylamine, triethylamine, pyridine, 2,6-lutidine, 2,4,6-collidine, and 4-dimethylaminopyridine, alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, and lithium hydroxide, alkoxy alkali metals such as potassium tert-butoxide, and alkali metal carbonates such as sodium carbonate, potassium carbonate, and lithium carbonate. Preferably, the base is N-diisopropylethylamine (DIPEA). The amount of base used is not particularly limited, and may be, for example, 0.1 to 10 times the molar amount of the starting compound. This reaction can be carried out in the presence of a suitable condensing agent.Non-limiting examples of suitable condensing agents include (1-cyano-2-ethoxy-2-oxoethylideneaminooxy) dimethylaminomorpholinocarbenium hexafluorophosphate, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorphonium chloride, O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, O-(6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, Examples of the condensing agent include (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate (COMU), or 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU). The amount of the condensing agent is not particularly limited, and may be, for example, 0.1 to 10 times the molar amount of the starting compound. The reaction temperature for this reaction may be, for example, −30° C. to 100° C., and the reaction time may be, for example, 5 minutes to 72 hours.

[0061] (Scheme 4) [ka]

[0062] In the formula, R 9 can be selected as described above in the [Compound] section. For example, R 9 Ga-CH2 CH 2 -, the starting compound for this reaction is lithocholic acid, which is commercially available. 10 , or R 11 may be selected from. The solvent used in this reaction is not particularly limited as long as it does not affect the reaction, and may be, for example, halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, aromatic hydrocarbons, etc., and is preferably toluene. The amount of the solvent is not particularly limited, and may be, for example, 1 to 1000 times (v / w) the amount of the starting compound. This reaction can be carried out in the presence of a suitable condensing agent. Non-limiting examples of suitable condensing agents include methyltrimethoxysilane (MTM), methyltriethoxysilane, etc. The amount of the condensing agent used is not particularly limited, and may be, for example, 0.1 to 10 times the molar amount of the starting compound. The reaction temperature for this reaction may be, for example, −30° C. to 50° C., and the reaction time may be, for example, 5 minutes to 48 hours.

[0063] (Scheme 5) [ka]

[0064] In the formula, R 9 can be selected as described above in the [Compound] section. For example, R 9 Ga-CH 2 CH 2 -, the starting compound for this reaction is lithocholic acid, which is commercially available. (1) is a dehydrogenation reaction of a hydroxy group between sterols. The solvent used in the reaction (1) is not particularly limited as long as it does not affect the reaction, and may be, for example, halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, aromatic hydrocarbons, etc., and preferably acetic acid, dichloromethane, etc. The amount of the solvent is not particularly limited, and may be, for example, 1 to 1000 times (v / w) the amount of the starting compound. The reaction of (1) can be carried out in the presence of a suitable oxidizing agent. Non-limiting examples of suitable oxidizing agents include sodium hypochlorite, sodium chlorite, potassium bromate, sodium periodate, tert-butyl hypochlorite, peracetic acid, hydrogen peroxide, sodium percarbonate, sodium perborate, potassium perborate, 3-chloroperbenzoic acid, dimethyldioxirane, chromium trioxide, manganese dioxide, manganese(III) acetate, potassium permanganate, potassium dichromate, divanadium(V) pentoxide, etc., and preferably, the oxidizing agent may be sodium hypochlorite. The amount of the oxidizing agent used is not particularly limited, and may be, for example, 0.1 to 10 times the molar amount of the starting compound. The reaction temperature for the reaction (1) may be, for example, −30° C. to 50° C., and the reaction time may be, for example, 5 minutes to 48 hours. The reaction (2) can be carried out using the compound obtained in the reaction (1) as a starting compound according to any one of Schemes 1 to 4. In the formula, d can be selected as described in Schemes 1 to 4.

[0065] (Scheme 6) [ka]

[0066] The starting compounds for the reaction of (3) can be the compounds obtained in Schemes 1 to 4. 9 is R described in Scheme 1 to Scheme 4. 9 or R 12In the formula, e can be selected as described in Scheme 1 to Scheme 4. (3) is an azidation reaction of the hydroxy group of the sterol ring. The solvent used in the reaction (3) is not particularly limited as long as it does not affect the reaction, and may be, for example, halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, aromatic hydrocarbons, etc., and preferably dichloromethane, etc. The amount of the solvent is not particularly limited, and may be, for example, 1 to 1000 times (v / w) the amount of the starting compound. The reaction of (3) can be carried out in the presence of a suitable azidation agent. Non-limiting examples of suitable azidation agents include trimethylsilyl azide, p-toluenesulfonyl azide, tosyl azide, diphenylphosphoryl azide, etc., and preferably, the azidation agent may be trimethylsilyl azide. The amount of the azidation agent used is not particularly limited, and may be, for example, 0.1 to 10 times the molar amount of the starting compound. The reaction temperature for the reaction (3) may be, for example, −30° C. to 50° C., and the reaction time may be, for example, 5 minutes to 48 hours. In the reaction of (3), the hydroxy group of the sterol ring may be in a protected state. The protecting group for the hydroxy group is an Ms group, a TBS group, a C 1-6 It may be an alkyl group, a Bn group, a PMB group, an Ac group, a Bz group, a triTMS group, a TES group, a TBDPS group, or the like, and is preferably an Ms group. The reaction of (4) is the reduction of the azide in the compound obtained by the reaction of (3). The reduction method is not particularly limited as long as it is a method known to those skilled in the art, and may be, for example, the Staudinger reaction using trimethylphosphine, catalytic reduction using palladium carbon, or hydride reduction using a hydride reducing agent. The solvent used in the reaction of (4) is not particularly limited as long as it does not affect the reaction, and may be, for example, halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, aromatic hydrocarbons, and the like, and may be preferably THF. The amount of the solvent is not particularly limited, and may be, for example, 1 to 1000 times (v / w) the amount of the starting compound. The reaction temperature for the reaction (4) may be, for example, −30° C. to 50° C., and the reaction time may be, for example, 5 minutes to 72 hours.

[0067] (Scheme 7) [ka]

[0068] The starting compound for the reaction of (5) and (6) can be a compound obtained according to Scheme 6, where R 7 can be selected as described above in the section on [Compounds]. 9 and e can be selected as described in Scheme 6. wherein X can be F, Cl, Br, or I. (5) is a reaction in which an acyl group is bonded to the amino group of a sterol ring. The solvent used in the reaction (5) is not particularly limited as long as it does not affect the reaction, and may be, for example, halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, aromatic hydrocarbons, etc. The amount of the solvent is not particularly limited, and may be, for example, 1 to 1000 times (v / w) the amount of the starting compound. The reaction temperature for the reaction (5) may be, for example, −30° C. to 50° C., and the reaction time may be, for example, 5 minutes to 48 hours. The reaction of (6) is a reaction in which an alkylsulfonyl group is bonded to the amino group of the sterol ring. The solvent used in the reaction of (6) is not particularly limited as long as it does not affect the reaction, and may be, for example, halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, aromatic hydrocarbons, etc. The amount of the solvent is not particularly limited, and may be, for example, 1 to 1000 times (v / w) the amount of the starting compound. The reaction temperature for the reaction (6) may be, for example, −30° C. to 50° C., and the reaction time may be, for example, 5 minutes to 48 hours.

[0069] [Application] In one embodiment, the present invention relates to a STING (Stimulator of interferon genes) pathway regulator comprising the compound of the present invention, its pharmacologically acceptable salt, or a prodrug thereof. In one embodiment, the STING pathway regulation is the suppression of the STING pathway in an activated state. In another embodiment, the STING pathway regulation is the activation of the STING pathway in an inactivated state. That is, the STING pathway regulation can be a partial activation of the STING pathway.

[0070] The STING pathway is activated in response to the appearance of DNA derived from pathogens such as viruses in cells, and is involved in immune responses to eliminate pathogens by inducing the production and secretion of inflammatory cytokines including interferon. Activation of the STING pathway may also be useful in immune responses to cancer cells. On the other hand, excessive activation or constant activation of the STING pathway can cause inflammatory diseases and autoimmune diseases. Therefore, STING pathway modulators that have partial agonistic effects on the STING pathway may be useful for the prevention and treatment of inflammatory diseases and autoimmune diseases by suppressing the STING pathway in an excessively activated state, and may have the advantage of being able to partially activate the STING pathway when the STING pathway is not activated, thereby stimulating the immune response.

[0071] The STING pathway regulator of the present invention may be intended to be administered in vivo or may not be intended to be administered in vivo, for example, but not limited to, intended for use in in vitro experiments.

[0072] In one embodiment, the present invention is a therapeutic agent for treating a STING-associated disease, comprising a compound of the present invention, a pharmacologically acceptable salt thereof, or a prodrug thereof. In the present invention, "treatment" includes prevention or therapy. In addition, "treatment" includes not only improving one or more symptoms of a disease, but also inhibiting its progression or worsening. In one embodiment, the STING-associated disease is an inflammatory disease or autoimmune disease caused by excessive activation or constitutive activation of the STING pathway, and non-limiting examples thereof include STING-associated vasculopathy (SAVI), COPA (Coatomer protein alpha) syndrome, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, Aicardi-Goutières syndrome, systemic lupus erythematosus, Parkinson's disease, or Huntington's disease.

[0073] In some embodiments, the STING pathway modulation or treatment of STING-related diseases is due to partial activation of the STING pathway. Partial activation of the STING pathway refers to suppressive action on the STING pathway in an activated state and partial agonistic action on the STING pathway in an inactivated state. Partial activation of the STING pathway can be confirmed by any method known to those skilled in the art. For example, the method may be to confirm that when a subject is subjected to a full STING pathway agonist (for example, DMXAA (vadimezan) in the case of mice) (hereinafter, an agonist may be referred to as an agonist, a full agonist may be referred to as a full agonist, and a partial agonist may be referred to as a partial agonist), weak activation of the STING pathway is observed compared to the full STING pathway agonist, whereas activation of the STING pathway by the full STING pathway agonist is suppressed when the subject is subjected to the full STING pathway agonist in the presence of the full STING pathway agonist. Activation of the STING pathway can be evaluated by any method known to those of skill in the art. For example, activation of the STING pathway can be evaluated by a method in which cells expressing a fluorescent protein or a luminescent protein are used to measure the luminescence generated when a target reagent is reacted with the cells.

[0074] In another embodiment, the STING pathway regulator or the treatment of STING-related diseases is by inhibiting the STING pathway. When referring to the inhibition of the STING pathway, it means that the STING pathway acts suppressively but does not act agonistically. The inhibition of the STING pathway can be confirmed by any method known to those skilled in the art. For example, the method may be to confirm that the activation of the STING pathway is not observed even when the subject's cells are acted on in the absence of a full STING pathway agonist in the method for confirming partial activation of the STING pathway described above.

[0075] The amount of the compound of the present invention, a pharmacologically acceptable salt thereof, or a prodrug thereof contained in the STING pathway regulator or the agent for treating a STING-related disease of the present invention is not particularly limited and may depend on the purpose of use.

[0076] The subject to which the STING pathway regulator or STING-associated disease treatment agent of the present invention is administered may be, for example, a mammal, preferably a human or mouse, more preferably a human (including infants, small children, adults, and elderly people). In addition, the subject to which the STING pathway regulator or STING-associated disease treatment agent of the present invention is administered is preferably suffering from a STING-associated disease.

[0077] The administration route of the STING pathway regulator and the STING-related disease treatment agent of the present invention is not particularly limited, and may be, for example, oral administration, intravenous administration, subcutaneous administration, intramuscular administration, rectal administration, intradermal administration, intrathecal administration, intraarterial administration, intranasal administration, etc. Examples of dosage forms suitable for oral administration include tablets, capsules, powders, fine granules, granules, liquids, syrups, etc. Examples of dosage forms suitable for administration routes other than oral administration include injections, drips, suppositories, inhalants, nasal drops, transdermal absorption materials, ointments, creams, patches, etc.

[0078] The STING pathway regulator and the STING-related disease treatment agent of the present invention may contain any additive within the range in which the effects of the present invention can be obtained. Examples of additives include excipients, disintegrants, binders, lubricants, coating agents, dyes, bases, solubilizers, isotonicity agents, pH regulators, preservatives, thickeners, antioxidants, flavoring agents, etc. Non-limiting examples of additives that can be used in the preparation of formulations for oral administration include excipients such as glucose, lactose, D-mannitol, starch, crystalline cellulose, etc.; disintegrants or disintegration aids such as carboxymethylcellulose, starch, and calcium carboxymethylcellulose, binders such as hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and gelatin, lubricants such as magnesium stearate and talc, coating agents such as hydroxypropylmethylcellulose, sucrose, polyethylene glycol, and titanium oxide, and bases such as petrolatum, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water, and hard fat. Non-limiting examples of additives that can be used in preparing formulations for injection or infusion include solubilizers or solubilizing agents that can constitute aqueous or ready-to-use injections, such as distilled water for injection, physiological saline, propylene glycol, surfactants, etc.; isotonicity agents, such as glucose, sodium chloride, D-mannitol, glycerin, etc.; and pH adjusters, such as inorganic acids, organic acids, inorganic bases, organic bases, etc.

[0079] The STING pathway regulator and the STING-related disease treatment agent of the present invention may contain other active ingredients other than the compound of the present invention, its pharmacologically acceptable salt, or their prodrugs. In addition, the STING pathway regulator and the STING-related disease treatment agent of the present invention may be administered simultaneously with other active ingredients, consecutively, or in the same treatment regimen. The other active ingredients may be, for example, immunosuppressants such as cyclosporine, mizoribine, cyclophosphamide, azathioprine, tacrolimus, mycophenolate mofetil, adrenal cortical hormones such as dexamethasone, betamethasone, prednisolone, and methylprednisolone, intravenous gamma globulin, methotrexate, fingolimod, etc., and are not particularly limited.

[0080] The dosage, frequency and number of administrations of the STING pathway regulator and the agent for treating a STING-related disease of the present invention can be arbitrarily determined by a physician depending on the purpose of treatment, severity of the disease, age, weight, sex, body surface area, general condition, excretory function, treatment history, interactions with other drugs, achievement of treatment, presence or absence of side effects, etc. Non-limiting examples of the dosage are in the range of 0.01 μg / kg to 10 g / kg of active ingredient per day for an adult, etc. Non-limiting examples of the dosage frequency and number of administrations are once a month, once a week, once a day, etc. EXAMPLES

[0081] The present invention will be described below with reference to examples, but the present invention is not limited to these.

[0082] (Abbreviations used in the examples) DMXAA: vadimezan (mouse STING agonist) DMEM: Dulbecco's modified Eagle's medium PBS: Phosphate-buffered saline EDTA: Ethylenediaminetetraacetic acid DMSO: Dimethyl sulfoxide 25HC (25-HC): 25-hydroxycholesterol FAB-MS: Fast atom bombardment mass spectrometry mNBA: m-nitrobenzyl alcohol MMR: Nuclear magnetic resonance THF: tetrahydrofuran DMF: Dimethylformamide DMAP: Dimethylaminopyridine COMU: (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate DIPEA: N-ethyldiisopropylamine TBS: tert-butyldimethylsilyl MTM: Methyltrimethoxysilane TBAF: Tetrabutylammonium fluoride HRMS-ESI-TOFMS: High Resolution Mass Spectrometry-Electrospray Ionization-Time of Flight Mass Spectrometry

[0083] <Example 1> Screening of a compound library in mouse STING-expressing reporter cells [Reporter cells transfected with RAW-Lucia ISG-mSTING] Using RAW-Lucia ISG-KO-STING cells (Invivogen) lacking endogenous STING and incorporating a reporter gene for monitoring activation of the STING pathway, a cell line (RAW-Lucia ISG-mSTING cells) stably incorporating mouse STING was used to verify whether a sterol derivative suppresses activation by the mouse STING agonist DMXAA (vadimezan).

[0084] Cells were cultured and maintained in DMEM (high glucose, containing phenol red, containing glutamine, containing pyruvate) containing 10% FBS in an incubator at 37 °C, 5% CO 2 of CO 2 incubator.

[0085] [Seeding of RAW-Lucia ISG-mSTING cells onto plates] RAW-Lucia ISG-mSTING cells cultured in a 10 cm dish were washed with PBS(-) (2 mL) after removing the medium, PBS containing 0.2 mM EDTA was added, the cells were detached from the dish with a scraper, and the cells were suspended by pipetting. The cells were collected by centrifugation at 3,000×g for 5 minutes, and the cells were suspended in the medium. The cell density was adjusted to 450,000 cells / mL, and seeded at 100 μL / well onto a sterile 96-well plate treated with tissue culture. The cells were cultured in an incubator for 20 - 24 hours.

[0086] [Compound treatment] Compound treatment of cells was performed by pre-diluting the compound in medium and replacing the medium in the well. A 10 mM DMSO solution or DMSO-ethanol (1:1) solution of the compound library was diluted with medium to give a final concentration of 10 μM or 30 μM on the cells. The cells were incubated in CO 2 After culturing in an incubator, the mouse STING agonist DMXAA (vadimezan) was added at 25 μg / mL and the cells were cultured for 20-24 hours.

[0087] [Measurement of luciferase activity] In this experimental system, activation of the STING pathway results in the secretion of Lucia luciferase into the medium. 20-24 hours after compound treatment, 5 μL of the medium supernatant was transferred to a Nunc F96 MicroWell white plate (ThermoFisher, 236107), and 25 μL / well of QUANTI-Luc reagent (Invivogen) was added as a substrate to measure luminescence, which was used as an index of STING pathway activation.

