JP6821909B2 - Quinoline derivative exhibiting human estrogen receptor activating action - Google Patents

Description

The present invention relates to a quinoline derivative exhibiting a human estrogen receptor activating action.

The estrogen receptor is a receptor that transmits the action of estrogen and is present in various cells. In addition to nuclear receptors, it is also partially expressed on cell membranes. There are alpha and beta types of estrogen receptors in the nucleus, and both types have DNA binding sites and react directly with DNA. The alpha type is present in the genitals such as the uterus and mammary glands. On the other hand, the beta type is widely distributed in bone cells and nerve cells.

The estrogen receptor transmits the action of estrogen, transmits the action of estrogen to the tissues of the whole body such as the genital organs and the skin, and causes the action of estrogen. The actions of estrogen include regeneration of skin cells, maintenance of ovaries, activation of nerve cells, increase of blood flow, lipolysis of adipocytes, activation of insulin receptors, lowering of blood glucose, and metabolic decomposition of cholesterol.

Estrogen secretion has a decline with age, and after about 40 years of age, symptoms due to estrogen lowering appear as menopausal symptoms. At the same time, the estrogen receptor also reduces the reactivity, and the estrogen cannot fully exert its function. One of the causes of the onset of menopause is not only a decrease in estrogen but also a decrease in the function of estrogen receptors.

There are few examples of inventions relating to estrogen receptor activation. For example, there is an invention relating to an isoflavonoid dimer, which is obtained by dimerizing an isoflavonoid ring system, and although there is an invention relating to a method for synthesizing a novel dimer compound and a composition containing the compound, The compound that activates the estrogen receptor has not been identified (see, for example, Patent Document 1).

Japanese Patent Application No. 2008-502191

The estrogen receptor activating effect of existing substances is mild, and there is a problem that industrial use is limited, and chemically synthesized substances have safety problems and their use is limited. ..

Therefore, a natural product having weak side effects and exhibiting an excellent estrogen receptor activating action is desired.

In order to achieve the above object, the invention according to claim 1 relates to a quinoline derivative exhibiting an estrogen receptor activating action represented by the following formula (1).

Since the present invention is configured as described above, the following effects are obtained.

The quinoline derivative according to claim 1 is excellent in estrogen receptor activating action.

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

The quinoline derivative exhibiting the estrogen receptor activating action has a structure represented by the following formula (1).

The quinoline derivative exhibiting the estrogen receptor activating action as described in the above formula (1) is composed of one molecule of quinoline, one molecule of p-coumaric acid and one molecule of valine (L-type-valine). All of these molecules and their bonds are of the natural form that exists in nature, and each molecule is bonded via an ester bond. It is a chemical formula of C30H31O6N2 and is formed from 30 carbon atoms, 31 hydrogen atoms, 6 oxygen atoms and 2 nitrogen atoms.

This quinoline derivative can be obtained by a chemical ester synthesis step using quinoline, p-coumaric acid and valine as raw materials. For example, commercially available chemicals and refined products are available for quinoline, p-coumaric acid and phenylalanine. It can be synthesized by an organic synthesis method while protecting functional groups.

On the other hand, its chemical synthesis results in a large loss of raw materials and high manufacturing costs, so its use in industry is limited. The highly pure quinoline derivative chemically synthesized here is used for quantitative and qualitative analysis.

It is preferable to analyze the structure of this quinoline derivative from the viewpoint that the target active ingredient can be identified. In addition, it is preferable because it can be used as an index of the content of the active ingredient when it is used for products and formulations and sold.

As an example of structural analysis of this quinoline derivative, when a chemically synthesized high-purity (purity 96% or more) standard substance is used and analyzed by 1H-NMR at 600 MHz in deuterated dimethyl sulfoxide, a peak is obtained. Is 1.84, 1.88, 1.90, 2.69, 3.33, 4.00, 5.06, 6.20, 6.65, 6.66, 6.74, 6.77, 6 It is found at .80, 6.91, 6.95, 7.96, 8.02 and 8.05 ppm.

In addition, in the analysis of C-NMR (13C-NMR), the peaks were 22.4, 22.6, 23.5, 36.0, 42.3, 49.7, 56.5, 71.3, 86.2. , 114.2, 116.4, 117.4, 117.9, 119.0, 119.4, 123.0, 124.3, 126.4, 131.7, 134.7, 136.5, 141 It is found at .5, 142.8, 146.0, 146.3, 146.9, 147.0, 173.9, 174.2 and 174.3 ppm.

