JP2015193581A - Novel sterol compound and cholesterol absorption inhibitor containing this - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel sterol compound and a cholesterol absorption inhibitor.SOLUTION: The invention provides a novel sterol compound represented by the following chemical formula (I), and a cholesterol absorption inhibitor containing this.

Description

  The present invention relates to a novel sterol compound. Furthermore, the present invention relates to a cholesterol absorption inhibitor containing a novel sterol compound.

Increased blood cholesterol is a major risk factor for coronary heart disease. In order to lower blood cholesterol, it is basically limited to low cholesterol food, bile acidic sequestrants (cholestyramine and colestipol), nicotinic acid (niacin), fibrates and probucol. It was. However, these drugs and therapies have limited treatment.
Recently, lovastatin, simvastatin and pravastatin (HMG-CoA reductase inhibitors) have been shown to delay the progression of atheromatous lesions in the coronary and carotid arteries. Furthermore, simvastatin and pravastatin were found to reduce the risk of coronary heart disease.

In arteriosclerotic diseases, cholesteryl esters are a major component of atherosclerotic lesions and a major storage form of cholesterol in arterial wall cells. The formation of this cholesteryl ester is also an important step in the intestinal absorption of dietary cholesterol.
It is known that cholesterol taken into small intestinal epithelial cells is converted into cholesteryl ester in the endoplasmic reticulum in small intestinal epithelial cells, and then formed into chylomicron and absorbed into the body. Therefore, it is speculated that the inhibition of cholesteryl ester formation prevents intestinal absorption of dietary cholesterol, and the concomitant reduction in blood cholesterol suppresses the progression of atheromatous lesion formation, and prevents or improves arteriosclerosis. It is thought to be connected.

Certain hydroxy-substituted azetidinones such as ezetimibe (US Pat. No. 5,767,115) may also be useful as cholesterol lowering agents in the treatment and prevention of atherosclerosis. Are known. The cholesterol-lowering action of ezetimibe is inhibited by binding of ezetimibe with Niemann Pick Type C 1 like 1 (hereinafter referred to as NPC1L1), which is a membrane protein responsible for cholesterol absorption in the small intestine, from studies on knockout mice and transgenic mice. Became clear (Patent Document 2: JP 2010-252798 A).
Therefore, a substance that inhibits the binding between ezetimibe and NPC1L1 is expected to inhibit cholesterol absorption in the gastrointestinal tract and improve atherosclerosis.

  In recent years, substances having a blood cholesterol lowering effect and those having an effect of preventing arteriosclerosis have been obtained from fruit bodies of mushrooms. In Patent Document 3, Pleurotus eringi var. It has been disclosed that mushrooms of the genus Pleurotus such as ferulae and Pleurotus ostreatus produce lovastatin, which is a therapeutic agent for hypercholesterolemia (Patent Document 3: JP 2003-520576 A). Further, Patent Document 4 discloses that basidiomycetous fungi, shiitake, jellyfish, maitake, and litchi promote secretion of adiponectin and show an effect on lowering cholesterol (Patent Document 4: JP 2008-297256 A). Thus, it has been known for a long time that the fruiting bodies of specific species of mushrooms have the effect of lowering cholesterol and reducing arteriosclerosis.

  The present inventors have already found that the extract of Kurosarokoshikatake, a kind of basidiomycete, binds to the NPC1L1 protein and suppresses absorption of cholesterol and is useful as a cholesterol lowering agent. (Japanese Patent Application Nos. 2013-033089 and 2013-215687).

US Pat. No. 5,767,115 JP 2010-252798 A Japanese translation of PCT publication No. 2003-520576 JP 2008-297256 A

  An object of the present invention is to provide a novel compound and a hypercholesterol absorption inhibitor, a hypercholesterolemia improving agent, and a hyperlipidemia improving agent containing the same.

  The present inventors conducted research on the cholesterol absorption inhibitory effect of black salmon mushroom, isolated a novel compound exhibiting cholesterol absorption inhibition, and determined the chemical structure.

