JP6292619B2 - Anti-obesity drugs - Google Patents

Description

  The present invention relates to a novel natural organic compound obtained from cyanobacteria and an anti-obesity drug containing such a natural organic compound as an active ingredient.

The ocean occupies about 71% of the surface area of the earth, and it is said that about 90% of all the creatures on the earth live in the ocean. Marine organisms live in a different environment compared to terrestrial organisms, exist in a high salt concentration system called seawater, have a relatively small temperature change, and are subject to high pressure. Chemical substances produced by organisms that grow in such unique environments have a variety of structures, some of which have various biological activities.
Currently, cyanobacteria are listed as promising marine organisms as a search source for bioactive substances. Cyanobacteria are prokaryotes that perform photosynthesis and grow in various environments such as freshwater lakes, brackish waters, oceans, and soil. It is known to produce a substance exhibiting a specific chemical structure or physiological activity depending on the growth environment. For example, Lyngbyoxin A (Non-patent Document 1) having an oncogenic promoter activity, Biselyngbiaside (Non-patent Document) that inhibits bone resorption activity 2) and Bisebromide (Non-patent Document 3), which shows potent protein kinase inhibitory activity, and the like, and cyanobacteria are expected as a search source for bioactive substances and their lead compounds.

Obesity caused by excessive fat accumulation in human body adipose tissue and various organs is caused by constitutional factors, dietary factors, mental factors, metabolic factors, lack of exercise, etc., resulting in calorie intake Is more than calories burned and fat is accumulated. Obesity is the cause of many lifestyle-related diseases such as diabetes, hypertension, and dyslipidemia. In particular, metabolic syndrome (visceral fat syndrome) that multiple lifestyle-related diseases such as hypertension and dyslipidemia are combined. It is called. According to the Ministry of Health, Labor and Welfare's 2006 National Health Survey, the number of people with metabolic syndrome aged 40 to 74 is about 9.6 million, and the number of reserve groups is about 9.8 million. Therefore, effective treatment and prevention methods are required.
The first cause of death among Japanese is cancer, the second is stroke (cerebral infarction and cerebral hemorrhage), the third is heart disease (myocardial infarction and angina), which is second or third? Arteriosclerosis, hypertension, dyslipidemia, etc. are major risk factors, but diet and obesity are greatly involved in these. In addition, diabetes, hyperuricemia, gout, fatty liver, pancreatitis, etc., which are rapidly increasing among Japanese, are said to be deeply related to obesity.
In the state of obesity, the amount of lipid droplets, that is, triglycerides present in fat cells in the body increases, and the cells are enlarged. Furthermore, recently, it has been reported that the number of fat cells increases even after becoming an adult. Therefore, an attempt to inhibit the progression of obesity is expected from both the viewpoint of inhibiting differentiation into fat cells and acting on fat droplets in fat cells.

The present inventors have continued research on drugs that exhibit an anti-obesity effect through inhibition of preadipocyte differentiation into adipocytes or inhibition of fat cell fat accumulation, and effective use of components extracted from specific mushrooms or plants Preadipocyte differentiation inhibitor as an ingredient (Patent Document 1), cyclic heptapeptide ternatin: cyclo [-D-Ile1- (N-Me) -L-Ala2- (N-Me) -L-Leu3-L A preadipocyte differentiation inhibitor (Patent Document 2) containing -Leu4- (N-Me) -L-Ala5- (N-Me) -D-Ala6-β-OH-D-Leu7-], and The inventors succeeded in finding a preadipocyte differentiation inhibitor (Patent Document 3) containing the plant component bisabolol oxide-A-β-glucoside as an active ingredient.
In addition, aureosin, which is a known natural organic compound having antibacterial action, has triglyceride biosynthesis inhibitory activity and has been reported to reduce the triglyceride concentration in blood (Patent Document 4).
However, the components extracted from natural products and natural organic compounds obtained up to now are not necessarily satisfactory as anti-obesity agents, especially from the viewpoint of anti-obesity agents by acting on lipid droplets in fat cells. However, there were no satisfactory ones, and it was essential to study natural organic compounds that exhibited better effects.