Furthermore, this quinoline derivative is analyzed by high performance liquid chromatography or mass spectrometer to identify its structure. This constituent, quinoline, is a naturally occurring compound that has been found in plants and microorganisms and has been confirmed to be safe.

In particular, mulberry leaves and lotus seeds, and lotus and magwa leaves originally contain quinoline, and it is possible to produce the desired quinoline derivative by fermenting the lotus seeds. It is preferable to use these vegetables, algae, fruits and grains as raw materials for producing the desired quinoline derivative.

Originally, this quinoline is demethylcochlorin, and this quinoline itself also has a function, in addition to estrogen receptor activating action, skin cell protective action, anti-inflammatory action, antitumor action, fat burning action, neuroprotection. The action and so on. However, quinoline itself was not sufficiently absorbed, pharmacodynamically and pharmacodynamically, and was poorly used industrially. Therefore, a substance having better absorption and stronger effect has been desired.

This quinoline derivative is preferable because it has excellent structural features in terms of both absorption and action. That is, this quinoline derivative has a hydrophobic region inside, while it has a hydroxyl group and an amino group on the outside and has excellent water solubility, and is amphipathic which can be both water-soluble and fat-soluble. Is the nature of. Therefore, it dissolves in an aqueous solution and easily passes through a fat-soluble portion such as a cell membrane. Due to this amphipathic property, it easily penetrates into cells as an estrogen receptor, penetrates into the nucleus, binds to DNA, and exerts an estrogen action.

Since this quinoline derivative is amphipathic, it is preferable that it easily passes through the mucosal layer of the intestinal tract and its absorption into the body is enhanced. Furthermore, the stratum corneum of the skin also easily penetrates into the barrier tissue between the keratinocytes and easily invades, and easily reaches the epidermis layer, that is, the skin stem cells in the stratum granulosum, the spinous layer and the basal layer. Further, since it exhibits amphipathic properties, it is preferable that the range of use as an aqueous solution and an oil is expanded.

In addition, the benzene ring of p-coumaric acid also contributes to hydrophobicity, and the amino group of valine exhibits a pH buffering effect and, when ingested as food, exhibits resistance to gastric acid. That is, it is preferable to prevent the decomposition of this derivative by gastric acid by relaxing the gastric acid by this pH buffering action.

The two hydroxyl groups of this quinoline derivative are free and exhibit antioxidant activity as the hydroxyl groups of polyphenols. It is preferable that this antioxidant action exerts a protective action against oxidative stress. For example, when applied to the skin, it has a function of preventing oxidation of the skin by ultraviolet rays, and it is preferable to protect the skin. Further, it is preferable that this derivative is pulverized and then reacted with a water-soluble solvent to generate hydrogen gas.

This quinoline derivative itself does not act as a ligand for the estrogen receptor, but acts near the active center of the nuclear estrogen receptor and exhibits an estrogen receptor activating effect. Further, it is preferable that the generated hydrogen gas assists the function of the receptor and has a stabilizing effect.

Further, as an estrogen receptor activating action by this quinoline derivative, the km value and the Vmax value of estrogen and the estrogen receptor are changed. That is, the km value is lowered to increase the binding property between estrogen and the estrogen receptor. Further, Vmax, which is the maximum number of bonds, is increased to increase the maximum bond value. This change in km and Vamx is an antagonistic reaction to estrogen, and the decrease in estrogen, which is a ligand, reduces the estrogen receptor activation effect of the quinoline derivative. That is, activation does not occur in the absence of estrogen. This antagonistic reaction is a reversible reaction and is highly safe because it does not strongly change the state of the estrogen receptor.

Valine and p-coumaric acid, which are constituents of this quinoline derivative, are natural products and are abundant in nature, and their safety has been confirmed. In addition, quinoline also exists as a component of amino acids in nature, and its safety is high.

As a result of the estrogen receptor activating action of this quinoline derivative, the cell proliferative action of skin cells, the proliferative action of osteoblasts, the lipolytic action of adipocytes, the metabolism increasing action of cholesterol and the activating action of nerve cells are confirmed. ..

Estrogen receptors are present in each organ, and their reactivity is organ-specific, and there are differences due to genetic polymorphisms in addition to age and gender.