The configuration of the present invention is as follows.
(1) A compound represented by the following chemical formula (I):
(2) A cholesterol absorption inhibitor containing the compound according to (1).
(3) A hypercholesterolemia improving agent comprising the compound according to (1).
(4) A hyperlipidemia improving agent comprising the compound according to (1).
(5) A pharmaceutical composition comprising the compound according to (1).
(6) Cosmetics containing the compound according to (1).
(7) A food or drink to which the compound according to (1) is added.

  According to the present invention, a novel sterol compound having an action of inhibiting cholesterol absorption is provided. Also provided are novel cholesterol absorption inhibitors and hypercholesterolemia-improving agents. An agent for improving hyperlipidemia is also provided.

It is a graph which shows that the compound of Chemical formula (I) inhibits the coupling | bonding of ezetimibe and NPC1L1. It is a graph which shows that the extract containing the compound of Chemical formula (I) has the improvement effect of cholesterolemia. It is a graph which shows that the extract containing the compound of Chemical formula (I) has an action for improving hyperlipidemia. It is a graph which shows that the compound of chemical formula (I) inhibits the absorption of cholesterol through NPC1L1 to Caco2 cells, which are cell lines derived from human colon cancer, like ezetimibe. It is a graph which shows that the compound of chemical formula (I) inhibits absorption of cholesterol via NPC1L1 to Caco2 cells, which are cell lines derived from human colon cancer, as in ezetimibe, and reduces intracellular cholesterol ester concentration. is there.

  The present invention is a novel sterol compound represented by the following chemical formula (I), a cholesterol absorption inhibitor and a hypercholesterolemia ameliorating agent comprising this compound as an active ingredient.

The IUPAC name of the novel sterol compound of the present invention is Acetic acid 15-hydroxy-17- (1-hydroxymethyl-5-methyl-hex-4-enyl) -4,4,10,13,14-pentamethyl-2, 3,4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta [α] phenanthren-3-yl ester. This compound can be extracted from the fruiting body or mycelium of Fomitopsis nigra. Moreover, when setting it as a cholesterol absorption inhibitor, the extract containing the compound of Chemical formula (I) can also be used as a cholesterol absorption inhibitor.
The extraction uses a solvent such as water, a lower aliphatic alcohol (such as methanol, ethanol, isopropyl alcohol), a lower aliphatic ketone (such as acetone), or hexane. Extraction is performed by a solvent containing the extract or a solvent containing these by an extraction process usually performed by those skilled in the art. The produced extract can be made into a concentrated extract or a dry extract by a treatment such as heat treatment, freeze-drying or reduced-pressure drying.

  When using the compound of the present invention or an extract containing this compound as a cholesterol absorption inhibitor or preventive or ameliorating agent, agents such as tablets, granules, powders, capsules, and liquids containing any commonly used optional ingredient It can be administered orally in the form. Examples of optional components include excipients, binders, coating agents, lubricants, sugar coatings, disintegrating agents, bulking agents, flavoring agents, emulsifying / solubilizing / dispersing agents, stabilizers, pH adjusting agents, and the like. Examples include tonicity agents. By treating these according to a conventional method, the cholesterol absorption inhibitor of the present invention can be produced.

The dose for use as a cholesterol absorption inhibitor or hypercholesterolemia-improving agent containing the compound of formula (I) of the present invention varies depending on age, sex, body weight, etc. The amount is about 0.01 mg to 1 g, preferably 1 mg to 500 mg per unit.
The agent includes not only pharmaceutical products but also quasi drugs and health foods.

