WO2015034010A1 - Intranuclear receptor liver x receptor agonist - Google Patents

Intranuclear receptor liver x receptor agonist Download PDF

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WO2015034010A1
WO2015034010A1 PCT/JP2014/073365 JP2014073365W WO2015034010A1 WO 2015034010 A1 WO2015034010 A1 WO 2015034010A1 JP 2014073365 W JP2014073365 W JP 2014073365W WO 2015034010 A1 WO2015034010 A1 WO 2015034010A1
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lxr
liver
receptor
agonist
lipid metabolism
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PCT/JP2014/073365
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French (fr)
Japanese (ja)
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瑞穂 宇根
裕介 井口
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学校法人常翔学園
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a novel agonist of a liver X receptor (Liver X Receptor: hereinafter also simply referred to as “LXR”), which is a nuclear receptor.
  • Liver X Receptor hereinafter also simply referred to as “LXR”
  • the nuclear receptor is a kind of intracellular protein, and is a kind of transcription factor that regulates DNA transcription in the cell nucleus by binding a fat-soluble compound in the living body such as a hormone as a ligand. It is involved in gene transcription related to life support such as development, homeostasis and metabolism (Zhang et al., Genome Res., 14 (4), 580-90 (2004)).
  • liver X receptor is a transcription that functions as an oxidized cholesterol (oxysterol) receptor and functions to process cholesterol accumulated in the body.
  • oxysterol oxidized cholesterol
  • LXR is activated with oxysterol as a ligand and plays an important role in maintaining lipid homeostasis (Non-Patent Document 1: Janowski (1996)).
  • LXR is highly expressed in energy-metabolizing organs such as liver and adipose tissue, such as enzymes involved in bile acid biosynthesis, which is also a catabolism and excretion pathway of cholesterol, and SREBP-1c that plays an important role in fatty acid biosynthesis Regulates gene expression.
  • LXR has also been reported to promote the conversion of sugars to fatty acids to lower blood sugar levels, and has also attracted attention for its effects on carbohydrate and insulin metabolism. Furthermore, LXR is also expressed in vascular constituent cells, particularly macrophages, and its influence on lipid metabolism at the site of arteriosclerotic lesions has attracted attention. LXR transports excess cholesterol to HDL (high density lipoprotein) and induces expression of ABCA1, which is responsible for cholesterol excretion. The small intestinal mucosa suppresses cholesterol absorption and promotes excretion. Therefore, compounds that activate LXR can be candidates for effective therapeutic agents for atherosclerosis and cardiovascular diseases.
  • Non-patent Document 2 Song (2000)
  • Non-Patent Document 3 Song (2000)
  • Patent Document 1 JP-A-2006-528200
  • Patent Document 2 JP-A-2008-179562
  • Patent Document 3 JP-A-2009-227615
  • a bile acid analog has not been reported so far, which covers oxysterol, which is an endogenous LXR agonist.
  • Non-patent document 4 Viennois® (2012)
  • Non-patent document 5 Schults® (2000)
  • the preparation process (synthesis, purification, and isolation) of these compounds is complicated, and a lot of time and labor are consumed at present.
  • non-patent document 5 reports that the administration of these synthetic LXR agonists to the living body causes an undesirable effect of increasing the triglyceride (TG) content in blood and liver. It has not reached.
  • TG triglyceride
  • Non-Patent Document 6 Zaepter ( 1972)
  • LXR agonist for the compound.
  • LDL cholesterol LDL cholesterol
  • HDL-C HDL cholesterol
  • neutral fat lipid components
  • LDL cholesterol LDL cholesterol
  • CETP inhibitors are listed as drugs that only increase HDL cholesterol levels, but some CETP inhibitors have been discontinued due to clinical trials confirming that blood pressure is also increased. Since HDL has antioxidation, anticoagulation, antiarrhythmic action, etc., and plays an important role in maintaining the cardiovascular system, a method of focusing on HDL has attracted attention. However, there is currently no drug that specifically acts on HDL, and development of a drug that specifically acts on HDL in vivo and has low side effects is desired.
  • An object of the present invention is to provide a novel LXR agonist having high specificity and high safety for LXR (LXR ⁇ and LXR ⁇ ).
  • a liver X receptor (LXR) agonist comprising a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof: (In the formula, R 1 and R 2 each independently represents a hydrocarbon group having 1 to 5 carbon atoms.) 2.
  • R 1 and R 2 each independently represents a compound representing an alkyl group having 1 to 3 carbon atoms, or a pharmaceutically acceptable salt thereof.
  • X receptor (LXR) agonist 3.
  • R 1 and R 2 are both a compound having a methyl group, an ethyl group or a propyl group, or a pharmaceutically acceptable salt thereof.
  • a lipid metabolism improving agent comprising the liver X receptor (LXR) agonist according to any one of 1 to 4 above. 6). 6.
  • a therapeutic or prophylactic agent for lipid metabolism abnormality or a disease associated with lipid metabolism abnormality comprising the lipid metabolism improving agent according to 5 or 6 as an active ingredient. 8). 5.
  • a pharmaceutical composition comprising the liver X receptor (LXR) agonist according to any one of items 1 to 4 as an active ingredient.
  • LXR liver X receptor
  • R 1 and R 2 are both a methyl group, an ethyl group, or a propyl group.
  • R 1 and R 2 are both a methyl group, an ethyl group, or a propyl group. 12
  • a lipid metabolism abnormality or lipid in a subject by administering to the subject the lipid metabolism improving agent according to 5 or 6 above, or the compound according to any one of 9 to 11 or a pharmaceutically acceptable salt thereof.
  • a method of activating liver X receptor (LXR) by contacting a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof with liver X receptor (LXR).
  • R 1 and R 2 are each independently a hydrocarbon group having 1 to 5 carbon atoms.
  • the present invention includes the following.
  • a food comprising the lipid metabolism improving agent according to item 7 as an active ingredient.
  • B A method of increasing the blood HDL cholesterol level of a subject by administering the lipid metabolism improving agent according to item 7 to the subject.
  • C A method for improving lipid metabolism in a subject by administering the lipid metabolism improving agent according to item 7 to the subject.
  • D A method of activating the liver X receptor (LXR) by administering a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof to a subject.
  • E A method of treating or preventing a disease associated with liver X receptor (LXR) by administering a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof to a subject.
  • the LXR agonist of the present invention for example, a compound represented by the formula (I) (24-Nor-5 ⁇ -cholestane-3 ⁇ , 6 ⁇ , 25-triol (24-Nor-5 ⁇ -cholestane-3 ⁇ , 6 ⁇ , 25-triol), hereinafter “ Abbreviation “24-Nor”) has excellent agonist activity among nucleic acid receptors, particularly LXR ⁇ and / or LXR ⁇ .
  • the LXR agonist compound of the present invention promoted the expression of ABCA1 and ABCG1 genes involved in lipid metabolism in vitro and in vivo, and the expression of SREBP1c involved in triglyceride synthesis was lower than that of existing drugs. .
  • the LXR agonist of the present invention has an excellent LXR agonist activity even compared to existing bile acid-derived components and derivatives thereof, and has an excellent function as a lipid metabolism improving agent with reduced side effects such as triglyceride synthesis. .
  • LXR ⁇ and LXR ⁇ nuclear receptors with bile acids synthesized from cholesterol as ligands (farnesoid X receptor: FXR, G protein-coupled receptors activated by bile acids: TGR5), vitamin D derivatives as ligands
  • FXR farnesoid X receptor
  • TGR5 G protein-coupled receptors activated by bile acids
  • vitamin D derivatives as ligands
  • Example 3 It is a figure which shows the result of having confirmed the influence which it has on expression of each gene of ABCG1 and SREBP1c in connection with lipid metabolism using 24-Hor-7 cells. (Example 3) It is a figure which shows the result of having confirmed the cholesterol excretion ability of the peripheral cell about 24-Nor.
  • Example 4 It is a figure which shows the result of having confirmed the influence with respect to a mouse
  • FIG. 4 is a view showing the results of confirming the influence on the amount of cholesterol in bile and the amount of neutral fat in feces when 24-Nor is administered in vivo.
  • FIG. 4 is a view showing the results of confirming the effect on the expression of liver ABCA1 and ABCG1 genes when 24-Nor is administered in vivo.
  • FIG. 4 is a view showing the results of confirming the effect of in vivo administration of 24-Nor on the expression of the small intestine NPC1L1 gene.
  • FIG. 6 is a view showing the results of confirming the effect of 24-Nor administered in vivo on the expression of SREBP1c, FAS, and SCD-1 genes in the liver.
  • Example 8) It is a figure which shows the result which confirmed the synthesis
  • Example 9 It is a figure which shows the result of having confirmed LXR (alpha) and LXR (beta) agonist activity of the various LXR agonist of this invention.
  • Example 10) It is a figure which shows the result of having confirmed LXR (alpha) and LXR (beta) agonist activity of the various LXR agonist of this invention.
  • Example 11 It is a figure which shows the result of having confirmed the agonist activity with respect to various nuclear receptors of the various LXR agonist of this invention.
  • Example 11 It is a figure which shows the result of having confirmed the agonist activity with respect to TGR5 of various LXR agonists in this invention.
  • Example 11 It is a figure which shows the synthetic scheme of the LXR agonist of this invention.
  • Example 12 It is a figure which shows the result of having confirmed LXR (alpha) and LXR (beta) agonist activity of the various LXR agonist of this invention.
  • Example 13 Example 13
  • the LXR agonist of the present invention comprises a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof.
  • R 1 and R 2 each independently represents a hydrocarbon group having 1 to 5 carbon atoms.
  • the hydrocarbon group in the general formula (I) of the present invention may be linear or branched and includes an alkyl group, an alkenyl group, and an alkynyl group.
  • the hydrocarbon group in the general formula (I) is preferably an alkyl group, and the alkyl group may be linear or branched, but is preferably linear. Further, the hydrocarbon group in the general formula (I) has 1 to 5 carbon atoms, preferably 1 to 3 carbon atoms.
  • R 1 and R 2 in the general formula (I) are selected from a methyl group, an ethyl group, and a propyl group (n-propyl group, iso-propyl group), and R 1 and R 2 are both the same group Particularly preferred.
  • R 1 and R 2 in Table 1 is R 1 and R 2 in the general formula (I).
  • each compound described in Table 2 may be represented by the name (24-Nor or the like) described in Table 2.
  • the compound described in the general formula (I) can be prepared with reference to the synthesis procedure shown in Non-Patent Document 6.
  • the production method of 24-Nor-Et, 24-Nor-nPr, 24-Nor-nBt, 24-Nor-nPe, 24-Nor-iPr is shown in Example 9 below, and 24-Nor-Me, Et The manufacturing method is described in Example 12 below.
  • Any compounds included in the scope of the present invention can be produced by appropriately modifying or altering the starting materials and reagents used in the methods shown in the Examples and the reaction conditions.
  • the compound 24-Nor represented by the formula (I) is 24-Nor-5 ⁇ -cholestane-3 ⁇ , 6 ⁇ , 25-triol, which is a known compound in Non-Patent Document 6. is there.
  • the compound can be synthesized according to the synthesis procedure shown in Non-Patent Document 6.
  • the LXR agonist of the present invention is selected from the compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof.
  • the present invention includes those isomers, and also includes solvates, hydrates and It includes crystals of various shapes.
  • the pharmaceutically acceptable salt includes general pharmacologically and pharmaceutically acceptable salts.
  • specific examples of such salts are as follows.
  • Examples of basic addition salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; ammonium salts; trimethylamine salts and triethylamine salts; dicyclohexylamine salts and ethanolamines.
  • Aliphatic amine salts such as salts, diethanolamine salts, triethanolamine salts and brocaine salts; aralkylamine salts such as N, N-dibenzylethylenediamine; and heterocyclic aromatics such as pyridine salts, picoline salts, quinoline salts and isoquinoline salts
  • tetramethylammonium salt tetraethylammonium salt, benzyltrimethylammonium salt, benzyltriethylammonium salt, benzyltributylammonium salt, methyltrioctylammonium salt, Quaternary ammonium salts such as tiger butyl ammonium salt; arginine; basic amino acid salts such as lysine salts.
  • the acid addition salt examples include inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate, carbonate, hydrogen carbonate, perchlorate; for example, acetate, propionate, lactate, maleate , Organic acid salts such as fumarate, tartrate, malate, citrate and ascorbate; sulfonic acids such as methanesulfonate, isethionate, benzenesulfonate and p-toluenesulfonate Salts; for example, acidic amino acids such as aspartate and glutamate.
  • inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate, carbonate, hydrogen carbonate, perchlorate
  • Organic acid salts such as fumarate, tartrate, malate, citrate and ascorbate
  • sulfonic acids such as methanesulfonate, isethionate, benzenesulfonate
  • the LXR agonist of the present invention is an existing oxysterol compound (22 (R) -hydroxycholesterol: 22 (R) -HC), 24-nor-5 ⁇ -cholestane-3 ⁇ , 7 ⁇ , 25 shown in the formula (II) of FIG.
  • the LXR agonist of the present invention has agonist activity specifically for LXR ⁇ and LXR ⁇ , but other nuclear receptors, for example, nuclear receptors (farnesoid X receptor: FXR) having a bile acid synthesized from cholesterol as a ligand. , G protein receptor: TGR5), and shows little agonist activity for nuclear receptor (vitamin D receptor: VDR) with vitamin D derivatives as ligands. 22 (R) -HC has agonist activity specifically for LXR ⁇ and LXR ⁇ , but also has slight agonist activity for other nuclear receptors such as FXR.
  • nuclear receptors farnesoid X receptor: FXR
  • TGR5 nuclear receptors
  • VDR vitamin D receptor
  • 22 (R) -HC has agonist activity specifically for LXR ⁇ and LXR ⁇ , but also has slight agonist activity for other nuclear receptors such as FXR.
  • chenodeoxycholic acid (CDCA), lithocholic acid (LCA: lithocholic acid: 7,12 ⁇ -dehydrated oxidized derivative of cholic acid) and 1,25-hydroxyvitamin D 3 (1,25-hydroxyvitamin D 3 ) are LXR ⁇ and LXR ⁇ . Does not show agonist activity and has agonist activity for each nuclear receptor such as FXR, TGR5 and VDR, and the activities are completely different.
  • LXR is a ligand-dependent nuclear receptor, and LXR ⁇ is highly expressed in the liver and macrophages, but LXR ⁇ is ubiquitously expressed throughout the body. These bind to a DNA sequence of about 10 bases called LXR response region (hereinafter referred to as LXRE) and activate transcription of the target gene in an oxysterol-dependent manner.
  • LXRE LXR response region
  • ABC ATP-Binding Cassette
  • SREBP1c sterol regulatory element binding protein 1c
  • ABC proteins are a family of membrane proteins having two ATP-binding domains per functional molecule, whose sequences are well conserved over 200 amino acids, and play important physiological functions in vivo.
  • ABCA1 a kind of ABC protein, is essential for the formation of high density lipoprotein (HDL).
  • HDL extracts cholesterol, which triggers arteriosclerosis, from peripheral cells and transports it to the liver. In the liver, cholesterol is converted into bile acids, which are excreted through the bile. This transport of cholesterol from peripheral cells to the liver by HDL is called a “reverse cholesterol transfer system”.
  • ABCA1 in the liver delivers cholesterol and phospholipids to apolipoprotein A-I (ApoA-I) to generate HDL particles.
  • Newborn HDL is carried to peripheral tissues by circulating blood, and cholesterol in peripheral tissues is received via ABCA1 existing in the peripheral tissues and ABCG1 which is one of ABC proteins, and mature HDL is formed.
  • ABCA1 mutations have been epidemiologically found to be an important risk factor for arteriosclerosis, and enhanced ABCA1 expression and activity are important targets for improving lipid homeostasis.
  • ⁇ Cholesterol carried to the liver is converted into bile acids and discharged through the bile.
  • cholesterol content in bile increases too much, cholesterol will crystallize and cause gallstones, so gallstones are a concern as a side effect when enhancing the reverse cholesterol transport system.
  • NPC1L1 Niemann-Pick C1Like1
  • NPC1L1 Niemann-Pick C1Like1
  • fatty acid synthesis is promoted by acetyl-CoA carboxylase, fatty acid synthase (hereinafter FAS), and SCD-1. Their expression is controlled by SREBP (sterol regulatory element binding protein).
  • SREBP sterol regulatory element binding protein
  • SREBP1a is said to control fatty acid and triglyceride synthesis, SREBP2 to synthesize cholesterol, and SREBP1a to regulate both fatty acid and cholesterol synthesis.
  • the LXR agonist of the present invention increases the expression of ABCA1 or ABCG1 in liver and peripheral tissues in vitro.