Since the estrogen receptor activating action of this quinoline derivative is expressed by two different mechanisms, it is possible to act on all states including gene polymorphisms and pathological states and all tissues.

In particular, it is preferable that the quinoline derivative has high reactivity with respect to the estrogen receptor of the stem cells of the basal layer of the skin and the stem cells of the nerve cells. Therefore, this quinoline derivative is excellent in the proliferative effect of skin and nerves, and the therapeutic and preventive effects of skin diseases and nerve diseases.

Quinoline derivatives are used as plant activators. Since this quinoline derivative is amphipathic, it passes through the cell membrane of animals and the cell wall of plants and yeast, and is easily absorbed into the cells. Of these, estrogen on plants has a plant hormone-like effect.

It is preferable that this quinoline derivative enhances the reactivity of auxin, cytokinin, abscisic acid, gibberellin and the like to promote the growth of plants.

It is preferable that this quinoline derivative can contribute to the development of the agricultural field and the increase of food production because it promotes the overall growth of plants such as germination, growth, flowering, fruiting and harvesting as a plant activator or plant growth agent. In particular, it is preferable that it can be used for growing precious flowers such as orchids and whisk ferns and bonsai.

In addition, quinoline derivatives are also used as microbial activators. In addition, this quinoline derivative can be used for shortening and rationalizing the fermentation process in that it can promote the growth of useful microorganisms. It is preferable that it can be used to increase production and shorten the production period for sake brewing, miso, soy sauce, natto, etc.

This quinoline derivative easily passes through the stratum corneum cell membrane of the skin, and it is preferable to maintain the barrier function of the stratum corneum from the viewpoint of skin health and beauty.

Further, since this quinoline derivative is amphipathic and exhibits a pH buffering action, it is preferable that it can be blended in both water-soluble lotions and creams.

It is preferable that this quinoline derivative promotes skin cell function by promoting collagen and elastin production through the proliferation of genital cells. Furthermore, it is preferable that a synergistic effect can be obtained by using in combination with EGF (Epidermal Growth Factor).

Nerve cells also exert an estrogen receptor activating action on nerve cells to activate the proliferation of nerve cells. This quinoline derivative restores the function of nerve cells through the estrogen receptor activating action against diseases such as dementia and Alzheimer's disease, and damages and phenomena of nerve cells caused by active oxygen and amyloid β protein. This action is preferred for the purpose of prevention, defense and recovery of neurological disorders.

In addition, it is preferable that this derivative promotes the release of neurotransmitters from nerve endings to enhance nerve transmission from the viewpoint of recovery of nerve function. Furthermore, it is preferable that a synergistic effect can be obtained by using in combination with NGF.

Increasing muscle contraction and increasing nerve and muscle activity by increasing the release of acetylcholine from the nerve endings of motor neurons is preferable from the viewpoint of muscle strengthening and prevention and treatment of bedtime elderly people.

In myocardial infarction, it is preferable that this quinoline derivative activates the estrogen receptor of cardiomyocytes to proliferate cardiomyocytes and promote regeneration of the damaged site even in a coronary artery infarction or an ischemic state.

In addition, when this quinoline derivative wants to strengthen an athlete or muscle, it is preferable to strengthen muscle tissue by exhibiting an estrogen receptor activating action of muscle cells and proliferating skeletal muscle cells. In particular, it is preferable to regenerate muscle cells against muscle cell loss such as muscle strain and muscle amputation.

This quinoline derivative is decomposed by renal and hepatic esterase in vivo and excreted in urine. It is decomposed into highly safe quinoline, p-coumaric acid and valine, which are constituents. Therefore, this quinoline derivative is not accumulated in the body, is decomposed by an enzyme in vivo, and the decomposed product is also a natural product, so that it is highly safe.

Examples of methods for producing this quinoline derivative include mulberry leaves, lotus seeds, lotus leaves, lotus flowers, algae, yusuraume fruits, citrus fruits such as grapefruit and satsuma mandarin, and soybean nuts. It is preferable to produce the desired quinoline derivative from the viewpoint of safety because the raw material is a natural product.

It is possible to extract this quinoline derivative from the above plants by purification. However, refining requires a large amount of raw materials, is expensive, and uses organic solvents, etc., which limits its use in the food industry.

Further, the production by the fermentation method is preferable because the production efficiency is high in that proteins can be decomposed and dietary fiber can be excluded, and it is suitable for the production of this quinoline derivative. When the obtained quinoline derivative is used as a pharmaceutical material, it is preferable to purify the target quinoline derivative from the viewpoint of increasing the purity of the target quinoline derivative and removing impurities.