Hereinafter, the present invention will be described more specifically with reference to examples and test examples.
(Production example of cholesterol absorption inhibitor)
Extracted 42 kg of dried fruit bodies of black moss mushroom (Fomitopsis nigra) collected from nature with about 4 times the amount of ethanol and an equal amount of acetone at room temperature, and then mixed with ethanol and acetone extract. Was used to concentrate the solvent under reduced pressure so that insolubles did not precipitate.
Next, 1 L of hexane was added to 500 ml of the concentrated liquid, and the liquid was partitioned using a separatory funnel. This operation was repeated a plurality of times, and the recovered hexane layer was concentrated and dried using a rotary evaporator to obtain 40.3 g of a hexane extraction part. Of these, 28.3 g of the hexane extractor was dissolved in 100 ml of methanol, 950 ml of hexane and 950 ml of 80% acetonitrile in water were added, and the mixture was partitioned and concentrated to dryness using a rotary evaporator. As a result, 6.8 g of a hexane layer, 5.2 g of an intermediate layer, and 13.6 g of an 80% acetonitrile layer were obtained. The fraction obtained by liquid-liquid distribution was subjected to the animal test described later.

Next, 11.6 g of 80% acetonitrile layer was subjected to YMC GEL ODS-A (manufactured by YMC) open chromatography (φ40 × 300 mm manufactured by Kiriyama Seisakusho). Elution was performed with 75% aqueous acetonitrile, and the mixture was separated into 8 fractions. The solvent was removed under reduced pressure, and 910.6 mg of the third fraction (Fr3) was recovered.
Next, 795.7 mg of this Fr3 was further subjected to YMC GEL ODS-A open chromatography (Kiriyama Seisakusho φ22 × 500 mm) and separated into 12 fractions with 65% acetonitrile water. Among them, the ninth fraction (Fr3-9) in this fraction was collected, and the solvent was removed under reduced pressure. 94.8 mg of this Fr3-9 was obtained.
Next, the entire amount of Fr3-9 was eluted with preparative high performance liquid chromatography (column: Develosil XG C30-M5: manufactured by Nomura Chemical) eluent acetonitrile: 0.1% formic acid aqueous solution (35:65), and 6 fractions were separated. The final sixth fraction (Fr3-9-6) was collected and the solvent was removed under reduced pressure to obtain 15.22 mg of Fr3-9-6. This total amount was eluted in two fractions using a Develosil XG C30-M5 column with eluent acetonitrile: 0.1% aqueous formic acid (40:60) and the second fraction (Fr3-9-6-2). Was recovered. The solvent was removed and 5.27 mg was isolated as a dry product of Fr3-9-6-2.

(Structure determination)
The structure of the isolated fraction (purified product) was determined by LC / MS and NMR.
LC / MS
Liquid chromatograph unit: Waters ACQUITY UPLC
Column: ACQUITY UPLC BEH C18 (2.1 mm ID × 100 mm) manufactured by Waters
Moving bed: A; 40% 0.1% aqueous formic acid solution, B; 60% acetonitrile
Detector: UV 210-400 nm

Mass separation unit Device: Synapt G2-S type manufactured by Waters Ionization method: Electrospray ionization method, negative mode Measurement range: m / z = 50-2000

NMR
Apparatus: AVANCE500 type (CryoProbe) manufactured by Bruker BioSpin
Nuclide: ¹ H, ¹³ C
Solvent: heavy methanol (hereinafter abbreviated as CD ₃ OD)
Temperature: Room temperature Measurement method: DEPT, COSY, HMQC, HMBC
Reference substance: TMS set to 0 ppm (internal standard)

In NMR spectrum analysis, chemical shift (δ value) is expressed in ppm, binding constant is expressed in Hz, abbreviations are used, and each is expressed by s; singlelet, dd; double doublet, t; triplet, tt; triple triplet. did. Furthermore, the peak assignment was expressed based on the carbon number of the triterpene skeleton.