Japanese Patent Application Laid-Open No. 2004-075640 Japanese Patent Laid-Open No. 2005-220074 JP 2006-213648 A Japanese Patent Laid-Open No. 9-194366

J. et al. H. Cardellina II, F.R. J. et al. Marner, R.M. E. Moore, Science, 204, 193 (1979) T.A. Yonezawa, N .; Mase, H .; Sasaki, T .; Teruya, S .; Hasegawa, B.I. Cha, K .; Yagasaki, K .; Suenaga, K .; Nagai, J .; Woo, Cell Biochem. , 2012,2,440 T.A. Teruya, H .; Sasaki, H .; Fukazawa, and K.K. Suenaga, Org. Lett. , 2009, 11, 5062

  An object of the present invention is to provide a novel natural organic compound having an activity of inhibiting the differentiation of preadipocytes into adipocytes and an activity of reducing lipid droplets in fat cells, and an active ingredient of such a natural organic compound in the background described above. It is in providing the pharmaceutical containing as.

（化学式（１）） The present inventors isolated a compound represented by the chemical formula (1), which is a novel natural organic compound, from a blue-green algae (Leptolingbya sp.) Collected in Ishigaki-jima Yonehara, Okinawa Prefecture, and such a compound becomes a fat cell of a preadipocyte. Have been found to have differentiation-inhibiting activity and reduction activity of lipid droplets in adipocytes, and the present invention has been completed.

(Chemical formula (1))

（化学式（１）） That is, the present invention relates to a compound represented by the chemical formula (1).

(Chemical formula (1))

  The present invention also relates to an anti-obesity drug comprising a compound represented by the above chemical formula (1) as an active ingredient.

  Furthermore, the present invention relates to an anti-obesity drug containing, as an active ingredient, a compound represented by the above chemical formula (1), characterized by reducing fat droplets in fat cells.

  According to the present invention, the novel compound represented by the chemical formula (1) has the activity of inhibiting the differentiation of preadipocytes into adipocytes, and also has the activity of reducing lipid droplets in adipocytes. It contributes to the prevention and improvement of obesity.

It is a figure which shows the fractionation / refinement | purification process of the compound shown by Chemical formula (1) based on this invention. It is a figure which shows the fractionation / refinement | purification process of the compound shown by Chemical formula (1) based on this invention. It is a graph which shows the differentiation inhibitory activity to the fat cell of the compound shown by Chemical formula (1) which concerns on this invention. It is a graph which shows the influence on the triglyceride level in the fat cell of the compound shown by Chemical formula (1) which concerns on this invention. It is a chart which shows the influence on the intracellular lactic acid level of the compound shown by Chemical formula (1) which concerns on this invention.

  Hereinafter, embodiments of the present invention will be described in detail.

  The compound represented by the chemical formula (1) according to the present invention can be obtained by extraction from cyanobacteria and purification. More specifically, the compound represented by the chemical formula (1) according to the present invention was obtained by extracting and purifying from a blue-green algae (Leptolingbya sp.) Collected at Ishigakijima Maibara, Okinawa Prefecture.

  Cyanobacteria are prokaryotes that carry out photosynthesis and grow widely under various environments such as freshwater lakes, brackish waters, oceans, and soils. The cyanobacteria used in the present invention is near Yonehara, Ishigaki Island, Okinawa Prefecture. It is preferable to use Leptolingbya sp. The blue-green algae (Leptolingbya sp.) Containing the compound represented by the chemical formula (1) according to the present invention or a portion thereof is a dried product obtained by drying itself, a pulverized product thereof, and a squeezed product obtained by compressing and extracting the product All of the crude extract with organic solvents such as juice, water or alcohol, ether, acetone, etc., and the extract fraction obtained by stepwise purification by various chromatographies such as column chromatography etc. Can be used. These may be used alone or in combination of two or more.