[0088] [result] Examples of the results of screening carried out using a library of sterol derivatives having a structure similar to that of 25HC are shown in Tables 1 and 2. Examples of the results of screening carried out using analogs having a structure similar to that of OG828, a sterol derivative that has been found to have a STING pathway inhibitory effect greater than that of 25HC, are shown in Tables 1 and 2. The smaller the values ​​shown in Tables 1 and 2, the more STING pathway activation was inhibited.

[0089] [Table 1]

[0090] [Table 2]

[0091] <Example 2> Synthesis of monoalkylcholenic acid amide derivatives having unsaturated bonds [Synthetic scheme] [ka]

[0092] 3β-Hydroxy-N-benzylchol-5-en-24-amide(FK077(C3)) 3β-Hydroxy-Δ5-cholenic acid (38.5 mg, 0.103 mmol) was dissolved in THF (8 mL), triethylamine (0.020 mL, 0.14 mmol) was added, and the mixture was stirred under ice cooling. Isobutyl chloroformate (0.020 mmol, 0.15 mmol) was added and stirred for 10 minutes, after which benzylamine (0.100 mL, 0.916 mmol) was added and the mixture was stirred at room temperature for 3 hours. The mixture was diluted with about 5 times the amount of AcOEt, washed with saturated aqueous ammonium chloride solution, saturated aqueous bicarbonate solution, and then saturated saline. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane:AcOEt 1:1) to obtain the title compound (45.5 mg, 0.0981 mmol, 95%) as a white solid.

[0093] FAB-MS(m / z, glycerol:mNBA 2:1): [M+H] + 464. 1H-NMR (500 MHz, CDCl 3 ) δ7.37-7.27(m, 5H), 5.84(t, J = 5.5 Hz, 1H), 5.37-5.32(m, 1H), 4.43(d, J = 5.5 Hz, 2H), 3.55-3.48(m, 1H), 1.00(s, 3H), 0.93(d, J = 6.7 Hz, 3H), 0.67(s, 3H). 13C-NMR (126 MHz, CDCl 3)δ173.40, 140.77, 138.43, 128.67(2C), 127.82(2C), 127.46, 121.59, 71.70, 56.72, 55.82, 50.07, 43.58, 42.35, 42.25, 39.75, 37.25, 36.47, 35.49, 33.60, 31.85, 31.81, 31.60, 28.17, 24.24, 21.05, 19.38, 18.41, 11.86.

[0094] 3β-Hydroxy-N-propargylchol-5-en-24-amide(FK074(C7)) 3β-Hydroxy-Δ5-cholenic acid (40.4 mg, 0.108 mmol) was dissolved in THF (8 mL), triethylamine (0.020 mL, 0.14 mmol) was added, and the mixture was stirred under ice cooling. Isobutyl chloroformate (0.020 mmol, 0.15 mmol) was added and stirred for 10 minutes, after which aniline (0.050 mL, 0.78 mmol) was added and the mixture was stirred at room temperature for 3 hours. The mixture was diluted with about 5 times the amount of AcOEt, washed with saturated aqueous ammonium chloride solution, saturated aqueous bicarbonate solution, and then saturated saline. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane:AcOEt 2:1) to obtain the title compound (36.8 mg, 0.0894 mmol, 83%) as colorless crystals.

[0095] Rf = 0.50 (Hexane:AcOEt 1:1). FAB-MS(m / z, glycerol:mNBA 2:1): [M+H] + 412. 1H-NMR (500 MHz, CDCl 3 :CD 3OD 10:1)δ6.27 (br s, 1H), 5.37-5.32 (m, 1H), 4.02(dd, J = 2.5, 0.6 Hz, 2H), 3.54-3.46(m, 1H), 2.50(t, J = 0.6 Hz, 1H), 1.00(s, 3H), 0.94(d, J = 6.7 Hz, 3H),0.68 (s, 3H). 13C-NMR (126 MHz, CDCl 3 :CD 3 OD 10:1)δ174.33, 141.41, 122.19, 80.25, 72.09, 72.01, 57.33, 56.41, 50.69, 42.98, 42.67, 40.36, 37.86, 37.10, 36.10, 33.82, 32.47, 32.23, 32.00, 29.72, 29.59, 28.75, 24.85, 21.67, 19.97, 18.97, 12.45.

[0096] 3β-Hydroxy-N-phenylchol-5-en-24-amide(FK079(C5)) 3β-Hydroxy-Δ5-cholenic acid (42.1 mg, 0.112 mmol) was dissolved in THF (8 mL), triethylamine (0.020 mL, 0.14 mmol) was added, and the mixture was stirred under ice cooling. Isobutyl chloroformate (0.020 mmol, 0.15 mmol) was added and stirred for 10 minutes, after which aniline (0.100 mL, 1.10 mmol) was added and the mixture was stirred at room temperature for 3 hours. The mixture was diluted with about 5 times the amount of AcOEt, washed with saturated aqueous ammonium chloride, saturated aqueous bicarbonate, and then saturated saline. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane:AcOEt 5:1 to 1:2) to obtain the title compound (46.5 mg, 0.103 mmol, 92%) as an off-white solid.

[0097] Rf = 0.75 (Hexane:AcOEt 1:3). FAB-MS(m / z, glycerol:mNBA 2:1): [M+H] + 450. 1H-NMR(500 MHz, CDCl 3 :CD 3 OD 10:1) δ7.53(d, J = 7.5 Hz, 2H), 7.30(dd, J = 7.5, 4.0 Hz, 2H), 7.08(t, J = 7.5 Hz, 1H), 5.36-5.32(m, 1H), 3.52-3.44(m, 1H), 1.01(s, 3H), 0.98(d, J = 6.1 Hz, 3H), 0.69(s, 3H). 13C-NMR(126 MHz, CDCl 3 :CD 3 OD 10:1) δ174.32, 141.97, 139.49, 129.92(2C), 125.14, 122.64, 121.13(2C), 72.47, 57.87, 57.05, 51.25, 43.51, 43.01, 40.90, 38.38, 37.62, 36.74, 35.44, 33.00, 32.97, 32.94, 32.32, 29.25, 25.37, 22.18, 20.43, 19.44, 12.93.

[0098] 3β-Hydroxy-N-(3-pyridyl)chol-5-en-24-amide(FK081(C11)) 3β-Hydroxy-Δ5-cholenic acid (40.0 mg, 0.107 mmol) was dissolved in THF (8 mL), triethylamine (0.020 mL, 0.14 mmol) was added, and the mixture was stirred under ice cooling. Isobutyl chloroformate (0.020 mmol, 0.15 mmol) was added and stirred for 10 minutes, after which 3-aminopyridine (64.5 mg, 0.685 mmol) was added and stirred at room temperature for 3 hours. The mixture was diluted with about 5 times the amount of AcOEt, washed with saturated aqueous ammonium chloride, saturated aqueous bicarbonate, and then saturated saline. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane:AcOEt 1:5) to obtain the title compound (27.7 mg, 0.0615 mmol, 58%) as an off-white solid.

[0099] Rf = 0.20 (Hexane:AcOEt 1:3). FAB-MS(m / z, glycerol:mNBA 2:1): [M+H] + 451. 1H-NMR (500 MHz, CDCl 3 :CD 3 OD 10:1)δ8.48(d, J = 2.4 Hz, 1H), 8.33-8.28(m, 1H), 8.22(dd, J = 4.9, 1.2 Hz, 1H), 7.32-7.28(m, 1H), 5.36-5.33(m, 1H), 3.53-3.44(m, 1H), 1.01(s, 3H), 0.99(d, J = 6.7 Hz, 3H), 0.70(s, 3H). 13C-NMR (126 MHz, CDCl 3 :CD 3OD 10:1)δ174.45, 144.38, 141.46, 140.96, 136.64, 128.29, 124.59, 122.15, 71.97, 57.36, 56.53, 50.73, 43.00, 42.52, 40.39, 37.87, 37.11, 36.23, 34.72, 32.49, 32.47, 32.23, 31.82, 28.74, 24.86, 21.67, 19.93, 18.93, 12.44.

[0100] Example 3: Synthesis of cholenic acid ester [Synthetic scheme] [ka]

[0101] [Experimental Procedure] 4-Pyridylmethyl 3β-hydroxychol-5-en-24-oate(ELN001(C12)) To a solution of 3β-hydroxy-Δ5-cholenic acid (43.1 mg, 0.115 mmol) in DMF (1 mL), potassium carbonate (64.1 mg, 0.464 mmol) and 4-(bromomethyl)pyridine hydrobromide (88.2 mg, 0.351 mmol) were added and stirred at room temperature overnight. After extraction with ethyl acetate, the organic layer was washed with water and saturated saline, and dried over magnesium sulfate. The solvent was removed and the residue was purified by silica gel chromatography (n-hexane:ethyl acetate 1:1) to obtain the title compound (16.7 mg, 0.0359 mol, 31%) as a white solid.

[0102] 1H-NMR (500 MHz, CDCl 3) δ 8.60 (d, J = 6.0 Hz, 2H), 7.25 (d, J = 6.2 Hz, 2H), 5.39 - 5.31 (m, 1H), 5.12 (s, 2H), 3.52 (tt, J = 11.1, 4.5 Hz, 1H), 2.46 (ddd, J = 15.3, 10.1, 5.1 Hz, 1H), 2.39 - 2.19 (m, 3H), 2.03 - 1.93 (m, 2H), 1.92 - 1.04 (m, 20H), 1.01 (s, 3H), 0.94 (d, J = 6.4 Hz, 3H), 0.67 (s, 3H). 13C-NMR(150 MHz, CDCl 3 ) δ 173.90, 150.09 (2C), 145.28, 140.92, 122.10 (2C), 121.74, 71.87, 64.21, 56.86, 55.87, 50.21, 42.52, 42.42, 39.88, 37.39, 36.62, 35.46, 32.01, 31.99, 31.77, 31.23, 31.10, 28.26, 24.37, 21.19, 19.53, 18.43, 12.00. HRMS-ESI-TOFMS (m / z): [M+H]+ calcd for C 30 H 43 NO 3 , 466.3316; found, 466.3316.

[0103] <Example 4> Synthesis of N-Methylaniline Cholate [Synthesis Scheme]

Chemical Formula

[0104] [Experimental Procedure] N-Methyl-N-phenyl-3β-hydroxychol-5-en-24-amide(ELN010(C13)) COMU (59.4 mg, 0.139 mmol) was added to a solution of 3β-hydroxy-Δ5-cholenic acid (50.4 mg, 0.135 mmol) dissolved in dimethylformamide (0.534 mL). DIPEA (0.0454 mL, 0.270 mmol) was added and stirred for 1 hour. N-methylaniline (0.0146 mL, 0.135 mmol) was added to the mixture and stirred for 2 hours. After extraction with ethyl acetate, the organic layer was washed with water and saturated saline. After drying over magnesium sulfate, the solvent was distilled off, and the obtained compound was purified by silica gel chromatography (n-hexane:ethyl acetate 3:1). Further recrystallization (dichloromethane-hexane) was performed to obtain the title compound (12.4 mg, 0.0267 mmol, 5%) as a yellow solid.

[0105] 1H-NMR (500 MHz, CDCl 3 ) δ 7.42 (t, J = 7.4 Hz, 2H), 7.34 (t, J = 7.2 Hz, 1H), 7.19 (d, J = 6.9 Hz, 2H), 5.34 (d, J = 5.2 Hz, 1H), 3.54-3.50 (m, 1H), 3.26 (s, 3H), 1.82 (d, J = 10.3 Hz, 3H), 0.70 (d, J = 4.6 Hz, 3H), 0.61 (s, 3H). 13C-NMR (150 MHz, CDCl3) δ 173.92, 144.23, 140.69, 129.70, 127.72, 127.32, 121.68, 71.75, 56.61, 55.78, 50.00, 42.26, 42.23, 39.62, 37.39, 37.20, 36.44, 35.52, 31.83, 31.81 (2C), 31.58, 31.21, 28.03, 24.22, 21.00, 19.37, 18.24, 11.81. HRMS-ESI-TOFMS (m / z): [M+H]+ calcd for C 31 H 45 NO 2, 464.3529; found, 464.3523. [M+Na]+ calcd 486.3341; found 486.3343.

[0106] <Example 5> Synthesis of anilide-type cholenoic acid derivatives [Synthetic scheme] [ka]

[0107] [Synthesis procedure] 3β-tert-Butyl(dimethyl)silyloxychol-5-en-24-oic acid(ELN026) To a solution of 3β-hydroxy-Δ5-cholenic acid (185.0 mg, 0.480 mmol) in DMF (4.13 mL), imidazole (266.4 mg, 3.91 mmol), 4-dimethylaminopyridine (121.0 mg, 0.990 mmol) and tert-butyldimethylchlorosilane (220.0 mg, 1.46 mmol) were added, and the mixture was stirred at room temperature overnight. After quenching with 1M hydrochloric acid, it was extracted with ethyl acetate, and the organic layer was washed with saturated saline and dried over magnesium sulfate. After distilling off the solvent under reduced pressure, methanol (4.13 mL), tetrahydrofuran (4.13 mL) and 10% aqueous potassium carbonate solution (2.36 mL) were added to the mixture. After stirring at room temperature for 20 minutes, the solvent was distilled off under reduced pressure. Saturated saline and hydrochloric acid were added to the obtained residue, and it was extracted with ethyl acetate. The organic layer was dried over magnesium sulfate, and the solvent was evaporated under reduced pressure to obtain the title compound (212.8 mg, 0.4344 mmol, 87%) as a white solid.

[0108] 1H-NMR (500 MHz, CDCl 3 )δ 5.33-5.31 (m, 1H), 3.52-3.45 (m, 1H), 1.00 (s, 3H), 0.94 (d, J = 6.9 Hz, 3H), 0.89 (s, 9H), 0.68 (s, 3H), 0.06 (s, 6H).

[0109] Amidation procedure with HATU A solution of the carboxylic acid in dichloromethane was added with 1 equivalent of the amine, 1.5 equivalents of DIPEA and 1.1 equivalents of HATU and stirred at room temperature overnight. After extraction with dichloromethane, the organic phase was washed twice with hydrochloric acid, followed by saturated saline and dried over magnesium sulfate. The solvent was evaporated and the resulting residue was purified by chromatography on silica gel.

[0110] N-(2-Methylphenyl)-3β-tert-Butyl(dimethyl)silyloxychol-5-en-24-amide(ELN027) Synthesized following the HATU amidation procedure described above: 29.7 mg, 0.0513 mmol, 70% yield. 1H-NMR (500 MHz, CDCl 3 )δ 7.82 (d, J = 8.0 Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H), 7.18 (d, J = 8.9 Hz, 1H), 7.08 (t, J = 7.3 Hz, 1H), 6.90 (s, 1H), 5.40 - 5.18 (m, 1H), 3.54 - 3.42 (m, 1H), 2.52 - 2.41 (m, 1H), 2.26 (s, 3H), 2.21 - 2.14 (m, 1H), 1.01 - 0.97 (m, 6H), 0.89 (s, 9H), 0.69 (s, 3H), 0.06 (s, 6H).

[0111] N-(3-Methylphenyl)-3β-tert-Butyl(dimethyl)silyloxychol-5-en-24-amide(ELN029) Synthesized following the HATU amidation procedure described above: 39.1 mg, 0.0675 mmol, 84% yield. 1H-NMR (500 MHz, CDCl 3) δ 7.39 (s, 1H), 7.27 (d, J = 7.0 Hz, 6H), 7.19 (t, J = 7.7 Hz, 1H), 7.03 (s, 1H), 6.91 (d, J = 7.0 Hz, 1H), 5.34 - 5.30 (m, 1H), 3.52 - 3.43 (m, 1H), 2.46 - 2.37 (m, 1H), 2.34 (s, 3H), 2.28 - 2.21 (m, 2H), 2.19 - 2.14 (m, 1H), 2.03 - 1.68 (m, 7H), 1.00 (s, 3H), 0.97 (d, J = 6.4 Hz, 3H), 0.89 (s, 9H), 0.68 (s, 3H), 0.06 (s, 6H).

[0112] N-(4-Benzoylphenyl)-3β-tert-Butyl(dimethyl)silyloxychol-5-en-24-amide(ELN030) Synthesized according to the amidation procedure with HATU above. 31.4 mg, 0.0470 mmol, yield 56%. 1H-NMR(500 MHz, CDCl 3 ) δ 7.82 (d, J = 8.7 Hz, 2H), 7.77 (dd, J = 8.2, 1.4 Hz, 2H), 7.64 (d, J = 8.5 Hz, 2H), 7.58 (tt, J = 7.2, 1.5 Hz, 1H), 7.48 (t, J = 7.7 Hz, 2H), 7.30 (s, 1H), 5.35 - 5.27 (m, 1H), 3.54 - 3.42 (m, 1H), 2.47 (ddd, J = 15.1, 10.3, 4.8 Hz, 1H), 2.36 - 2.23 (m, 2H), 2.21 - 2.13 (m, 1H), 2.07 - 1.69 (m, 7H), 1.00 (s, 3H), 0.98 (d, J = 6.3 Hz, 3H), 0.89 (s, 9H), 0.69 (s, 3H), 0.06 (s, 6H).