The obtained mass spectrum data and NMR data are as follows.
NMR spectrum:
1) ¹ H-NMR (CD ₃ OD); δ 0.80 ( ³ H, s, Me-18), 0.88 ( ³ H, s, Me-29), 0.95 ( ³ H, s, Me-28), 0.97 ( ³ H, s, Me-30), 1.04 ( ³ H, s, Me-19), 1.36 ( ¹ H, H ₂ -22), 1.44 ( ¹ H, H ₂ -22), 1.50 ( ¹ H, H ₂ -1), 1.53 ( ¹ H, H-5), 1.53 ( ² H, H ₂ -6), 1.54 ( ¹ H, H ₂ -12), 1.60 ( ³ H, s, Me-26), 1.62 ( ¹ H, H ₂ -2), 1.62 ( ¹ H, H-20), 1.67 ( ¹ H, H ₂ -12), 1.67 ( ³ H, s, Me-27), 1.78 ( ¹ H, H ₂ -16), 1.92 ( ¹ H, H ₂ -1), 1.93 ( ¹ H, H ₂ -2), 1.94 ( ¹ H, H ₂ -16), 1.95 ( ¹ H, H ₂ -23), 1.98 ( ¹ H, H-17), 2.05 ( ¹ H, H ₂ -23), 2.05 ( ³ H, s, COCH ₃ ), 2.26 ( ² H, H ₂ -11), 4.04 ( ¹ H, dd, J = 5.2, 11.3 Hz, H ₂ -21), 4.19 ( ¹ H, dd, J = 6.0, 9.4 Hz, H-5), 2.17 ( ² H, H ₂ -7) 4.29 ( ¹ H, dd, J = 3.1, 11.3 Hz, H ₂ -21), 4.64 ( ¹ H, t, J = 2.7 Hz, H-3), 5.10 ( ¹ H, tt, J = 1.3, 7.2 Hz, H-24)

2) ¹³ C-NMR (CD ₃ OD); δ 16.9 (C-18), 17.8 (C-26), 17.8 (C-30), 19.2 (C-6), 19.4 (C-19), 21.2 ( COCH ₃ ), 21.9 (C-2), 22.3 (C-28), 24.2 (C-7), 25.7 (C-23), 25.9 (C-27), 27.7 (C-11), 28.2 (C- 29) 31.2 (C-22), 31.9 (C-1), 32.1 (C-12), 37.8 (C-4), 38.2 (C-10), 39.6 (C-16), 41.0 (C-20) , 44.8 (C-17), 45.9 (C-13), 46.7 (C-5), 53.1 (C-14), 66.4 (C-21), 73.8 (C-15), 79.6 (C-3), 125.7 (C-24), 132.4 (C-25), 135.4 (C-8), 136.4 (C-9), 172.6 (COCH ₃ )

MS spectrum:
High resolution ESIMS m / z 499.3788 [M−H] ⁻ , C ₃₂ H ₅₁ O ₄ , req
uires 499.3787
From the above data, the chemical formula was determined as follows.

The IUPAC name of this novel sterol compound is Acetic acid 15-hydroxy-17- (1-hydroxymethyl-5-methyl-hex-4-enyl) -4,4,10,13,14-pentamethyl-2,3, 4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta [α] phenanthren-3-yl ester.

(Test 1: Ezetimibe binding inhibition test)
The compound of the formula (I) obtained in the production example was subjected to a test for evaluating the binding inhibitory activity between NPC1L1 and ezetimibe by the following test method.
(1) Cloning of rat NPC1L1 gene By PCR using iProof High-Fidelity DNA polymerase (manufactured by Bio-Rad) using rat jejunum-derived single-stranded cDNA (manufactured by Geno Staff) as a template, Hind at the 5 'end III The full length of rNPC1L1 with Xba I added at the 3 ′ end was amplified and cloned into the pCR4 Blunt TOPO vector using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen). The cloned DNA sequence is shown in SEQ ID NO: 1 in the sequence listing.
The primers and PCR conditions used for rNPC1L1 full-length amplification are as follows.
Sense: 5'- aagcttacatgggcaggctgcctggctgggaggg-3 '(SEQ ID NO: 2)
Antisense: 5'- tctagattagaactttttgggtactttgggg-3 '(SEQ ID NO: 3)

PCR conditions 1.98 ° C. for 30 seconds, 2.98 ° C. for 10 seconds, 3.68 ° C. for 30 seconds, 4.72 ° C. for 1.5 minutes, 5.72 ° C. for 10 minutes, 35 cycles of 2-4 heating cycles To do.