As a pharmaceutical product, it is used as an injection or an oral preparation or a parenteral preparation such as a coating agent, and as a quasi-drug, it is used by being blended in tablets, capsules, drinks, soaps, coating agents, gel agents, toothpaste, etc. Toothpaste.

Examples of the oral preparation include tablets, capsules, powders, syrups, drinks and the like. When mixed with the above-mentioned tablets and capsules, it can be used together with a binder, an excipient, a leavening agent, a lubricant, a sweetener, a flavoring agent and the like. The tablets can also be coated with shellac, sugar or the like.

Further, in the case of the above-mentioned capsule, the above-mentioned material can further contain a liquid carrier such as oil and fat. In the case of the above-mentioned syrup and drink, sweeteners, preservatives, pigment flavors and the like can be added.

Examples of parenteral preparations include injections in addition to external preparations such as ointments, creams and liquids. As the base material of the external preparation, petrolatum, paraffin, oils and fats, lanolin, macrogold and the like are used, and ointments, creams and the like can be prepared by a usual method.

Injections include liquids and lyophilizers. When used, it is aseptically dissolved in distilled water for injection, physiological saline, or the like.

In addition, as a food preparation, it can be used for foods for the purpose of tissue regeneration such as skin regeneration, nerve regeneration, and muscle regeneration. It is preferable to use it as a food with a nutritional function or a food for specified health use as a food with a health function.

When the obtained food preparation is used for pets such as dogs and cats and domestic animals, it is used as a feed or a supplement for pets for the purpose of recovering skin disorders and neuropathy, suppressing aging and improving athletic ability.

As a cosmetic, it can be used together with a surfactant, a solvent, a thickener, an excipient and the like according to a conventional method. For example, it can be in the form of a cream, a gel for hair, a face wash, a beauty essence, a lotion, or the like. Since it exhibits amphipathic properties, it is preferable to expand the range of use as an aqueous solution and an oil.

The form of the cosmetic is arbitrary and can be used as a solution, cream, paste, gel, gel, solid or powder.

The cosmetics produced here are used for the purpose of enhancing estrogen action to repair damaged skin, increasing collagen and elastin, and maintaining skin health.

In addition, this quinoline derivative can be used in dentifrices, mouthwashes, dentifrice pastes, etc. for the purpose of maintaining the proliferation and function of osteoblasts and gingival cells that have decreased due to aging.

As a method for producing this quinoline derivative, for example, it is preferable to ferment the seeds of sacred lotus, treat it with protease, and sterilize the filtered solution.

The lotus (scientific name: Nelumbo nucifera) is cultivated in various parts of Japan, and the seeds of this lotus are collected as fruits after the flowers bloom. Organically cultivated products are preferable because they have a low risk of contamination by pesticides. In addition, it is preferable from the viewpoint of safety that the lotus seed has a track record of being used as a raw material for cosmetics and foods.

Lotus seeds are cultivated in Tatsuta Village, Ama District, Aichi Prefecture, and can be purchased at trading companies that handle Kampo, such as Kojuken in Nihonbashi. In addition, proteases for food processing can be used. For example, Protease N manufactured by Amano Enzyme is preferable because it has a long track record for food processing and is highly safe.

Lotus seeds are placed in clean tanks and dispersed with purified water. After heating this to 37 to 43 ° C., protease N is added and the protein is decomposed for 3 to 6 hours with stirring.

The weight of sacred lotus seeds is preferably 300 g to 1 kg, and the weight of protease N added is preferably 10 to 30 g with respect to 10 L of purified water.

After this reaction, it is preferable to collect the filtrate by filtration with a filter paper because the residue of undecomposed lotus seeds can be removed. Further, boiling this filtrate at a temperature of 95 ° C. or higher is preferable for the purpose of inactivating protease and sterilizing.

Furthermore, it is preferable to obtain the desired quinoline derivative by fermenting the seeds of sacred lotus. For example, a fermentation method of fermenting soybeans with Bacillus natto is preferable because it has abundant technical knowledge and experience.

The soybean used as a fermentation raw material can be used from any of the soybeans produced in Japan, China, the United States, Russia, etc., but it is preferable that the soybeans are produced in Japan, which has reliable traceability and a clear producer.