[0113] N-(-Dibenzofuranyl)-3β-tert-Butyl(dimethyl)silyloxychol-5-en-24-amide(ELN033) Synthesized following the HATU amidation procedure described above: 39.6 mg, 0.0605 mmol, 71% yield. 1H-NMR (500 MHz, CDCl 3 )δ 8.35 (d, J = 2.3 Hz, 1H), 7.93 (d, J = 7.4 Hz, 1H), 7.55 (d, J = 8.3 Hz, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7.46 (td, J = 7.7, 1.3 Hz, 1H), 7.36 (dd, J = 8.8, 2.1 Hz, 1H), 7.33 (t, J = 7.2 Hz, 1H), 7.27 (s, 1H), 5.35 - 5.29 (m, 1H), 3.52 - 3.44 (m, 1H), 2.53 - 2.44 (m, 1H), 2.36 - 2.22 (m, 2H), 2.21 - 2.13 (m, 1H), 2.05 - 1.67 (m, 7H), 1.01 (d, J = 5.2 Hz, 3H), 1.00 (s, 3H), 0.89 (s, 9H), 0.70 (s, 3H), 0.06 (s, 6H).

[0114] N-(3-Benzoylphenyl)-3β-tert-Butyl(dimethyl)silyloxychol-5-en-24-amide(ELN034) Synthesized following the HATU amidation procedure described above. 55.3 mg, 0.0828 mmol, 91% yield. 1H-NMR (500 MHz, CDCl 3)δ 7.97 (d, J = 7.6 Hz, 1H), 7.81 (d, J = 6.8 Hz, 2H), 7.76 (t, J = 2.0 Hz, 1H), 7.60 (t, J = 7.4 Hz, 1H), 7.53 - 7.47 (m, 3H), 7.44 (t, J = 7.9 Hz, 1H), 7.22 (s, 1H), 5.34 - 5.30 (m, 1H), 3.51 - 3.44 (m, 1H), 2.49 - 2.39 (m, 1H), 2.32 - 2.22 (m, 2H), 2.20 - 2.13 (m, 1H), 2.04 - 1.85 (m, 5H), 1.81 (dt, J = 13.3, 3.1 Hz, 1H), 1.75 - 1.67 (m, 1H), 1.00 (s, 3H), 0.97 (d, J = 6.3 Hz, 3H), 0.89 (s, 9H), 0.68 (s, 3H), 0.06 (s, 6H).

[0115] N-(3-Trifluoromethylphenyl)-3β-tert-Butyl(dimethyl)silyloxychol-5-en-24-amide(ELN038) Synthesized according to the amidation procedure with the above HATU. 35.9 mg, 0.0568 mmol, yield 89%. 1H-NMR(500 MHz, CDCl 3 )δ 7.81 (s, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H), 7.35 (d, J = 7.8 Hz, 1H), 7.23 (s, 1H), 5.34 - 5.29 (m, 1H), 3.53 - 3.43 (m, 1H), 2.44 (ddd, J = 15.1, 10.5, 4.9 Hz, 1H), 2.33 - 2.22 (m, 2H), 2.21 - 2.12 (m, 1H), 2.04 - 1.68 (m, 6H), 1.00 (s, 3H), 0.98 (d, J = 6.4 Hz, 3H), 0.89 (s, 9H), 0.69 (s, 3H), 0.06 (s, 6H).

[0116] N-(3-Cyanolphenyl)-3β-tert-Butyl(dimethyl)silyloxychol-5-en-24-amide(ELN043) Synthesized following the HATU amidation procedure described above. 19.5 mg, 0.0331 mmol, 54% yield. 1H-NMR (500 MHz, CDCl 3 )δ 7.93 (s, 1H), 7.72 (d, J = 8.1 Hz, 1H), 7.41 (t, J = 7.8 Hz, 1H), 7.38 (dt, J = 7.7, 1.4 Hz, 1H), 7.34 (s, 1H), 5.35 - 5.27 (m, 1H), 3.53 - 3.42 (m, 1H), 2.45 (ddd, J = 15.0, 10.4, 4.8 Hz, 1H), 2.33 - 2.22 (m, 2H), 2.17 (ddd, J = 13.3, 4.9, 2.2 Hz, 1H), 2.04 - 1.68 (m, 7H), 1.65 - 1.24 (m, 18H), 1.00 (s, 3H), 0.97 (d, J = 6.4 Hz, 3H), 0.89 (s, 9H), 0.68 (s, 3H), 0.06 (s, 6H).

[0117] N-(3-(((1,1-dimethylethoxy)carbonyl)amino)phenyl)-3β-tert-Butyl(dimethyl)silyloxychol-5-en-24-amide(ELN072) Synthesized following the HATU amidation procedure described above. 164.6 mg, 0.2424 mmol, 69% yield. 1H-NMR (400 MHz, CDCl 3)δ 7.66 (s, 1H), 7.28 (d, J = 8.6 Hz, 1H), 7.22 (t, J = 8.0 Hz, 1H), 7.09 (s, 1H), 7.01 (d, J = 8.3 Hz, 1H), 6.49 (s, 1H), 5.34 - 5.28 (m, 1H), 3.54 - 3.42 (m, 2H), 2.39 (ddd, J = 15.0, 10.3, 4.8 Hz, 1H), 2.32 - 2.13 (m, 4H), 2.06 - 1.68 (m, 7H), 1.51 (s, 9H), 1.00 (s, 3H), 0.97 (d, J = 6.4 Hz, 3H), 0.89 (s, 9H), 0.68 (s, 3H), 0.06 (s, 6H).

[0118] [Synthetic scheme] [ka]

[0119] [Synthesis procedure] TBS deprotection procedure To a solution of the TBS-protected compound in tetrahydrofuran, add 12 equivalents of 1M TBAF H 2 The mixture was stirred at room temperature overnight. After extraction with ethyl acetate, the mixture was washed twice with ethyl acetate and with saturated saline. The organic phase was dried over magnesium sulfate, and the solvent was removed by distillation. The resulting residue was purified by silica gel chromatography.

[0120] N-(2-Methylphenyl)-3β-hydroxychol-5-en-24-amide(ELN028(C18)) Synthesized following the TBS deprotection procedure above. 10.2 mg, 0.022 mmol, 47% yield. 1H-NMR (500 MHz, CDCl 3)δ 7.81 (d, J = 8.1 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H), 6.93 (s, 1H), 5.42 - 5.28 (m, 1H), 3.60 - 3.46 (m, 1H), 2.53 - 2.40 (m, 1H), 2.34 - 2.27 (m, 2H), 2.26 (s, 3H), 2.25 - 2.19 (m, 1H), 2.07 - 1.79 (m, 7H), 1.01 (s, 3H), 1.00 (d, J = 6.1 Hz, 3H), 0.70 (s, 3H).; 13C-NMR (150 MHz, CDCl 3 )δ171.87, 140.85, 135.82, 130.57, 128.91, 126.94, 125.20, 123.23, 121.81, 71.91, 56.84, 55.95, 50.16, 42.52, 42.39, 39.87, 37.35, 36.60, 35.64, 34.66, 31.98 (2C), 31.96, 31.74, 28.35, 24.39, 21.19, 19.54, 18.59, 17.96, 12.04. HRMS-ESI-TOFMS (m / z): [M+H]+ calcd for C 31 H 45 NO 2 , 464.3532; found, 464.3523. [M+Na]+ calcd 486.3352; found 486.3343.

[0121] N-(3-Methylphenyl)-3β-hydroxychol-5-en-24-amide(ELN031(C14)) The above-mentioned TBS deprotection hand is easy to synthesize. 20.1 mg, 0.0433 mmol, yield 69%. 1H-NMR (500 MHz, CDCl 3)δ 7.39 (s, 1H), 7.27 (d, J = 7.6 Hz, 1H), 7.22 (s, 1H), 7.19 (t, J = 7.8 Hz, 1H), 6.91 (d, J = 7.0 Hz, 1H), 5.37 - 5.32 (m, 1H), 3.57 - 3.49 (m, 1H), 2.41 (ddd, J = 15.0, 10.4, 4.9 Hz, 1H), 2.33 (s, 3H), 2.32 - 2.18 (m, 3H), 2.05 - 1.79 (m, 6H), 1.01 (s, 3H), 0.97 (d, J = 6.5 Hz, 3H), 0.68 (s, 3H).; 13C-NMR(150 MHz, CDCl 3 )δ 171.84, 140.69, 138.89, 137.88, 128.77, 124.92, 121.67, 120.38, 116.75, 71.76, 56.68, 55.79, 50.01, 42.34, 42.23, 39.71, 37.21, 36.45, 35.48, 34.64, 31.82 (2C), 31.61, 31.58, 28.18, 24.24, 21.50, 21.03, 19.39, 18.44, 11.88.

[0122] N-(4-Benzoylphenyl)-3β-hydroxychol-5-en-24-amide(ELN032(C20)) Synthesized following the TBS deprotection procedure above. 23.5 mg, 0.0424 mmol, 98% yield. 1H-NMR (500 MHz, CDCl 3)δ 7.81 (d, J = 8.7 Hz, 2H), 7.78 - 7.75 (m, 2H), 7.73 (s, 1H), 7.66 (d, J = 8.7 Hz, 2H), 7.58 (ddt, J = 7.9, 6.9, 1.3 Hz, 1H), 7.48 (t, J = 7.6 Hz, 2H), 5.38 - 5.32 (m, 1H), 3.57 - 3.50 (m, 1H), 2.46 (ddd, J = 15.1, 10.5, 4.8 Hz, 1H), 2.35 - 2.19 (m, 3H), 2.03 - 1.80 (m, 6H), 1.01 (s, 3H), 0.97 (d, J = 6.3 Hz, 3H), 0.68 (s, 3H).; 13C-NMR(150 MHz, CDCl3)δ 195.79, 172.32, 142.11, 140.68, 137.77, 132.69, 132.26, 131.65 (2C), 129.87 (2C), 128.27 (2C), 121.66, 118.66 (2C), 71.77, 56.68, 55.74, 50.87, 50.00, 42.35, 42.23, 39.71, 37.21, 36.45, 35.45, 34.66, 31.82 (2C), 31.58, 31.43, 28.19, 24.23, 21.03, 19.39, 18.45, 11.88.

[0123] N-(2-Dibenzofuranyl)-3β-hydroxychol-5-en-24-amide(ELN035(C21)) Synthesized following the TBS deprotection procedure above. 29.0 mg, 0.0537 mmol, 96% yield. 1H-NMR (500 MHz, CDCl 3)δ 8.33 (s, 1H), 7.89 (d, J = 7.7 Hz, 1H), 7.59 - 7.51 (m, 2H), 7.49 - 7.41 (m, 2H), 7.37 (d, J = 8.7 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H), 5.37 - 5.32 (m, 1H), 3.58 - 3.48 (m, 1H), 2.52 - 2.42 (m, 1H), 2.35 - 2.18 (m, 3H), 1.00 (s, 3H), 0.98 (d, J = 6.0 Hz, 3H), 0.68 (s, 3H). 13C-NMR (150 MHz, CDCl 3 )δ 172.11, 156.85, 152.96, 140.81, 133.29, 127.48, 124.73, 124.29, 122.82, 121.80, 121.03, 119.87, 112.71, 111.80, 111.76, 71.90, 56.80, 55.92, 50.13, 42.48, 42.36, 39.84, 37.33, 36.57, 35.65, 34.68, 31.95, 31.80, 31.72, 28.34, 24.37, 21.16, 19.52, 18.61, 14.32, 12.02.

[0124] N-(3-Benzoylphenyl)-3β-hydroxychol-5-en-24-amide(ELN036(C17)) The above-mentioned TBS's deprotection hand is smooth and easy to synthesize. 32.1 mg, 0.0580 mmol, yield 72%. 1H-NMR (500 MHz, CDCl 3)δ 7.99 (d, J = 8.0 Hz, 1H), 7.81-7.80 (m, 2H), 7.78 (br s, 1H), 7.61- 7.58 (m, 1H), 7.50-7.42 (m, 5H), 5.36-5.34 (m, 1H), 3.53-3.49 (m, 1H), 1.01 (s, 3H), 0.97 (d, J = 6.3 Hz, 3H), 0.68 (s, 3H); 3 ) δ 196.50, 172.20, 140.84, 138.34 (2C), 137.42, 132.76, 130.21(2C), 129.16, 128.48(2C), 125.97, 124.02, 121.80, 120.95, 71.91, 56.83, 55.91, 50.16, 42.51, 42.40, 39.87, 37.36, 36.60, 35.62, 34.74, 31.98 (2C), 31.75, 31.66, 28.34, 24.38, 21.19, 19.53, 18.60, 12.03. HRMS-ESI-TOFMS (m / z): [MH]- calcd for C37H47NO3, 552.3507; found, 552.3483.

[0125] N-(3-Trifluoromethylphenyl)-3β-hydroxychol-5-en-24-amide(ELN040(C19)) Synthesized following the TBS deprotection procedure above. 25.7 mg, 0.0496 mmol, 90% yield. 1H-NMR (500 MHz, CDCl 3 / MeOD(10:1))δ 7.87 (s, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.42 (t, J = 8.0 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 5.35-5.33 (m, 1H), 3.50-3.46 (m, 1H), 1.01 (s, 3H), 0.98 (d, J = 6.3 Hz, 3H), 0.69 (s, 3H).; 13C-NMR(150 MHz, CDCl 3 / MeOD(10:1))δ173.43, 140.78, 139.05, 131.04 (q, J = 32.3 Hz), 129.32, 125.75 (q, J = 271.8 Hz), 122.76, 121.55, 120.24 (q, J = 3.9 Hz), 116.37 (q, J = 4.1 Hz), 71.32, 56.69, 55.86, 50.04, 42.35, 41.85, 39.72, 37.21, 36.46, 35.59, 34.20, 31.82 (2C), 31.62, 31.16, 28.11, 24.22, 21.00 (d, J = 5.9 Hz), 19.31, 18.28, 11.80.

[0126] N-(3-Cyanolphenyl)-3β-hydroxychol-5-en-24-amide(ELN044(C15)) Synthesized following the TBS deprotection procedure above. 13.6 mg, 0.0287 mmol, 89% yield. 1H-NMR (500 MHz, CDCl 3 / MeOD(10:1))δ 7.90 (s, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.40-7.31 (m, 1H), 7.27 (d, J = 8.0 Hz, 1H), 5.26 (s, 1H), 3.41-3.37 (m, 1H), 0.92 (s, 3H), 0.90 (d, J = 6.3 Hz, 3H), 0.61 (s, 3H); 13C-NMR(150 MHz, CDCl 3 / MeOD(10:1)) δ 173.71, 140.72, 139.53, 129.61, 126.96, 123.91, 122.71, 121.41, 118.65, 112.18, 71.15, 56.61, 55.80, 49.96, 42.25, 41.69, 39.63, 37.12, 36.36, 35.52, 34.02, 31.74, 31.72, 31.52, 30.99, 28.02, 24.12, 20.92, 19.18, 18.13, 11.68.

[0127] N-(3-(((1,1-dimethylethoxy)carbonyl)amino)phenyl)-3β-hydroxychol-5-en-24-amide (ELN074(C16)) Synthesized according to the above-mentioned TBS deprotection procedure. 16.5 mg, 0.0292 mmol, yield 56%. 1H-NMR(500 MHz, CDCl 3 ) δ.65 (s, 1H), 7.28 (d, J = 8.9 Hz, 1H), 7.24 (s, 1H), 7.21 (t, J = 8.0 Hz, 1H), 7.02 (d, J = 7.9 Hz, 1H), 6.57 (s, 1H), 5.40 - 5.30 (m, 1H), 3.59 - 3.47 (m, 1H), 2.39 (ddd, J = 15.1, 10.5, 5.1 Hz, 1H), 2.30 (dd, J = 13.2, 5.0 Hz, 1H), 2.27 - 2.16 (m, 2H), 2.15 - 1.78 (m, 9H), 1.51 (s, 9H), 1.01 (s, 3H), 0.96 (d, J = 6.4 Hz, 3H), 0.69 (s, 3H). ; 13C-NMR(150 MHz, CDCl 3)δ 172.02, 152.87, 140.83, 139.04, 138.80, 129.65, 121.82, 114.28, 114.03, 109.58, 80.82, 71.92, 56.83, 55.93, 50.17, 42.50, 42.38, 39.86, 37.36, 36.60, 35.63, 34.87, 31.98 (2C), 31.76, 31.73, 28.45 (3C), 28.33, 24.39, 21.19, 19.53, 18.59, 12.03.

[0128] [Synthetic scheme] [ka]

[0129] [Synthesis procedure] N-(3-Aminophenyl)-3β-hydroxychol-5-en-24-amide(ELN75(C22)) To a solution of ELN72 (65.1 mg, 0.0959 mmol) in dichloromethane was added 85% phosphoric acid (200 μL, 0.320 mmol) and stirred at room temperature for 3 h. Water was added, followed by aqueous sodium hydroxide at 0 °C until the pH reached 8. After extraction with dichloromethane, the organic layer was washed with saturated brine, dried over magnesium sulfate, and filtered. The solvent was removed and the resulting compound was chromatographed on silica gel eluting with dichloromethane / ethyl acetate (7:1→1:1) to give the title compound (1.8 mg, 0.00387 mmol, 4%).

[0130] 1H-NMR (500 MHz, CDCl3) δ 7.21 (s, 1H), 7.06 (t, J = 7.9 Hz, 1H), 6.99 (s, 1H), 6.62 (d, J = 8.4 Hz, 1H), 6.42 (d, J = 7.9 Hz, 1H), 5.38 - 5.33 (m, 1H), 3.57 - 3.47 (m, 1H), 2.39 (ddd, J = 15.0, 10.3, 5.1 Hz, 1H), 2.33 - 2.15 (m, 5H), 2.03 - 1.81 (m, 8H), 1.01 (s, 3H), 0.97 (d, J = 6.4 Hz, 3H), 0.69 (s, 3H); 13C-NMR (150 MHz, CDCl 3 ) δ 171.83, 147.36, 140.86, 139.13, 129.82, 121.84, 111.02, 109.58, 106.58, 71.93, 56.86, 55.98, 50.19, 42.52, 42.41, 39.89, 37.37, 36.62, 35.64, 34.95, 32.00 (2C), 31.76 (2C), 28.35, 24.41, 21.20, 19.54, 18.61, 12.05. HRMS-ESI-TOFMS (m / z): [M+H]+ calcd for C30H44N2O2, 465.3477; found, 465.3476.