(2) Preparation of rat NPC1L1 stably expressing cells The pCR4 Blunt TOPO rNPC1L1 vector was treated with Hind III and Xba I, and after agarose electrophoresis, the vector for rNPC1L1 was extracted using QIAquick Gel Extraction Kit (manufactured by QIAGEN). .1 (manufactured by Invitrogen) was subcloned into the Hind III and Xba I sites. The sequence of the prepared pcDNA3.1 rNPC1L1 was confirmed using a premix sequence analysis service (manufactured by Takara Bio Inc.).
Sequencer primer
Sense: 5'-cactgcttactgggcttatcg-3 '(SEQ ID NO: 4)
Sense: 5'-gccttttattcaggcgcacttt-3 '(SEQ ID NO: 5)
Sense: 5′-aactctaccaccataccatc-3 ′ (SEQ ID NO: 6)
Sense: 5′-cagtacttccagaacaaccg-3 ′ (SEQ ID NO: 7)
Sense: 5'-ttctactcctacctggggtgt-3 '(SEQ ID NO: 8)
Sense: 5'-ctttttccgcaagatatacgc-3 '(SEQ ID NO: 9)
Sense: 5′-ccacagcggagaacattttcat-3 ′ (SEQ ID NO: 10)
Sense: 5′-tcccccatcaactttgtcac-3 ′ (SEQ ID NO: 11)
Sense: 5'-gtccatgctttctgtctgatg-3 '(SEQ ID NO: 12)

  Subsequently, pcDNA3.1 rNPC1L1 was transfected into human kidney-derived HEK293 cells (ATCC) using Fugene HD (Roche), and selection was performed at a final concentration of 1 mg / ml G418-sulfate (Wako). Further, selection was performed by a limiting dilution method to obtain a single clone of HEK / rNPC1L1 stably expressing cells.

(3) Preparation of membrane fraction HEK293 / rNPC1L1 stably expressing cells are seeded on φ150 mm dish (Nunc), cultured in DMEM (Nacalai tesque) + 10% FBS (Gibco) until confluent. The cells after treatment for 24 hours at a concentration of 4 mM Sodium Butyrate (manufactured by Sigma Aldrich) were collected using a Cell Dissociation Buffer (manufactured by Invitrogen). After centrifugation at 800 rpm for 3 minutes, the remaining cells were suspended in 20 mM HEPES / Tris Buffer (pH 7.4) + proteinase inhibitor (Roche) containing 8% sucrose and disrupted using ultrasonic waves. After centrifugation at 4000 rpm, 4 ° C. for 10 minutes, the supernatant was ultracentrifuged at 48000 rpm (125000 G), 4 ° C. for 3 hours, and the residue was collected as a membrane fraction.
The membrane fraction was resuspended in 20 mM HEPES / Tris Buffer containing 160 mM NaCl and 5% Glycerol, and the protein concentration was quantified with BCA assay (manufactured by Thermo Scientific Pierce) and stored at −80 ° C. until use.

(4) Binding inhibition experiment 96 well plate (manufactured by Nunc) with final concentration of 25 nM [ ³ H] Ezetimibe-glucuronide (manufactured by American Radiolabeled Chemicals, Inc.), 1.25 mg / ml HEK / rNPC1L1-derived membrane protein The substance was added to buffer A (26 mM NaHCO ₃ , 0.96 mM NaH ₂ PO ₄ , 5 mM HEPES, 5.5 mM Glucose, 117 mM NaCl, 5.4 mM KCl, 0.03% Sodium taurocholate, 0.05% Digitonin, pH 7.4). ) And a total volume of 30 μl, and reacted at room temperature for 1 hour. The reaction solution was trapped in UniFilter-96 GF / C (manufactured by PerkinElmer) using Harvester (manufactured by PerkinElmer), and free [ ³ H] Ezetimibe-glucuronide was washed using MilliQ water (manufactured by Millipore). After drying, 15 μl / well of Microscint-20 (PerkinElmer) was added, and the radioactivity of [ ³ H] Ezetimibe-glucuronide bound to rNPC1L1 was measured with Topcount (PerkinElmer).