  For example, the cyanobacteria are collected and stored frozen, crushed and extracted with water-containing methanol to obtain a crude extract, the crude extract is distributed into an ethyl acetate layer and an aqueous layer, and an ethyl acetate layer as an organic layer is further obtained. Can be fractionated by ODS silica gel column chromatography.

  The compound represented by the chemical formula (1) according to the present invention is a novel compound, and has been found by the present invention to be excellent in the differentiation inhibiting activity of preadipocytes and the activity of reducing lipid droplets in fat cells. is there.

  The compound represented by the chemical formula (1) according to the present invention can be used as an anti-obesity agent because it has excellent activity of inhibiting the differentiation of preadipocytes into adipocytes and the activity of reducing lipid droplets in fat cells. .

  Since the compound represented by the chemical formula (1) according to the present invention has the activity of reducing lipid droplets in fat cells, it can be used as an anti-obesity drug by reducing the lipid droplets in fat cells.

The pharmaceutical composition containing the compound represented by the chemical formula (1) according to the present invention as an active ingredient can be used as an anti-obesity agent. Here, “obesity” generally means that adipose tissue is constant in the body. A state where a large amount is accumulated. In the present specification, “obesity” is to be interpreted broadly, and the concept includes obesity. “Obesity” refers to a medical condition that is or is predicted to have a health disorder (complication) due to or related to obesity, and that requires medical weight loss.
As a method for determining obesity, a method based on BMI (Body Mass Index), which is widely used internationally, is usually used. BMI is a numerical value obtained by dividing body weight (kg) by the square of height (m) (BMI = weight (kg) / height (m) ² ). BMI <18.5 is low weight, 18.5 ≦ BMI <25 is normal weight, 25 ≦ BMI <30 is obesity (1 degree), 30 ≦ BMI <35 is obesity (2 degrees), 35 ≦ BMI <40 is Obesity (3 degrees), 40 ≦ BMI is determined to be obese (4 degrees). However, since the standard body weight (ideal body weight) is different for each individual due to differences in gender, age, occupation or lifestyle, obesity is uniformly determined by this method, and the anti-obesity drug of the present invention is used. It is not intended to be strictly applied.

  Formulation of the anti-obesity drug of the present invention can be performed according to a conventional method. In the case of formulating, other pharmaceutically acceptable ingredients (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological Saline solution and the like). As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the stabilizer, propylene glycol, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.

The dosage form for formulation is not particularly limited, and the medicament of the present invention can be provided as tablets, powders, fine granules, granules, capsules, syrups, injections, external preparations, suppositories, and the like.
The pharmaceutical composition of the present invention contains an active ingredient in an amount necessary for obtaining an expected therapeutic effect (including a preventive effect) (that is, a therapeutically effective amount). The amount of the active ingredient in the pharmaceutical composition of the present invention generally varies depending on the dosage form, but the amount of the active ingredient is set within a range of, for example, about 0.1 wt% to about 95 wt% so as to achieve a desired dose. .

  The anti-obesity drug of the present invention is applied to a subject by oral administration or parenteral administration (intravenous, intraarterial, subcutaneous, intramuscular or intraperitoneal injection, transdermal, nasal, transmucosal, etc.) depending on the dosage form. The The “subject” here is not particularly limited, and includes humans and non-human mammals (including pet animals, farm animals, and laboratory animals). In a preferred embodiment, the antiobesity agent of the present invention is applied to humans.

  The dose of the anti-obesity drug (active ingredient) of the present invention is set so as to obtain the expected therapeutic effect. In setting a therapeutically effective dose, symptoms, patient age, sex, weight, etc. are generally considered. A person skilled in the art can set an appropriate dose in consideration of these matters. For example, the amount of active ingredient per day for an adult (body weight: about 60 kg) is about 1 mg to about 6 g. The dose can be set to be preferably about 6 mg to about 600 mg. As the administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. In preparing the administration schedule, the patient's medical condition and the duration of effect of the active ingredient can be taken into consideration.