[0131] [Synthesis Scheme] [Chemical Structure]

[0132] [Experimental Procedure] 1,1-Dimethylethyl N-[4-(4-aminobenzoyl)phenyl]carbamate (ELN045) Di-tert-butyl dicarbonate (156.2 μL, 0.68 mmol) was added to a solution of 4,4'-aminobenzophenone (144.3 mg, 0.68 mmol) in THF, and the mixture was stirred under reflux for 5 hours under nitrogen. The mixture was extracted with ethyl acetate, and then washed with water and saturated saline. The organic layer was dried over magnesium sulfate, and the solvent was removed. The residue was purified by silica gel chromatography (hexane:dichloromethane 3:2) to obtain the title compound (47.7 mg, 0.153 mmol, 22%) as a pale yellow solid.

[0133] 1H-NMR (500 MHz, CDCl 3 :CD 3 OD 10:1)δ7.52 (d, J = 8.6 Hz, 2H), 7.49 (d, J = 8.6 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 6.53 (d, J = 8.6 Hz, 2H), 1.38 (s, 9H).

[0134] 1,1-Dimethylethyl N-(4-(4-(acetylamino)benzoyl)phenyl)carbamate(ELN046) Acetic anhydride (21.8 μL, 0.220 mmol) was added to a pyridine solution of ELN045 (45.2 mg, 0.145 mmol) and stirred at 70°C for 3 hours. After extraction with ethyl acetate, the mixture was washed with saturated aqueous hydrogen carbonate and saturated saline. The organic layer was dried over magnesium sulfate, the solvent was removed, and the residue was purified by silica gel chromatography (hexane:dichloromethane:ethyl acetate 1:1:1) to obtain the title compound (24.2 mg, 0.0683 mmol, 47%) as a white solid.

[0135] 1H-NMR (500 MHz, CDCl 3 :CD 3OD 10:1)δ7.75 (d, J = 8.7 Hz, 4H), 7.68 (d, J = 8.7 Hz, 2H), 7.51 (d, J = 8.3 Hz, 2H), 2.19 (s, 3H), 1.54 (s, 9H).

[0136] N-(4-(4-Aminobenzoyl)phenyl)acetamide(ELN048) Trifluoroacetic acid (200 μL, 2.61 mmol) was added to a dichloromethane solution of ELN046 (20.0 mg, 0.0564 mmol) and stirred at room temperature overnight. After extraction with dichloromethane, the mixture was washed with saturated aqueous bicarbonate and saturated saline. The organic layer was dried over magnesium sulfate, the solvent was removed, and the residue was purified by silica gel chromatography (hexane:dichloromethane:ethyl acetate 1:1:3) to obtain the title compound (10.3 mg, 0.0405 mmol, 72%) as a white solid.

[0137] 1H-NMR (500 MHz, CDCl 3 :CD 3 OD 10:1)δ7.76 - 7.64 (m, 6H), 6.69 (d, J = 8.7 Hz, 2H), 2.18 (s, 3H).

[0138] N-(4-(4-(((1,1-dimethylethoxy)carbonyl)amino)benzoyl)phenyl)-3β-tert-butyl(dimethyl)silyloxychol-5-en-24-amide(ELN060) Synthesized following the HATU amidation procedure described above: 51.3 mg, 0.0580 mmol, 55% yield. 1H-NMR (500 MHz, CDCl 3) δ 7.80 - 7.74 (m, 4H), 7.63 (d, J = 8.9 Hz, 2H), 7.47 (d, J = 8.6 Hz, 2H), 7.28 (s, 1H), 6.68 (s, 1H), 5.35 - 5.27 (m, 1H), 3.51 - 3.44 (m, 1H), 2.46 (ddd, J = 15.1, 10.4, 4.9 Hz, 1H), 2.34 - 2.13 (m, 4H), 2.03 - 1.68 (m, 7H), 1.54 (s, 9H), 1.00 (s, 3H), 0.98 (d, J = 6.3 Hz, 3H), 0.89 (s, 9H), 0.69 (s, 3H), 0.06 (s, 6H).

[0139] N-(4-(4-((1,1-dimethylethoxy)carbonyl)amino)benzoyl)phenyl)-3β-hydroxychol-5-en-24-amide (ELN062 (C23)) Synthesized according to the above-mentioned TBS deprotection procedure. 11.0 mg, 0.01644 mmol, yield 61%. 1H-NMR(500 MHz, DMSO-d 6 ) δ 10.23 (s, 1H), 9.80 (s, 1H), 7.74 (d, J = 8.8 Hz, 2H), 7.68 (d, J = 8.7 Hz, 2H), 7.66 (d, J = 8.7 Hz, 2H), 7.61 (d, J = 8.8 Hz, 2H), 5.30 - 5.22 (m, 1H), 4.60 (d, J = 4.6 Hz, 1H), 3.28 - 3.22 (m, 1H), 2.43 - 2.34 (m, 1H), 2.31 - 2.22 (m, 1H), 2.17 - 1.60 (m, 10H), 1.49 (s, 9H), 0.96 - 0.91 (m, 6H), 0.65 (s, 3H). ; 13C-NMR(150 MHz, CDCl 3 / MeOD (10:1))δ 195.68, 173.72, 153.02, 143.22, 142.56, 140.72, 132.61, 131.48 (2C), 131.35, 131.21 (2C), 121.43, 118.70, 117.29, 80.80, 71.16, 56.61, 55.81, 49.96, 42.26, 41.69, 39.63, 37.12, 36.37, 35.54, 34.21, 31.74, 31.72, 31.56, 30.99, 29.56, 28.06 (3C), 24.13, 20.92, 19.18, 18.16, 11.69. HRMS-ESI-TOFMS (m / z): [MH]- calcd for C42H56N2O5, 667.4136; found, 667.4116.

[0140] N-(4-(4-aminobenzoyl)phenyl)- 3β-hydroxychol-5-en-24-amide(ELN063(C24)) To a solution of ELN060 (28.8 mg, 0.0368 mmol) in dichloromethane, p-toluenesulfonic acid (6.2 mg, 0.0326 mmol) was added and stirred at room temperature overnight. After extraction with ethyl acetate, the organic layer was washed with saturated sodium bicarbonate solution and saturated brine, dried over magnesium sulfate, and filtered. The solvent was removed and the resulting compound was chromatographed on silica gel eluting with n-hexane / dichloromethane / ethyl acetate (1:1:2) to give the title compound (17.2 mg, 0.0302 mmol, 92%) as a light brown solid.

[0141] 1H-NMR (500 MHz, CDCl 3 / MeOD(10:1)) δ 7.73 - 7.53 (m, 8H), 5.29 - 5.24 (m, 1H), 2.45 - 2.32 (m, 1H), 2.29 - 2.13 (m, 4H), 0.93 (s, 3H), 0.91 (d, J = 6.4 Hz, 3H), 0.62 (s, 3H).; 13C-NMR(150 MHz, CDCl 3 / MeOD(10:1)) δ 195.53, 173.41, 140.79, 132.98, 131.65, 131.41, 131.37, 130.99, 130.93, 122.92, 121.57, 118.72 (2C), 113.56, 71.35, 56.70, 55.87, 50.03, 42.36, 41.86, 39.72, 37.20, 36.46, 35.61, 34.38, 31.82 (2C), 31.59, 31.16, 28.15, 24.24, 21.02, 19.33, 18.31, 11.83. HRMS-ESI-TOFMS (m / z): [M-H]- calcd for C37H48N2O3, 567.3611; found, 567.3592.

[0142] N-(4-(4-(acetylamino)benzoyl)phenyl)-3β-hydroxychol-5-en-24-amide(ELN049(C25)) Amidation was carried out according to the above amidation procedure with HATU, and concentrated hydrochloric acid was added before organic solvent extraction to obtain the titled compound with the TBS group deprotected (9.1 mg, 0.0149 mmol, 49%).

[0143] 1H-NMR(500 MHz, CDCl 3 / MeOD(10:1)) δ 7.67 (d, J = 8.6 Hz, 4H), 7.63 (d, J = 7.7 Hz, 4H), 5.29 - 5.23 (m, 1H), 3.71 (s, 1H), 3.44 - 3.33 (m, 1H), 2.44 - 2.32 (m, 1H), 2.27 - 2.12 (m, 3H), 2.10 (s, 3H), 1.99 - 1.68 (m, 7H), 0.94 - 0.90 (m, 6H), 0.62 (s, 3H).; 13C-NMR(150 MHz, CDCl 3 / MeOD(10:1)) δ 196.07, 174.22, 170.78, 143.15, 142.95, 141.14, 133.02, 132.82, 131.67 (2C), 131.64(2C), 121.81, 119.13 (4C), 71.54, 57.03, 56.22, 50.39, 42.67, 42.09, 40.05, 37.53, 36.77, 35.95, 34.59, 32.16, 32.13, 31.98, 31.38, 28.43, 24.53, 24.10, 21.33, 19.57, 18.54, 12.08.

[0144] <Example 6> Synthesis of OGK4007 [Synthesis Scheme]

Chem.

[0145] [Experimental Procedure] 5-Iodo-1,3-diaminobenzene (OGK4002) 1-Iodo-3,5-dinitrobenzene (197 mg, 0.670 mmol) was dissolved in ethanol (6.0 mL) and acetic acid (1.0 mmol), iron powder (703 mg, 12.6 mmol) was added, and the atmosphere was replaced with argon. The mixture was stirred at 80°C for 3 hours. The mixture was neutralized with 10% aqueous sodium hydroxide solution and saturated aqueous sodium bicarbonate solution at room temperature, and ethyl acetate was added. Insoluble matter was removed by filtration through a cotton plug, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with 0.5 M aqueous sodium bicarbonate solution, followed by saturated saline, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (hexane:AcOEt 1:1 to 1:2) to give the title compound (136 mg, 0.581 mmol, 87%) as pale yellow needle crystals.

[0146] Rf = 0.25(hexane:AcOEt = 1:1). 1H-NMR (400 MHz, CDCl 3 )δ6.49(d, J = 2.0 Hz, 2H), 5.97(t, J = 2.0 Hz, 1H), 3.58(br s, 4H).

[0147] 1,3-Diamino-5-(2-(trimethylsilyl)ethynyl)benzene(OGK4004) OGK4002 (133 mg, 0.566 mmol) and triethylamine (0.40 mL, 2.8 mmol) were dissolved in dry THF (6 mL) and degassed under argon. Trimethylsilylacetylene (0.32 mL, 2.3 mmol), copper(I) iodide (11.7 mg, 0.0614 mmol), Pd(PPh 3 ) 4(36.4 mg, 0.0315 mmol) was added and stirred at room temperature for 18 hours. After further heating at 80°C for 4 hours, AcOEt and water were added. The organic layer was washed with water and saturated saline, dried over magnesium sulfate, and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (hexane:AcOEt = 2:1 to 1:1) to obtain the title compound (111 mg, 0.544 mmol, 96%) as a brown solid.

[0148] Rf = 0.22(hexane:AcOEt = 1:1). 1H-NMR (400 MHz, CDCl 3 )δ6.24(d, J = 2.0 Hz, 2H), 5.98(t, J = 2.0 Hz, 1H), 3.56(br s, 4H), 0.22(s, 9H). 13C-NMR (101 MHz, CDCl 3 )δ147.18(2C), 124.25, 109.44(2C), 105.52, 102.41, 92.65, -0.02(3C).

[0149] 3β-Hydroxy-N-(3-amino-5-(2-(trimethylsilyl)ethynyl)phenyl)chol-5-en-24-amide(OGK4005(C26)) 3β-hydroxy-Δ 5-cholenic acid (91.7 mg, 0.245 mmol) was dissolved in DMF (3.0 mL), DIPEA (0.066 mL, 0.381 mmol) and HATU (146 mg, 0.385 mmol) were added, and a solution of OGK4004 (52.0 mg, 0.254 mmol) dissolved in DMF (1.0 mL) was added while stirring at room temperature. After stirring at room temperature for 3 hours, the organic layer obtained by diluting with AcOEt was washed with water and saturated saline. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure to obtain a brown oily substance. This residue was purified by silica gel chromatography (hexane:AcOEt = 1:1) to obtain the title compound (77.1 mg, 0.137 mmol, 56%) as an off-white solid.

[0150] Rf = 0.45(hexane:AcOEt = 1:2). HRMS-ESI-TOFMS(m / z): [M+H] + calcd for C 35 H 52 N 2 O 2 Si, 561.3871; found 561.3872. 1H-NMR (400 MHz, CDCl 3 )δ7.20(s, 1H), 7.03(s, 1H), 6.76(s, 1H), 6.52(s, 1H), 5.36-5.35(m, 1H), 3.70(s, 2H), 3.57-3.49(m, 1H), 2.42-2.17(m, 4H), 2.03-1.82(m, 6H),1.01(s, 3H), 0.96(d, J = 6.3 Hz, 3H), 0.68(s, 3H), 0.22(s, 9H). 13C-NMR (101 MHz, CDCl 3)δ171.74, 147.01, 140.68, 138.81, 124.07, 121.66, 113.94, 112.98, 106.83, 104.73, 93.64, 71.76, 56.68, 55.79, 50.01, 42.35, 42.24, 39.71, 37.20, 36.45, 35.45, 34.73, 31.83(2C), 31.60, 31.54, 28.18, 24.24, 21.04, 19.38, 18.43, 11.89, -0.07(3C).

[0151] 3β-Hydroxy-N-(3-azido-5-(2-(trimethylsilyl)ethynyl)phenyl)chol-5-en-24-amide(OGK4006(C27)) OGK4005 (73.4 mg, 0.131 mmol) was dissolved in acetic acid (1.8 mL) and water (0.2 mL), and sodium nitrite (14.9 mg, 0.216 mmol) was added in an ice bath and stirred for 10 minutes. Sodium azide (23.2 mg, 0.357 mmol) was added, and the mixture was removed from the ice bath and stirred for another hour. The organic layer obtained by diluting the reaction solution with AcOEt was washed with water, saturated aqueous sodium bicarbonate solution, and saturated saline, dried over magnesium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (hexane:AcOEt = 2:1) to obtain the title compound (61.4 mg, 0.108 mmol, 82%) as a pale yellow solid.

[0152] Rf = 0.60(hexane:AcOEt = 1:1). HRMS-ESI-TOFMS(m / z): [M+H] + calcd for C 35 H 50 N 4 O 2 Si, 587.3776; found 587.3775. 1H-NMR (400 MHz, CDCl 3)δ7.60 (s, 1H), 7.35-7.33(m, 2H), 6.85(s, 1H), 5.35-5.34(m, 1H), 3.57-3.49(m, 1H), 2.41(ddd, J = 15.0, 10.4, 4.7 Hz, 1H), 2,33-2.20(m, 3H), 1.98-1.83(m, 8H), 1.62-1.43(m, 8H), 1.00(s, 3H), 0.95(d, J = 6.2 Hz, 3H), 0.68(s, 3H), 0.23(s, 9H). 13C-NMR (101 MHz, CDCl 3 )δ172.15, 140.85, 140.61, 139.32, 124.96, 121.63, 119.19, 117.56, 110.51, 103.41, 95.51, 71.75, 56.64, 55.69, 49.99, 42.31, 42.21, 39.68, 37.19, 36.41, 35.38, 34.44, 31.79(2C), 31.56, 31.41, 28.14, 24.20, 21.01, 19.35, 18.40, 11.84, -0.20(3C).

[0153] 3β-Hydroxy-N-(3-azido-5-ethynylphenyl)chol-5-en-24-amide(OGK4007(C28)) OGK4006 (60.1 mg, 0.106 mmol) was dissolved in methanol (3 mL), potassium carbonate (18.5 mg, 0.134 mmol) was added, and the mixture was stirred at room temperature for 2 hours. A saturated aqueous solution of ammonium chloride was added, and the mixture was extracted with AcOEt. The organic layer was washed with water and saturated saline, dried over magnesium sulfate, and concentrated under reduced pressure. The resulting pale yellow solid was dissolved in heated AcOEt, and about three times the amount of hexane was gently added, and the mixture was allowed to stand overnight. The resulting crystals were filtered with a Kiriyama funnel under suction to obtain the title compound (34.1 mg, 0.0663 mmol, 63%) as pale yellow crystals.

[0154] Rf = 0.20(hexane:AcOEt = 2:1). HRMS-ESI-TOFMS(m / z): [M+H] + Calculate for C 32 H 42 N 4 O 2 , 515.3381; found 515.3382. 1H-NMR(400 MHz, DMSO-D6)δ 10.11(s, 1H), 7.49(s, 1H), 7.46(s, 1H), 6.82(s, 1H), 5.25(s, 1H), 4.62(d, J = 4.4 Hz, 1H), 4.27(s, 1H), 3.27-3.20(m, 1H), 2.35-0.98(m, 24H), 0.92(s, 3H), 0.91(d, J = 7.7 Hz, 3H), 0.63(s, 3H). 13C-NMR(101 MHz, DMSO-D6) δ 172.30, 141.25, 141.00, 140.37, 123.41, 120.45, 118.33, 116.37, 109.66, 82.49, 81.54, 70.04, 56.24, 55.32, 49.62, 42.25, 41.91, 36.96, 36.09, 34.97, 33.40, 31.48, 31.45, 31.39, 31.13, 27.69, 23.92, 20.66, 19.19, 18.35, 11.73.