  • the existing LXR agonist TO901317 N- [4- (1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl) phenyl] -N- (2,2,2- Although slightly inferior to (trifluoroethyl) benzenesulfonamide), it has the ability to excrete cholesterol from peripheral tissues (increase in the amount of cholesterol delivered to ApoA-I).
  • the LXR agonist of the present invention increases the amount of HDL cholesterol (HDL-C) in blood in vivo.
  • LXR agonists Similar to ABCA1 and ABCG1, known synthetic LXR agonists, such as TO901317, increase triglycerides in blood through increased expression of genes related to neutral fat synthesis, such as SREBP1c, which is a LXR-controlled gene, and fatty acid synthase (FAS). It is known to increase levels.
  • SREBP1c which is a LXR-controlled gene
  • FAS fatty acid synthase
  • the LXR agonist of the present invention does not have a high effect on triglyceride synthesis and can be said to be a safer drug. Further, the LXR agonist of the present invention can suppress the NPC1L1 mRNA expression level in the small intestine and reduce cholesterol absorption without increasing the cholesterol content in bile.
  • the LXR agonist of the present invention has agonist activity specifically for LXR, more specifically LXR ⁇ and LXR ⁇ .
  • the LXR agonist of the present invention can be used as an active ingredient of a therapeutic or prophylactic agent for diseases related to LXR (lipid metabolism disorders, atherosclerosis, cardiovascular disorders, etc.).
  • the LXR agonist of the present invention can be used as a lipid metabolism improving agent.
  • the present invention also extends to a therapeutic or prophylactic agent for any disease, such as dyslipidemia, atherosclerosis and cardiovascular disorder, comprising the lipid metabolism improving agent as an active ingredient.
  • a pharmaceutical composition comprising a lipid metabolism improving agent comprising the LXR agonist of the present invention as an active ingredient is also included in the scope of the present invention.
  • the pharmaceutical composition can be used as a therapeutic or ameliorating agent for various disorders associated with abnormal lipid metabolism and abnormal lipid metabolism.
  • dyslipidemia include dyslipidemia such as hypercholesterolemia, high LDL cholesterolemia, low HDL cholesterolemia, and hypertriglyceridemia.
  • various diseases associated with abnormal lipid metabolism are not particularly limited, and examples thereof include atherosclerosis and cardiovascular disorders.
  • the present invention extends to the above-described lipid metabolism abnormality and methods for treating and / or preventing various diseases associated with lipid metabolism abnormality.
  • the present invention is a pharmaceutical composition
  • a pharmaceutical composition comprising the LXR agonist of the present invention as an active ingredient, such as tablets, capsules, powders, granules, pills, liquids, syrups, or the like, injections, external preparations, suppositories Depending on the form of a parenteral preparation such as eye drops, it can be administered orally or parenterally. As an administration route, oral administration is more preferable.
  • the LXR agonist of the present invention may be administered as it is, but it is preferably administered as an oral or parenteral pharmaceutical composition.
  • the pharmaceutical composition of the present invention is manufactured according to a conventional method, and can be manufactured using pharmaceutical additives that are available to those skilled in the art, that is, pharmacologically and pharmaceutically acceptable carriers.
  • Examples of the pharmaceutical composition suitable for oral administration include tablets, capsules, powders, fine granules, granules, liquids, and syrups.
  • Examples of the pharmaceutical composition suitable for parenteral administration include Injections, drops, suppositories, inhalants and the like.
  • Examples of pharmacologically and pharmaceutically acceptable carriers used in the production of the above pharmaceutical composition include, for example, excipients, disintegrating agents or disintegrating aids, binders, lubricants, coating agents, dyes, Diluents, bases, solubilizers or solubilizers, isotonic agents, pH adjusters, stabilizers, propellants, adhesives and the like can be mentioned.
  • the dosage of the pharmaceutical composition of the present invention can be appropriately determined according to the degree of symptoms, administration route, administration subject, age of the user, body weight, and the like.
  • the pharmaceutical composition of the present invention may be administered several times, divided into multiple courses, and the number of administrations per course, the administration interval, etc. can be arbitrarily set.
  • an LXR agonist as an active ingredient can be used in the range of about 0.01 to 1000 mg per adult day.
  • the LXR agonist of the present invention may be used as an additive for foods and the like in addition to the pharmaceutical composition.
  • a food containing an effective amount of the LXR agonist of the present invention can be used for health foods such as foods for specified health use, and is expected to prevent and improve lipid metabolism abnormalities and associated diseases when taken daily. can do.
  • the dose can be appropriately determined according to the degree of symptoms, administration route, administration subject, age of the user, body weight, and the like.
  • Example 1 Evaluation of LXR agonist activity of 24-Nor
  • the LXR agonist activity of 24-Nor represented by the following formula (I) was confirmed.
  • 24-Nor was synthesized and obtained by the method of Example 9 described later.
  • LXR agonist activity was confirmed using LXR ⁇ or LXR ⁇ -expressing COS-7 cells (African green monkey kidney-derived cells) transduced with LXR ⁇ or LXR ⁇ expression plasmid and a luciferase-expressing plasmid (pLXREx4-tk-Luc) as a reporter protein .
  • Example 2 Evaluation of agonist activity for various nuclear receptors of 24-Nor
  • nuclear receptors using bile acids as endogenous ligands farnesoid X receptor: FXR, G protein
  • Receptor TGR5
  • 24-Nor agonist activity against nuclear receptor vitamin D receptor: VDR
  • Agonist activity against LXR ⁇ and LXR ⁇ was confirmed by measuring luciferase activity by the same method as described in Example 2.
  • Agonist activity against FXR and TGR5 was measured according to the method described in the literature (Iguchi Y. et al., J. Lipid Res., 51, 1432-1441, 2010).
  • Agonist activity against VDR is expressed in human VDR and human RXR ⁇ expression plasmid, VDR-responsive luciferase expression plasmid (pCYP3A4-ER6x3-tk-Luc), and HEK293T cells transduced with Renilla luciferase plasmid as a control plasmid for transduction (human embryonic kidney) Cell).
  • the results are shown in FIG.
  • the 24-Nor of the present invention was confirmed to have agonist activity specifically for LXR ⁇ and LXR ⁇ , and only showed the same effect as the negative control for other nuclear receptors. From this result, it was confirmed that 24-Nor of the present invention acts specifically for LXR ⁇ and LXR ⁇ .
  • LXR ⁇ and ⁇ Effect of factors related to lipid metabolism on gene expression
  • LXR ⁇ and ⁇ are ligand-dependent nuclear receptors, and LXR ⁇ is particularly expressed in the liver, macrophages and the like. These bind to a DNA sequence of about 10 bases called LXR response region (hereinafter referred to as LXRE) and activate transcription of the target gene in an oxysterol-dependent manner.
  • LXRE LXR response region
  • THP-1 cells human and monocyte-derived cells
  • Huh-7 cells human and hepatocellular carcinoma-derived cells
  • 24-Nor, 22 (R) -HC or TO901317 for 24 hours, and the total RNA of each cell Extracted.
  • the mRNA amounts of ABCA1, ABCG1 and SREBP1c were measured by RT-PCR method.
  • the negative control was treated with DMSO alone.
  • 24-Nor of the present invention increased ABCA1 and ABCG1 mRNAs at the same level as or higher than those treated with 22 (R) -HC (FIG. 4).
  • 24-Nor increased ABCG1 mRNA expression level as confirmed in THP-1 cells, but did not change SREBP1c mRNA expression level (Fig. 5). Since SREBP1c mRNA expression level was increased by treatment with TO901317, it was suggested that 24-Nor (I) did not cause the increase in triglyceride observed with TO901317 administration, but only showed an HDL raising effect. In this respect, it was confirmed that the LXR agonist of the present invention, ie, 24-Nor is superior to TO901317.
  • Cholesterol transport / excretion ability Cholesterol transport from peripheral cells to the liver begins with the delivery of cholesterol to ApoA-I via ABCA1 and ABCG1. ApoA-I that receives cholesterol from peripheral cells becomes HDL and transports cholesterol to the liver.
  • the ability to excrete cholesterol from peripheral cells was confirmed for 24-Nor.
  • the mechanism of cholesterol excretion independent of ApoA-I has also been confirmed, so the cholesterol excretion ability is the system containing 10 ⁇ g / mL ApoA-I (+ ApoA-I) and the system not containing (-ApoA-I)
  • THP-1 cells human monocyte-derived cells
  • the cholesterol level was measured using a commercially available kit, Amplex (R) Red Cholesterol Assay Kit (Invitrogen).
  • DMSO was used as a negative control
  • TO901317 an existing LXR agonist
  • mice C57BL / 6N mice (male, 8 weeks old) were administered with physiological saline (negative control: NC), TO901317 10 mg / kg and 50 mg / kg, 24-Nor 10 mg / kg. The group was divided into 5 groups and a 50 mg / kg administration group, each of which was orally administered (as a physiological saline suspension) once a day for 3 days. After fasting overnight, mice were euthanized by cervical dislocation, body weight was measured, liver was collected and liver weight (wet weight) was measured.
  • physiological saline negative control: NC
  • TO901317 10 mg / kg and 50 mg / kg 24Nor 10 mg / kg.
  • the group was divided into 5 groups and a 50 mg / kg administration group, each of which was orally administered (as a physiological saline suspension) once a day for 3 days. After fasting overnight, mice were euthanized by cervical dislocation, body weight was measured, liver was collected and liver weight (wet weight) was
  • liver enlargement was observed because liver weight was significantly increased by TO901317 treatment, but it was not observed in 24-Nor. Since 24-Nor exhibits an LXR-specific agonistic action, it is considered that hepatic hypertrophy was not observed.
  • Example 6 Confirmation of influence on cholesterol level by in vivo administration of 24-Nor
  • 24-Nor was administered to mice, and feces were collected every day. Mice were euthanized in the same manner as in Example 5, and blood, bile, and liver were collected. The total cholesterol levels in serum, bile, liver and feces were measured using a commercially available kit Amplex® Red Cholesterol Assay Kit (invitrogen). Serum HDL-cholesterol level was measured by LipoSEARCH contract analysis service.
  • FIG. 8 shows the results of measuring total cholesterol (TC) and HDL-cholesterol (HDL-C) levels in serum and liver of each group.
  • TO901317 treatment significantly increased serum total cholesterol (serum TC) and HDL-cholesterol (serum HDL-C).
  • 24-Nor treatment gave almost the same result as TO901317.
  • the increase in total cholesterol was almost equal to the increase in HDL-cholesterol.
  • mice it is known that the proportion of HDL-cholesterol is as high as 80% of the total cholesterol amount.
  • the total amount of cholesterol in the liver was decreased by TO901317 treatment and 24-Nor treatment. This is presumably due to the result of accelerated cholesterol excretion in bile.
  • Example 7 Confirmation of influence on expression of LXR-controlled gene by in vivo administration of 24-Nor
  • 24-Nor was administered to mice.
  • Mice were euthanized as in Example 5, and blood, bile, liver, and small intestine were collected.
  • the mRNA expression levels of LXR-controlled genes in the liver and small intestine were measured by real time-RT-PCR after extracting total RNA from each organ.
  • FIGS. FIG. 10 shows the results of evaluating the expression level of each gene under the LXR control in the liver.
  • Liver ABCA1 and ABCG1 mRNA expression levels showed an increasing tendency with 24-Nor. This result suggests that the 24-Nor treatment may promote the reverse cholesterol transfer system.
  • LXR agonists are known to reduce cholesterol absorption by suppressing the expression of NPC1L1, which is a cholesterol transporter. Therefore, whether 24-Nor has a similar effect was examined.
  • the results are shown in FIG. 24-Nor was shown to significantly reduce NPC1L1 mRNA expression levels. Therefore, it was suggested that 24-Nor reduces the absorption of cholesterol present in the small intestine.
  • Example 8 Confirmation of effect on blood triglyceride level by in vivo administration of 24-Nor
  • 24-Nor was administered to mice and the mice were euthanized. Liver was collected. The amount of triglyceride (TG) in serum and liver was measured using a commercially available kit, Triglyceride assay kit (Biovision).
  • mRNA expression levels of genes (SREBP1c, FAS, SCD-1) involved in fatty acid synthesis in the liver were measured by real time-RT-PCR after extracting total RNA from the liver.
  • FIG. 12 shows the measurement results of blood triglyceride (TG) level and liver triglyceride level
  • FIG. 13 shows the expression levels of various genes.
  • TO901317 promoted the expression of genes involved in fatty acid synthesis such as liver SREBP1c, FAS, and SCD-1, and also increased blood triglyceride levels.
  • 24-Nor did not change the expression of these genes and the blood triglyceride level.
  • GC-MS Gas Chromatograph Mass Spectrometer
  • LC-MS Liquid Chromatograph Mass Spectrometer
  • GCMS-QP2010 (SHIMADZU) was used as the GC-MS apparatus.
  • GC-MS conditions were as follows. ⁇ GC condition Column oven temperature: 240 °C ⁇ 2 °C / min. ⁇ 300 °C (5min. Hold) total 35min. Carrier gas: Helium Column: HP-5MS (Agilent Technologies, film thickness 0.25 ⁇ m, length 30m, inner diameter 0.25mm) ⁇ MS condition Ionization method: EI (electron impact ionization method) Ion source temperature: 200 ° C Interface temperature: 280 °C
  • LC-MS Confirmation by LC-MS was performed as follows. First, 1 mg of the compound was dissolved in methanol and filtered through a 0.45 ⁇ m filter to obtain a sample for LC-MS.
  • the LC-MS apparatus used was JMS-T-100LC (JEOL).
  • LC-MS conditions were as follows. ⁇ HPLC conditions Flow rate: 0.2mL / min.
  • Mobile solvent methanol MS conditions
  • Ionization method ESI (electrospray method)
  • Table 3 shows the results of GC-MS and LC-MS analysis of each compound.
  • a trimethylsilyl derivative was used as a sample for GC-MS. These compounds were hardly recognized except the fragments shown in Table 3. It can be inferred that these fragments were generated by breaking carbon-carbon bonds at the 23 and 25 positions. It can be considered that each compound could be synthesized from the fact that m / z increased by 28 as the added alkyl group increased.
  • LC-MS analysis confirmed that most of the fragments were the fragments from which hydrogen ions were released from the compounds and the fragments to which chlorine ions were added.
  • Example 10 Confirmation of LXR agonist activity of various compounds
  • 24-Nor, 24-Nor-Et, and 24-Nor-nPr were LXR agonists. The activity was confirmed.
  • the treatment concentration of each compound is as shown in FIG. DMSO treatment was used as a negative control (NC).
  • Example 11 Confirmation of LXR agonist activity of various compounds
  • DMSO treatment was used as a negative control (NC).
  • the compound synthesized in Example 9 was confirmed to have an action on RXR ⁇ , PPAR ⁇ , FXR, VDR, and TGR5.
  • DMSO treatment was used as a negative control (NC)
  • agonists 9-cis-RA, GW1929, GW4064, 1,25-OH-VD3, and LCA for each receptor were used as positive controls.
  • the treatment concentration of each compound for RXR ⁇ , PPAR ⁇ , FXR and VDR is 1 ⁇ M
  • the treatment concentration of each compound for TGR5 is as shown in FIG.
  • Example 12 Synthesis of LXR Agonist
  • a compound in which R 1 was CH 3 and R 2 was CH 2 CH 3 was synthesized by the following method (FIG. 19).
  • Hyodeoxycholate (HDCA, 1) dissolved in methanol, 28% aqueous ammonia solution and 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methyl-morpholium (DMT-MM) was added and stirred at room temperature to obtain Compound (IV).
  • Compound (IV) was dissolved in anhydrous Tetrahydrofuran (THF), and while stirring at 0 ° C., triethylamine and trifluoroacetic anhydride were slowly added, and the temperature was gradually raised to reflux to obtain Compound (V).
  • Compound (V) was obtained by dissolving Compound (V) in anhydrous THF, slowly adding Grignard reagent (CH3CH2MgX), and refluxing for reaction.
  • Compound (VII) 24-Nor-Me, Et was able to be obtained by dissolving Compound (VI) in anhydrous THF, slowly adding Grignard reagent (CH3MgX), and reacting under reflux.
  • Table 4 below shows GC-MS results of 24-Nor-Me, Et.
  • Sample preparation and GC-MS method for GC-MS analysis were performed in the same manner as in Example 9. Similar to the compound described in Example 9, a fragment ([C (CH 3 ) (CH 2 CH 3 ) OTMS] + ) in which the carbon-carbon bonds at positions 23 and 25 were broken was detected as a reference peak, Most other mass fragments were not recognized. Further, since the m / z value was 14 larger than 24-Nor and 14 smaller than 24-Nor-Et, the synthesis of 24-Nor-Me, Et could be confirmed.