  EXAMPLES Hereinafter, although an Example is given and the specific aspect of this invention is shown, the technical scope of this invention is not limited at all by description of an Example.

Isolation / Purification / Structural Determination of Compound Represented by Chemical Formula (1) According to the Present Invention 600 g (wet weight) of blue-green algae (Leptolingya sp.) Collected from Ishigakijima Maibara, Okinawa Prefecture was used. After collection, the sample was stored frozen, pebbles were removed, and the mixture was pulverized in water-containing methanol with a blender. The extract was filtered with suction, and the filtrate was concentrated using an evaporator. The crude extract was partitioned between ethyl acetate (400 ml × 3) and water (400 ml). The obtained ethyl acetate layer was concentrated under reduced pressure, and the concentrate was degreased with 80% aqueous methanol solution (250 ml) and hexane (250 ml × 3). The obtained 80% methanol aqueous solution layer was concentrated under reduced pressure, and the concentrate was subjected to ODS column chromatography [Cosmosil 75C ₁₈ -OPN (φ 20 × 85 mm), methanol / water (20/80 → 40/60 → 60/40 → 80). / 20 → methanol)] was separated into 5 fractions (Fr. 1 to 5).
(See Figure 1)
Among each of the obtained elution fractions, 15.2 mg of 60% methanol elution fraction (Fr.3) was subjected to reverse phase high performance liquid chromatography [Develosil ODS HG-5 (φ 10 × 250 mm), acetonitrile / water (10 / 90) → (gradient 30 min) → (100/0), detection UV 215 nm, flow rate 4.6 mL / min], and separated into 9 fractions (Fr. 3-1 to 9). Since the obtained fraction (Fr. 3-5) showed activity of inhibiting differentiation into adipocytes, this fraction was purified. The obtained Fr. 3-5 was subjected to reverse phase high performance liquid chromatography [Develosil ODS HG-5 (φ 10 × 250 mm), acetonitrile / water (30/70) → (gradient 30 min) → (55/45) → (gradient 5 min) → (100/0), detection UV 215 nm, flow rate 4.6 mL / min], and separated into 9 fractions (Fr. 3-5-1 to 3-5-9). Inhibitory activity was observed in the obtained fractions (Fr. 3-5-5, 7). Fr. 3-5-7 (1.0 mg, amorphous white powder) was a compound represented by the chemical formula (1) according to the present invention.
(See Figure 2)

With respect to the compound represented by the chemical formula (1) according to the present invention, which is an amorphous white powder obtained by the operation shown in FIG. 1 and FIG. 2, the molecular ion peak value [m / z387.1633 (M + Na) +, Δ + 1.6 mmu], the molecular formula was determined and found to be a compound of C ₂₁ H ₃₂ O ₅ .
Furthermore, by measuring ¹ H-NMR, ¹³ C-NMR and HMBC, and analyzing the obtained spectrum, the structure of a compound having an amorphous white powder and having a molecular formula of C ₂₁ H ₃₂ O ₅ is shown below. It was decided as follows.