(5) Results The novel sterol compound (the compound of the chemical formula (I)) extracted and isolated from the black salmon mushroom is dissolved in dimethyl sulfoxide (DMSO), and a 12-fold three-fold dilution series is prepared from a final concentration of 4 μM. Then, the radioactivity was measured by the above procedure, and IC50 was calculated from the result. IC50 inhibited the binding of NPC1L1 and ezetimibe at a low concentration of 35.6 μM.

The inhibition of the dilution series for which the IC50 was calculated is shown in FIG.
The novel sterol compound isolated from Black Sarcophagus strongly inhibited the reaction of ezetimibe binding to NPC1L1. Therefore, like ezetimibe, it inhibits the function of NPC1L1 and inhibits intestinal absorption of cholesterol.

(Test 2: Cholesterol reduction test using hypercholesterolemic rats)
Using the extract containing the compound of the present invention (hexane layer, intermediate layer, 80% acetonitrile layer described in Production Examples), the therapeutic effect on hypercholesterolemia that occurs when a high cholesterol-containing diet is ingested was confirmed. .

1. Test animals After acclimatization for 1 week, body weights were measured, and 8-week-old Wistar male rats (Japan SLC) grouped by stratified continuous randomization were used for the experiment.
Hypercholesterolemia was induced by giving a high cholesterol diet (1% cholesterol and 0.5% cholic acid added based on CRF-1; Oriental Yeast Co., Ltd.). In order to confirm hypercholesterolemia, a normal group fed with a normal diet (CRF-1; Oriental Yeast Co., Ltd.) was set up.
The group setting is a control group in which corn oil is administered in a state in which hypercholesterolemia is induced in addition to a normal group given a normal diet, 0.3 mg / kg ezetimibe (Cosmo Bio Inc.) The ezetimibe group of the positive control to which was administered, the black sarcoma mushroom hexane extraction part (before separation), the hexane layer, the intermediate layer, and the 80% acetonitrile layer produced by the above method were each administered at 500 mg / kg (setting of 6 animals in each group).

2. Test method Regular diet, high-cholesterol diet, and water are allowed to freely take for 3 days, the test substance is dissolved in corn oil, once a day for 2 days (no administration on the first day) from the second day of the test, stomach tube Forcibly orally (2 ml / kg). The normal group and the control group were administered corn oil. The breeding was individual breeding (1 animal / cage), and food intake for 3 days was recorded. In addition, the floor (Palsoft; Oriental Yeast Co., Ltd.) was replaced on the second day after the start of test substance administration. The blood was fasted for 2 hours after the final administration of the test substance (water was freely ingested) and then collected using a vacuum blood collection tube (Terumo Corporation) heparinized from the abdominal aorta under diethyl ether anesthesia. The collected blood was immediately centrifuged at 3,000 rpm for 15 minutes at 4 ° C., and the obtained plasma was stored at −80 ° C. until analysis. The amount of cholesterol in plasma was measured using Cholesterol E Test Wako (Wako Pure Chemical Industries, Ltd.), and the neutral fat was measured using Triglyceride E Test Wako (Wako Pure Chemical Industries, Ltd.).