  • Example 13 Confirmation of LXR agonist activity The agonist activity of 24-Nor-Me, Et synthesized in Example 9 against LXR ⁇ and ⁇ was examined. The experiment was performed in the same manner as in Example 10. The results are shown in FIG.
  • 24-Nor-Me and Et had agonistic activity against LXR ⁇ and ⁇ , similar to 24-Nor and 24-Nor-Et.
  • the strength of activity was the same as that of 24-Nor, which was slightly smaller than 24-Nor-Et.
  • the LXR agonist of the present invention has an excellent agonist activity particularly against LXR ⁇ and / or LXR ⁇ among the nuclear receptors. Further, the LXR agonist of the present invention promoted the expression of ABCA1 and ABCG1 genes involved in lipid metabolism, and showed a lower value than the existing drugs in promoting the expression of SREBP1c involved in triglyceride synthesis. Therefore, the LXR agonist of the present invention has a function as an excellent lipid metabolism improving agent with reduced side effects such as triglyceride synthesis. Further, it was confirmed that the cholesterol transport ability was excellent depending on the concentration.
  • the LXR agonist of the present invention was confirmed to act specifically on LXR ⁇ and LXR ⁇ , and as a result, was confirmed to have a function of regulating lipid metabolism. Although it is slightly inferior to TO901317, which is an existing LXR agonist, in terms of cholesterol transport / excretion, etc., it is not highly effective in triglyceride synthesis and is useful as a safer drug.
  • LXR agonists of the present invention 24-Nor is a known compound that is reported in Non-Patent Document 6 (Zepter (1972)) and registered in the database in CAS # 40551-80-2. Compounds other than are novel compounds. Synthetic LXR agonists such as TO901317 are generally complicated in the preparation process (synthesis / purification / isolation) and consume a lot of time and labor. However, the LXR agonist of the present invention is synthesized / purified / simple. The point that the separation method is easy also shows an advantageous effect.
  • the LXR agonist of the present invention increases the level of HDL cholesterol in blood and enhances the expression level of ABCA1 and ABCG1 mRNA in the liver when administered in vivo. From this, it is considered that the LXR agonist of the present invention contributes to the production of neoplastic HDL and increases the blood HDL cholesterol level. In recent years, since the low level of HCL cholesterol has been established as an independent risk factor for abnormal lipid metabolism, the LXR agonist of the present invention has fewer side effects and is used as a safer drug that increases only HDL cholesterol level. It is expected.

Abstract

The present invention was completed based on the finding that a compound represented by general formula (I) [wherein R1 and R2 independently represent a hydrocarbon group having 1-5 carbon atoms] or a pharmaceutically acceptable salt thereof exerts an agonistic action on LXRs (LXRα and LXRβ), has a high specificity and is capable of improving lipid metabolism. The LXR agonist according to the present invention has a higher LXR agonist activity than existing components derived from bile acid and derivatives thereof and functions as an excellent lipid metabolism-improving agent with reduced side effects such as the synthesis of triglycerides.

Description

核内受容体肝臓Ｘ受容体アゴニストNuclear receptor liver X receptor agonist
　本発明は、核内受容体である肝臓Ｘ受容体（Liver X Receptor: 以下、単に「LXR」ともいう。）の新規アゴニストに関する。 The present invention relates to a novel agonist of a liver X receptor (Liver X Receptor: hereinafter also simply referred to as “LXR”), which is a nuclear receptor.
　本出願は、参照によりここに援用されるところの日本出願特願2013-183522号優先権を請求する。 This application claims the priority of Japanese Patent Application No. 2013-183522, which is incorporated herein by reference.
　核内受容体とは細胞内タンパク質の一種であり、生体内の脂溶性化合物、例えばホルモンなどがリガンドとして結合することで細胞核内でのDNA転写を調節する転写因子の一種である。発生、恒常性、代謝など生命維持の根幹に係わる遺伝子転写に関与している（Zhang et al., Genome Res., 14(4), 580-90 (2004)）。 The nuclear receptor is a kind of intracellular protein, and is a kind of transcription factor that regulates DNA transcription in the cell nucleus by binding a fat-soluble compound in the living body such as a hormone as a ligand. It is involved in gene transcription related to life support such as development, homeostasis and metabolism (Zhang et al., Genome Res., 14 (4), 580-90 (2004)).
　多くの種類の核内受容体が公知であり、その一つである肝臓Ｘ受容体（LXR）は、酸化コレステロール（オキシステロール）受容体として、また体内に蓄積したコレステロールを処理する機能を担う転写因子として知られている。LXRはオキシステロールをリガンドとして活性化され、脂質の恒常性を維持するために重要な役割を果たしている（非特許文献１：Janowski (1996)）。LXRは肝、脂肪組織等エネルギー代謝臓器において多く発現しており、コレステロールの異化・排泄経路でもある胆汁酸生合成に関与する酵素群や、脂肪酸生合成に重要な役割を果たすSREBP-1cなどの遺伝子発現を調節している。またLXRは糖の脂肪酸への変換を促進して血糖値を下げる作用も報告されており、糖質及びインスリン代謝への影響も注目されている。さらに、LXRは血管構成細胞、特にマクロファージにおいても発現しており、動脈硬化病変部位に対する脂質代謝への影響も注目されている。LXRは、過剰なコレステロールをHDL（high density lipoprotein）へ輸送してコレステロール排出を担うABCA1を発現誘導する。小腸粘膜ではコレステロールの吸収を抑制し、排出を促進する。従って、LXRを活性化する化合物はアテローム性動脈硬化症や心血管疾患の有効な治療薬の候補となり得る。 Many types of nuclear receptors are known, and one of them, liver X receptor (LXR), is a transcription that functions as an oxidized cholesterol (oxysterol) receptor and functions to process cholesterol accumulated in the body. Known as a factor. LXR is activated with oxysterol as a ligand and plays an important role in maintaining lipid homeostasis (Non-Patent Document 1: Janowski (1996)). LXR is highly expressed in energy-metabolizing organs such as liver and adipose tissue, such as enzymes involved in bile acid biosynthesis, which is also a catabolism and excretion pathway of cholesterol, and SREBP-1c that plays an important role in fatty acid biosynthesis Regulates gene expression. LXR has also been reported to promote the conversion of sugars to fatty acids to lower blood sugar levels, and has also attracted attention for its effects on carbohydrate and insulin metabolism. Furthermore, LXR is also expressed in vascular constituent cells, particularly macrophages, and its influence on lipid metabolism at the site of arteriosclerotic lesions has attracted attention. LXR transports excess cholesterol to HDL (high density lipoprotein) and induces expression of ABCA1, which is responsible for cholesterol excretion. The small intestinal mucosa suppresses cholesterol absorption and promotes excretion. Therefore, compounds that activate LXR can be candidates for effective therapeutic agents for atherosclerosis and cardiovascular diseases.
　ヒオデオキシコール酸（HDCA）やケノデオキシコール酸（CDCA）のような天然に存在する胆汁酸及びそれらの類縁体がLXR活性化能を有することが報告されている（非特許文献２：Song (2000)、非特許文献３：Song (2000)、特許文献１：特開2006-528200号公報、特許文献２：特開2008-179562号公報、特許文献３：特開2009-227615号公報）。しかしながら、胆汁酸類縁体であっても、内因性のLXRアゴニストであるオキシステロールに及ぶ物質はこれまでに報告されていない。また、現在までにTO901317、LXR-623、WYE-672など様々な非ステロイド系合成LXRアゴニストが創製されており、内因性LXRリガンドであるオキシステロールよりも高い活性を有する化合物も見出されている（非特許文献４：Viennois (2012)、非特許文献５：Schults (2000)）。しかし、これら化合物はその調製過程（合成・精製・単離）が煩雑で、多くの時間と労力を費やしているのが現状である。また、これら合成LXRアゴニストの生体への投与により血中、及び肝臓中のトリグリセリド（triglyceride:TG）含量を増加させるという望ましくない作用を惹起することが非特許文献５に報告されており、実用化には至っていない。 It has been reported that naturally occurring bile acids such as hyodeoxycholic acid (HDCA) and chenodeoxycholic acid (CDCA) and their analogs have LXR activation ability (Non-patent Document 2: Song (2000) Non-Patent Document 3: Song (2000), Patent Document 1: JP-A-2006-528200, Patent Document 2: JP-A-2008-179562, Patent Document 3: JP-A-2009-227615). However, even a bile acid analog has not been reported so far, which covers oxysterol, which is an endogenous LXR agonist. In addition, various nonsteroidal synthetic LXR agonists such as TO901317, LXR-623, and WYE-672 have been created so far, and compounds having higher activity than oxysterol, which is an endogenous LXR ligand, have also been found. (Non-patent document 4: Viennois® (2012), Non-patent document 5: Schults® (2000)). However, the preparation process (synthesis, purification, and isolation) of these compounds is complicated, and a lot of time and labor are consumed at present. Further, non-patent document 5 reports that the administration of these synthetic LXR agonists to the living body causes an undesirable effect of increasing the triglyceride (TG) content in blood and liver. It has not reached.
　胆汁酸類縁体の１種である24-ノル-5β-コレスタン-3α,6α,25-トリオール（24-Nor-5β-cholestane-3α,6α,25-triol）は、非特許文献６（Zepter (1972)）に報告されており、CAS#40551-80-2においてデータベースに登録されている公知の化合物である。しかしながら、前記化合物については、LXRアゴニストに関する報告はない。 One of the bile acid analogs, 24-nor-5β-cholestane-3α, 6α, 25-triol (24-Nor-5β-cholestane-3α, 6α, 25-triol) is described in Non-Patent Document 6 (Zepter ( 1972)) and is a known compound registered in the database in CAS # 40551-80-2. However, there is no report regarding the LXR agonist for the compound.
　脂質代謝異常においては、総コレステロール値が高いことだけを問題とするのではなく、LDLコレステロール（LDL-C）やHDLコレステロール（HDL-C）、中性脂肪といった脂質成分の質も重要であることが臨床上の共通の認識となっている。またFramingham研究をはじめとする多くの疫学調査により、HDLコレステロールの低値が独立した冠動脈疾患危険因子として確立されている。HDLコレステロール値のみを増やす薬剤としてCETP阻害薬が挙げられるが、CETP阻害薬には、臨床試験において血圧も上昇させてしまうことが確認され、開発が中止になったものある。HDLは、抗酸化、抗凝固、抗不整脈作用などを持ち、心血管系の維持に重要な働きをしていることから、HDLに集中してアプローチを行う方法が着目されている。しかしながら、HDLに特異的に作用する薬剤がないのが現状であり、生体内でHDLに特異的に作用し、かつ副作用の低い薬剤の開発が望まれている。 In lipid metabolism disorders, not only the high total cholesterol level is a problem, but also the quality of lipid components such as LDL cholesterol (LDL-C), HDL cholesterol (HDL-C), and neutral fat is important. Has become a common clinical recognition. Many epidemiological studies, including the Framingham study, have established low HDL cholesterol levels as independent risk factors for coronary artery disease. CETP inhibitors are listed as drugs that only increase HDL cholesterol levels, but some CETP inhibitors have been discontinued due to clinical trials confirming that blood pressure is also increased. Since HDL has antioxidation, anticoagulation, antiarrhythmic action, etc., and plays an important role in maintaining the cardiovascular system, a method of focusing on HDL has attracted attention. However, there is currently no drug that specifically acts on HDL, and development of a drug that specifically acts on HDL in vivo and has low side effects is desired.
特開2006-528200号公報JP 2006-528200 A 特開2008-179562号公報JP 2008-179562 A 特開2009-227615号公報JP 2009-227615 A
　本発明は、LXR（LXRα及びLXRβ）に対して特異性が高く、安全性の高い新規なLXRアゴニストを提供することを課題とする。 An object of the present invention is to provide a novel LXR agonist having high specificity and high safety for LXR (LXRα and LXRβ).
　本発明者らは、上記課題を解決するために鋭意検討を重ねた結果、胆汁酸類縁体の一種であって、従来LXRアゴニスト活性が一切報告されていない化合物に着目した。この化合物および当該化合物に類似の構造を有する化合物は、LXR（LXRα及びLXRβ）に対して特異性が高く、脂質代謝改善能を有するものであることを確認し、本発明を完成した。 As a result of intensive studies in order to solve the above-mentioned problems, the present inventors have focused on a compound that is a kind of bile acid analog and has not reported any LXR agonist activity. It was confirmed that this compound and a compound having a structure similar to that compound had high specificity for LXR (LXRα and LXRβ) and had an ability to improve lipid metabolism, thereby completing the present invention.
　即ち本発明は、以下よりなる。
１．以下の一般式(I)で表される化合物又はその薬学的に許容される塩からなる、肝臓Ｘ受容体（LXR）アゴニスト。
Figure JPOXMLDOC01-appb-C000004
 （式中、R1およびR2は、それぞれ独立して、炭素原子数１～５の炭化水素基を表す。）
２．一般式(I)において、R1およびR2が、それぞれ独立して、炭素原子数１～３のアルキル基を表す化合物、又はその薬学的に許容される塩からなる、前項１に記載の肝臓Ｘ受容体（LXR）アゴニスト。
３．一般式(I)において、R1およびR2がともに、メチル基、エチル基、プロピル基のいずれかである化合物、又はその薬学的に許容される塩からなる、前項１または２に記載の肝臓Ｘ受容体（LXR）アゴニスト。
４．前記肝臓Ｘ受容体（LXR）が、LXRα及び／又はLXRβである、前項１～３のいずれか１に記載の肝臓Ｘ受容体（LXR）アゴニスト。
５．前項１～４のいずれか１に記載の肝臓Ｘ受容体（LXR）アゴニストからなる脂質代謝改善剤。
６．肝臓Ｘ受容体（LXR）アゴニストが血中HDLコレステロールを上昇させるものである、前項５に記載の脂質代謝改善剤。
７．前項５又は６に記載の脂質代謝改善剤を有効成分として含む、脂質代謝異常又は脂質代謝異常に伴う疾患の治療又は予防剤。
８．前項１～４のいずれかに記載の肝臓Ｘ受容体（LXR）アゴニストを有効成分として含む、医薬組成物。
９．以下の一般式(I)で表される化合物又はその薬学的に許容される塩。
Figure JPOXMLDOC01-appb-C000005
 （式中、R1およびR2は、それぞれ独立して、炭素原子数１～５の炭化水素基を表す。ただし、R1およびR2がともに、メチル基である場合を除く。）
１０．一般式(I)において、R1およびR2が、それぞれ独立して、炭素原子数１～３のアルキル基を表す、前項９に記載の化合物、又はその薬学的に許容される塩。
１１．一般式(I)において、R1およびR2がともに、メチル基、エチル基、プロピル基のいずれかである、前項９又は１０に記載の化合物、又はその薬学的に許容される塩。
１２．前項５又は６に記載の脂質代謝改善剤、又は、前項９～１１のいずれか１に記載の化合物又はその薬学的に許容される塩を対象に投与することにより、対象の脂質代謝異常又は脂質代謝異常に伴う疾患を治療又は予防する方法。
１３．以下の一般式(I)で表される化合物又はその薬学的に許容される塩を肝臓Ｘ受容体（LXR）と接触させることにより、肝臓Ｘ受容体（LXR）を作動させる方法。
Figure JPOXMLDOC01-appb-C000006
  
（式中、R1およびR2は、それぞれ独立して、炭素原子数１～５の炭化水素基である。） That is, this invention consists of the following.
1. A liver X receptor (LXR) agonist comprising a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof:
Figure JPOXMLDOC01-appb-C000004
 (In the formula, R 1 and R 2 each independently represents a hydrocarbon group having 1 to 5 carbon atoms.)
2. 2. The liver according to item 1 above , wherein in general formula (I), R 1 and R 2 each independently represents a compound representing an alkyl group having 1 to 3 carbon atoms, or a pharmaceutically acceptable salt thereof. X receptor (LXR) agonist.
3. The liver according to the above item 1 or 2, wherein in the general formula (I), R 1 and R 2 are both a compound having a methyl group, an ethyl group or a propyl group, or a pharmaceutically acceptable salt thereof. X receptor (LXR) agonist.
4). 4. The liver X receptor (LXR) agonist according to any one of items 1 to 3, wherein the liver X receptor (LXR) is LXRα and / or LXRβ.
5. 5. A lipid metabolism improving agent comprising the liver X receptor (LXR) agonist according to any one of 1 to 4 above.
6). 6. The lipid metabolism improving agent according to 5 above, wherein the liver X receptor (LXR) agonist increases blood HDL cholesterol.
7). 7. A therapeutic or prophylactic agent for lipid metabolism abnormality or a disease associated with lipid metabolism abnormality comprising the lipid metabolism improving agent according to 5 or 6 as an active ingredient.