¹ H-NMR and ¹³ C-NMR spectrum data of the compound represented by the chemical formula (1) of the present invention, which is an amorphous white powder obtained by the operations shown in FIGS. 1 and 2, are shown below.
¹ H-NMR (C ₆ D ₆ , 500 MHz)
d 5.36 (d, 1H, J = 8.5, H-14), 5.16 (t, 1H, J = 7.0, H-8), 4.38 (q, 1H, J = 6) .5, 8.5, H-15), 3.29 (dd, 1H, J = 5.5, 7.5, H-12), 3.23 (s, 3H, 2-OMe), 3. 03 (s, 3H, 12-OMe), 2.86 (d, 2H, J = 7.0, H-7), 2.13 (s, 3H, 3-Me), 2.10 (m, 1H) , H-10a), 2.03 (m, 1H, H-10b), 2.01 (s, 3H, 5-Me), 1.79 (m, 1H, H-11a), 1.58 (m , 1H, H-11b), 1.48 (s, 3H, 13-Me), 1.48 (s, 3H, 9-Me), 1.13 (d, 3H, J = 6.5, 15- Me)
¹³ C-NMR (C ₆ D ₆ , 125 MHz)
d 179.9 (C-4), 161.7 (C-2), 156.7 (C-6), 138.2 (C-5), 135.0 (C-13), 134.0 ( C14), 119.9 (C9), 118.0 (C-8), 100.1 (C-3), 86.4 (C-12), 64.0 (C-15), 52.8 ( 12-OMe), 51.8 (2-OMe), 35.9 (C-10), 32.3 (C-7), 32.1 (C-11), 23.8 (15-Me), 15.9 (9-Me), 10.5 (13-Me), 10.0 (5-Me), 7.1 (3-Me)

Biological activity of the compound represented by the chemical formula (1) of the present invention obtained in Example 1 According to the method described in Example 1, as the biological activity of the compound represented by the chemical formula (1) of the present invention obtained, fat Inhibition of cell differentiation, cytotoxicity due to cell viability, reduction of triglycerides from adipocytes, and increase of lactic acid were investigated.
In the examples, mouse fibroblast 3T3-L1 cells, which have been demonstrated to be efficiently differentiated into adipocytes, were used as prepared in the following (A) to (D).
(A) Growth and storage of 3T3-L1 cells 3T3-L1 cells were cultured on a 100 mm dish in a DMEM growth medium supplemented with 5% bovine serum at 37 ° C. under 5% carbon dioxide-air and saturated water vapor. In addition, 3T3-L1 cells were first cultured in large quantities, and dispensed and stored frozen according to a conventional method.
(B) Passage Cells were washed with 5 ml of PBS (−), 1 ml of 0.25% trypticin 1 mM EDTA-containing physiological saline was added, and the mixture was allowed to stand at 37 ° C. for 3 minutes. The cells were suspended in 5 ml of growth medium, transferred to a 15 ml centrifuge tube, and precipitated by centrifugation at 1500 rpm for 3 minutes. Dilute from 1/4 to 1/5 and go to dish.
(C) Preparation of cells for examples The cells peeled from the dish and centrifuged by the same method as described above were suspended in an appropriate amount of growth medium, and the number of cells was counted. Based on this, it was seeded in a 96-well plate at 5 × 10 ⁴ cells / well and cultured for 48 hours.
(D) Preparation of differentiation induction medium Into 100 ml of the above growth medium, 1 ml of 1 mM dexamethasone solution, 11.1 mg of 3-isobutyl-1-methylxanthine, 0.1 ml of 5 mg / ml insulin solution, and antibiotic solution (317 mg of penicillin G potassium salt) The solution was adjusted by adding 0.45 ml and dissolving 500 mg of streptomycin in 25 ml of sterile physiological saline.