Of these, soybeans cultivated organically or without pesticides are more preferable because they do not contain harmful pesticides or metals.

When using soybeans, Nara Machinery Co., Ltd.'s free mill, super free mill, sample mill, goblin, super clean mill, micros, small vacuum dryer made by Toyo Riko Co., Ltd., small vacuum dryer made by Matsui Co., Ltd. It is dried and crushed by a vacuum heat transfer dryer DPTH-40, a clean dry VD-7 and VD-20 manufactured by AKUM Kyushu Technos Co., Ltd., and a DM-6 manufactured by Nakayama Institute of Technology. As a result, the fermentation process is likely to proceed efficiently.

The natto bacterium used has the scientific name Bacillus subtilis and is a useful microorganism used in the production of natto. Bacillus natto powder from Natto Motopo is preferable because it has good quality and is suitable for fermentation.
First, lotus seeds are crushed by a crusher or a mixer. By being crushed, it becomes easier to process. Soybeans are crushed by a crusher and suspended by adding clean water. 60 to 200 g of soybean is added to 100 g of lotus seeds, and the mixture is stirred with water such as purified water and 3 to 10 liters in a clean container.

Lotus seeds and soybeans are sterilized by boiling and added to the fermentation tank. Sterilization prevents the contamination of germs and promotes fermentation by Bacillus natto.

Fermentation may be carried out by either a static method or a stirring method, but the stirring method is preferable because the fermentation can be carried out in a short time. Fermentation is preferably carried out at 40-44 ° C. for 20-80 hours. If the temperature is low and the time is short, fermentation will not proceed, and if the temperature is high and the time is long, the target quinoline derivative may be decomposed.

Purification is preferred for higher purity. For example, the desired quinoline derivative can be obtained by being separated by a separation carrier or a resin and separated. As the separation carrier or resin, a porous polysaccharide, a silicon oxide compound, polyacrylamide, polystyrene, polypropylene, a styrene-vinylbenzene copolymer or the like whose surface is coated as described later is used. Those having a particle size of 0.1 to 300 μm are preferable, and the finer the particle size, the higher the accuracy of separation, but there is a drawback that the separation time is long.

For example, a reversed-phase carrier or a resin whose surface is coated with a hydrophobic compound is used for separating highly hydrophobic substances. Those coated with a cationic substance are suitable for separating anionically charged substances. In addition, those coated with an anionic substance are suitable for separating cationically charged substances. When coated with a specific antibody, it is used as an affinity carrier or resin that separates only specific substances.

The affinity carrier or resin is utilized for the specific preparation of the antigen by utilizing the antigen-antibody reaction. Distributable carriers or resins, such as silica gel (manufactured by Merck & Co., Inc.), are used for isolation of substances when there is a difference in partition coefficient between the substance and the solvent for separation. It is preferable to repeat the purification operation in order to obtain a highly pure purified product.

Of these, an adsorptive carrier or resin, a distributable carrier or resin, a molecular sieve carrier or resin, and an ion exchange carrier or resin are preferable from the viewpoint of reducing the production cost. Further, a reversed-phase carrier or resin and a distributable carrier or resin are more preferable because the difference in partition coefficient with respect to the separation solvent is large.

When an organic solvent is used as the separation solvent, a carrier or resin resistant to the organic solvent is used. In addition, carriers or resins used for pharmaceutical production or food production are preferable.

From these points, Diaion HP20 (manufactured by Mitsubishi Chemical Corporation) and XAD-2 or XAD-4 (manufactured by Roam & Haas) as an adsorptive carrier, and Sephadex LH-20 (manufactured by Amasham Pharmacia) as a carrier for molecular sieves. ), Silica gel as the distribution carrier, IRA-410 (manufactured by Roam & Haas) as the ion exchange carrier, and DM1020T (manufactured by Fuji Silicia) as the reversed-phase carrier are more preferable.

Of these, Mitsubishi Chemical's Diaion HP20, Sephadex LH-20 and DM1020T are even more preferable.

The resulting extract is dissolved in a separation carrier or solvent for swelling the resin prior to separation. The amount thereof is preferably 2 to 40 times, more preferably 4 to 20 times the weight of the extract from the viewpoint of separation efficiency. The separation temperature is preferably 10 to 30 ° C., more preferably 12 to 25 ° C. from the viewpoint of substance stability.