8). 5. A pharmaceutical composition comprising the liver X receptor (LXR) agonist according to any one of items 1 to 4 as an active ingredient.
9. A compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof:
Figure JPOXMLDOC01-appb-C000005
 (In the formula, R 1 and R 2 each independently represents a hydrocarbon group having 1 to 5 carbon atoms, except when both R 1 and R 2 are methyl groups.)
10. 10. The compound according to item 9 or a pharmaceutically acceptable salt thereof, wherein in general formula (I), R 1 and R 2 each independently represents an alkyl group having 1 to 3 carbon atoms.
11. 11. The compound according to item 9 or 10, or a pharmaceutically acceptable salt thereof, wherein, in general formula (I), R 1 and R 2 are both a methyl group, an ethyl group, or a propyl group.
12 A lipid metabolism abnormality or lipid in a subject by administering to the subject the lipid metabolism improving agent according to 5 or 6 above, or the compound according to any one of 9 to 11 or a pharmaceutically acceptable salt thereof. A method of treating or preventing a disease associated with metabolic abnormality.
13. A method of activating liver X receptor (LXR) by contacting a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof with liver X receptor (LXR).
Figure JPOXMLDOC01-appb-C000006
(In the formula, R 1 and R 2 are each independently a hydrocarbon group having 1 to 5 carbon atoms.)
　上記の他、本発明は以下も包含されるものである。
（Ａ）前項７に記載の脂質代謝改善剤を有効成分として含む食品。
（Ｂ）前項７に記載の脂質代謝改善剤を対象に投与することにより、対象の血中HDLコレステロール量を上昇させる方法。
（Ｃ）前項７に記載の脂質代謝改善剤を対象に投与することにより、対象の脂質代謝を改善する方法。
（Ｄ）一般式(I)で表される化合物又はその薬学的に許容される塩を、対象に投与することにより、肝臓Ｘ受容体（LXR）を作動させる方法。
（Ｅ）一般式(I)で表される化合物又はその薬学的に許容される塩を、対象に投与することにより、肝臓Ｘ受容体（LXR）に関連する疾患を治療または予防する方法。 In addition to the above, the present invention includes the following.
(A) A food comprising the lipid metabolism improving agent according to item 7 as an active ingredient.
(B) A method of increasing the blood HDL cholesterol level of a subject by administering the lipid metabolism improving agent according to item 7 to the subject.
(C) A method for improving lipid metabolism in a subject by administering the lipid metabolism improving agent according to item 7 to the subject.
(D) A method of activating the liver X receptor (LXR) by administering a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof to a subject.
(E) A method of treating or preventing a disease associated with liver X receptor (LXR) by administering a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof to a subject.
　本発明のLXRアゴニスト、例えば式(I)に示す化合物（24-ノル-5β-コレスタン-3α,6α,25-トリオール（24-Nor-5β-cholestane-3α,6α,25-triol）、以下「24-Nor」と略する。）は、核酸受容体のうち、特にLXRα及び／又はLXRβに対して優れたアゴニスト活性を有する。また、本発明のLXRアゴニスト化合物は、in　vitroおよびin　vivoにおいて、脂質代謝に関わるABCA1及びABCG1遺伝子の発現を促進し、トリグリセリド合成に関わるSREBP1cの発現は、既存の薬剤よりも低い値を示した。また、用量依存的に優れたコレステロール排出能を示すことが確認された。 The LXR agonist of the present invention, for example, a compound represented by the formula (I) (24-Nor-5β-cholestane-3α, 6α, 25-triol (24-Nor-5β-cholestane-3α, 6α, 25-triol), hereinafter “ Abbreviation “24-Nor”) has excellent agonist activity among nucleic acid receptors, particularly LXRα and / or LXRβ. Moreover, the LXR agonist compound of the present invention promoted the expression of ABCA1 and ABCG1 genes involved in lipid metabolism in vitro and in vivo, and the expression of SREBP1c involved in triglyceride synthesis was lower than that of existing drugs. . Moreover, it was confirmed that the cholesterol excretion ability was excellent in a dose-dependent manner.
　本発明のLXRアゴニストは、既存の胆汁酸由来成分やその誘導体と比較しても優れたLXRアゴニスト活性を有し、トリグリセリド合成等の副作用が軽減された優れた脂質代謝改善剤としての機能を有する。 The LXR agonist of the present invention has an excellent LXR agonist activity even compared to existing bile acid-derived components and derivatives thereof, and has an excellent function as a lipid metabolism improving agent with reduced side effects such as triglyceride synthesis. .
本発明のLXRアゴニストのうち、24-Norの合成スキームを示す図である。また、比較例として各実施例で使用される類縁化合物についても示す図である。It is a figure which shows the synthetic scheme of 24-Nor among the LXR agonists of this invention. Moreover, it is a figure which shows also about the related compound used by each Example as a comparative example. 24-NorのLXRα及びLXRβアゴニスト活性を確認した結果を示す図である。（実施例１）It is a figure which shows the result of having confirmed LXR (alpha) and LXR (beta) agonist activity of 24-Nor. (Example 1) 24-Norの各種核内受容体に対するアゴニスト活性を確認した結果を示す図である。LXRα及びLXRβの他、コレステロールから合成される胆汁酸をリガンドとする核内受容体（farnesoid X receptor: FXR、胆汁酸によって活性化されるGタンパク質結合型受容体：TGR5）、ビタミンD 誘導体をリガンドとする核内受容体（vitamin D receptor: VDR）に対するアゴニスト活性を確認した結果を示す図である。（実施例２）It is a figure which shows the result of having confirmed the agonist activity with respect to various nuclear receptors of 24-Nor. In addition to LXRα and LXRβ, nuclear receptors with bile acids synthesized from cholesterol as ligands (farnesoid X receptor: FXR, G protein-coupled receptors activated by bile acids: TGR5), vitamin D derivatives as ligands It is a figure which shows the result of having confirmed the agonist activity with respect to the nuclear receptor (vitamin (D) receptor: (VDR)) made into. (Example 2) 24-Norについて、脂質代謝に関わるABCA1及びABCG1の各遺伝子の発現に及ぼす影響をTHP-1細胞を用いて確認した結果を示す図である。（実施例３）It is a figure which shows the result which confirmed the influence which acts on expression of each gene of ABCA1 and ABCG1 in connection with lipid metabolism using THP-1 cells. (Example 3) 24-Norについて、脂質代謝に関わるABCG1及びSREBP1cの各遺伝子の発現に及ぼす影響をHuh-7細胞を用いて確認した結果を示す図である。（実施例３）It is a figure which shows the result of having confirmed the influence which it has on expression of each gene of ABCG1 and SREBP1c in connection with lipid metabolism using 24-Hor-7 cells. (Example 3) 24-Norについて、末梢細胞のコレステロール排出能を確認した結果を示す図である。（実施例４）It is a figure which shows the result of having confirmed the cholesterol excretion ability of the peripheral cell about 24-Nor. Example 4 24-Norをin　vivo投与した場合の、マウス体重及び肝重量に対する影響を確認した結果を示す図である。（実施例５）It is a figure which shows the result of having confirmed the influence with respect to a mouse | mouth body weight and a liver weight when 24-Nor is administered in vivo. (Example 5) 24-Norをin　vivo投与した場合の、血清中及び肝臓のコレステロール量に対する影響を確認した結果を示す図である。（実施例６）It is a figure which shows the result of having confirmed the influence with respect to the amount of cholesterol in a blood serum when 24-Nor is administered in vivo. (Example 6) 24-Norをin　vivo投与した場合の、胆汁中コレステロール量及び糞中中性脂肪量に対する影響を確認した結果を示す図である。（実施例６）FIG. 4 is a view showing the results of confirming the influence on the amount of cholesterol in bile and the amount of neutral fat in feces when 24-Nor is administered in vivo. (Example 6) 24-Norをin　vivo投与した場合の、肝臓ABCA1及びABCG1の各遺伝子の発現に及ぼす影響を確認した結果を示す図である。（実施例７）FIG. 4 is a view showing the results of confirming the effect on the expression of liver ABCA1 and ABCG1 genes when 24-Nor is administered in vivo. (Example 7) 24-Norをin　vivo投与した場合の、小腸NPC1L1遺伝子の発現に及ぼす影響を確認した結果を示す図である。（実施例７）FIG. 4 is a view showing the results of confirming the effect of in vivo administration of 24-Nor on the expression of the small intestine NPC1L1 gene. (Example 7) 24-Norをin　vivo投与した場合の、血清及び肝臓中のトリグリセリド量に対する影響を確認した結果を示す図である。（実施例８）It is a figure which shows the result of having confirmed the influence with respect to the amount of triglycerides in a serum and the liver at the time of administering 24-Nor in vivo. (Example 8) 24-Norをin　vivo投与した場合の、肝臓中のSREBP1c、FAS、SCD-1の各遺伝子の発現に及ぼす影響を確認した結果を示す図である。（実施例８）FIG. 6 is a view showing the results of confirming the effect of 24-Nor administered in vivo on the expression of SREBP1c, FAS, and SCD-1 genes in the liver. (Example 8) 24-Norについて、NMRにて合成を確認した結果を示す図である。（実施例９）It is a figure which shows the result which confirmed the synthesis | combination by NMR about 24-Nor. Example 9 本発明の各種LXRアゴニストのLXRα及びLXRβアゴニスト活性を確認した結果を示す図である。（実施例１０）It is a figure which shows the result of having confirmed LXR (alpha) and LXR (beta) agonist activity of the various LXR agonist of this invention. (Example 10) 本発明の各種LXRアゴニストのLXRα及びLXRβアゴニスト活性を確認した結果を示す図である。（実施例１１）It is a figure which shows the result of having confirmed LXR (alpha) and LXR (beta) agonist activity of the various LXR agonist of this invention. (Example 11) 本発明の各種LXRアゴニストの各種核内受容体に対するアゴニスト活性を確認した結果を示す図である。（実施例１１）It is a figure which shows the result of having confirmed the agonist activity with respect to various nuclear receptors of the various LXR agonist of this invention. (Example 11) 本発明に各種LXRアゴニストのTGR5に対するアゴニスト活性を確認した結果を示す図である。（実施例１１）It is a figure which shows the result of having confirmed the agonist activity with respect to TGR5 of various LXR agonists in this invention. (Example 11) 本発明のLXRアゴニストの合成スキームを示す図である。（実施例１２）It is a figure which shows the synthetic scheme of the LXR agonist of this invention. (Example 12) 本発明の各種LXRアゴニストのLXRα及びLXRβアゴニスト活性を確認した結果を示す図である。（実施例１３）It is a figure which shows the result of having confirmed LXR (alpha) and LXR (beta) agonist activity of the various LXR agonist of this invention. (Example 13)
　本発明のLXRアゴニストは、以下の一般式(I)で表される化合物又はその薬学的に許容される塩からなる。
一般式(I)： 
Figure JPOXMLDOC01-appb-C000007
 　一般式(I)中、R1及びR2は、それぞれ独立して、炭素原子数１～５の炭化水素基を表す。 The LXR agonist of the present invention comprises a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof.
Formula (I):
Figure JPOXMLDOC01-appb-C000007
 In general formula (I), R 1 and R 2 each independently represents a hydrocarbon group having 1 to 5 carbon atoms.
　本発明の一般式(I)における炭化水素基は、直鎖状であっても分岐状であってもよく、アルキル基、アルケニル基、アルキニル基を含む。一般式(I)における炭化水素基は、好ましくはアルキル基であり、当該アルキル基は直鎖状であっても分岐状であってもよいが、好ましくは直鎖状である。さらに、一般式(I)における炭化水素基の炭素原子数は１～５であり、好ましくは１～３である。一般式(I)におけるR1及びR2は、メチル基、エチル基、プロピル基（n-プロピル基、iso-プロピル基）から選択され、R1及びR2がともに同一の基であることが特に好ましい。 The hydrocarbon group in the general formula (I) of the present invention may be linear or branched and includes an alkyl group, an alkenyl group, and an alkynyl group. The hydrocarbon group in the general formula (I) is preferably an alkyl group, and the alkyl group may be linear or branched, but is preferably linear. Further, the hydrocarbon group in the general formula (I) has 1 to 5 carbon atoms, preferably 1 to 3 carbon atoms. R 1 and R 2 in the general formula (I) are selected from a methyl group, an ethyl group, and a propyl group (n-propyl group, iso-propyl group), and R 1 and R 2 are both the same group Particularly preferred.
　本発明の具体的な化合物として、以下の表１に示す化合物が例示される。表１におけるR1及びR2は、一般式(I)におけるR1及びR2である。
Figure JPOXMLDOC01-appb-T000008
 　本明細書では表２に記載の各化合物を、表２に記載の名称（24-Nor等）で表すこともある。 Specific compounds of the present invention are exemplified by the compounds shown in Table 1 below. R 1 and R 2 in Table 1 is R 1 and R 2 in the general formula (I).
Figure JPOXMLDOC01-appb-T000008
 In this specification, each compound described in Table 2 may be represented by the name (24-Nor or the like) described in Table 2.
　上記一般式(I)に記載の化合物は、非特許文献６に示される合成手順を参照して作製することができる。一例として、24-Nor-Et、24-Nor-nPr、24-Nor-nBt、24-Nor-nPe、24-Nor-iPrの製造方法を下記実施例９に、24-Nor-Me,　Etの製造方法を下記実施例１２に記載する。実施例に示す方法において用いられた出発原料及び試薬、並びに反応条件などを適宜修飾ないし改変することにより、本発明の範囲に包含される化合物はいずれも製造可能である。 The compound described in the general formula (I) can be prepared with reference to the synthesis procedure shown in Non-Patent Document 6. As an example, the production method of 24-Nor-Et, 24-Nor-nPr, 24-Nor-nBt, 24-Nor-nPe, 24-Nor-iPr is shown in Example 9 below, and 24-Nor-Me, Et The manufacturing method is described in Example 12 below. Any compounds included in the scope of the present invention can be produced by appropriately modifying or altering the starting materials and reagents used in the methods shown in the Examples and the reaction conditions.
　一般式(I)に示す化合物のうち、式(I)に示す化合物24-Norは、24-Nor-5β-cholestane-3α,6α,25-triolであり、非特許文献６において公知の化合物である。当該化合物は、非特許文献６に示す合成手順に従って合成することができる。 Among the compounds represented by the general formula (I), the compound 24-Nor represented by the formula (I) is 24-Nor-5β-cholestane-3α, 6α, 25-triol, which is a known compound in Non-Patent Document 6. is there. The compound can be synthesized according to the synthesis procedure shown in Non-Patent Document 6.
　本発明のLXRアゴニストは、一般式(I)に示す化合物又はその薬学的に許容される塩から選択される。上記化合物又はその塩において、異性体（例えば光学異性体、幾何異性体及び互換異性体）などが存在する場合は、本発明はそれらの異性体を包含し、また溶媒和物、水和物及び種々の形状の結晶を包含するものである。 The LXR agonist of the present invention is selected from the compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof. In the above compounds or salts thereof, when isomers (for example, optical isomers, geometric isomers and interchangeable isomers) and the like exist, the present invention includes those isomers, and also includes solvates, hydrates and It includes crystals of various shapes.
　本発明において、薬学的に許容される塩とは、薬理学的及び製剤学的に許容される一般的な塩が挙げられる。そのような塩として、具体的には以下が例示される。
　塩基性付加塩としては、例えばナトリウム塩、カリウム塩等のアルカリ金属塩；例えばカルシウム塩、マグネシウム塩等のアルカリ土類金属塩；例えばアンモニウム塩；例えばトリメチルアミン塩、トリエチルアミン塩；ジシクロヘキシルアミン塩、エタノールアミン塩、ジエタノールアミン塩、トリエタノールアミン塩、ブロカイン塩等の脂肪族アミン塩；たとえばＮ，Ｎ－ジベンジルエチレンジアミン等のアラルキルアミン塩；例えばピリジン塩、ピコリン塩、キノリン塩、イソキノリン塩等の複素環芳香族アミン塩；例えばテトラメチルアンモニウム塩、テトラエチルアモニウム塩、ベンジルトリメチルアンモニウム塩、ベンジルトリエチルアンモニウム塩、ベンジルトリブチルアンモニウム塩、メチルトリオクチルアンモニウム塩、テトラブチルアンモニウム塩等の第４級アンモニウム塩；アルギニン塩；リジン塩等の塩基性アミノ酸塩等が挙げられる。 In the present invention, the pharmaceutically acceptable salt includes general pharmacologically and pharmaceutically acceptable salts. Specific examples of such salts are as follows.