[0155] <Example 7> Synthesis of OGK4007-like body [Synthesis スキーム]

change

[0156] [Synthetic Hands] N-(3-Amino-5-trimethylsilylethynylphenyl)-3β-tert-butyl(dimethyl)silyloxychol-5-en-24-amide(ELN050B) OGK4004 (51.0 mg, 0.250 mmol), DIPEA (53 μL, 0.312 mmol), and HATU (87.0 mg, 0.229 mmol) were added to a dichloromethane solution of ELN026 (101.9 mg, 0.208 mmol), and the mixture was stirred at room temperature overnight. After extraction with dichloromethane, the mixture was washed with water and saturated saline. The organic layer was dried over magnesium sulfate, and the solvent was removed. The residue was purified by silica gel chromatography (hexane:dichloromethane:ethyl acetate 6:4:1 to 0:4:1) to give the title compound (98.4 mg, 0.146 mmol, 70%) as a pale yellow solid.

[0157] 1H-NMR (500 MHz, CDCl 3 )δ7.21(1H, s), 6.95(1H, s), 6.76(1H, s), 6.54(1H, s), 5.32(1H, s), 3.50-3.46(1H, m), 1.00(3H, s), 0.96(3H, d, J = 6.3 Hz), 0.89(9H, s), 0.68(3H, s), 0.22(9H, s), 0.06(6H, s).

[0158] N-(3-Acetylamino-5-trimethylsilylethynylphenyl)-3β-hydroxychol-5-en-24-amide(ELN052) Acetic anhydride (130.6 μL, 2.19 mmol) was added to a pyridine solution of ELN050B (48.1 mg, 0.0712 mmol) and stirred at room temperature for 4 hours. After removing the solvent, the mixture was extracted with ethyl acetate and washed with saturated saline. The organic layer was dried over magnesium sulfate, the solvent was distilled off, and the residue was purified by silica gel chromatography (dichloromethane:ethyl acetate 6:0.7) to obtain the title compound (35.9 mg, 0.0500 mmol, 70%) as a white solid.

[0159] 1H-NMR (600 MHz, CDCl 3)δ7.79 (s, 1H), 7.44 (s, 1H), 7.34 (t, J = 1.7 Hz, 1H), 7.13 (s, 1H), 7.10 (s, 1H), 5.31 (d, J = 5.2 Hz, 1H), 3.52 - 3.44 (m, 1H), 0.99 (s, 3H), 0.96 (d, J = 6.5 Hz, 3H), 0.89 (s, 9H), 0.68 (s, 3H), 0.22 (s, 9H), 0.05 (s, 6H).

[0160] N-(3-Acetylamino-5-ethynylphenyl)-3β-hydroxychol-5-en-24-amide(ELN053(C29)) ELN052 (32.5 mg, 0.0453 mmol) was dissolved in 1M TBAF H 2 O (600 μL, 0.600 mmol) was added and stirred at room temperature overnight. After extraction with ethyl acetate, the mixture was washed twice with ethyl acetate and with saturated saline. The organic layer was dried over magnesium sulfate, the solvent was removed, and the residue was purified by silica gel chromatography (dichloromethane:ethyl acetate 1:2) to obtain the title compound (20.9 mg, 0.0394 mmol, 87%) as a white solid.

[0161] 1H-NMR (600 MHz, CDCl 3 :CD 3 OD 10:1)δ7.64 (s, 1H), 7.42 - 7.33 (m, 2H), 5.30 - 5.24 (m, 1H), 3.44 - 3.38 (m, 1H), 2.99 (s, 1H), 2.39 - 2.27 (m, 1H), 2.25 - 2.10 (m, 3H), 2.06 (s, 3H), 1.98 - 1.70 (m, 7H), 0.95 - 0.89 (m, 6H), 0.61 (s, 3H), 3 :CD 3OD 10:1) δ173.45, 170.01, 140.79, 138.78, 138.72, 122.88, 121.57, 118.93, 118.83, 111.73, 83.17, 77.20, 71.36, 56.70, 55.85, 50.04, 42.35, 41.89, 39.72, 37.22, 36.46, 35.60, 34.24, 31.83 (2C), 31.68, 31.20, 28.13, 24.24, 23.91, 21.03, 19.34, 18.32, 11.83. HRMS-ESI-TOFMS (m / z): [M+H]+ calcd for C 34 H 46 N 2 O 3 , 531.3600; found, 531.3581. [M-H2O+H]+ calcd 513.3481; found 513.3476. [M+Na]+ calcd 553.3407; found 553.3401.

[0162] N-(3-Amino-5-ethynylphenyl)-3β-hydroxychol-5-en-24-amide(ELN054(C30)) ELN050B (48.1 mg, 0.0712 mmol) was dissolved in 1M TBAF H 2 O (853 μL, 0.853 mmol) was added and stirred at room temperature overnight. After extraction with ethyl acetate, the mixture was washed twice with ethyl acetate and with saturated saline. The organic layer was dried over magnesium sulfate, the solvent was removed, and the residue was purified by silica gel chromatography (dichloromethane:ethyl acetate 1:1) to obtain the title compound (29.2 mg, 0.0598 mmol, 87%) as a white solid.

[0163] 1H-NMR (600 MHz, CDCl 3 :CD 3OD 10:1)δ7.02 (t, J = 2.0 Hz, 1H), 6.81 (t, J = 1.6 Hz, 1H), 6.44 (t, J = 1.9 Hz, 1H), 5.25 - 5.21 (m, 1H), 3.40 - 3.32 (m, 1H), 2.92 (s, 1H), 2.27 (ddd, J = 15.3, 10.3, 4.8 Hz, 1H), 2.18 - 2.06 (m, 4H), 0.88 (s, 3H), 0.85 (d, J = 6.6 Hz, 3H), 0.57 (s, 3H); 13C-NMR (150 MHz, CDCl 3 :CD 3 OD 10:1)δ173.34, 146.98, 140.72, 139.20, 122.81, 121.45, 114.23, 113.64, 107.59, 83.55, 76.33, 71.20, 56.62, 55.80, 49.97, 42.26, 41.73, 39.64, 37.14, 36.38, 35.53, 34.20, 31.75(2C), 31.66, 31.04, 28.04, 24.14, 20.94, 19.22, 18.18, 11.71. HRMS-ESI-TOFMS (m / z): [M+H]+ calcd for C 32 H 44 N 2 O 2 , 489.3488; found, 489.3476. [M+Na]+ calcd 511.3312; found 511.3295.

[0164] <Example 8> Synthesis of ELN025 [Synthesis スキーム]

change

[0165] [Synthetic Hands] N-(4-(4-Aminobenzoyl)phenyl)-4-pentynamide(ELN024) A solution of 4-pentynoic acid (149.6 mg, 1.52 mmol) in THF (3 mL) was added dropwise with dimethylformamide under nitrogen and stirred at 0°C, then oxalyl chloride (128.4 μL, 1.49 mmol) was added and stirred at room temperature for 30 minutes (A). A solution of 4,4'-diaminobenzophenone (214.8 mg, 1.01 mmol) and triethylamine (139.4 μL, 1.00 mmol) in tetrahydrofuran (3 mL) / dimethylformamide (1 mL) was stirred at room temperature under nitrogen. A was added dropwise to this mixture and stirred overnight under nitrogen. After extraction with dichloromethane, the organic phase was washed with saturated sodium bicarbonate solution and water, dried over magnesium sulfate, and filtered. The solvent was removed and the resulting compound was chromatographed on silica gel eluting with n-hexane / ethyl acetate (2:1→1:2) to give 78.0 mg (0.267 mmol, 27%) of the title compound as a yellow solid.

[0166] 1H-NMR (500 MHz, CDCl 3 -CD 3 OD 10:1)δ 7.67-7.44 (m, 6H), 6.56-6.51(m, 2H), 2.47(tt, J = 10.9, 3.6 Hz, 4H), 1.92-1.91(m, 1H).

[0167] 3β-Hydroxy-N-(4-(4-((1-oxo-4-pentyn-1-yl)amino)benzoyl)phenyl)chol-5-en-24-amide(ELN025(C31)) To a solution of 3β-hydroxy-Δ5-cholenic acid (83.2 mg, 0.22 mmol) in tetrahydrofuran (17 mL) was added triethylamine (44 μL, 0.33 mmol) and isobutyl chloroformate (29.3 μL, 0.22 mmol). After stirring at 0 °C, the mixture was added with ELN024 (78.0 mg, 0.267 mmol) and stirred at room temperature overnight. After extraction with dichloromethane, the organic phase was washed three times with aqueous ammonium chloride and saturated brine, dried over magnesium sulfate, and filtered. The solvent was removed and the resulting compound was chromatographed on silica gel eluting with n-hexane / dichloromethane / ethyl acetate (1:1:1). 11.0 mg (0.0170 mmol, 8%) of the title compound was obtained as a white solid.

[0168] 1H-NMR (500 MHz, CDCl 3 )δ 7.69 (dd, J = 8.7, 1.9 Hz, 4H), 7.65 - 7.60 (m, 4H), 5.30 - 5.26 (m, 1H), 3.48 - 3.38 (m, 1H), 2.62 - 2.51 (m, 4H), 2.40 (ddd, J = 15.1, 10.4, 4.9 Hz, 1H), 2.29 - 2.10 (m, 3H), 2.00 (t, J = 2.3 Hz, 1H), 1.98 - 1.71 (m, 7H), 1.58 - 0.96 (m, 16H), 0.95 (s, 3H), 0.93 (d, J = 6.4 Hz, 3H), 0.90 - 0.84 (m, 1H), 0.63 (s, 3H). 13C-NMR (150 MHz, CDCl 3)δ 195.44, 173.35, 170.50, 142.54, 142.27, 140.81, 132.88, 132.65, 131.45 (2C), 131.42 (2C), 121.62, 118.83 (2C), 118.76 (2C), 82.80, 71.43, 69.40, 56.74, 55.89, 50.07, 42.40, 41.94, 39.76, 37.24, 36.50, 35.91, 35.62, 34.45, 31.86 (2C), 31.62, 31.26, 28.19, 24.28, 21.07, 19.38, 18.38, 14.61, 11.88. HRMS-ESI-TOFMS (m / z): [M+Na]+ calcd for C 42 H 52 N 2 O 4 , 671.3826; found, 671.3819.

[0169] Example 9: Saturated alkyl chain of ELN025 [Synthetic scheme] [ka]

[0170] [Synthesis procedure] 1,1-Dimethylethyl N-(4-(4-(pentanoylamino)benzoyl)phenyl)carbamate(ELN065) To a solution of ELN050 (78.1 mg, 0.250 mmol) in DMF, pentanoic acid (57.9 μL, 0.533 mmol), DIPEA (102 μL, 0.600 mmol), and HATU (444.0 mg, 1.17 mmol) were added and stirred at room temperature overnight. After extraction with dichloromethane, the mixture was washed three times with saturated saline. The organic layer was dried over magnesium sulfate, the solvent was removed, and the residue was purified by silica gel chromatography (hexane:dichloromethane:ethyl acetate 3:3:4) to obtain the title compound (98.4 mg, 0.248 mmol, 99%) as a white solid.

[0171] 1H-NMR (400 MHz, CDCl 3 )δ7.79 - 7.71 (m, 4H), 7.48 - 7.44 (m, 2H), 7.44 - 7.40 (m, 2H), 7.27 (s, 1H), 6.68 (s, 1H), 2.37 - 2.31 (m, 2H), 1.74 - 1.69 (m, 2H), 1.38 - 1.34 (m, 2H), 0.95 (d, J = 7.0 Hz, 3H)

[0172] N-(4-(4-Aminobenzoyl)phenyl)-4-pentanamide(ELN067(C32)) Trifluoroacetic acid (400 μL, 5.22 mmol) was added to ELN065 (96.9 mg, 0.244 mmol) and stirred at room temperature overnight. After extraction with dichloromethane, it was washed with saturated saline. After drying the organic layer over magnesium sulfate, the solvent was distilled off, and the residue was purified by silica gel chromatography (hexane:dichloromethane:ethyl acetate 1:1:2) to obtain the title compound (33.5 mg, 0.113 mmol, 46%) as a yellow solid.

[0173] 1H-NMR(400 MHz, CDCl3)δ7.73 (d, J = 8.8 Hz, 2H), 7.69 (d, J = 8.8 Hz, 2H), 7.63 (s, 1H), 7.61 (s, 1H), 7.53 (s, 1H), 6.67 (d, J = 8.7 Hz, 2H), 2.44 - 2.35 (m, 2H), 1.79 - 1.67 (m, 2H), 1.48 - 1.35 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H).

[0174] 3β-Hydroxy-N-(4-(4-((1-oxopentan-1-yl)amino)benzoyl)phenyl)chol-5-en-24-amide(ELN068) To a solution of 3β-hydroxy-Δ5-cholenic acid (42.3 mg, 0.113 mmol) in anhydrous THF (8 mL) and anhydrous DMF (2 mL), triethylamine (66 μL, 0.47 mmol) and isobutyl chloroformate (15 μL, 0.116 mmol) were added. After stirring at 0 °C for 4 hours, a THF solution of ELN067 (33.5 mg, 0.113 mmol) was added to the mixture and stirred at room temperature overnight. After extraction with ethyl acetate, the mixture was washed three times with saturated saline. The organic layer was dried over magnesium sulfate, the solvent was removed, and the residue was purified by silica gel chromatography (hexane:dichloromethane:ethyl acetate 1:1:1) to obtain the title compound (9.3 mg, 0.0142 mmol, 13%) as a pale yellow solid.

[0175] 1H-NMR (500 MHz, CDCl 3 :CD 3 OD 10:1)δ7.70 (d, J = 8.9 Hz, 4H), 7.66 - 7.58 (m, 4H), 5.35 - 5.24 (m, 1H), 3.50 - 3.40 (m, 1H), 2.45 - 2.38 (m, 1H), 2.35 (d, J = 7.7 Hz, 2H), 2.30 - 2.19 (m, 2H), 2.00 - 1.74 (m, 6H), 1.67 (p, J = 7.7 Hz, 3H), 1.60 - 1.24 (m, 12H), 1.17 - 1.00 (m, 5H), 0.96 (s, 3H), 0.94 (d, J = 6.4 Hz, 3H), 0.91 (t, J = 7.3 Hz, 3H), 0.65 (s, 3H); 13C-NMR(150 MHz, CDCl 3 :CD 3OD 10:1) δ 195.33, 173.14, 172.80, 142.39 (2C), 140.81, 132.78 (2C), 131.46 (4C), 121.66, 118.75 (4C), 71.51, 56.76, 55.91, 50.09, 42.42, 42.03, 39.79, 37.31, 37.26, 36.52, 35.63, 34.52, 31.89 (2C), 31.61, 31.35, 28.22, 27.65, 24.30, 22.42, 21.09, 19.41, 18.43, 13.84, 11.91. HRMS-ESI-TOFMS (m / z): [M+H]+ calcd for C 42 H 56 N 2 O 4 , 653.4313; found, 653.4313. [M+Na]+ calcd 675.4143; found 675.4132.

[0176] <Example 10> cis form of AB ring [Synthetic scheme] [Chemical formula]

[0177] [Synthetic procedure] N-(3-Methylphenyl)-3β-hydroxy-5β-cholan-24-amide(ELN071(C33)) To a solution of lithocholic acid (113.0 mg, 0.300 mmol) in dehydrated toluene, m-toluidine (33.2 μL, 0.300 mmol) and MTM (130.3 μL, 0.900 mmol) were added, and the mixture was stirred at reflux for 4 hours at 0 °C under argon. The solvent was removed under reduced pressure, and 0.3 M aqueous sodium hydroxide solution (300 μL) and THF were added, followed by stirring at room temperature for 2 hours. The mixture was extracted with ethyl acetate, and then washed with hydrochloric acid and saturated saline. The organic layer was dried over magnesium sulfate, the solvent was removed, and the residue was purified by silica gel chromatography (dichloromethane:ethyl acetate 4:1) to obtain the title compound (16.6 mg, 0.0356 mmol, 12%) as a white solid.

[0178] 1H-NMR (400 MHz, CDCl 3 :CD 3 OD 10:1)δ7.39 (s, 1H), 7.30 (d, J = 9.0 Hz, 1H), 7.19 (t, J = 7.8 Hz, 1H), 6.91 (d, J = 7.6 Hz, 1H), 3.63 - 3.54 (m, 1H), 2.46 - 2.35 (m, 1H), 2.33 (s, 3H), 2.29 - 2.17 (m, 1H), 2.02 - 1.70 (m, 6H), 1.69 - 1.01 (m, 20H), 0.97 (d, J = 6.3 Hz, 3H), 0.92 (s, 3H), 0.66 (s, 3H). 13C-NMR (100 MHz, CDCl 3 :CD 3 OD 10:1)δ173.14, 138.66, 138.12, 128.60, 124.79, 120.61, 117.02, 71.42, 56.49, 56.01, 42.70, 42.04, 40.43, 40.16, 35.96, 35.80, 35.53, 35.27, 34.52, 34.21, 31.74, 30.06, 28.16, 27.14, 26.38, 24.16, 23.31, 21.32, 20.77, 18.23, 11.95.