(1) Inhibition of differentiation by rate of induction of differentiation into adipocytes Induction of differentiation of 3T3-L1 cells into adipocytes was performed by replacing the culture medium with a differentiation-inducing medium. At this time, the compound represented by the chemical formula (1) adjusted to a specific concentration was added to the differentiation induction medium, and only the differentiation induction medium was added to the blank and the control. The plate was cultured at 37 ° C. under 5% carbon dioxide gas-air and saturated water vapor. After 48 hours, it was replaced with fresh growth medium supplemented with insulin. Thereafter, the medium was replaced with fresh growth medium every two days.
After culturing for 7 to 10 days from the start of differentiation induction by the above method, the cells were observed with a microscope and evaluated when the control cells were sufficiently differentiated.
The induction rate of differentiation into fat cells was calculated by quantifying the amount of triglyceride accumulated in the cells. The cells were washed with PBS (−) and air-dried, and Lab Assay Triglyceride Kit Wako was added thereto at 100 μL / well. The mixture was allowed to stand at room temperature for 30 to 60 minutes, and the absorbance at 630 nm was measured to quantify triglycerides.
The results are shown in FIG. The compound represented by the chemical formula (1) according to the present invention inhibited the differentiation of 3T3-L1 cells into adipocytes by 50% at a concentration of 420 nM. On the other hand, Fr. The compound isolated from 3-5-5 (Fr. 3-5-5-2, 3-5-5-3) did not show an activity of inhibiting differentiation into adipocytes.

(2) Cytotoxicity by cell viability Cytotoxicity was evaluated using cell viability as an index. Cells that were detached and centrifuged by the method of (C) above and the number of cells was counted were seeded on a 96-well plate at 1 × 10 ⁴ cells / well. After culturing for 16 hours, the culture medium was replaced with a growth medium containing a compound represented by the chemical formula (1) adjusted to a specific concentration, and culturing was continued for further 48 hours. Cell Counting Kit-8 was added in an amount of 5 μL / well and cultured for 1 hour, and then the absorbance at a wavelength of 450 nm was measured to calculate the cell viability.
The compound represented by the chemical formula (1) according to the present invention did not show significant toxicity to 3T3-L1 cells even at a concentration of 50 μM. Similarly, in experiments using Hela cells, no significant toxicity was observed at a concentration of 50 μM.

(3) Adipocyte triglyceride reduction In order to confirm the reduction of lipid droplets in fat cells, the reduction of fat cell glycerides was examined. The 3T3-L1 adipocytes fully differentiated by the above method are cultured in the presence of the compound represented by the chemical formula (1) diluted to a specific concentration in a growth medium, and the medium is fresh with a sample every 3 days. I replaced it with something. Seven to nine days later, the amount of triglycerides in the cells was quantified using the above-described laboratory assay triglyceride kit Wako. The results are shown in FIG. Since the compound represented by the chemical formula (1) according to the present invention reduces the triglyceride level in the fat cells, the compound represented by the chemical formula (1) according to the present invention is effective for reducing the lipid droplets in the fat cells. It has been found.

(4) Increase in lactic acid Adipocytes or Hela cells differentiated from 3T3-L1 were seeded in 1 × 10 ⁵ cells in a 60 mm dish, cultured for 16 hours, and then the compound represented by the chemical formula (1) at an arbitrary concentration The culture was continued for an additional 72 hours in the presence. The culture solution was collected and centrifuged at 15000 rpm for 15 minutes to remove cell debris. When this was analyzed by HPLC (Develosil ODS HG-5 4.6 × 250 mm), it was observed that the lactic acid peak increased depending on the concentration of the compound represented by the chemical formula (1). The results are shown in FIG. From the bottom, the chart shows fat cells differentiated from 3T3-L1 treated with the compound represented by chemical formula (1) of 0, 0.05 μM and 0.1 μM, respectively, and Hela cells similarly treated with 0.1 μM. Results are shown. The peak after 11 minutes indicates lactic acid, and the lactic acid peak increases depending on the concentration of the compound represented by the chemical formula (1) regardless of the cell type.

  The compound represented by the chemical formula (1) of the present invention has an activity of inhibiting differentiation into adipocytes, and also has an activity of reducing triglyceride levels in adipocytes, and thus has a reduction activity of lipid droplets in adipocytes. It is useful as a pharmaceutical composition as an anti-obesity agent.

Claims (3)

The compound shown by following Chemical formula (1).

(Chemical formula (1))   An anti-obesity drug comprising the compound according to claim 1 as an active ingredient.   3. The anti-obesity drug according to claim 2, wherein cell droplets in fat cells are reduced.

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