Examples of basic addition salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; ammonium salts; trimethylamine salts and triethylamine salts; dicyclohexylamine salts and ethanolamines. Aliphatic amine salts such as salts, diethanolamine salts, triethanolamine salts and brocaine salts; aralkylamine salts such as N, N-dibenzylethylenediamine; and heterocyclic aromatics such as pyridine salts, picoline salts, quinoline salts and isoquinoline salts For example, tetramethylammonium salt, tetraethylammonium salt, benzyltrimethylammonium salt, benzyltriethylammonium salt, benzyltributylammonium salt, methyltrioctylammonium salt, Quaternary ammonium salts such as tiger butyl ammonium salt; arginine; basic amino acid salts such as lysine salts.
　酸付加塩としては、例えば塩酸塩、硫酸塩、硝酸塩、リン酸塩、炭酸塩、炭酸水素塩、過塩素酸塩等の無機酸塩；例えば酢酸塩、プロピオン酸塩、乳酸塩、マレイン酸塩、フマール酸塩、酒石酸塩、リンゴ酸塩、クエン酸塩、アスコルビン酸塩等の有機酸塩；例えばメタンスルホン酸塩、イセチオン酸塩、ベンゼンスルホン酸塩、ｐ－トルエンスルホン酸塩等のスルホン酸塩；例えばアスパラギン酸塩、グルタミン酸塩等の酸性アミノ酸等を挙げることができる。 Examples of the acid addition salt include inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate, carbonate, hydrogen carbonate, perchlorate; for example, acetate, propionate, lactate, maleate , Organic acid salts such as fumarate, tartrate, malate, citrate and ascorbate; sulfonic acids such as methanesulfonate, isethionate, benzenesulfonate and p-toluenesulfonate Salts; for example, acidic amino acids such as aspartate and glutamate.
　本発明のLXRアゴニストは、既存のオキシステロール化合物（22(R)-hydroxycholesterol: 22(R)-HC）、図１の式(II)に示す24-ノル-5β-コレスタン-3α,7α,25-トリオール（24-Nor-5β-cholestane-3α,7α,25-triol）、及び図１の式(III)に示すHDC-OHに比べて、LXRα及びLXRβのいずれに対しても優れたアゴニスト作用を示す。さらに、本発明のLXRアゴニストは、LXRα及びLXRβ特異的にアゴニスト活性を有するが、他の核内受容体、例えばコレステロールから合成される胆汁酸をリガンドとする核内受容体（farnesoid X receptor: FXR、Gタンパク質受容体：TGR5）、ビタミンD 誘導体をリガンドとする核内受容体（vitamin D receptor: VDR）に対するアゴニスト活性はほとんど示さない。22(R)-HCは、LXRα及びLXRβ特異的にアゴニスト活性を有するが、他の核内受容体、例えばFXRに対するアゴニスト活性もわずかではあるが有している。また、ケノデオキシコール酸（CDCA）、リトコール酸（LCA：lithocholic acid：コール酸の7,12α-脱水酸化誘導体）及び1,25-ヒドロキシビタミンD3（1,25- hydroxyvitamin D3）は、LXRα及びLXRβにはアゴニスト活性を示さず、FXR、TGR5やVDRなどの各核内受容体に対するアゴニスト活性を有するものであり、活性は全く相違する。 The LXR agonist of the present invention is an existing oxysterol compound (22 (R) -hydroxycholesterol: 22 (R) -HC), 24-nor-5β-cholestane-3α, 7α, 25 shown in the formula (II) of FIG. Agonist action superior to both LXRα and LXRβ in comparison with HDC-OH represented by 2-triol (24-Nor-5β-cholestane-3α, 7α, 25-triol) and formula (III) in FIG. Indicates. Furthermore, the LXR agonist of the present invention has agonist activity specifically for LXRα and LXRβ, but other nuclear receptors, for example, nuclear receptors (farnesoid X receptor: FXR) having a bile acid synthesized from cholesterol as a ligand. , G protein receptor: TGR5), and shows little agonist activity for nuclear receptor (vitamin D receptor: VDR) with vitamin D derivatives as ligands. 22 (R) -HC has agonist activity specifically for LXRα and LXRβ, but also has slight agonist activity for other nuclear receptors such as FXR. In addition, chenodeoxycholic acid (CDCA), lithocholic acid (LCA: lithocholic acid: 7,12α-dehydrated oxidized derivative of cholic acid) and 1,25-hydroxyvitamin D 3 (1,25-hydroxyvitamin D 3 ) are LXRα and LXRβ. Does not show agonist activity and has agonist activity for each nuclear receptor such as FXR, TGR5 and VDR, and the activities are completely different.
　LXRはリガンド依存性核内受容体であり、LXRαは肝臓やマクロファージに多く発現しているが、LXRβは全身にユビキタスに発現している。これらはLXR応答領域（以下LXRE）とよばれる10塩基程度のDNA配列に結合し、標的遺伝子の転写を及びオキシステロール依存的に活性化する。 LXR is a ligand-dependent nuclear receptor, and LXRα is highly expressed in the liver and macrophages, but LXRβ is ubiquitously expressed throughout the body. These bind to a DNA sequence of about 10 bases called LXR response region (hereinafter referred to as LXRE) and activate transcription of the target gene in an oxysterol-dependent manner.
　脂質代謝を制御する因子として、ABC（ATP-Binding Cassette）タンパク質、SREBP1c（sterol regulatory element binding protein 1c）が知られている。ABCタンパク質は、200アミノ酸に渡って配列がよく保存されたATP結合ドメインを１機能分子あたり２つ有する膜タンパク質ファミリーであり、生体内で重要な生理機能を果たしている。ABCタンパク質の一種であるABCA1は、高密度リポ蛋白質（HDL）の形成に必須である。HDLは、動脈硬化等の引き金となるコレステロールを末梢細胞から引き出して肝臓に運ぶ。肝臓ではコレステロールが胆汁酸に転換され、胆汁を経て体外に排出される。このようなHDLによる末梢細胞から肝臓へのコレステロールの輸送を「コレステロール逆転送系」と呼ぶ。肝臓のABCA1は、アポリポプロテインA-I（ApoA-I）にコレステロールとリン脂質を受け渡し、HDL粒子を新生する。新生HDLは循環血により末梢組織に運ばれ、末梢組織に存在するABCA1や、ABCタンパク質の１種であるABCG1を介して末梢組織中のコレステロールを受け取り、成熟型HDLが形成される。ABCA1の変異は動脈硬化症の重要な危険因子であることが疫学的に判明しており、ABCA1の発現や活性の増強は脂質恒常性の改善の重要なターゲットである。 As factors controlling lipid metabolism, ABC (ATP-Binding Cassette) protein and SREBP1c (sterol regulatory element binding protein 1c) are known. ABC proteins are a family of membrane proteins having two ATP-binding domains per functional molecule, whose sequences are well conserved over 200 amino acids, and play important physiological functions in vivo. ABCA1, a kind of ABC protein, is essential for the formation of high density lipoprotein (HDL). HDL extracts cholesterol, which triggers arteriosclerosis, from peripheral cells and transports it to the liver. In the liver, cholesterol is converted into bile acids, which are excreted through the bile. This transport of cholesterol from peripheral cells to the liver by HDL is called a “reverse cholesterol transfer system”. ABCA1 in the liver delivers cholesterol and phospholipids to apolipoprotein A-I (ApoA-I) to generate HDL particles. Newborn HDL is carried to peripheral tissues by circulating blood, and cholesterol in peripheral tissues is received via ABCA1 existing in the peripheral tissues and ABCG1 which is one of ABC proteins, and mature HDL is formed. ABCA1 mutations have been epidemiologically found to be an important risk factor for arteriosclerosis, and enhanced ABCA1 expression and activity are important targets for improving lipid homeostasis.
　肝臓に運ばれたコレステロールは、胆汁酸に転換され、胆汁を経て体外に排出される。しかしながら胆汁におけるコレステロール含有量が増えすぎた場合は、コレステロールが結晶化し、胆石の原因となってしまうことが知られていることから、コレステロール逆転送系を亢進させる場合は、胆石が副作用として懸念される。 ¡Cholesterol carried to the liver is converted into bile acids and discharged through the bile. However, it is known that if the cholesterol content in bile increases too much, cholesterol will crystallize and cause gallstones, so gallstones are a concern as a side effect when enhancing the reverse cholesterol transport system. The
　また、小腸におけるコレステロールの吸収の分子機構として、ニーマンピック(Niemann-Pick)C1Like1（NPC1L1）が関与することが知られており、NPC1L1を阻害することにより、食物由来のコレステロールの吸収を阻害すると考えられている。小腸におけるコレステロール吸収阻害は、生体外へのコレステロール排出促進に寄与し得ると考えられ、脂質代謝改善において望ましい。 In addition, it is known that Niemann-Pick C1Like1 (NPC1L1) is involved in the molecular mechanism of cholesterol absorption in the small intestine, and by inhibiting NPC1L1, it is thought to inhibit the absorption of food-derived cholesterol. It has been. Inhibition of cholesterol absorption in the small intestine is thought to contribute to the promotion of cholesterol excretion outside the body and is desirable in improving lipid metabolism.
　また肝臓においては、アセチルCoAカルボキシラーゼや脂肪酸合成酵素（以下FAS）、SCD-1により、脂肪酸の合成が促進されることが知られている。これらの発現はSREBP（sterol regulatory element binding protein）によって制御される。SREBPにはSREBP1a、SREBP1c、SREBP2の３つのアイソフォームが存在し、SREBP1cは脂肪酸、トリグリセリド合成、SREBP2はコレステロール合成、SREBP1aは脂肪酸・コレステロール合成の両方を制御するといわれている。従来の合成LXRアゴニストはSREBP1c及びABCA1の発現をともに亢進方向に制御していたが、脂質代謝を改善するためには、トリグリセリド合成を促進するSREBP1c及び、その下流のFAS、SCD-1は発現抑制方向に、コレステロール輸送・排出作用や抗動脈硬化症作用を有するABCA1は発現亢進方向に制御するのが好ましいと考えられる。 Also, in the liver, it is known that fatty acid synthesis is promoted by acetyl-CoA carboxylase, fatty acid synthase (hereinafter FAS), and SCD-1. Their expression is controlled by SREBP (sterol regulatory element binding protein). SREBP has three isoforms, SREBP1a, SREBP1c, and SREBP2. SREBP1c is said to control fatty acid and triglyceride synthesis, SREBP2 to synthesize cholesterol, and SREBP1a to regulate both fatty acid and cholesterol synthesis. Conventional synthetic LXR agonists both upregulated the expression of SREBP1c and ABCA1, but in order to improve lipid metabolism, SREBP1c that promotes triglyceride synthesis and its downstream FAS and SCD-1 were suppressed. It is considered that ABCA1 having cholesterol transport / excretion action and anti-arteriosclerosis action is preferably controlled in the direction of enhanced expression.
　本発明のLXRアゴニストは、in　vitroにおいて肝及び末梢組織のABCA1もしくはABCG1の発現を増加させる。また、既存のLXRアゴニストであるTO901317（N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-N-(2,2,2-trifluoroethyl)benzenesulfonamide）よりはやや劣るものの、末梢組織からのコレステロール排出能（ApoA-Iへのコレステロール受け渡し量の増加作用）を有する。また、本発明のLXRアゴニストは、in　vivoにおいて血中のHDLコレステロール（HDL-C）量を増加させる。
　TO901317などの既知の合成LXRアゴニストはABCA1やABCG1と同様にLXR支配下遺伝子であるSREBP1c、及びそれに伴う脂肪酸合成酵素（FAS）などの中性脂肪合成に関わる遺伝子発現増加を介して、血中トリグリセリドレベルを増加させることが知られている。本発明のLXRアゴニストは、トリグリセリド合成等に関する作用は高くなく、より安全な薬剤ということができる。また、本発明のLXRアゴニストは胆汁中のコレステロール含有量を上昇させることもなく、小腸におけるNPC1L1 mRNA発現レベルを抑制し、コレステロール吸収を低減させることも可能である。 The LXR agonist of the present invention increases the expression of ABCA1 or ABCG1 in liver and peripheral tissues in vitro. In addition, the existing LXR agonist TO901317 (N- [4- (1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl) phenyl] -N- (2,2,2- Although slightly inferior to (trifluoroethyl) benzenesulfonamide), it has the ability to excrete cholesterol from peripheral tissues (increase in the amount of cholesterol delivered to ApoA-I). In addition, the LXR agonist of the present invention increases the amount of HDL cholesterol (HDL-C) in blood in vivo.
Similar to ABCA1 and ABCG1, known synthetic LXR agonists, such as TO901317, increase triglycerides in blood through increased expression of genes related to neutral fat synthesis, such as SREBP1c, which is a LXR-controlled gene, and fatty acid synthase (FAS). It is known to increase levels. The LXR agonist of the present invention does not have a high effect on triglyceride synthesis and can be said to be a safer drug. Further, the LXR agonist of the present invention can suppress the NPC1L1 mRNA expression level in the small intestine and reduce cholesterol absorption without increasing the cholesterol content in bile.
　本発明のLXRアゴニストは、LXR、より詳しくはLXRα及びLXRβ特異的にアゴニスト活性を有する。本発明のLXRアゴニストは、LXRに関連する疾患（脂質代謝異常症、アテローム性動脈硬化及び心血管障害等）の治療又は予防剤の有効成分として使用することができる。そして、本発明のLXRアゴニストは脂質代謝改善剤として使用することができる。本発明は、前記脂質代謝改善剤を有効成分として含む、脂質代謝異常症、アテローム性動脈硬化及び心血管障害等、いずれかの疾患の治療又は予防剤にもおよぶ。 The LXR agonist of the present invention has agonist activity specifically for LXR, more specifically LXRα and LXRβ. The LXR agonist of the present invention can be used as an active ingredient of a therapeutic or prophylactic agent for diseases related to LXR (lipid metabolism disorders, atherosclerosis, cardiovascular disorders, etc.). The LXR agonist of the present invention can be used as a lipid metabolism improving agent. The present invention also extends to a therapeutic or prophylactic agent for any disease, such as dyslipidemia, atherosclerosis and cardiovascular disorder, comprising the lipid metabolism improving agent as an active ingredient.
　本発明のLXRアゴニストからなる脂質代謝改善剤を有効成分とする医薬組成物も本発明の範囲に含まれる。医薬組成物は、脂質代謝異常及び脂質代謝異常に伴う各種疾患の治療剤又は改善剤として使用することができる。脂質代謝異常としては、脂質異常症（dyslipidemia）、例えば高コレステロール血症、高LDLコレステロール血症、低HDLコレステロール血症、高トリグリセリド血症等が挙げられる。脂質代謝異常に伴う各種疾患の具体例としては特に限定されないが、例えば、アテローム性動脈硬化及び心血管障害などが挙げられる。さらに、本発明は、上述した脂質代謝異常及び脂質代謝異常に伴う各種疾患の治療方法及び／又は予防方法にも及ぶ。 A pharmaceutical composition comprising a lipid metabolism improving agent comprising the LXR agonist of the present invention as an active ingredient is also included in the scope of the present invention. The pharmaceutical composition can be used as a therapeutic or ameliorating agent for various disorders associated with abnormal lipid metabolism and abnormal lipid metabolism. Examples of dyslipidemia include dyslipidemia such as hypercholesterolemia, high LDL cholesterolemia, low HDL cholesterolemia, and hypertriglyceridemia. Specific examples of various diseases associated with abnormal lipid metabolism are not particularly limited, and examples thereof include atherosclerosis and cardiovascular disorders. Furthermore, the present invention extends to the above-described lipid metabolism abnormality and methods for treating and / or preventing various diseases associated with lipid metabolism abnormality.
　本発明は、本発明のLXRアゴニストを有効成分とする医薬組成物は、錠剤、カプセル剤、散剤、顆粒剤、丸剤、液剤、シロップ剤等の経口投与剤、注射剤、外用剤、坐剤、点眼剤等の非経口投与剤などの形態に応じて、経口投与又は非経口投与することができる。投与経路としては経口投与がより好適である。本発明のLXRアゴニストをそのまま投与してもよいが、経口用あるいは非経口用の医薬組成物として投与することが好ましい。本発明の医薬組成物は、常法に従って製造され、当業者に利用可能な製剤用添加物、即ち薬理学的及び製剤学的に許容しうる担体を用いて製造することができる。 The present invention is a pharmaceutical composition comprising the LXR agonist of the present invention as an active ingredient, such as tablets, capsules, powders, granules, pills, liquids, syrups, or the like, injections, external preparations, suppositories Depending on the form of a parenteral preparation such as eye drops, it can be administered orally or parenterally. As an administration route, oral administration is more preferable. The LXR agonist of the present invention may be administered as it is, but it is preferably administered as an oral or parenteral pharmaceutical composition. The pharmaceutical composition of the present invention is manufactured according to a conventional method, and can be manufactured using pharmaceutical additives that are available to those skilled in the art, that is, pharmacologically and pharmaceutically acceptable carriers.