[0179] <Example 11> AB ring cis ketone [Synthetic scheme] [ka]

[0180] [Synthesis procedure] 3-Oxo-5β-cholan-24-oic acid(OGK4106(C34)) Lithocholic acid (1222 mg, 3.245 mmol) was dissolved in acetic acid (8 mL) and dichloromethane (8 mL), and while stirring in a water bath at about 20°C, aqueous sodium hypochlorite solution (12% w / w, 3.0 mL, 4.9 mmol) was slowly added dropwise. After 1 hour and 1.5 hours, aqueous sodium hypochlorite solution (1.0 mmol, 1.6 mmol) was added dropwise, and the mixture was stirred for another 30 minutes. Isopropanol (1 mL) was added to consume excess sodium hypochlorite, and the mixture was concentrated under reduced pressure. The residue was partitioned between water and chloroform, and the chloroform layer was washed with water, saturated aqueous sodium thiosulfate solution, and then saturated saline, dried over magnesium sulfate, and the solvent was distilled off. The resulting colorless solid was dissolved in methyl-tert-butyl ether (6 mL), and hexane (12 mL) was slowly added. The resulting crystals were collected by suction filtration, washed with hexane, and then dried under reduced pressure to obtain the title compound (642 mg, 1.71 mmol, 53%) as a colorless solid.

[0181] Rf = 0.25 (dichloromethane:AcOEt:AcOH 100:10:1). 1H-NMR (500 MHz, CDCl 3)δ 2.70(t, J = 14.3 Hz, 1H), 2.44-2.37(m, 1H), 2.37-2.30(m, 1H), 2.29-2.23(m, 1H), 2.20-2.13(m, 1H), 2.05-2.01(m, 3H), 1.92-1.79(m, 4H), 1.63-1.58(m, 1H), 1.53-1.05(m, 15H), 1.02(s, 3H), 0.94(d, J = 6.9 Hz, 3H), 0.69(s, 3H). 13C-NMR(126 MHz, CDCl3)δ 213.55, 179.13, 56.42, 55.94, 44.31, 42.78, 42.35, 40.72, 40.04, 37.21, 37.00, 35.52, 35.29, 34.88, 30.81, 30.74, 28.14, 26.60, 25.75, 24.14, 22.64, 21.18, 18.24, 12.07.

[0182] N-(3-Methylphyenyl)-3-oxo-5β-cholan-24-amide(ELN076) To a solution of OGK4106 (200.9 mg, 0.537 mmol) in dehydrated dichloromethane (2 mL), m-toluidine (70.5 μL, 0.645 mmol), DIPEA (280 μL, 1.61 mmol), and HATU (408.2 mg, 1.07 mmol) were added and stirred overnight at room temperature under nitrogen. After extraction with ethyl acetate, the mixture was washed with water and saturated saline. The organic layer was dried over magnesium sulfate, the solvent was removed, and the residue was purified by silica gel chromatography (dichloromethane:ethyl acetate 10:0.7) to obtain the title compound (235.3 mg, 0.507 mmol, 95%) as a white solid.

[0183] 1H-NMR (400 MHz, CDCl 3 :CD 3OD 10:1)δ8.11 (s, 1H), 7.42 (s, 1H), 7.32 (d, J = 8.9 Hz, 1H), 7.15 (t, J = 7.8 Hz, 1H), 6.88 (d, J = 7.4 Hz, 1H), 2.69 (dd, J = 15.1, 13.3 Hz, 1H), 2.46 - 2.31 (m, 2H), 2.29 (s, 3H), 2.28 - 2.11 (m, 2H), 1.01 (s, 3H), 0.94 (d, J = 6.4 Hz, 3H), 0.67 (s, 3H). 13C-NMR (151 MHz, CDCl 3 ) δ 213.77, 172.10, 138.73, 138.11, 128.68, 124.79, 120.47, 116.87, 56.37, 56.02, 44.29, 42.74, 42.35, 40.65, 40.01, 37.21, 36.97, 35.45 (2C), 34.84, 34.52, 31.62, 28.18, 26.57, 25.73, 24.14, 22.62, 21.49, 21.15, 18.39, 12.07. HRMS-ESI-TOFMS (m / z): [M+H]+ calcd for C 31 H 45 NO 2 , 464.3532; found, 464.3523. [M+Na]+ calcd 486.3350; found 486.3343.

[0184] <Example 12> Aminori derivative [Synthesis スキーム]

change

[0185] [Synthetic Hands] 3β-Hydroxy-N-(3-methylphenyl)chol-5-en-24-amide(TYK1014) 3β-Hydroxy-Δ5-cholenic acid (200.2 mg, 0.534 mmol) was dissolved in DMF (6 mL), m-methylaniline (57.8 μL, 0.534 mmol), HATU (304.6 mg, 0.801 mmol), and DIPEA (137 μL, 0.806 mmol) were added, and the mixture was stirred at room temperature for 18 hours. The mixture was diluted with about 5 times the amount of AcOEt and washed with saturated saline. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane:dichloromethane:AcOEt 2:3:1) to obtain the title compound (190 mg, 0.410 mmol, 77%) as a pale yellow solid.

[0186] Rf=0.55 (Hexane: EtOAc 2:1) 1H-NMR (500 MHz, Chloroform-d) δ 7.38 (s, 1H), 7.26 (d, J = 7.0 Hz, 1H), 7.19 (t, J = 7.9 Hz, 1H), 7.03 (s, 1H), 6.91 (d, J = 7.7 Hz, 1H), 5.37 - 5.34 (m, 1H), 3.55 - 3.49 (m, 1H), 2.34 (s, 3H), 1.01 (s, 3H), 0.97 (d, J = 6.2 Hz, 3H), 0.69 (s, 3H).

[0187] 3β-Hydroxy-N-(3-methylphenyl)chol-5-en-24-amide methanesulfonate(TYK1015) TYK1014 (119 mg, 0.256 mmol) was dissolved in anhydrous dichloromethane under argon, and triethylamine (149 μL, 1.07 mmol) and mesyl chloride (62 μL, 0.80 mmol) were added under ice cooling and stirred for 30 minutes, then the temperature was returned to room temperature and stirred for 24 hours. The mixture was diluted with about 5 times the amount of AcOEt and washed with saturated saline. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane:dichloromethane:AcOEt 4:2:0.7) to obtain the title compound (57.7 mg, 0.106 mmol, 41%) as a white solid.

[0188] Rf=0.10 (Hexane:Dichloromethane:EtOAc 4:2:0.7) 1H-NMR (500 MHz, Chloroform-d) δ 7.38 (s, 1H), 7.26 (d, J = 8.0 Hz, 1H), 7.19 (t, J = 7.7 Hz, 1H), 7.03 (s, 1H), 6.91 (d, J = 7.4 Hz, 1H), 5.44 - 5.39 (m, 1H), 4.56 - 4.49 (m, 1H), 3.01 (s, 3H), 2.56 - 2.35 (m, 2H), 2.34 (s, 3H), 2.29 - 2.20 (m, 1H), 1.02 (s, 3H), 0.97 (d, J = 6.6 Hz, 3H), 0.69 (s, 3H).

[0189] 3β-Azido-N-(3-methylphenyl)chol-5-en-24-amide(TYK1020) TYK1015 (57.7 mg, 0.106 mmol) was dissolved in anhydrous dichloromethane (0.5 mL) with trimethylsilyl azide (14.5 μL, 0.144 mmol) and boron trifluoride diethyl etherate (27.0 μL, 0.169 mmol) and stirred for 1.5 hours. Since the reaction did not proceed, trimethylsilyl azide (14.5 μL, 0.144 mmol) and boron trifluoride diethyl etherate (27.0 μL, 0.169 mmol) were added and stirred for 1.5 hours. Trimethylsilyl azide (29.0 μL, 0.288 mmol) and boron trifluoride diethyl etherate (27.0 μL, 0.388 mmol) were further added and stirred for 1.5 hours. The mixture was neutralized with 5M NaOH (2 mL), diluted with about 5 times the amount of dichloromethane, and washed with saturated saline. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane:dichloromethane:AcOEt 8:2:1) to obtain the title compound (19.8 mg, 0.0405 mmol, 38%) as a white solid.

[0190] Rf=0.30 (Hexane:Dichloromethane:AcOEt 8:2:1) 1H-NMR (500 MHz, Chloroform-d) δ 7.38 (s, 1H), 7.26 - 7.24 (m, 1H), 7.19 (t, J = 7.7 Hz, 1H), 7.03 (s, 1H), 6.91 (d, J = 7.4 Hz, 1H), 5.41 - 5.36 (m, 1H), 3.25 - 3.15 (m, 1H), 2.45 - 2.36 (m, 1H), 2.34 (s, 3H), 2.32 - 2.18 (m, 3H), 2.04 - 1.81 (m, 5H), 1.00 (s, 3H), 0.97 (d, J = 6.4 Hz, 3H), 0.69 (s, 3H).

[0191] 3β-Amino-N-(3-methylphenyl)chol-5-en-24-amide(TYK1021(C35)) TYK1020 (19.0 mg, 0.0389 mmol) in THF (0.5 mL), H 2 The mixture was dissolved in 2H2O (0.075 mL), triphenylphosphine (12.4 mg, 0.0447 mmol) was added, and the mixture was stirred for 18 hours. Triphenylphosphine (18.4 mg, 0.0702 mmol) was further added, and the mixture was stirred for 3 hours and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (dichloromethane:methanol:ammonia water 7:1:0.05) to give the title compound (7.4 mg, 0.0160 mmol, 41%) as an off-white solid.

[0192] Rf=0.30 (dichloromethane:methanol:ammonia water 7:1:0.05) 1H-NMR (500 MHz, Methanol-d4) δ 7.37 (s, 1H), 7.32 (d, J = 7.9 Hz, 1H), 7.17 (t, J = 7.8 Hz, 1H), 6.91 (d, J = 7.6 Hz, 1H), 5.36 - 5.33 (m, 1H), 2.31 (s, 3H), 1.04 - 1.00 (m, 6H), 0.74 (s, 3H). 13C-NMR (151 MHz, CD 3 OD) δ 175.28, 142.64, 139.97, 139.83, 129.76, 125.97, 122.18, 121.98, 118.55, 58.32, 57.44, 53.01, 51.89, 43.69, 42.74, 41.27, 39.43, 37.83, 37.10, 35.16, 33.37 (2C), 33.12, 32.26, 29.37, 25.45, 22.26, 21.71, 20.01, 19.10, 12.46. HRMS-ESI-TOFMS (m / z): [M+H]+ calcd for C 31 H 46 N 2 O, 463.3688; found, 463.3683.

[0193] [Synthetic scheme] [ka]

[0194] [Synthesis procedure] Methyl 3β-hydroxy-chol-5-en-24-oate(TYK1024) 3β-Hydroxy-Δ5-cholenic acid (500.5 mg, 1.37 mmol) was dissolved in methanol (10 mL), and sulfuric acid (0.5 mL) was added dropwise under ice cooling and stirred for 18 hours. The solution was diluted with chloroform and washed with saturated aqueous sodium bicarbonate and saturated saline. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure to obtain the title compound (487.9 mg, 1.26 mmol, 95%) as a white solid.

[0195] Rf=0.55 (Hexane:Dichloromethane:AcOEt 1:1:1) 1H-NMR (500 MHz, Chloroform-d) δ 5.36 - 5.34 (m, 1H), 3.66 (s, 3H), 3.58 - 3.47 (m, 1H), 2.40 - 2.18 (m, 4H), 2.03 - 1.94 (m, 1H), 1.90 - 1.77 (m, 3H), 1.53 - 1.39 (m, 6H), 1.37 - 1.25 (m, 2H), 1.20 - 1.04 (m, 4H), 1.01 (s, 3H), 0.93 (d, J = 6.4 Hz, 3H), 0.68 (s, 3H).

[0196] Methyl 3β-hydroxy-chol-5-en-24-oate methanesulfonate(TYK1027) Pyridine (304 μL, 3.77 mmol) was added to TYK1024 (487.9 mg, 1.26 mmol) and dissolved in anhydrous dichloromethane (3 mL). A solution of mesyl chloride (292 μL, 3.77 mmol) dissolved in anhydrous dichloromethane (0.5 mL) was added dropwise under ice cooling and stirred for 30 minutes under ice cooling. The mixture was returned to room temperature and stirred for 18 hours. Pyridine (406 μL, 5.03 mmol) and mesyl chloride (388 μL, 5.02 mmol) were added under ice cooling and stirred for 8 hours. The mixture was diluted with about 5 times the amount of dichloromethane and washed with saturated saline. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane:dichloromethane:AcOEt 7:7:1) to obtain the title compound (228.1 mg, 0.489 mmol, 39%) as a pale yellow solid.

[0197] Rf=0.75 (Hexane:Dichloromethane:AcOEt 5:5:2) 1H-NMR (500 MHz, Chloroform-d) δ 5.44 - 5.40 (m, 1H), 4.56 - 4.48 (m, 1H), 3.66 (s, 3H), 3.01 (s, 3H), 2.57 - 2.46 (m, 2H), 2.40 - 2.31 (m, 2H), 2.22 (ddd, J = 15.8, 9.8, 6.6 Hz, 1H), 2.07 - 1.73 (m, 5H), 1.02 (s, 3H), 0.92 (d, J = 6.4 Hz, 3H), 0.68 (s, 3H).

[0198] Methyl 3β-azido-chol-5-en-24-oate(TYK1031) TYK1027 (228.1 mg, 0.489 mmol) was dissolved in anhydrous dichloromethane (2 mL) with trimethylsilyl azide (254 μL, 1.94 mmol) and boron trifluoride diethyl etherate (243 μL, 1.93 mmol) and stirred for 2 hours. Trimethylsilyl azide (254 μL, 1.94 mmol) and boron trifluoride diethyl etherate (243 μL, 1.93 mmol) were added and stirred for 1.5 hours. The solution was neutralized with 5M NaOH (1 mL), diluted with approximately 5 times the amount of dichloromethane, and washed with saturated saline. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane:dichloromethane:AcOEt 10:2:0.2) to obtain the title compound (114.2 mg, 0.276 mmol, 56%) as a white solid.

[0199] Rf=0.20 (Hexane:Dichloromethane:AcOEt 10:2:0.2) 1H-NMR (500 MHz, Chloroform-d) δ 5.40 - 5.37 (m, 1H), 3.66 (s, 3H), 3.24 - 3.16 (m, 1H), 2.35 (ddd, J = 15.4, 10.2, 5.2 Hz, 1H), 2.31 - 2.26 (m, 2H), 2.22 (ddd, J = 15.7, 9.8, 6.5 Hz, 1H), 2.03 - 1.95 (m, 2H), 1.92 - 1.74 (m, 4H), 1.59 (d, J = 10.0 Hz, 2H), 1.52 - 1.25 (m, 7H), 1.20 - 1.04 (m, 4H), 1.00 (s, 3H), 0.93 (d, J = 6.5 Hz, 3H), 0.68 (s, 3H).

[0200] 3β-Azido-chol-5-en-24-oic acid(TYK1039) TYK1031 (112.2 mg, 0.271 mmol) was dissolved in THF (0.75 mL) and methanol (0.3 mL) was added. 5 M aqueous sodium hydroxide solution (0.20 mL) was added dropwise under ice cooling, and the mixture was stirred for 15 minutes and then stirred at room temperature for 3 hours. 5 M hydrochloric acid (0.2 mL) was added dropwise under ice cooling, and the mixture was extracted with ethyl acetate at room temperature. The organic layer was washed with water and saturated saline, dried over magnesium sulfate, and concentrated under reduced pressure to obtain the title compound (93.5 mg, 0.234 mmol, 85%) as a white solid.

[0201] Rf=0-0.25 (Hexane:Dichloromethane:AcOEt 10:1:0.3) 1H-NMR (500 MHz, Chloroform-d) δ 5.42 - 5.35 (m, 1H), 3.25 - 3.16 (m, 1H), 2.40 (ddd, J = 15.6, 10.2, 5.2 Hz, 1H), 2.33 - 2.22 (m, 3H), 2.00 (d, J = 12.3 Hz, 2H), 1.93 - 1.76 (m, 4H), 1.68 - 1.25 (m, 11H), 1.22 - 1.01 (m, 4H), 1.00 (s, 3H), 0.94 (d, J = 6.5 Hz, 3H), 0.69 (s, 3H).

[0202] N-(3-Methylphenyl)-3β-azido-chol-5-en-24-amide(TYK1040) TYK1040 (92.5 mg, 0.231 mmol) was dissolved in DMF (1.0 mL), and m-methylaniline (23.8 μL, 0.220 mmol), HATU (125.0 mg, 0.329 mmol), and DIPEA (56 μL, 0.329 mmol) were added. The mixture was stirred at room temperature for 21 h. After extraction with AcOEt, the organic layer was washed with water and saturated brine, dried over magnesium sulfate, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (hexane:dichloromethane:AcOEt 10:1:0.85) to give the title compound (98.7 mg, 0.202 mmol, 87%) as a white solid.

[0203] Rf = 0.25 (hexane:dichloromethane:AcOEt 10:1:0.85) 1H-NMR (500 MHz, Chloroform-d) δ 7.38 (s, 1H), 7.29 - 7.23 (m, 1H), 7.19 (t, J = 7.7 Hz, 1H), 7.03 (s, 1H), 6.91 (d, J = 7.5 Hz, 1H), 5.41 - 5.35 (m, 1H), 3.25 - 3.15 (m, 1H), 2.41 (ddd, J = 15.0, 10.3, 5.0 Hz, 1H), 2.34 (s, 3H), 2.31 - 2.20 (m, 3H), 2.03 - 1.95 (m, 2H), 1.93 - 1.84 (m, 4H), 1.00 (s, 3H), 0.98 (d, J = 6.4 Hz, 3H), 0.69 (s, 3H).