3. Test results All results are mean ± S. Indicated by E. Statistical analysis was performed on 5 groups with high cholesterol diet intake, excluding the normal group, using Excel Statistics 2010. Each measurement data was subjected to a one-dimensional analysis of variance and Dunnett's multiple test. The significance level was less than 5% on both sides in all cases.
The measurement result of blood total cholesterol is shown in FIG. 2, and the measurement result of blood triglyceride is shown in FIG. As a result of the test, there was no difference in food intake among the five groups fed with a high cholesterol diet, and there was no change in body weight. From this, it was evaluated that induction of hypercholesterolemia was performed without any difference between groups.
Blood cholesterol was significantly different from the plasma cholesterol of the control group in the positive control ezetimibe administration group (Eze), the pre-separation group and the 80% acetonitrile layer group (ACN layer), and the cholesterol lowering effect was clear It became. No significant cholesterol lowering was observed in the hexane layer (HEX layer) and the intermediate layer.
Moreover, as a result of measuring neutral fat, a significant difference was observed between the pre-separation group and the 80% acetonitrile group, similar to the cholesterol measurement result.
Therefore, it was revealed that the layer (ACN layer) containing the compound of the formula (I) isolated from black Sarcophagus has a cholesterol lowering action and a triglyceride lowering action in plasma.
From the above test results, it was revealed that the novel sterol compound compound (I) having an inhibitory action on NPC1L1 is useful as an agent for improving dietary hypercholesterolemia and hyperlipidemia.

(Test 3: Inhibition test of cholesterol uptake using Caco-2 cells)
The test was performed using human colon cancer-derived cell line Caco-2 cells.
1. Preparation of Lentiviral Solution and Caco2 / mock, Caco2 / rNPC1L1 Stable Expression Cells Using the SwaI and SgfI sites of the lentiviral vector pCDH-CMV-MCS-EF1-Puro (System Biosciences), the same restriction enzyme sites were added. The full length of rNPC1L1 was amplified by PCR and subcloned (hereinafter referred to as pCDH-rNPC1L1). Three vectors, pCDH-rNPC1L1, envelope vector pMD2.G (Addgene) and packing vector psPAX2 (Addgene) were transfected into 293T (ATCC) cells using lipofection reagent LTX (invitrogen), and 96 hours later The medium was collected. Centrifugation was performed twice at 2500 rpm for 10 minutes, and the obtained supernatant was passed through a 0.45 μm filter (Millipore) to prepare a lentivirus solution.
Infection of human colon cancer-derived Caco2 cells (ATCC) with the addition of lentivirus solution, and further selection using puromycin (Sigma Aldrich) with a final concentration of 5 μg / ml using limiting dilution method, stable expression of Caco2 / rNPC1L1 cells A single clone was made. In addition, a single clone was prepared using the pCDH-CMV-MCS-EF1-Puro vector containing no insert DNA by the same method, and designated as Caco2 / mock.

2-1. Evaluation test using [ ³ H] labeled cholesterol
Each of the Caco2 / mock and Caco2 / rNPC1L1 stably expressing cells is seeded on a 12-well plate (Falcon) at 5 × 10 ⁵ cells / well, and the medium DMEM + 10% FBS is changed every 2 days. The state was maintained for 14 days and differentiated into small intestine-like cells. After washing the cells with DMEM, final concentrations of 2 mM sodium taurocholate (Wako), 50 μM phosphatidylcholine (Sigma Aldrich), 1 μM cholesterol (Sigma Aldrich), 1 μCi / ml [ ³ H] cholesterol (PerkinElmer), 1 ml / well of DMEM micelle solution containing Ezetimibe or the compound of formula (I) was added and treated at 37 ° C. for 1 hour. After washing the cells twice with 1 ml / well PBS +, add 1 ml / well of 0.1% lithium dodecyl sulfate (WAKO) and 0.2N sodium hydroxide (Nacalai tesque) in distilled water and treat for 48 hours at room temperature. The protein concentration was measured. In addition, add 100 μl of the cell lysate to 2 ml of Clearsol II (Nacalai tesque), measure the radioactivity of total [ ³ H] cholesterol using a liquid scintillation counter (ALOKA), and convert it into an amount per protein. The amount.