　経口投与に適する医薬組成物としては、例えば、錠剤、カプセル剤、散剤、細粒剤、顆粒剤、液剤、及びシロップ剤等を挙げることができ、非経口投与に適する医薬組成物としては、例えば、注射剤、点滴剤、坐剤、吸入剤等を挙げることができる。上記の医薬組成物の製造に用いられる薬理学的及び製剤学的に許容しうる担体としては、例えば、賦形剤、崩壊剤ないし崩壊補助剤、結合剤、滑沢剤、コーティング剤、色素、希釈剤、基剤、溶解剤ないし溶解補助剤、等張化剤、pH調節剤、安定化剤、噴射剤、及び粘着剤等を挙げることができる。 Examples of the pharmaceutical composition suitable for oral administration include tablets, capsules, powders, fine granules, granules, liquids, and syrups. Examples of the pharmaceutical composition suitable for parenteral administration include Injections, drops, suppositories, inhalants and the like. Examples of pharmacologically and pharmaceutically acceptable carriers used in the production of the above pharmaceutical composition include, for example, excipients, disintegrating agents or disintegrating aids, binders, lubricants, coating agents, dyes, Diluents, bases, solubilizers or solubilizers, isotonic agents, pH adjusters, stabilizers, propellants, adhesives and the like can be mentioned.
　本発明の医薬組成物の投与量は、症状の程度、投与経路、投与対象、服用者の年齢、体重などに応じて適宜決定することができる。本発明の医薬組成物は、数回にわたり投与してもよいし、複数回のクールに分け、一クール当たりの投与回数、投与間隔などを任意に設定することができる。例えば、経口投与の場合には有効成分としてのLXRアゴニストを成人一日あたり0.01～1000mg程度の範囲で用いることができる。 The dosage of the pharmaceutical composition of the present invention can be appropriately determined according to the degree of symptoms, administration route, administration subject, age of the user, body weight, and the like. The pharmaceutical composition of the present invention may be administered several times, divided into multiple courses, and the number of administrations per course, the administration interval, etc. can be arbitrarily set. For example, in the case of oral administration, an LXR agonist as an active ingredient can be used in the range of about 0.01 to 1000 mg per adult day.
　本発明のLXRアゴニストは医薬組成物の他、食品等の添加剤として使用してもよい。本発明のLXRアゴニストを有効量含む食品は、特定保健用食品等の健康食品に使用することができ、日常的に服用することで、脂質代謝異常や、それに伴う疾患の予防及び改善効果を期待することができる。服用量については、症状の程度、投与経路、投与対象、服用者の年齢、体重などに応じて適宜決定することができる。 The LXR agonist of the present invention may be used as an additive for foods and the like in addition to the pharmaceutical composition. A food containing an effective amount of the LXR agonist of the present invention can be used for health foods such as foods for specified health use, and is expected to prevent and improve lipid metabolism abnormalities and associated diseases when taken daily. can do. The dose can be appropriately determined according to the degree of symptoms, administration route, administration subject, age of the user, body weight, and the like.
　以下、理解を深めるために、実施例により本発明について具体的に説明するが、本発明は以下の実施例に限定されないことはいうまでもない。 Hereinafter, in order to deepen the understanding, the present invention will be specifically described with reference to examples. However, it is needless to say that the present invention is not limited to the following examples.
（実施例１）24-NorのLXRアゴニスト活性の評価
　本実施例では、以下の式(I)に示す24-NorのLXRアゴニスト活性を確認した。24-Norは後述する実施例９の手法により合成して入手した。LXRα又はLXRβ発現プラスミド及びレポータータンパク質としてルシフェラーゼを発現するプラスミド（pLXREx4-tk-Luc）を形質導入したLXRα、又はLXRβ発現COS-7細胞（アフリカミドリザル腎臓由来細胞）を用いてLXRアゴニスト活性を確認した。ジメチルスルホキシド（DMSO）に溶解した24-Norをさらに培地で希釈することにより調製した各種濃度の24-Norを用いて、LXRα又はLXRβ発現COS-7細胞を24時間処理し、ルシフェラーゼ活性を測定することで24-NorのLXRリガンド活性を評価した。既存のオキシステロール（22(R)-hydroxycholesterol: 22(R)-HC）、及び24-Norに類似した分子構造を有する以下の式(II)に示す24-Nor-5β-cholestane-3α,7α,25-triolと式(III)に示すHDC-OHを比較例とした。測定は３回行った。 (Example 1) Evaluation of LXR agonist activity of 24-Nor In this example, the LXR agonist activity of 24-Nor represented by the following formula (I) was confirmed. 24-Nor was synthesized and obtained by the method of Example 9 described later. LXR agonist activity was confirmed using LXRα or LXRβ-expressing COS-7 cells (African green monkey kidney-derived cells) transduced with LXRα or LXRβ expression plasmid and a luciferase-expressing plasmid (pLXREx4-tk-Luc) as a reporter protein . Treatment of LXRα or LXRβ-expressing COS-7 cells with 24-Nor of various concentrations prepared by further diluting 24-Nor dissolved in dimethyl sulfoxide (DMSO) with a medium and measuring luciferase activity Thus, the LXR ligand activity of 24-Nor was evaluated. Existing oxysterol (22 (R) -hydroxycholesterol: 22 (R) -HC) and 24-Nor-5β-cholestane-3α, 7α shown in the following formula (II) having a molecular structure similar to 24-Nor 25-triol and HDC-OH represented by formula (III) were used as comparative examples. The measurement was performed 3 times.
Figure JPOXMLDOC01-appb-C000009
Figure JPOXMLDOC01-appb-C000009
Figure JPOXMLDOC01-appb-C000010
Figure JPOXMLDOC01-appb-C000010
Figure JPOXMLDOC01-appb-C000011
Figure JPOXMLDOC01-appb-C000011
　結果を図２に示す。本発明の24-Norは、LXRα及びLXRβのいずれに対しても内因性LXRアゴニストであるオキシステロール（22(R)-HC）に比べて、優れたアゴニスト作用を示すことが確認された。 The results are shown in FIG. It was confirmed that 24-Nor of the present invention exhibits an excellent agonistic effect on both LXRα and LXRβ as compared with oxysterol (22 (R) -HC) which is an endogenous LXR agonist.
（実施例２）24-Norの各種核内受容体に対するアゴニスト活性の評価
　本実施例ではLXRα及びLXRβの他、胆汁酸を内因性リガンドとする核内受容体（farnesoid X receptor: FXR、Gタンパク質受容体：TGR5）、ビタミンD 誘導体をリガンドとする核内受容体（vitamin D receptor: VDR）に対する24-Norのアゴニスト活性も確認した。 (Example 2) Evaluation of agonist activity for various nuclear receptors of 24-Nor In this example, in addition to LXRα and LXRβ, nuclear receptors using bile acids as endogenous ligands (farnesoid X receptor: FXR, G protein) Receptor: TGR5), 24-Nor agonist activity against nuclear receptor (vitamin D receptor: VDR) with vitamin D derivative as a ligand was also confirmed.
　LXRα及びLXRβに対するアゴニスト活性は、実施例２に記載の方法と同手法により、ルシフェラーゼ活性を測定することにより確認した。FXR及びTGR5に対するアゴニスト活性は、文献（Iguchi Y. et al., J. Lipid Res., 51, 1432-1441, 2010）に記載の方法に従って測定した。 VDRに対するアゴニスト活性は、ヒトVDR及びヒトRXRα発現プラスミド、VDR応答性ルシフェラーゼ発現プラスミド（pCYP3A4-ER6x3-tk-Luc）、及び形質導入のコントロールプラスミドとしてRenillaルシフェラーゼプラスミドを形質導入したHEK293T細胞（ヒト胎児腎臓由来細胞）を用いて測定した。1μMの24-Nor含有培地で上記細胞を24時間処理した後、それらのルシフェラーゼ活性を既知の方法で測定し、24-Norの各受容体への作用を評価した。また、DMSOを陰性コントロールとし、22(R)-HC、ケノデオキシコール酸（CDCA）、リトコール酸（LCA：lithocholic　acid：コール酸の7,12α-脱水酸化誘導体）、及び1,25-ヒドロキシビタミンD3（1,25-hydroxyvitamin D3）をそれぞれLXRα/β、FXR、TGR5、VDRの陽性コントロールとした。 Agonist activity against LXRα and LXRβ was confirmed by measuring luciferase activity by the same method as described in Example 2. Agonist activity against FXR and TGR5 was measured according to the method described in the literature (Iguchi Y. et al., J. Lipid Res., 51, 1432-1441, 2010). Agonist activity against VDR is expressed in human VDR and human RXRα expression plasmid, VDR-responsive luciferase expression plasmid (pCYP3A4-ER6x3-tk-Luc), and HEK293T cells transduced with Renilla luciferase plasmid as a control plasmid for transduction (human embryonic kidney) Cell). After the cells were treated with 1 μM 24-Nor-containing medium for 24 hours, their luciferase activity was measured by a known method, and the effect of 24-Nor on each receptor was evaluated. DMSO as a negative control, 22 (R) -HC, chenodeoxycholic acid (CDCA), lithocholic acid (LCA: lithocholic acid: 7,12α-dehydrated oxidized derivative of cholic acid), and 1,25-hydroxyvitamin D 3 (1,25-hydroxyvitamin D 3 ) was used as a positive control for LXRα / β, FXR, TGR5, and VDR, respectively.
　結果を図３に示す。本発明の24-Norは、LXRα及びLXRβに対し特異的にアゴニスト活性を有することが確認され、その他の核内受容体に対しては陰性コントロールと同程度の作用を示したのみであった。この結果より、本発明の24-Norは、LXRα及びLXRβ特異的に作用することが確認された。 The results are shown in FIG. The 24-Nor of the present invention was confirmed to have agonist activity specifically for LXRα and LXRβ, and only showed the same effect as the negative control for other nuclear receptors. From this result, it was confirmed that 24-Nor of the present invention acts specifically for LXRα and LXRβ.
（実施例３）脂質代謝に関連する因子の遺伝子発現に及ぼす影響について
　LXRα及びβはリガンド依存性核内受容体であり、特にLXRαは肝臓やマクロファージなどに多く発現している。これらはLXR応答領域（以下LXRE）とよばれる10塩基程度のDNA配列に結合し、標的遺伝子の転写をオキシステロール依存的に活性化する。 (Example 3) Effect of factors related to lipid metabolism on gene expression LXRα and β are ligand-dependent nuclear receptors, and LXRα is particularly expressed in the liver, macrophages and the like. These bind to a DNA sequence of about 10 bases called LXR response region (hereinafter referred to as LXRE) and activate transcription of the target gene in an oxysterol-dependent manner.
　本実施例ではLXR支配下遺伝子であるABCA1、ABCG1及びSREBP1cのmRNA発現に及ぼす24-Norの影響を確認した。既存のオキシステロール化合物である22(R)-HC及び既存のLXRアゴニストであるTO901317（N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-N-(2,2,2-trifluoroethyl)benzenesulfonamide）を比較例とした。THP-1細胞（ヒト・単球由来細胞）及びHuh-7細胞（ヒト・肝細胞癌由来細胞）を24-Nor、22(R)-HC又はTO901317で24時間処理し、各細胞のtotal RNAを抽出した。ABCA1、ABCG1及びSREBP1cのmRNA量をRT-PCR法により測定した。DMSOのみで処理したときを陰性コントロールとした。 In this example, the influence of 24-Nor on the mRNA expression of ABCA1, ABCG1 and SREBP1c, which are LXR-controlled genes, was confirmed. The existing oxysterol compound 22 (R) -HC and the existing LXR agonist TO901317 (N- [4- (1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl) phenyl] -N- (2,2,2-trifluoroethyl) benzenesulfonamide) was used as a comparative example. THP-1 cells (human and monocyte-derived cells) and Huh-7 cells (human and hepatocellular carcinoma-derived cells) are treated with 24-Nor, 22 (R) -HC or TO901317 for 24 hours, and the total RNA of each cell Extracted. The mRNA amounts of ABCA1, ABCG1 and SREBP1c were measured by RT-PCR method. The negative control was treated with DMSO alone.
　結果を図４及び図５に示す。mRNA量は、DMSOで処理したときを1とし、相対量で示した。THP-1細胞において本発明の24-Norは、22(R)-HC処理時と同等もしくはそれ以上ABCA1及びABCG1 のmRNAを増加させた（図４）。一方、Huh-7細胞において、24-NorはTHP-1細胞で確認されたようにABCG1 mRNA発現レベルを増加させたが、SREBP1c mRNA発現レベルを変化させなかった（図５）。TO901317処理によってSREBP1c mRNA発現レベルは増加していたことから、24-Nor(I)はTO901317投与で観察されたトリグリセリド上昇を引き起こさず、HDL上昇作用のみを示すことが示唆された。この点において本発明のLXRアゴニスト、すなわち24-NorがTO901317よりも優れていることが確認された。 The results are shown in FIGS. The amount of mRNA was 1 when treated with DMSO, and expressed as a relative amount. In THP-1 cells, 24-Nor of the present invention increased ABCA1 and ABCG1 mRNAs at the same level as or higher than those treated with 22 (R) -HC (FIG. 4). On the other hand, in Huh-7 cells, 24-Nor increased ABCG1 mRNA expression level as confirmed in THP-1 cells, but did not change SREBP1c mRNA expression level (Fig. 5). Since SREBP1c mRNA expression level was increased by treatment with TO901317, it was suggested that 24-Nor (I) did not cause the increase in triglyceride observed with TO901317 administration, but only showed an HDL raising effect. In this respect, it was confirmed that the LXR agonist of the present invention, ie, 24-Nor is superior to TO901317.
（実施例４）コレステロールの輸送・排出能について
　末梢細胞から肝臓へのコレステロール輸送はABCA1及びABCG1を介したApoA-Iへのコレステロール受け渡しから始まる。末梢細胞からコレステロールを受け取ったApoA-IはHDLとなり、肝臓へコレステロールを輸送する。 (Example 4) Cholesterol transport / excretion ability Cholesterol transport from peripheral cells to the liver begins with the delivery of cholesterol to ApoA-I via ABCA1 and ABCG1. ApoA-I that receives cholesterol from peripheral cells becomes HDL and transports cholesterol to the liver.
　本実施例では24-Norについて、末梢細胞からのコレステロールの排出能を確認した。近年、ApoA-I非依存的なコレステロール排出機構も確認されているため、コレステロール排出能は、10μg/mLのApoA-Iを含む系（+ApoA-I）と含まない系（-ApoA-I）でTHP-1細胞（ヒト・単球由来細胞）を、1μMの24-Norで24時間処理し、培地中に放出されたコレステロール量の測定により確認した。コレステロール量の測定は市販のキットであるAmplex(R) Red Cholesterol Assay Kit (Invitrogen)を用いて行った。DMSOを陰性コントロールとし、既存のLXRアゴニストであるTO901317を比較例とした。 In this example, the ability to excrete cholesterol from peripheral cells was confirmed for 24-Nor. In recent years, the mechanism of cholesterol excretion independent of ApoA-I has also been confirmed, so the cholesterol excretion ability is the system containing 10 μg / mL ApoA-I (+ ApoA-I) and the system not containing (-ApoA-I) Then, THP-1 cells (human monocyte-derived cells) were treated with 1 μM 24-Nor for 24 hours and confirmed by measuring the amount of cholesterol released into the medium. The cholesterol level was measured using a commercially available kit, Amplex (R) Red Cholesterol Assay Kit (Invitrogen). DMSO was used as a negative control, and TO901317, an existing LXR agonist, was used as a comparative example.
　結果を図６に示す。本発明の24-Norは、既存のLXRアゴニストであるTO901317よりはやや劣るものの、ApoA-Iの有無に関わらず、濃度依存的にコレステロールの輸送能を増加させることが認められた。しかしながら、実施例３で示したように、TO901317は、ABCA1、ABCG1だけでなくSREBP1c mRNAについても強い発現能を示した。このことからTO901317はトリグリセリド合成も促進することとなり、副作用が高い薬剤といえる。一方、24-Norは必要な活性は有するものの、トリグリセリド合成等に関する作用は高くなく、より安全な薬剤ということができる。 The results are shown in FIG. Although 24-Nor of the present invention is slightly inferior to TO901317, which is an existing LXR agonist, it was observed that the cholesterol transport ability was increased in a concentration-dependent manner regardless of the presence or absence of ApoA-I. However, as shown in Example 3, TO901317 showed strong expression ability not only for ABCA1 and ABCG1, but also for SREBP1c mRNA. This indicates that TO901317 also promotes triglyceride synthesis and is a drug with high side effects. On the other hand, although 24-Nor has the necessary activity, it does not have a high effect on triglyceride synthesis and can be said to be a safer drug.