[0204] N-(3-Methylphenyl)-3β-amino-chol-5-en-24-amide (TYK1041) TYK1040 (98.7 mg, 0.202 mmol) was dissolved in THF (0.25 mL), H 2The mixture was dissolved in 2H2O (0.03 mL), triphenylphosphine (154.2 mg, 0.588 mmol) was added, and the mixture was stirred at room temperature for 25 hours and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methanol:saturated aqueous ammonia solution 7:1:0.05) to give the title compound (54.0 mg, 0.113 mmol, 57%) as a white solid.

[0205] Rf=0.30 (dichloromethane:methanol:saturated aqueous ammonia solution 7:1:0.05) 1H-NMR (500 MHz, Chloroform-d) δ 7.38 (s, 1H), 7.26 - 7.26 (m, 1H), 7.19 (t, J = 7.8 Hz, 1H), 7.03 (s, 1H), 6.91 (d, J = 7.5 Hz, 1H), 5.33 - 5.30 (m, 1H), 2.65 - 2.56 (m, 1H), 2.44 - 2.37 (m, 1H), 2.34 (s, 3H), 2.28 - 2.20 (m, 1H), 2.18 - 2.12 (m, 1H), 2.09 - 1.86 (m, 6H), 1.83 (dd, J = 13.2, 3.4 Hz, 1H), 1.73 - 1.67 (m, 1H), 0.99 (s, 3H), 0.98 (d, J = 6.4 Hz, 3H), 0.69 (s, 3H).

[0206] N-(3-Methylphenyl)-3β-acetylamino-chol-5-en-24-amide(TYK1043(C36)) TYK1041 (25.0 mg, 0.0540 mmol) was dissolved in chloroform (0.5 mL) and 0.5 M aqueous sodium bicarbonate (0.5 mL), and acetyl chloride (17.8 μL, 2.71 mmol) was added dropwise under ice cooling and stirred for 30 minutes. After returning to room temperature and stirring for 18 hours, isopropanol (0.6 mL) was added, acetyl chloride (17.8 μL, 2.71 mmol) was added dropwise, and the mixture was stirred for 3 hours and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (dichloromethane:AcOEt:methanol 1:2:0.1) to obtain the title compound (13.37 mg, 0.026 mmol, 49%) as an off-white solid.

[0207] Rf=0.62 (dichloromethane:AcOEt:methanol 1:2:0.1) 1H-NMR (500 MHz, Methanol-d4) δ 7.36 (s, 1H), 7.31 (d, J = 9.0 Hz, 1H), 7.16 (t, J = 7.8 Hz, 1H), 6.90 (d, J = 6.6 Hz, 1H), 5.35 (d, J = 5.1 Hz, 1H), 4.54 (s, 1H), 3.58 - 3.45 (m, 1H), 2.41 (ddd, J = 14.0, 10.1, 5.3 Hz, 1H), 2.30 (s, 3H), 2.29 - 2.22 (m, 1H), 2.21 - 1.92 (m, 5H), 1.90 (s, 3H), 1.02 (s, 3H), 1.01 (d, J = 6.6 Hz, 3H), 0.73 (s, 3H). 13C-NMR (151 MHz, DMSO-d 6) δ 172.02, 168.53, 141.29, 139.77, 138.20, 128.92, 124.03, 121.13, 119.96, 116.62, 56.63, 55.78, 50.02, 49.26, 42.32, 40.85, 39.14, 38.06, 36.57, 35.41, 33.74, 31.86, 31.79, 31.76, 28.77, 28.10, 24.33, 23.24, 21.66, 20.97, 19.48, 18.79, 12.16. HRMS-ESI-TOFMS (m / z): [M+H]+ calcd for C 33 H 48 N 2 O 2 , 505.3799; found, 505.3789.

[0208] N-(3-Methylphenyl9-3β-methanesulfonylamino-chol-5-en-24-amide(TYK1044(C37)) Triethylamine (9.65 μL, 0.0692 mmol) was added to TYK1041 (22.0 mg, 0.0463 mmol) and dissolved in anhydrous dichloromethane (1.0 mL). A solution of mesyl chloride (4.65 μL, 0.125 mmol) mixed with anhydrous dichloromethane (0.5 mL) in argon was added dropwise under ice cooling, stirred for 30 minutes, and then stirred at room temperature for 3 hours. After extraction with AcOEt, the organic layer was washed with saturated saline, dried over magnesium sulfate, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane:dichloromethane:AcOEt 1:1:3) to obtain the title compound (11.33 mg, 0.2095 mmol, 45%) as an off-white solid.

[0209] Rf=0.70 (Hexane:Dichloromethane:AcOEt 1:1:3) 1H-NMR (500 MHz, Chloroform-d) δ 7.38 (s, 1H), 7.26 (d, J = 8.0 Hz, 1H), 7.19 (t, J = 7.8 Hz, 1H), 7.12 (s, 1H), 6.91 (d, J = 7.5 Hz, 1H), 5.41 - 5.35 (m, 1H), 4.22 (d, J = 7.4 Hz, 1H), 3.27 - 3.17 (m, 1H), 2.98 (s, 3H), 2.45 - 2.34 (m, 2H), 2.33 (s, 3H), 2.28 - 2.16 (m, 2H), 2.03 - 1.84 (m, 7H), 0.98 (s, 3H), 0.97 (d, J = 6.4 Hz, 3H), 0.68 (s, 3H). 13C-NMR (151 MHz, Chloroform-d) δ 171.83, 139.91, 139.07, 138.02, 128.94, 125.10, 122.69, 120.52, 116.88, 56.77, 55.98, 54.31, 50.14, 42.51, 42.25, 40.79, 39.81, 37.95, 36.52, 35.62, 34.84, 31.91 (2C), 31.76, 30.70, 28.32, 24.37, 21.64, 21.08, 19.41, 18.61, 12.05. HRMS-ESI-TOFMS (m / z): [M+H]+ calcd for C 32 H 48 N 2 O 3 S, 541.3468; found, 541.3458.

[0210] <Example 13> AB ring cis type Aminotron derivative [Synthesis スキーム]

change

[0211] [Synthetic Hands] Methyl lithocholate(Methyl 3α-hydroxy-5β-cholan-24-oate)(TYK1025) Lithocholic acid (2001.5 mg, 5.32 mmol) was dissolved in methanol (45 mL), and sulfuric acid (2.2 mL) was added dropwise under ice cooling and stirred for 20 hours. 180 mL of ice water was added, and the resulting white solid was suction filtered and washed with water:methanol 4:1. Concentration under reduced pressure gave the title compound (2084.4 mg, 5.34 mmol, 100%) as a white solid.

[0212] Rf=0.60 (dichloromethane:AcOEt 10:1) 1H-NMR (500 MHz, Chloroform-d) δ 3.66 (s, 3H), 3.65 - 3.59 (m, 1H), 2.35 (ddd, J = 15.4, 10.2, 5.2 Hz, 1H), 2.22 (ddd, J = 15.7, 9.8, 6.5 Hz, 1H), 1.99 - 1.92 (m, 1H), 1.90 - 1.62 (m, 6H), 1.45 - 1.01 (m, 15H), 0.97 (td, J = 14.2, 3.4 Hz, 1H), 0.92 (s, 3H), 0.91 (d, J = 6.7 Hz, 3H), 0.64 (s, 3H).

[0213] Methyl 3α-hydroxy-5β-cholan-24-oate methanesulfonate(TYK1026) Pyridine (429μL, 5.31 mmol) was added to TYK1025 (1001.5 mg, 2.56 mmol) and dissolved in anhydrous dichloromethane (5 mL) under argon. A solution of mesyl chloride (411 μL, 5.31 mmol) dissolved in anhydrous dichloromethane (1.0 mL) under argon was added dropwise under ice cooling and stirred for 30 minutes under ice cooling. The mixture was returned to room temperature and stirred for 18 hours. The mixture was diluted with about 5 times the amount of dichloromethane and washed with saturated saline. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane:dichloromethane 1:1 to hexane:dichloromethane:AcOEt 5:5:0.8) to obtain the title compound (1058.9 mg, 2.26 mmol, 39%) as a white solid.

[0214] Rf=0.65 (Hexane:Dichloromethane:AcOEt 5:5:0.8) 1H-NMR (500 MHz, Chloroform-d) δ 4.69 - 4.61 (m, 1H), 3.66 (s, 3H), 3.00 (s, 3H), 2.35 (ddd, J = 15.3, 10.2, 5.2 Hz, 1H), 2.22 (ddd, J = 15.7, 9.8, 6.5 Hz, 1H), 2.10 - 2.01 (m, 1H), 1.99 - 1.94 (m, 1H), 1.91 - 1.57 (m, 7H), 1.53 - 1.51 (m, 1H), 1.48 - 0.99 (m, 13H), 0.93 (s, 3H), 0.91 (d, J = 6.4 Hz, 3H), 0.64 (s, 3H).

[0215] Methyl 3β-azido-5β-cholan-24-oate(TYK1030) TYK1026 (566.4 mg, 1.21 mmol) was added with DMPU (4.5 mL) and sodium azide (378.0 mg, 5.81 mmol) and stirred at 60°C for 17 hours. After returning to room temperature, DMPU (2 mL) and sodium azide (161.0 mg, 2.48 mmol) were added and stirred at room temperature for 22 hours. The mixture was diluted with about 5 times the amount of t-butyl methyl ether and washed with water and saturated saline. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane:AcOEt 10:1) to obtain the title compound (402.9 mg, 0.969 mmol, 81%) as a white solid.

[0216] Rf=0.38 (Hexane:AcOEt 10:1) 1H-NMR (500 MHz, Chloroform-d) δ 3.97 - 3.92 (m, 1H), 3.66 (s, 3H), 2.35 (ddd, J = 15.4, 10.3, 5.2 Hz, 1H), 2.21 (ddd, J = 15.7, 9.8, 6.5 Hz, 1H), 2.04 - 1.93 (m, 2H), 1.93 - 1.74 (m, 3H), 1.63 (s, 4H), 1.54 - 1.50 (m, 2H), 1.46 - 0.99 (m, 15H), 0.95 (s, 3H), 0.91 (d, J = 6.5 Hz, 3H), 0.65 (s, 3H).

[0217] 3β-Azido-5β-cholan-24-oic acid(TYK1034) TYK1031 (402.9 mg, 0.969 mmol) was dissolved in THF (4 mL) and methanol (2 mL) was added. 5 M aqueous sodium hydroxide solution (1.0 mL) was added dropwise under ice cooling, and the mixture was stirred under ice cooling for 4 hours. 5 M hydrochloric acid (1.0 mL) was added dropwise under ice cooling, and the mixture was extracted with ethyl acetate at room temperature. The organic layer was washed with water and saturated saline, and dried over magnesium sulfate. The mixture was concentrated under reduced pressure to obtain the title compound (382.2 mg, 0.952 mmol, 98%) as a white solid.

[0218] Rf=0.15 (Hexane:AcOEt 5:1) 1H-NMR (500 MHz, Chloroform-d) δ 3.97 - 3.91 (m, 1H), 2.40 (ddd, J = 15.6, 10.2, 5.2 Hz, 1H), 2.26 (ddd, J = 16.0, 9.8, 6.5 Hz, 1H), 2.03 - 1.94 (m, 2H), 1.93 - 1.76 (m, 2H), 1.66 - 0.99 (m, 20H), 0.95 (s, 3H), 0.93 (d, J = 6.5 Hz, 3H), 0.65 (s, 3H).

[0219] N-(3-Methylphenyl)-3β-azido-5β-cholan-24-amide(TYK1037) TYK1034 (382.2 mg, 0.952 mmol) was dissolved in DMF (1.5 mL), m-methylaniline (102.9 μL, 0.951 mmol), HATU (544.2 mg, 1.43 mmol), and DIPEA (242.3 μL, 1.42 mmol) were added, and the mixture was stirred at room temperature for 18 hours. After extraction with about 5 times the amount of AcOEt, the organic layer was washed with water and saturated saline, dried over magnesium sulfate, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane:dichloromethane:AcOEt 10:2:1) to obtain the title compound (390.9 mg, 0.797 mmol, 87%) as a white solid.

[0220] Rf=0.30 (Hexane:Dichloromethane:AcOEt 10:2:1) 1H-NMR (500 MHz, Chloroform-d) δ 7.38 (s, 1H), 7.26 (d, J = 6.4 Hz, 1H), 7.19 (t, J = 7.9 Hz, 1H), 7.04 (s, 1H), 6.91 (d, J = 7.2 Hz, 1H), 3.97 - 3.92 (m, 1H), 2.40 (ddd, J = 14.9, 10.3, 4.9 Hz, 1H), 2.33 (s, 3H), 2.23 (ddd, J = 14.9, 9.7, 6.2 Hz, 1H), 2.03 - 1.93 (m, 2H), 1.88 (tt, J = 9.3, 4.6 Hz, 3H), 0.98 - 0.93 (m, 6H), 0.66 (s, 3H).

[0221] N-(3-Methylphenyl)-3β-amino-5β-cholan-24-amide(TYK1038) TYK1037 (390.9 mg, 0.797 mmol) in THF (1.0 mL), H 2 The mixture was dissolved in 2H2O (0.12 mL), triphenylphosphine (630.1 mg, 2.40 mmol) was added, and the mixture was stirred at room temperature for 18 hours and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane:dichloromethane:EtOAc 2:1:1) to give the title compound (15.4 mg, 0.0331 mmol, 4.2%) as a white solid.

[0222] Rf=0.20 (Hexane:Dichloromethane:EtOAc 2:1:1) 1H-NMR (500 MHz, CDCl 3 :CD 3 OD 10:1) δ 7.35 (s, 1H), 7.24 (d, J = 7.0 Hz, 1H), 7.16 (t, J = 7.7 Hz, 1H), 6.88 (d, J = 7.4 Hz, 1H), 3.92 (s, 1H), 2.31 (s, 3H), 0.93 (d, J = 5.4 Hz, 6H), 0.63 (s, 3H).

[0223] N-(3-Methylphenyl)-3β-acetylamino-5β-cholan-24-amide(TYK1042) TYK1038 (15.4 mg, 0.0331 mmol) was dissolved in chloroform (0.5 mL) and 0.5 M aqueous sodium bicarbonate (0.5 mL). Acetyl chloride (4.73 μL, 0.0663 mmol) was added dropwise under ice cooling, and the mixture was stirred for 3 hours under ice cooling. Acetyl chloride (7.10 μL, 0.0994 mmol) was added dropwise, and the mixture was stirred at room temperature for 18 hours. Acetyl chloride (9.47 μL, 0.133 mmol) was added dropwise, and the mixture was stirred at room temperature for 3 hours. The mixture was extracted with chloroform and washed with saturated saline. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane:dichloromethane:AcOEt 2:1:3) to obtain the title compound (13.45 mg, 0.02654 mmol, 80%) as an off-white solid.

[0224] Rf=0.55 (Hexane:Dichloromethane:AcOEt 2:1:3) 1H-NMR (500 MHz, Chloroform-d) δ 7.37 (s, 1H), 7.26 - 7.24 (m, 1H), 7.18 (t, J = 7.8 Hz, 1H), 6.90 (d, J = 7.5 Hz, 1H), 3.93 (s, 1H), 2.43 - 2.36 (m, 1H), 2.32 (s, 3H), 2.27 - 2.16 (m, 1H), 2.05 (s, 3H), 0.95 (d, J = 5.8 Hz, 6H), 0.64 (s, 3H).

[0225] Example 14: Activity evaluation of derivatives in mouse STING-expressing cells [RAW-Lucia ISG-mSTING-transfected reporter cells] We used a cell line (RAW-Lucia ISG-mSTING cells) in which mouse STING was stably incorporated into RAW-Lucia ISG-KO-STING cells (Invivogen), which lack endogenous STING and contain a reporter gene to monitor activation of the STING pathway, to examine whether sterol derivatives suppress activation of mouse STING by the agonist DMXAA (vadimezan).

[0226] The cells were cultured in DMEM (high glucose, phenol red, glutamine, pyruvate) containing 10% FBS at 37°C and 5% CO 2 CO 2 The cells were cultured and maintained in an incubator.

[0227] [Seeding RAW-Lucia ISG-mSTING cells onto plates] RAW-Lucia ISG-mSTING cells cultured in a 10 cm dish were washed with PBS(-) (2 mL) after removing the medium, and PBS containing 0.2 mM EDTA was added. The cells were scraped off from the dish with a scraper and suspended by pipetting. The cells were collected by centrifugation at 400 × g for 5 minutes and suspended in medium. The cell density was adjusted to 450,000 cells / mL and plated at 100 μL / well in a tissue culture-treated sterile 96-well plate. The cells were cultured in an incubator for 20-24 hours.

[0228] [Compound processing] Compound treatment of cells was performed by pre-diluting the compound in medium and adding 20 μL of the diluted solution to the well containing the cells. Typically, cells were treated with a 3.16-fold dilution series of compounds from 10 μM to 10 nM final concentration, and DMXAA (vadimezan) was added at 20 μM to activate the STING pathway. The test compound was frozen and stored as a 10 mM DMSO solution, and DMXAA was frozen and stored as a 20 mM DMSO solution. The test was performed under the condition that the medium contained 0.2% DMSO.

[0229] [Measurement of luciferase activity] In this experimental system, activation of the STING pathway results in the secretion of Lucia luciferase, a secreted luciferase, into the medium. 20-24 hours after compound treatment, 10 μL of the medium supernatant was dispensed into a white half-area plate, and 20 μL / well of QUANTI-Luc reagent (Invivogen) was added as a substrate. After 1 minute, luminescence was measured using GloMax Explorer (Promega). Luminescence values ​​were used as an index of STING pathway activation, and a sigmoid curve was fitted to the concentration-dependent data for each compound using the nonlinear least squares method to obtain IC 50 The value was calculated.