As the separation solvent, water, a lower alcohol containing water, a hydrophilic solvent, or a lipophilic solvent is used. As the lower alcohol, methanol, ethanol, propanol and butanol are used, but ethanol used for food is preferable.

When Sephadex LH-20 is used, a lower alcohol is preferable as the separation solvent. When silica gel is used, chloroform, methanol, acetic acid or a mixture thereof is preferable as the separation solvent.

When Diaion HP20 and DM1020T are used, the separation solvent is preferably a lower alcohol such as methanol or ethanol or a mixed solution of lower alcohol and water.

It is preferable to collect a fraction containing the quinoline derivative, dry it or vacuum dry it to remove the solvent, and obtain the desired quinoline derivative as a powder or a concentrated solution because the influence of the solvent can be excluded. Since it exhibits amphipathic properties, it is preferable to expand the range of use as an aqueous solution and an oil.

On the other hand, it is also possible to carry out the final extraction with oils and fats used for cooking oils and cosmetics. For example, it can be extracted with soybean oil, rice bran oil, grape seed oil, olive oil, and jojoba oil. In other words, by making it oily, the range of use is expanded.

Further, it is preferable to pulverize this quinoline derivative by a spray-drying method or a freeze-drying method for the purpose of preserving and extending the storage period.

It is preferable to perform structural analysis of the quinoline derivative produced in this manner by HPLC or NMR because it is possible to provide a high quality quinoline derivative.

Hereinafter, the embodiment will be specifically described with reference to Examples and Test Examples. These are just examples, and it is possible to change the conditions within the range of common sense according to the difference in the material, raw material, and sample.

First, an example of production by the fermentation method will be described. That is, 1 kg of lotus seeds (fruits) purchased at Nihonbashi Kojuken (Tokyo) was purchased. After washing this with water, it was suspended in a crusher (super free mill manufactured by Nara Machinery Co., Ltd.) together with purified water to obtain 0.88 kg of a suspension.

The seeds of the lotus were analyzed for pesticides in advance, and it was confirmed that there were no pesticides resident. It was frozen and stored until use.

Furthermore, 1 kg of soybeans from Hokkaido was purchased from Miwa Bussan and used. After washing this, it was pulverized by a pulverizer to obtain about 920 g of a pulverized product.

500 g of lotus seed suspension and 500 g of soybean powder were transferred to a clean stainless steel barrel, 5 liters of purified water was added and suspended by a stirrer.

This was sterilized by boiling at 95-99 ° C. for 1 hour. After cooling, these were transferred to a stirring fermentation tank (FP211) manufactured by Yokogawa Electric Co., Ltd. having a capacity of 100 kg, and 18 liters of sterilized purified water was added.

To this, 3 g of powdered natto bacteria manufactured by Yuzo Takahashi Laboratory Co., Ltd., which is the main office of natto bacteria, was purchased. This natto bacterium was suspended in 100 g of sterilized water, powdered soybean powder was added thereto, and the mixture was pre-cultured by heating at 37 ° C. for 1 hour.

This natto bacterium solution was added to the stirring fermentation tank and fermented at a temperature of 40 to 43 ° C. for 42 hours. The state of fermentation was monitored by the degradability of soybean powder and the quantification of dissolved protein (Buret method).

After fermentation, the supernatant of the obtained fermentation broth was roughly filtered through a filter cloth to obtain a filtrate.

This filtrate was freeze-dried with a freeze-dryer (Freeze Trap VA-140S manufactured by Titec Co., Ltd.) to obtain 136 g of the desired powder. This was designated as sample 1. When 10 mL of purified water was added to 0.1 g of this powder, hydrogen gas was detected at a concentration of 1.6 ppm.

The obtained sample 1 was subjected to a purification step. That is, 80 g of this powder was suspended in 320 mL of purified water and subjected to 500 g of Diaion HP20 (manufactured by Mitsubishi Chemical Corporation) swollen with 8% ethanol. After washing with 1200 mL of 8% ethanol, further washing with 1 liter of 50% ethanol.

To this, 800 mL of 80% ethanol was added, and a fraction of the target quinoline derivative was collected. This purification step was repeated 3 times to increase the purity. The temperature at which this purification step was carried out was 18 to 22 ° C. The final fraction obtained in these three purification steps was dried with a vacuum dryer (manufactured by Nippon Biocon Co., Ltd.) to obtain 12 g of powder. This powder was used as sample 2. When 10 mL of purified water was added to 0.1 g of the powder of this sample 2, as a result of analysis by gas chromatography (manufactured by Shimadzu Corporation), generation of 1.6 ppm of hydrogen gas was detected.