（実施例５）24-Norのin　vivo投与による影響の確認
　マウスを用いてin vivoにおける24-Norの作用について検討を行った。方法として、C57BL/6Nマウス（雄、8週齢）を、生理食塩水投与群（陰性コントロール：N.C.）、TO901317の10mg/kg投与群及び50mg/kg投与群、24-Norの10mg/kg投与群及び50mg/kg投与群の5群に分け、それぞれ3日間、1日1回経口投与（生理食塩水の懸濁液として）した。1晩絶食後頚椎脱臼によりマウスを安楽死させ、体重を測定し、肝臓を採取して肝重量（湿重量）を測定した。 (Example 5) Confirmation of the effect of in vivo administration of 24-Nor The effect of 24-Nor in vivo was examined using mice. As a method, C57BL / 6N mice (male, 8 weeks old) were administered with physiological saline (negative control: NC), TO901317 10 mg / kg and 50 mg / kg, 24-Nor 10 mg / kg. The group was divided into 5 groups and a 50 mg / kg administration group, each of which was orally administered (as a physiological saline suspension) once a day for 3 days. After fasting overnight, mice were euthanized by cervical dislocation, body weight was measured, liver was collected and liver weight (wet weight) was measured.
　その結果を、図７に示す。各群間に有意な体重の差異は確認されなかった。また、TO901317処理により肝重量が有意に増加していたことから肝肥大が認められたが、24-Norでは認められなかった。24-Norは、LXR特異的なアゴニスト作用を示すことから、肝肥大が認められなかったと考えられる。 The result is shown in FIG. There was no significant difference in body weight between groups. In addition, liver enlargement was observed because liver weight was significantly increased by TO901317 treatment, but it was not observed in 24-Nor. Since 24-Nor exhibits an LXR-specific agonistic action, it is considered that hepatic hypertrophy was not observed.
（実施例６）24-Norのin　vivo投与によるコレステロール量に対する影響の確認
　実施例５の方法と同様にして、マウスに24-Norを投与し、1日毎に糞を回収した。実施例５と同様にしてマウスを安楽死させ、血液、胆汁、肝臓を採取した。血清、胆汁、肝臓及び糞中の総コレステロール量は、市販のキットであるAmplex(R) Red Cholesterol Assay Kit (invitrogen)を用いて測定した。血清中HDL-コレステロール量はLipoSEARCH受託解析サービスにより測定を行った。 (Example 6) Confirmation of influence on cholesterol level by in vivo administration of 24-Nor In the same manner as in Example 5, 24-Nor was administered to mice, and feces were collected every day. Mice were euthanized in the same manner as in Example 5, and blood, bile, and liver were collected. The total cholesterol levels in serum, bile, liver and feces were measured using a commercially available kit Amplex® Red Cholesterol Assay Kit (invitrogen). Serum HDL-cholesterol level was measured by LipoSEARCH contract analysis service.
　結果を図８及び図９に示す。
　図８に各群の血清中及び肝臓中の総コレステロール（T-C）及びHDL-コレステロール（HDL-C）レベルを測定した結果を示す。TO901317処理により、血清中の総コレステロール量（serum　T-C）、HDL-コレステロール量（serum　HDL-C）は有意に増加した。24-Nor処理でもTO901317とほぼ同様の結果が得られた。24-Nor処理においては総コレステロールの増加量はHDL-コレステロール量の増加量にほぼ等しかった。マウスにおいては、総コレステロール量のうち、HDL-コレステロールの割合が80%と高いことが知られている。また、肝臓中の総コレステロール量はTO901317処理及び24-Nor処理により減少していた。これは、胆汁中へのコレステロール排泄が促進された結果によるものと推測される。
　図９に胆汁中の総コレステロール量と３日目の糞中の中性ステロール量を測定した結果を示す。TO901317投与群では用量依存的に増加したが、24-Nor投与群については10mg/kg投与群で増加していたものの50mg/kgでは減少していた。糞中への中性ステロール排泄量は24-Nor投与群においても有意に増加していた。これらの結果から、24-Norはコレステロールの胆汁中での貯留時間を減少させ、速やかに消化管中に排泄する可能性が考えられる。 The results are shown in FIGS.
FIG. 8 shows the results of measuring total cholesterol (TC) and HDL-cholesterol (HDL-C) levels in serum and liver of each group. TO901317 treatment significantly increased serum total cholesterol (serum TC) and HDL-cholesterol (serum HDL-C). 24-Nor treatment gave almost the same result as TO901317. In the 24-Nor treatment, the increase in total cholesterol was almost equal to the increase in HDL-cholesterol. In mice, it is known that the proportion of HDL-cholesterol is as high as 80% of the total cholesterol amount. In addition, the total amount of cholesterol in the liver was decreased by TO901317 treatment and 24-Nor treatment. This is presumably due to the result of accelerated cholesterol excretion in bile.
FIG. 9 shows the results of measuring the total cholesterol content in bile and the neutral sterol content in feces on the third day. In the TO901317-administered group, it increased in a dose-dependent manner, but in the 24-Nor-administered group, it increased in the 10 mg / kg-administered group, but decreased at 50 mg / kg. Neutral sterol excretion in feces was also significantly increased in the 24-Nor group. These results suggest that 24-Nor reduces the retention time of cholesterol in bile and can be rapidly excreted in the digestive tract.
（実施例７）24-Norのin　vivo投与によるLXR支配下遺伝子の発現に対する影響の確認
　実施例５の方法と同様にして、マウスに24-Norを投与した。実施例５と同様にしてマウスを安楽死させ、血液、胆汁、肝臓、小腸を採取した。肝臓及び小腸のLXR支配下遺伝子（肝臓はABCA1、ABCG1、小腸はNPC1L1）のmRNA発現レベルを、各臓器のtotal RNAを抽出後、Real time-RT-PCR法で測定した。 (Example 7) Confirmation of influence on expression of LXR-controlled gene by in vivo administration of 24-Nor In the same manner as in Example 5, 24-Nor was administered to mice. Mice were euthanized as in Example 5, and blood, bile, liver, and small intestine were collected. The mRNA expression levels of LXR-controlled genes in the liver and small intestine (liver ABCA1, ABCG1, small intestine NPC1L1) were measured by real time-RT-PCR after extracting total RNA from each organ.
　結果を図１０及び図１１に示す。
　図１０に、肝臓におけるLXR支配下の各遺伝子の発現レベルを評価した結果を示す。肝ABCA1、ABCG1の各mRNA発現レベルは24-Norによって増加傾向を示した。この結果より、24-Nor処理によりコレステロール逆転送系が促進された可能性が示唆された。
　小腸においては、LXRアゴニストがコレステロールトランスポーターであるNPC1L1の発現を抑制させることでコレステロールの吸収を低下させることが知られているため、24-Norが同様の効果を有するか検討した。結果を図１１に示す。24-NorはNPC1L1 mRNA発現レベルを有意に低下させることが示された。よって、24-Norは小腸管内に存在するコレステロールの吸収を低下させることも示唆された。 The results are shown in FIGS.
FIG. 10 shows the results of evaluating the expression level of each gene under the LXR control in the liver. Liver ABCA1 and ABCG1 mRNA expression levels showed an increasing tendency with 24-Nor. This result suggests that the 24-Nor treatment may promote the reverse cholesterol transfer system.
In the small intestine, LXR agonists are known to reduce cholesterol absorption by suppressing the expression of NPC1L1, which is a cholesterol transporter. Therefore, whether 24-Nor has a similar effect was examined. The results are shown in FIG. 24-Nor was shown to significantly reduce NPC1L1 mRNA expression levels. Therefore, it was suggested that 24-Nor reduces the absorption of cholesterol present in the small intestine.
（実施例８）24-Norのin　vivo投与による血中トリグリセリド量に対する影響の確認
　実施例５の方法と同様にして、マウスに24-Norを投与し、マウスを安楽死させた後、血液及び肝臓を採取した。血清及び肝臓中の中性脂肪量（TG）は市販のキットであるTriglyceride assay kit (Biovision)を用いて行った。また肝臓中の脂肪酸合成に関与する遺伝子（SREBP1c、FAS、SCD-1）のmRNA発現レベルを、肝臓のtotal RNAを抽出後、Real time-RT-PCR法で測定した。 (Example 8) Confirmation of effect on blood triglyceride level by in vivo administration of 24-Nor In the same manner as in Example 5, 24-Nor was administered to mice and the mice were euthanized. Liver was collected. The amount of triglyceride (TG) in serum and liver was measured using a commercially available kit, Triglyceride assay kit (Biovision). In addition, mRNA expression levels of genes (SREBP1c, FAS, SCD-1) involved in fatty acid synthesis in the liver were measured by real time-RT-PCR after extracting total RNA from the liver.
　結果を図１２及び図１３に示す。図１２に血中トリグリセリド（TG）量及び肝臓トリグリセリド量、図１３に各種遺伝子の発現量の測定結果を示す。TO901317は、肝SREBP1c、FAS、SCD-1などの脂肪酸合成に関与する遺伝子発現を促進させ、さらに血中トリグリセリド量も増加させた。一方、24-Norはこれらの遺伝子発現及び血中トリグリセリド量を変化させないことがわかった。これらのことから、24-Norはコレステロール代謝を選択的に制御するLXRアゴニストであることが示された。 The results are shown in FIG. 12 and FIG. FIG. 12 shows the measurement results of blood triglyceride (TG) level and liver triglyceride level, and FIG. 13 shows the expression levels of various genes. TO901317 promoted the expression of genes involved in fatty acid synthesis such as liver SREBP1c, FAS, and SCD-1, and also increased blood triglyceride levels. On the other hand, it was found that 24-Nor did not change the expression of these genes and the blood triglyceride level. These results indicate that 24-Nor is an LXR agonist that selectively regulates cholesterol metabolism.
（実施例９）LXRアゴニストの合成
　一般式（Ｉ）におけるR1及びR2が、以下の表２に示される基である化合物を、図１に示す合成経路に準じてグリニャール反応を用いて合成した。表２におけるR1及びR2は、一般式(I)におけるR1及びR2である。
Figure JPOXMLDOC01-appb-T000012
 (Example 9) Synthesis of LXR agonist A compound in which R 1 and R 2 in the general formula (I) are groups shown in Table 2 below was synthesized using a Grignard reaction according to the synthesis route shown in FIG. did. R 1 and R 2 in Table 2 is R 1 and R 2 in the general formula (I).
Figure JPOXMLDOC01-appb-T000012
　得られた化合物の確認を、Gas Chromatograph Mass Spectrometer (GC-MS)及びLiquid Chromatograph Mass Spectrometer (LC-MS)にて行った。
　GC-MSについて、まず化合物 1mgをピリジン0.5mLに溶解し、1,1,1,3,3,3-hexamethyldisilazane 0.2mL及びchlorotrimethylsilane 10dropsを加え、80℃、2時間反応させた。窒素気流下で溶媒を留去した後、ヘプタン0.5mLを加えた。懸濁、遠心した上清をGC-MS用サンプルとした。GC-MS装置はGCMS-QP2010 (SHIMADZU)を使用した。GC-MS条件は以下の通りで行った。
・GC条件
　カラムオーブン温度：240℃→ 2℃/ min.→ 300℃ (5min. Hold)　total 35min.
　キャリアガス：ヘリウム
　カラム：HP-5MS (Agilent Technologies, 膜厚0.25μm, 長さ 30m, 内径 0.25mm)
・MS条件
　イオン化法：EI (電子衝撃イオン化法)
　イオン源温度：200℃
　インターフェース温度：280℃ Confirmation of the obtained compound was performed by Gas Chromatograph Mass Spectrometer (GC-MS) and Liquid Chromatograph Mass Spectrometer (LC-MS).
For GC-MS, 1 mg of compound was first dissolved in 0.5 mL of pyridine, 0.2 mL of 1,1,1,3,3,3-hexamethyldisilazane and 0.2 mL of chlorotrimethylsilane were added, and reacted at 80 ° C. for 2 hours. After the solvent was distilled off under a nitrogen stream, 0.5 mL of heptane was added. The suspended and centrifuged supernatant was used as a sample for GC-MS. GCMS-QP2010 (SHIMADZU) was used as the GC-MS apparatus. GC-MS conditions were as follows.
・ GC condition Column oven temperature: 240 ℃ → 2 ℃ / min. → 300 ℃ (5min. Hold) total 35min.
Carrier gas: Helium Column: HP-5MS (Agilent Technologies, film thickness 0.25μm, length 30m, inner diameter 0.25mm)
・ MS condition Ionization method: EI (electron impact ionization method)
Ion source temperature: 200 ° C
Interface temperature: 280 ℃
　LC-MSによる確認は以下の通り行った。まず、化合物1mgをメタノールに溶解し、0.45μmフィルターでろ過したものをLC-MS用サンプルとした。LC-MS装置はJMS-T-100LC (JEOL)を使用した。LC-MS条件は以下の通りで行った。
・HPLC条件
　流量：0.2mL/min.
　カラム：XBridge C18 (WATERS, 粒子径 3.5μm, 長さ 100mm, 内径 2.1mm)
　移動溶媒：メタノール
 ・MS条件
　イオン化法：ESI (エレクトロスプレー法)  Confirmation by LC-MS was performed as follows. First, 1 mg of the compound was dissolved in methanol and filtered through a 0.45 μm filter to obtain a sample for LC-MS. The LC-MS apparatus used was JMS-T-100LC (JEOL). LC-MS conditions were as follows.
・ HPLC conditions Flow rate: 0.2mL / min.
Column: XBridge C18 (WATERS, particle size 3.5μm, length 100mm, inner diameter 2.1mm)
Mobile solvent: methanol MS conditions Ionization method: ESI (electrospray method)
　各化合物のGC-MS及びLC-MSによる分析結果を、表３に示す。各化合物の揮発性を高めることを目的として、トリメチルシリル誘導体化したものをGC-MS用サンプルとした。これらの化合物は表３に示したフラグメント以外はほとんど認められなかった。これらのフラグメントは23位と25位の炭素-炭素結合が切れ生じたものと推測できる。付加したアルキル基が増加するに従ってm/zが28ずつ増加していることからも各化合物が合成できたと考えられる。
　また、LC-MS分析では化合物から水素イオンが離脱したフラグメント、及び塩素イオンが付加したフラグメントがほとんどであったことが確認された。
Figure JPOXMLDOC01-appb-T000013
 Table 3 shows the results of GC-MS and LC-MS analysis of each compound. For the purpose of increasing the volatility of each compound, a trimethylsilyl derivative was used as a sample for GC-MS. These compounds were hardly recognized except the fragments shown in Table 3. It can be inferred that these fragments were generated by breaking carbon-carbon bonds at the 23 and 25 positions. It can be considered that each compound could be synthesized from the fact that m / z increased by 28 as the added alkyl group increased.
In addition, LC-MS analysis confirmed that most of the fragments were the fragments from which hydrogen ions were released from the compounds and the fragments to which chlorine ions were added.
Figure JPOXMLDOC01-appb-T000013
　24-NorのNMRの結果を、図１４上段に示した。24-NorはHyodeoxycholic acidのメチルエステル体（HDC-Me）のグリニア反応により合成されるため、HDC-MeのNMR結果についても併せて示した（図１４下段）。
　24-Norの側鎖末端に存在する2つのメチル基は1.199ppm及び1.204ppmに示された（3H, s, 25-CH3, 26-CH3）。若干のppmの違いは20位の不斉炭素の存在によるものと推測された。また、HDC-Meで認められる3.668ppm付近のピークはメチルエステルに由来するピークであり、このピークは24-Norでは確認できなかった。NMRの結果からも、24-Norが合成できたことが確認された。 The results of 24-Nor NMR are shown in the upper part of FIG. Since 24-Nor is synthesized by the Grineer reaction of methyl ester form (HDC-Me) of Hydeoxycholic acid, the NMR results of HDC-Me are also shown (lower part of FIG. 14).
Two methyl groups present at the side chain ends of 24-Nor were shown at 1.199 ppm and 1.204 ppm (3H, s, 25-CH 3 , 26-CH 3 ). The slight difference in ppm was presumed to be due to the presence of the 20th asymmetric carbon. In addition, the peak near 3.668 ppm observed in HDC-Me is a peak derived from methyl ester, and this peak could not be confirmed in 24-Nor. From the NMR results, it was confirmed that 24-Nor could be synthesized.