2-2. result
For each of the Caco2 / mock and Caco2 / rNPC1L1 cells, the total of the Contorol group to which the solvent was added, the Ezetimibe group to which the final concentrations of 3, 10, and 30 μM were added, and the compound I group to which the final concentrations of 1, 3, 10, and 30 μM were added [ ³ H The measurement results of cholesterol are shown in FIG. 4 below.
Ezetimibe and the compound addition group of chemical formula (I) both significantly reduced the amount of [ ³ H] cholesterol incorporated and markedly suppressed cholesterol uptake compared to the respective controls of mock and rNPC1L1 cells.

3-1. Evaluation test using [ ³ H] labeled cholesterol ester
Caco2 / mock and Caco2 / rNPC1L1 stably expressing cells are seeded on a 12-well plate (Falcon) at 5 × 10 ⁵ cells / well, and the medium DMEM + 10% FBS is changed every 2 days to ensure overconfluence. Maintained for 14 days and differentiated into small intestine-like cells. After washing cells with DMEM, final concentrations of 5 mM sodium taurocholate (Wako), 500 μM oleic acid (Sigma Aldrich), 10 μM cholesterol (Sigma Aldrich), 1 μCi / ml [ ³ H] cholesterol (PerkinElmer), test substances at each concentration 1 ml / well of DMEM micelle solution containing was added and treated at 37 ° C. for 1 hour. After washing with DMEM, the sample was replaced with serum-free cocktail ITS (Thermo Fisher Scientific) -containing DMEM, and further incubated at 37 ° C. for 8 hours to induce esterification. After washing the cells with PBS +, lipids were extracted from the cells using a mixture of hexane: isopropanol (3: 2) (Nacalai tesque) and replaced with chloroform: methanol (2: 1) (Nacalai tesque). Spotted on (Roche) and developed using hexane: diethyl ether: acetic acid (60: 40: 1) (Nacalai tesque). The developed TLC is colored with iodine (Nacalai tesque), scraped the separated [ ³ H] cholesterol and [ ³ H] cholesterol ester, soaked in Clearsol II (Nacalai tesque), and using a liquid scintillation counter (ALOKA) Radioactivity was measured.
In addition, 0.1% lithium dodecyl sulfate (WAKO) and 0.2N sodium hydroxide (Nacalai tesque) -containing distilled water were added to the cells after lipid extraction, and after treatment for 48 hours at room temperature, the protein concentration was measured by BCA assay. Cholesterol taken up into cells and esterified was converted to the amount per protein and taken as the amount taken up.

3-2. result
[ ³ H] of the Contorol group to which the solvent was added, the Ezetimibe group to which the final concentrations of 3, 10, and 30 μM were added, and the compound I group to which the final concentrations of 1, 3, 10, and 30 μM were added, respectively for the Caco2 / mock and Caco2 / rNPC1L1 cells. The measurement result of the amount of cholesterol ester is shown in FIG.
Ezetimibe and the compound addition group of chemical formula (I) suppressed the amount of [ ³ H] cholesterol ester produced after uptake of cholesterol into cells in a concentration-dependent manner, as compared with the respective controls of mock and rNPC1L1 cells. In addition, the amount of cholesterol uptake into cells was significantly reduced.
From the above two tests, it has been clarified that the compound of the formula (I) inhibits the uptake of cholesterol into Caco2, which is a cell of the intestinal tract wall which is the absorption site of cholesterol.

Claims (7)

A compound represented by the following chemical formula (I):
  A cholesterol absorption inhibitor comprising the compound according to claim 1.   A hypercholesterolemia-improving agent comprising the compound according to claim 1.   The hyperlipidemia improving agent containing the compound of Claim 1.   A pharmaceutical composition comprising the compound according to claim 1.   A cosmetic comprising the compound according to claim 1.   The food-drinks which added the compound of Claim 1.