The test methods and results for structural analysis of quinoline derivatives will be described below.
(Test Example 1)

The sample 2 obtained as described above was dissolved in ethanol and analyzed by high performance liquid chromatography (HPLC, Shimadzu Corporation) equipped with a mass spectrometer.

Further, as a result of analyzing this with a nuclear magnetic resonance apparatus (600 MHz, H-NMR and C-NMR, manufactured by Bruker), the target quinoline derivative was identified from Specimen 1 and Specimen 2.

That is, as a result of 1 H-NMR measurement in deuterated dimethyl sulfoxide of sample 2, the peak positions were 1.84, 1.88, 1.90, 2.69, 3.33, 4.00, 5.06. , 6.20, 6.65, 6.66, 6.74, 6.77, 6.80, 6.91, 6.95, 7.96, 8.02 and 8.05 ppm.

As a result of C-NMR measurement of Specimen 2, 22.4, 22.6, 23.5, 36.0, 42.3, 49.7, 56.5, 71.3, 86.2, 114. 2, 116.4, 117.4, 117.9, 119.0, 119.4, 123.0, 124.3, 126.4, 131.7, 134.7, 136.5, 141.5, Peaks were observed at 142.8, 146.0, 146.3, 146.9, 147.0, 173.9, 174.2 and 174.3 ppm.

The chart of the analysis result of C-NMR is shown below. (The horizontal axis unit is ppm, and the vertical axis unit is peak intensity.)

From the above analysis results, it was found that the product has the same structure as the chemically synthesized standard product. That is, it was confirmed from the sample 2 that it was the target quinoline derivative in which one molecule of quinoline, one molecule of valine and one molecule of p-coumaric acid were bound. Moreover, in the HPLC analysis of the sample 2, the peak was one, and the purity was 99.1%. The purity of sample 1 was 77.1%.

The estrogen receptor activation test using human skin epidermal cells will be described below. This test method is a reproducible method that can biochemically verify the effect of the active ingredient.
(Test Example 2)

Human-derived epidermal cells (epidermal-derived, epidermis) purchased from Kurabo Industries were used. 1000 cells cultured in MEM medium containing 5% fetal bovine serum (manufactured by Sigma) as a culture medium were seeded in a 35 mm culture dish (manufactured by FALCON) and cultured at 37 ° C. under 5% carbon dioxide gas. This was irradiated with ultraviolet rays of 280 nm for 1 hour by an ultraviolet irradiation device (manufactured by Quark Technology). To this, EGF (manufactured by Funakoshi, epidermal growth factor) was added to each of the above-mentioned Specimen 1, Specimen 2 and as a positive control at a final concentration of 10 mg / ml. At the same time, the function of the combined use with EGF (manufactured by Funakoshi, product code NO20) was investigated. This was cultured for 48 hours and tested.

After collecting the culture medium, the survival rate of epidermal cells was counted by the trypan blue method. Then, a suspension of epidermal cells was prepared. The activated state of the estrogen receptor was measured using this cell suspension. The binding affinity between the estrogen receptor and the sample was analyzed by the nuclear receptor reporter assay kit (INDIGO Biosciences, Inc.). This Citesum is an estrogen receptor assay system mediated by cell culture and has been used extensively.

Furthermore, mRNA was extracted from the cell suspension using a nucleic acid extraction kit (manufactured by Funakoshi). According to a conventional method, the amount of estrogen receptor mRNA was quantified by the RT-PCR method (Takara Bio Inc., microRNA Ready-to-Use PCR Panel) using a primer manufactured by Takara Bio.

The average value of the petri dish was calculated using five petri dishes. It was compared with the solvent control group to which the solvent was added.

As a result, the number of human-derived epidermal cells increased to 177% on average as compared with the solvent control group by adding 10 mg / ml of Specimen 1. In sample 2, it increased to 206%. On the other hand, it was 136% with the addition of EGF. As a result, Specimen 1 and Specimen 2 exhibited a better human-derived epidermal cell proliferation effect than EGF. Furthermore, the combined use of Specimen 2 and EGF increased the ratio to 773% as compared with the solvent control group, and a synergistic effect with EGF was observed.