（実施例１０）各種化合物のLXRアゴニスト活性の確認
　実施例１と同様にして、実施例９にて合成した化合物のうち、24-Nor、24-Nor-Et、24-Nor-nPrについてLXRアゴニスト活性を確認した。各化合物の処理濃度は、図１５に記載の通りである。DMSO処理を陰性コントロール（N.C.）とした。 (Example 10) Confirmation of LXR agonist activity of various compounds In the same manner as in Example 1, among the compounds synthesized in Example 9, 24-Nor, 24-Nor-Et, and 24-Nor-nPr were LXR agonists. The activity was confirmed. The treatment concentration of each compound is as shown in FIG. DMSO treatment was used as a negative control (NC).
　結果を図１５に示す。上記化合物は、LXRα及びLXRβのいずれに対しても、優れたアゴニスト作用を示すことが確認された。特にエチル基を付加させた24-Nor-Etは、24-Norよりも低濃度で高い活性を有することが示された。 The results are shown in FIG. The above compound was confirmed to exhibit an excellent agonistic effect on both LXRα and LXRβ. In particular, it was shown that 24-Nor-Et to which an ethyl group was added had higher activity at a lower concentration than 24-Nor.
（実施例１１）各種化合物のLXRアゴニスト活性の確認
　実施例１と同様にして、実施例９にて合成した化合物についてLXRアゴニスト活性を確認した。各化合物の処理濃度は、図１６に記載の通りである。DMSO処理を陰性コントロール（N.C.）とした。
　また、実施例２と同様にして、実施例９にて合成した化合物について、RXRα、PPARγ、FXR、VDR、及びTGR5に対する作用を確認した。DMSO処理を陰性コントロール（N.C.）とし、各受容体に対するアゴニスト9-cis-RA、GW1929、GW4064、1,25-OH-VD3、LCAを陽性コントロールとして用いた。RXRα、PPARγ、FXR及びVDRに対する各化合物の処理濃度は１μMであり、TGR5に対する各化合物の処理濃度は図１８に記載の通りである。 (Example 11) Confirmation of LXR agonist activity of various compounds In the same manner as in Example 1, LXR agonist activity was confirmed for the compound synthesized in Example 9. The treatment concentration of each compound is as shown in FIG. DMSO treatment was used as a negative control (NC).
Further, in the same manner as in Example 2, the compound synthesized in Example 9 was confirmed to have an action on RXRα, PPARγ, FXR, VDR, and TGR5. DMSO treatment was used as a negative control (NC), and agonists 9-cis-RA, GW1929, GW4064, 1,25-OH-VD3, and LCA for each receptor were used as positive controls. The treatment concentration of each compound for RXRα, PPARγ, FXR and VDR is 1 μM, and the treatment concentration of each compound for TGR5 is as shown in FIG.
　LXRアゴニスト活性を確認した結果を図１６に示す。24-Nor-Et、24-Nor-nPr、及び24-Nor-iPrに強いアゴニスト活性が認められた。またLXR以外の受容体であるRXRα、PPARγ、VDR、及びFXRに対しては、いずれの化合物もアゴニスト活性を示さなかった（図１７）。なお、24-Nor-nPrについては、TGR5に対するアゴニスト活性が認められた（図１８）。  The result of confirming the LXR agonist activity is shown in FIG. Strong agonist activity was observed for 24-Nor-Et, 24-Nor-nPr, and 24-Nor-iPr. In addition, none of the compounds showed agonist activity against RXRα, PPARγ, VDR, and FXR, which are receptors other than LXR (FIG. 17). For 24-Nor-nPr, agonist activity against TGR5 was observed (FIG. 18).
（実施例１２）LXRアゴニストの合成
　一般式(I)において、R1がCH3でR2がCH2CH3の化合物を以下の方法により合成した（図１９）。
　hyodeoxycholate(HDCA, 1)をメタノールに溶解し、28%アンモニア水溶液、及び4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholium (DMT-MM)を加え室温で攪拌させることで化合物（IV）を得た。化合物（IV）を無水Tetrahydrofuran(THF)に溶解し、0℃で撹拌しながら、トリエチルアミン、無水トリフルオロ酢酸をゆっくり加え、徐々に温度を上げた還流させ、化合物（V）を得た。化合物（V）を無水THFに溶解し、Grignard試薬(CH3CH2MgX)ゆっくり加え、還流し反応させることで化合物（VI）を得た。化合物（VI）を無水THFに溶解し、Grignard試薬(CH3MgX)ゆっくり加え、還流し反応させることで化合物（VII）（24-Nor-Me, Et）を得ることができた。 Example 12 Synthesis of LXR Agonist In general formula (I), a compound in which R 1 was CH 3 and R 2 was CH 2 CH 3 was synthesized by the following method (FIG. 19).
Hyodeoxycholate (HDCA, 1) dissolved in methanol, 28% aqueous ammonia solution and 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methyl-morpholium (DMT-MM) Was added and stirred at room temperature to obtain Compound (IV). Compound (IV) was dissolved in anhydrous Tetrahydrofuran (THF), and while stirring at 0 ° C., triethylamine and trifluoroacetic anhydride were slowly added, and the temperature was gradually raised to reflux to obtain Compound (V). Compound (V) was obtained by dissolving Compound (V) in anhydrous THF, slowly adding Grignard reagent (CH3CH2MgX), and refluxing for reaction. Compound (VII) (24-Nor-Me, Et) was able to be obtained by dissolving Compound (VI) in anhydrous THF, slowly adding Grignard reagent (CH3MgX), and reacting under reflux.
　以下の表４に、24-Nor-Me, EtのGC-MS結果を示した。GC-MS分析のためのサンプル調製及びGC-MS方法は実施例９と同様に行った。実施例９に記載の化合物と同様に、23位と25位の炭素-炭素結合が切れ生じたフラグメント（[C(CH3)(CH2CH3)OTMS]+）が基準ピークとして検出され、それ以外のマスフラグメントはほとんど認められなかった。また、24-Norより14大きく、24-Nor-Etよりも14小さいm/z値であることから、24-Nor-Me, Etの合成を確認できた。
Figure JPOXMLDOC01-appb-T000014
 Table 4 below shows GC-MS results of 24-Nor-Me, Et. Sample preparation and GC-MS method for GC-MS analysis were performed in the same manner as in Example 9. Similar to the compound described in Example 9, a fragment ([C (CH 3 ) (CH 2 CH 3 ) OTMS] + ) in which the carbon-carbon bonds at positions 23 and 25 were broken was detected as a reference peak, Most other mass fragments were not recognized. Further, since the m / z value was 14 larger than 24-Nor and 14 smaller than 24-Nor-Et, the synthesis of 24-Nor-Me, Et could be confirmed.
Figure JPOXMLDOC01-appb-T000014
（実施例１３）LXRアゴニスト活性の確認
　実施例９にて合成した24-Nor-Me, EtのLXRα及びβに対するアゴニスト活性について検討した。実験は、実施例１０と同様にして行った。結果を図２０に示す。 (Example 13) Confirmation of LXR agonist activity The agonist activity of 24-Nor-Me, Et synthesized in Example 9 against LXRα and β was examined. The experiment was performed in the same manner as in Example 10. The results are shown in FIG.
　24-Nor-Me, Etは、24-Nor及び24-Nor-Etと同様に、LXRα及びβに対してアゴニスト活性を有していた。活性の強さは24-Norと同等であり、24-Nor-Etよりは若干小さいものであった。 24-Nor-Me and Et had agonistic activity against LXRα and β, similar to 24-Nor and 24-Nor-Et. The strength of activity was the same as that of 24-Nor, which was slightly smaller than 24-Nor-Et.
　以上詳述したように、本発明のLXRアゴニストは、核内受容体のうち、特にLXRα及び／又はLXRβに対して優れたアゴニスト活性を有する。また、本発明のLXRアゴニストは、脂質代謝に関わるABCA1及びABCG1遺伝子の発現を促進し、トリグリセリド合成に関わるSREBP1cの発現促進については、既存の薬剤よりも低い値を示した。このことから、本発明のLXRアゴニストはトリグリセリド合成等の副作用が軽減された優れた脂質代謝改善剤としての機能を有する。また、濃度依存的に優れたコレステロール輸送能を示すことが確認された。つまり、本発明のLXRアゴニストは、LXRα及びLXRβ特異的に作用することが確認され、その結果、脂質代謝調節機能を有することが確認された。コレステロールの輸送・排出能等に関しては、既存のLXRアゴニストであるTO901317よりはやや劣るものの、トリグリセリド合成等に関する作用は高くなく、より安全な薬剤として有用である。 As described in detail above, the LXR agonist of the present invention has an excellent agonist activity particularly against LXRα and / or LXRβ among the nuclear receptors. Further, the LXR agonist of the present invention promoted the expression of ABCA1 and ABCG1 genes involved in lipid metabolism, and showed a lower value than the existing drugs in promoting the expression of SREBP1c involved in triglyceride synthesis. Therefore, the LXR agonist of the present invention has a function as an excellent lipid metabolism improving agent with reduced side effects such as triglyceride synthesis. Further, it was confirmed that the cholesterol transport ability was excellent depending on the concentration. That is, the LXR agonist of the present invention was confirmed to act specifically on LXRα and LXRβ, and as a result, was confirmed to have a function of regulating lipid metabolism. Although it is slightly inferior to TO901317, which is an existing LXR agonist, in terms of cholesterol transport / excretion, etc., it is not highly effective in triglyceride synthesis and is useful as a safer drug.
　本発明のLXRアゴニストのうち、24-Norは、非特許文献６（Zepter (1972)）に報告されており、CAS#40551-80-2においてデータベースに登録されている公知の化合物であり、それ以外の化合物は新規化合物である。TO901317などの合成LXRアゴニストは、一般に調製過程（合成・精製・単離）が煩雑で、多くの時間と労力を費やしているのが現状であるが、本発明のLXRアゴニストは合成・精製・単離方法が容易である点も有利な効果を示すものである。 Among the LXR agonists of the present invention, 24-Nor is a known compound that is reported in Non-Patent Document 6 (Zepter (1972)) and registered in the database in CAS # 40551-80-2. Compounds other than are novel compounds. Synthetic LXR agonists such as TO901317 are generally complicated in the preparation process (synthesis / purification / isolation) and consume a lot of time and labor. However, the LXR agonist of the present invention is synthesized / purified / simple. The point that the separation method is easy also shows an advantageous effect.
　また本発明のLXRアゴニストは、in　vivo投与した際に、血中のHDLコレステロール量を増大させるとともに、肝臓においてABCA1、ABCG1 mRNA発現レベルを亢進させることが確認されている。このことから、本発明のLXRアゴニストは新生HDLの産生に寄与し、血中HDLコレステロール量を増大させると考えられる。近年、HCLコレステロールの低値が脂質代謝異常の独立した危険因子として確立されていることから、本発明のLXRアゴニストは副作用が少なく、より安全な薬剤として、HDLコレステロール値のみを増やす薬剤として使用することが期待される。 Further, it has been confirmed that the LXR agonist of the present invention increases the level of HDL cholesterol in blood and enhances the expression level of ABCA1 and ABCG1 mRNA in the liver when administered in vivo. From this, it is considered that the LXR agonist of the present invention contributes to the production of neoplastic HDL and increases the blood HDL cholesterol level. In recent years, since the low level of HCL cholesterol has been established as an independent risk factor for abnormal lipid metabolism, the LXR agonist of the present invention has fewer side effects and is used as a safer drug that increases only HDL cholesterol level. It is expected.

Claims (13)

  1. 以下の一般式(I)で表される化合物又はその薬学的に許容される塩からなる、肝臓Ｘ受容体（LXR）アゴニスト。
    Figure JPOXMLDOC01-appb-C000001
     （式中、R1およびR2は、それぞれ独立して、炭素原子数１～５の炭化水素基を表す。）     A liver X receptor (LXR) agonist comprising a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof:
    Figure JPOXMLDOC01-appb-C000001
     (In the formula, R 1 and R 2 each independently represents a hydrocarbon group having 1 to 5 carbon atoms.)
  2. 一般式(I)において、R1およびR2が、それぞれ独立して、炭素原子数１～３のアルキル基を表す化合物、又はその薬学的に許容される塩からなる、請求項１に記載の肝臓Ｘ受容体（LXR）アゴニスト。 The general formula (I), wherein R 1 and R 2 are each independently a compound representing an alkyl group having 1 to 3 carbon atoms, or a pharmaceutically acceptable salt thereof. Liver X receptor (LXR) agonist.
  3. 一般式(I)において、R1およびR2がともに、メチル基、エチル基、プロピル基のいずれかである化合物、又はその薬学的に許容される塩からなる、請求項１または２に記載の肝臓Ｘ受容体（LXR）アゴニスト。 The general formula (I), wherein R 1 and R 2 are both a methyl group, an ethyl group, or a propyl group, or a pharmaceutically acceptable salt thereof. Liver X receptor (LXR) agonist.
  4. 前記肝臓Ｘ受容体（LXR）が、LXRα及び／又はLXRβである、請求項１～３のいずれか１に記載の肝臓Ｘ受容体（LXR）アゴニスト。 The liver X receptor (LXR) agonist according to any one of claims 1 to 3, wherein the liver X receptor (LXR) is LXRα and / or LXRβ.
  5. 請求項１～４のいずれか１に記載の肝臓Ｘ受容体（LXR）アゴニストからなる脂質代謝改善剤。 A lipid metabolism improving agent comprising the liver X receptor (LXR) agonist according to any one of claims 1 to 4.
  6. 肝臓Ｘ受容体（LXR）アゴニストが血中HDLコレステロールを上昇させるものである、請求項５に記載の脂質代謝改善剤。 6. The lipid metabolism improving agent according to claim 5, wherein the liver X receptor (LXR) agonist increases blood HDL cholesterol.
  7. 請求項５又は６に記載の脂質代謝改善剤を有効成分として含む、脂質代謝異常又は脂質代謝異常に伴う疾患の治療又は予防剤。 A therapeutic or prophylactic agent for lipid metabolism abnormality or a disease associated with lipid metabolism abnormality, comprising the lipid metabolism improving agent according to claim 5 or 6 as an active ingredient.
  8. 請求項１～４のいずれかに記載の肝臓Ｘ受容体（LXR）アゴニストを有効成分として含む、医薬組成物。 A pharmaceutical composition comprising the liver X receptor (LXR) agonist according to any one of claims 1 to 4 as an active ingredient.
  9. 以下の一般式(I)で表される化合物又はその薬学的に許容される塩。
    Figure JPOXMLDOC01-appb-C000002
     （式中、R1およびR2は、それぞれ独立して、炭素原子数１～５の炭化水素基を表す。ただし、R1およびR2がともに、メチル基である場合を除く。）     A compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof:
    Figure JPOXMLDOC01-appb-C000002
     (In the formula, R 1 and R 2 each independently represents a hydrocarbon group having 1 to 5 carbon atoms, except when both R 1 and R 2 are methyl groups.)
  10. 一般式(I)において、R1およびR2が、それぞれ独立して、炭素原子数１～３のアルキル基を表す、請求項９に記載の化合物、又はその薬学的に許容される塩。 The compound according to claim 9, or a pharmaceutically acceptable salt thereof, wherein in the general formula (I), R 1 and R 2 each independently represents an alkyl group having 1 to 3 carbon atoms.
  11. 一般式(I)において、R1およびR2がともに、メチル基、エチル基、プロピル基のいずれかである、請求項９又は１０に記載の化合物、又はその薬学的に許容される塩。 The compound according to claim 9 or 10, or a pharmaceutically acceptable salt thereof, wherein, in the general formula (I), both R 1 and R 2 are any of a methyl group, an ethyl group, and a propyl group.
  12. 請求項５又は６に記載の脂質代謝改善剤、又は、請求項９～１１のいずれか１に記載の化合物又はその薬学的に許容される塩を対象に投与することにより、対象の脂質代謝異常又は脂質代謝異常に伴う疾患を治療又は予防する方法。 A lipid metabolism abnormality of a subject by administering to the subject the lipid metabolism improving agent of claim 5 or 6, or the compound of any one of claims 9 to 11 or a pharmaceutically acceptable salt thereof. Alternatively, a method for treating or preventing a disease associated with abnormal lipid metabolism.
  13. 以下の一般式(I)で表される化合物又はその薬学的に許容される塩を肝臓Ｘ受容体（LXR）と接触させることにより、肝臓Ｘ受容体（LXR）を作動させる方法。
    Figure JPOXMLDOC01-appb-C000003
      
    （式中、R1およびR2は、それぞれ独立して、炭素原子数１～５の炭化水素基である。）     A method of activating liver X receptor (LXR) by contacting a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof with liver X receptor (LXR).
    Figure JPOXMLDOC01-appb-C000003
    (In the formula, R 1 and R 2 are each independently a hydrocarbon group having 1 to 5 carbon atoms.)
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