[0230] [result] As an example of the concentration-dependence evaluation, the graphs for OG828, ELN025, and H151 are shown in Figure 1. The IC 50 The values ​​are shown in Tables 3 to 6.

[0231] [Table 3]

[0232] [Table 4]

[0233] [Table 5]

[0234] [Table 6]

[0235] Example 15: Evaluation of STING pathway activation state by WB Activation of the STING pathway leads to the activation and autophosphorylation of TBK1, phosphorylation of STING, and phosphorylation of the transcription factor IRF3. In the reporter gene assay, IRF3 activation is detected as luciferase enzyme activity, but in this experiment, the phosphorylation states of STING, TBK1, and IRF3 were evaluated as upstream activation states.

[0236] [Western Blotting (WB)] Mouse embryonic fibroblasts (MEFs) were cultured in DMEM containing 10% FBS at 5% CO 2 The cells were cultured at 37°C and 30°C. The cells were pretreated with sterol derivatives at 30 μM for 16 hours, and then the medium was replaced with sterol derivative-added medium containing mouse STING agonist DMXAA (vadimezan) at 25 μg / mL, and cultured for another hour. Proteins obtained by solubilizing the cells were separated by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinyl difluoride (PVDF) membrane. The following primary antibodies were used: Rabbit anti-TBK1 antibody: Abcam ab40676, diluted 1:1000. Rabbit anti-phosphorylated TBK1 antibody: Cell signaling D52C2, diluted 1:1000. Rabbit anti-phosphorylated STING antibody: Cell signaling D8F4W, diluted 1:1000. Rabbit anti-STING antibody: Proteintech 19851-1-AP, diluted 1:1000. The secondary antibody used was a goat anti-rabbit IgG (H+L) HRP-conjugated antibody (Southern Biotech 4050-05, diluted 1:10000). Bands were visualized by enhanced chemiluminescence (ECL) and detected by Fusion SOLO 7S EDGE (Vilber-Lourmat).

[0237] As shown in Figure 2, 25-hydroxycholesterol (25HC) and sterol derivatives (OGK4005, OGK4006, OGK4007) were confirmed to suppress the phosphorylation of TBK1, STING, and IRF3 by DMXAA. In particular, the degree of phosphorylation of phosphorylated STING (pSTING) confirmed that the sterol derivatives had superior activity to 25HC.

[0238] Example 16: Activity evaluation of derivatives in human STING SAVI mutant expressing cells [HEK293T cells transfected with hSTING SAVI mutant] We verified by reporter gene assay that sterol derivatives suppressed activation of the STING pathway in HEK293T cells expressing constitutively activated human STING due to SAVI mutation.

[0239] HEK293T cells that do not express endogenous STING were stably expressing human STING with the V155M mutation (replacement of valine at position 155 with methionine) as a SAVI mutation (HEK293T-hSTING-V155M) by selecting HEK293T cells with puromycin that had been transfected with pBabe-puro plasmid containing the hSTING-V155M sequence, as reported in Non-Patent Documents 4 and 7. The cells were cultured at 37°C in 5% CO using DMEM (high glucose, phenol red, glutamine, pyruvic acid) medium containing 10% FBS. 2 The cells were maintained by subculturing every 3-4 days.

[0240] [Seeding HEK293T-hSTING-V155M cells onto plates] HEK293T-hSTING-V155M cells cultured in a 10 cm dish were washed with PBS(-) (2 mL) after removing the medium, and detached from the dish by adding 0.5 mL of 0.25% trypsin-EDTA solution and incubating at 37°C for 3 minutes, and then suspended in medium (10 mL). This cell suspension was diluted to 400,000 cells / mL and plated at 100 μL / well in a Greiner clear-bottom white 96-well plate.

[0241] [Transfection] After culturing for 20-24 hours, the reporter plasmid (ISRE-luciferase) and β-galactosidase expression plasmid (pCMX-βGal) for transfection efficiency correction were transfected per plate as follows: ISRE-luciferase (58 μL of 200 ng / μL) and pCMX-βGal (2 μL of 200 ng / μL) were added to PBS (480 μL), and PEI Max (1 mg / mL in water, 60 μL) was added and immediately mixed with a vortex mixer. The plate was incubated at room temperature for 20 minutes, mixed again with a vortex mixer, and then added to the cells in the 96-well plate at 5 μL / well. The plate was then incubated for 20 minutes at room temperature, mixed again with a vortex mixer, and then cooled to 37°C. 2 The cells were cultured in an incubator.

[0242] [Compound processing] Compound treatment was performed 6 hours after transfection. Compounds were diluted in 20 μL of DMEM and added to the cells. As standard conditions, compounds were added in a dilution series of 3.16-fold dilutions starting from a final concentration of 10 μM. Experiments were performed in triplicate for each compound and concentration. Since the compounds were used in 10 mM DMSO solutions, each well was treated to contain 0.1% DMSO. After compound addition, the cells were incubated in a CO 2 The cells were cultured in an incubator for 20-24 hours.

[0243] [Measurement of luciferase and β-galactosidase activity] The medium was removed from the plate, and 50 μL of luciferase substrate solution (1% Triton-X100, 250 μM D-luciferin, 1.5 mM ATP, 0.5 mM coenzyme A, 100 mM 3-mercapto-1,2-propanediol, 30 mM Tricine-NaOH pH 7.8) was added, and luminescence was measured using a GloMax Explorer plate reader (Promega). Subsequently, β-galactosidase substrate solution (1.2 mM o-nitrophenyl-β-D-galactopyranoside, 100 mM 3-mercapto-1,2-propanediol, 100 mM sodium phosphate buffer pH 7.0, 8 mM KCl, 1.7 mM MgCl 2 ) was added at 125 μL / well, and the absorbance at 405 nm was measured over time, and the initial velocity was calculated for each well. The luminescence amount of luciferase was normalized by the initial velocity of β-galactosidase, and relative luminescence unit (RLU) was calculated and used as an index for the activation of the STING pathway. For the concentration-dependent data of each compound, a sigmoid curve was fitted by the non-linear least squares method, and the IC 50 value was calculated.

[0244] [Results] As an example of the evaluation of concentration dependence, the data when using existing STING pathway antagonists (H-151, SN-011) and when using ELN025 (C29) are shown in Figure 3. Also, the IC 50 values calculated from the results when using each compound are shown in Tables 7 to 9.

[0245] [Table 7]

[0246] [Table 8]

[0247] [Table 9]

[0248] <Example 17> Partial agonist-like effect of derivatives in mouse STING-expressing cells [Experimental Method] The same procedures as in Example 16 were used, except that HEK293 cells stably expressing mouse STING were used, and compound treatment was performed with various concentrations of ELN025 alone or in the presence of 25 μM vadimezan.

[0249] [result] Data showing partial activation of the STING pathway by ELN025(C29) alone and reduced levels of STING pathway activity following treatment with ELN025(C29) compared to vadimezan activation are shown in Figure 4.

Claims

1. A compound represented by the following general formula (I) or (II), a pharmacologically acceptable salt thereof, or a prodrug thereof: 【Chemistry 1】 (In the formula, R 1 -OR 4 , and -NHR 5 selected from the group consisting of: 【Chemistry 2】 teeth, 【Chemistry 3】 selected from the group consisting of: R 2 ' is CH 2 and NH; R 2 " is selected from CH; R 3 teeth, 【Chemistry 4】 selected from the group consisting of: R 4 -H, and -C(=O)R 6 selected from the group consisting of: R 5 -H, -C(=O)R 7 , and -S(=O) 2 R 7 selected from the group consisting of: R 6 teeth, 【Chemistry 5】 and W 1 , W 2 , and W 3 are each independently 8 , and N.R. 8 selected from the group consisting of: R 7 -C 1-6 is alkyl; R 8 -H, and -C 1-6 selected from the group consisting of alkyl; X is 【Chemistry 6】 selected from the group consisting of: R 9 , R 12 , and R 14 are each independently -C 1-6 alkyl or absent; R 10 , R 11 , R 13 , and R 15 are each independently -H, -C 1-6 Alkyl, -R 16 C.C.H. 【Chemistry 7】 (wherein R 1 is -OH, 【Chemistry 8】 but, 【Chemistry 9】 and R 2 ' is CH 2 In the case where (i) R 10 and R 11 All of the above are -CH 3 (ii) R 10 and R 11 All of the above are -CH 2 CH 3 and (iii) R 10 and R 11 One of them is -H and the other is -CH 3 (It is not possible); Y 1 , Y 2 , and Y 3 are each independently 22 , and N.R. 22 selected from the group consisting of: Z 1 , Z 2 , and Z 3 are each independently selected from the group consisting of CH and N; R 16 -C 1-6 alkyl or absent; R 17 , and R 18 are each independently -NH 2 , -COOH, -C(=O)NH 2 , -NHC(=O)CH 3 , -NHCOOC(CH 3 ) 3 , -NHS(=O) 2 CH 3 , -OH, -C 1-3 Alkyl, -CCH, -CCSi(CH 3 ) 3 , -NHC(=O)CH 2 CH 2 CCH, -NHC(=O)CH 2 CH 2 CH 2 CH 3 , -N 3 , -F, -Cl, -Br, -I, -CN, and -CF 3 or absent; Z 4 , NH, NCH 3 , O, and S; R 19 -F, -Cl, -Br, -I, -CH 3 , -COOR 23 , and -C(=O)NR 24 R 25 or absent; R 20 , and R 21 are each independently -H, -NH 2 , -NHC(=O)CH 3 , -NHCOOC(CH 3 ) 3 , -NHC(=O)CH 2 CH 2 CH 2 CH 3 , and -NHC(=O)CH 2 CH 2 CCH or absent; R 22 , R 23 , R 24 , and R 25 are each independently -H, and -C 1-6 is selected from the group consisting of alkyl.

2. R 3 but, 【Chemistry 10】 The compound according to claim 1, or a pharmacologically acceptable salt thereof, or a prodrug thereof, selected from the group consisting of:

3. 3. A compound according to claim 1 or 2, a pharmacologically acceptable salt thereof, or a prodrug thereof, comprising: W 1 , and W 2 But, CH 2 Selected from: W 3 But NR 8 Selected from: R 7 , R 8 , R 9 , and R 12 However, each independently, -C 1-3 alkyl; X, 【Chemistry 11】 selected from the group consisting of: R 10 , R 11 , R 13 , and R 15 are independently -H, -C 1-3 Alkyl, -R 16 C.C.H. 【Chemistry 12】 (wherein R 1 is -OH, 【Chemistry 13】 but, 【Chemistry 14】 and R 2 ' is CH 2 In the case where R 10 and R 11 All of the above are -CH 3 (It is not possible); R 14 But -C 1-5 alkyl; Y 1 , Y 2 , and Y 3 But CHR 22 Selected from: R 16 But -C 1-3 alkyl or absent; R 17 , and R 18 are each independently -NH 2 , -NHC(=O)CH 3 , -NHCOOC(CH 3 ) 3 , -CH 3 , -CH 2 CH 3 , -CCH, -CCSi(CH 3 ) 3 , -NHC(=O)CH 2 CH 2 CCH, -NHC(=O)CH 2 CH 2 CH 2 CH 3 , -N 3 , -CN, and -CF 3 or absent; R 20 , and R 21 are each independently -H, -NH 2 , -NHC(=O)CH 3 , -NHCOOC(CH 3 ) 3 , -NHC(=O)CH 2 CH 2 CH 2 CH 3 , and -NHC(=O)CH 2 CH 2 CCH or absent; R 22 But -H, and -C 1-3 A compound, a pharmacologically acceptable salt thereof, or a prodrug thereof, selected from the group consisting of alkyl.

4. 4. A compound according to claim 3, a pharmacologically acceptable salt thereof, or a prodrug thereof, comprising: R 8 But -CH 3 Selected from: 【Chemistry 15】 but, 【Chemistry 16】 selected from the group consisting of: 【Chemistry 17】 but, 【Chemistry 18】 selected from the group consisting of: 【Chemistry 19】 but, 【Chemistry 20】 selected from the group consisting of: 【Chemistry 21】 but, 【Chemical 22】 Selected from: R 22 is selected from -H, or a pharmacologically acceptable salt thereof, or a prodrug thereof.

5. 4. A compound according to claim 3, a pharmacologically acceptable salt thereof, or a prodrug thereof, comprising: X, 【Chemistry 23】 【change】 A compound selected from the group consisting of:

6. Any of the following compounds, a pharmacologically acceptable salt thereof, or a prodrug thereof: 【Chemistry 24】 【change】 【change】

7. A STING pathway regulator or a STING-related disease treatment agent, comprising a compound represented by the following general formula (I) or (II), a pharmacologically acceptable salt thereof, or a prodrug thereof: 【Chemistry 25】 (In the formula, R 1 -OR 4 , and -NHR 5 selected from the group consisting of: 【Chemistry 26】 teeth, 【Chemical 27】 selected from the group consisting of: R 2 ' is CH 2 and NH; R 2 " is CH; R 3 teeth, 【Chemistry 28】 selected from the group consisting of: R 4 -H, and -C(=O)R 6 selected from the group consisting of: R 5 -H, -C(=O)R 7 , and -S(=O) 2 R 7 selected from the group consisting of: R 6 teeth, 【Chemical 29】 and W 1 , W 2 , and W 3 are each independently 8 , and N.R. 8 selected from the group consisting of: R 7 -C 1-6 is alkyl; R 8 -H, and -C 1-6 selected from the group consisting of alkyl; X is 【Chemistry 30】 selected from the group consisting of: R 9 , R 12 , and R 14 are each independently -C 1-6 alkyl or absent; R 10 , R 11 , R 13 , and R 15 are each independently -H, -C 1-6 Alkyl, -R 16 C.C.H. 【Chemistry 31】 selected from the group consisting of: Y 1 , Y 2 , and Y 3 are each independently 22 , and N.R. 22 selected from the group consisting of: Z 1 , Z 2 , and Z 3 are each independently selected from the group consisting of CH and N; R 16 -C 1-6 alkyl or absent; R 17 , and R 18 are each independently -NH 2 , -COOH, -C(=O)NH 2 , -NHC(=O)CH 3 , -NHCOOC(CH 3 ) 3 , -NHS(=O) 2 CH 3 , -OH, -C 1-3 Alkyl, -CCH, -CCSi(CH 3 ) 3 , -NHC(=O)CH 2 CH 2 CCH, -NHC(=O)CH 2 CH 2 CH 2 CH 3 , -N 3 , -F, -Cl, -Br, -I, -CN, and -CF 3 or absent; Z 4 , NH, NCH 3 , O, and S; R 19 -F, -Cl, -Br, -I, -CH 3 , -COOR 23 , and -C(=O)NR 24 R 25 or absent; R 20 , and R 21 are each independently -H, -NH 2 , -NHC(=O)CH 3 , -NHCOOC(CH 3 ) 3 , -NHC(=O)CH 2 CH 2 CH 2 CH 3 , and -NHC(=O)CH 2 CH 2 CCH or absent; R 22 , R 23 , R 24 , and R 25 are each independently -H, and -C 1-6 is selected from the group consisting of alkyl.

8. R 3 but, 【Chemistry 32】 The agent according to claim 7, which is selected from the group consisting of

9. 9. The agent according to claim 7 or 8, comprising: W 1 , and W 2 But, CH 2 Selected from: W 3 But NR 8 Selected from: R 7 , R 8 , R 9 , and R 12 However, each independently, -C 1-3 alkyl; X, 【Chemical 33】 selected from the group consisting of: R 10 , R 11 , R 13 , and R 15 are independently -H, -C 1-3 Alkyl, -R 16 C.C.H. 【Chemical 34】 selected from the group consisting of: R 14 But -C 1-5 alkyl; R 16 But -C 1-3 alkyl or absent; R 17 , and R 18 are each independently -NH 2 , -NHC(=O)CH 3 , -NHCOOC(CH 3 ) 3 , -CH 3 , -CH 2 CH 3 , -CCH, -CCSi(CH 3 ) 3 , -NHC(=O)CH 2 CH 2 CCH, -NHC(=O)CH 2 CH 2 CH 2 CH 3 , -N 3 , -CN, and -CF 3 or absent; R 20 , and R 21 are each independently -H, -NH 2 , -NHC(=O)CH 3 , -NHCOOC(CH 3 ) 3 , -NHC(=O)CH 2 CH 2 CH 2 CH 3 , and -NHC(=O)CH 2 CH 2 CCH or absent; R 22 But -H, and -C 1-3 9. The agent according to claim 7 or 8, selected from the group consisting of alkyl.

10. 10. The agent according to claim 9, comprising: R 8 But -CH 3 Selected from: R 22 is selected from -H; 【Chemistry 35】 but, 【Chemical 36】 selected from the group consisting of: 【Chemical 37】 but, 【Chemical 38】 selected from the group consisting of: 【Chemical Formula 39】 but, 【Chemistry 40】 selected from the group consisting of: 【Chemistry 41】 but, 【Chemistry 42】 An agent selected from:

11. 10. The agent according to claim 9, comprising: X, 【Chemistry 43】 【change】 An agent selected from the group consisting of:

12. A STING pathway regulator or a STING-related disease treatment agent, comprising any one of the following compounds, a pharmacologically acceptable salt thereof, or a prodrug thereof: 【Chemistry 44】 【change】 【change】

13. The agent according to any one of claims 7, 8 or 12, wherein the modulation of the STING pathway or the treatment of a STING-associated disease is by partial activation of the STING pathway.

14. The agent according to any one of claims 7, 8 and 12, wherein the STING-associated disease is STING-associated vasculitis, COPA (Coatomer protein alpha) syndrome, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, Aicardi-Goutieres syndrome, systemic lupus erythematosus, Parkinson's disease, or Huntington's disease.

Images