As a result of measuring the binding affinity with the estrogen receptor by the nuclear receptor reporter assay kit, the value of Vmax increased to 180% as an average value when the sample 1 was added as compared with the solvent control group. In sample 2, it increased to 311%. On the other hand, it was 110% with the addition of EGF. Since Vmax shows the maximum binding ability between estrogen and estrogen receptor, Specimen 1 and Specimen 2 showed better binding to estrogen receptor than EGF. This means that the reactivity with the estrogen receptor was enhanced, which is the activation of the estrogen receptor. Furthermore, the combined use of Specimen 2 and EGF resulted in 709%, and a synergistic effect was observed when used in combination with EGF.

On the other hand, the km value (that is, 50% concentration) indicating the binding concentration of the estrogen receptor to estrogen decreased to 77% on average as compared with the solvent control group. In sample 2, it decreased to 65%. On the other hand, it was 98% with the addition of EGF. Since the Km value shows the affinity of the binding between estrogen and the estrogen receptor, the sample 1 and the sample 2 showed the superior affinity with the estrogen receptor at a lower concentration than the EGF. This means that the reactivity with the estrogen receptor was increased, and since the km was decreased, this reaction was the activation of the estrogen receptor. Furthermore, the combined use of Specimen 2 and EGF resulted in 52%, and the synergistic effect of the combined use with EGF was observed.

Furthermore, the binding property of Specimen 1 and Specimen 2 was reduced by treating the estrogen at a high concentration. This reduction indicates that the activating action is antagonistic activation.

The mRNA expression level (number of copies) of the estrogen receptor in the above cells was 19 copies in the solvent control group, 58 copies in the sample 1 treatment group, 388 copies in the sample 2 treatment group, and 39 copies in the EGF treatment group. .. Furthermore, the combined use of Specimen 2 and EGF resulted in 820 copies. The results of this combination indicate that Specimen 2 and EGF acted synergistically.

From this result, the mRNA expression level of the estrogen receptor was remarkable in Specimen 1 and Specimen 2, and was superior to EGF. This showed the mRNA-inducing action of the estrogen receptor by Specimen 1 and Specimen 2. In addition, a synergistic effect was observed when the sample 2 and EGF were used in combination.

On the other hand, as a part of the safety test, a skin irritation test using EpiSkin (manufactured by SkinEthic), which is an artificial skin, was performed. As a result, no irritation was observed in normal cells due to the addition of Specimen 1 and Specimen 2, and the safety of Specimen 1 and Specimen 2 was confirmed. The safety test method using artificial skin is a skin irritation test evaluation method using cells, and has been established as an alternative method for animal experiments without using animals, and is a highly reliable method.

The effect test for the disorder using the disorder model of human nerve cells is described below. This test method is a reproducible conventional method that can verify the action of the active ingredient biochemically and molecularly, has abundant test results, and is highly reliable.
(Test Example 3)

Human neurons (Human Neurons (HN), 1520 type) purchased from Cosmo Bio were used. 1000 cells cultured using a special culture medium (nerve cell growth medium) as a culture medium were seeded in a 35 mm culture dish and cultured at 37 ° C. under 5% carbon dioxide gas. A 1% aqueous solution of acrylamide, which is a neurotoxin, was added to stimulate nerve cells.

To this, Specimens 1 and 2 obtained in Example 1 above, and NGF (Funakoshi Co., Ltd., human type) as a positive control were all added at a final concentration of 10 mg / ml. This was cultured for 48 hours. Furthermore, NGF (manufactured by Funakoshi, β type, product code 450-01) and sample 2 were used in combination for the experiment.

After completion of the culture, the number of nerve cells was counted microscopically. Furthermore, the function of the estrogen receptor (nuclear receptor reporter assay kit) and the expression level of mRNA are measured by the RT-PCR method (Takara Bio Co., Ltd., microRNA Ready-to-Use PCR Panel) by the same method as described above. did.

As a result, the addition of 10 mg / ml of Specimen 1 increased the number of nerve cells to 132% on average as compared with the solvent control group. In sample 2, it increased to 199%. On the other hand, it was 112% in NGF. As a result, Specimen 1 and Specimen 2 exhibited a better cell activating effect than NGF. In addition, the combined use of NGF and Specimen 2 resulted in a cell number of 442%, and Specimen 2 and NGF showed synergistic proliferation.

As a result of measuring the binding affinity with the estrogen receptor by the nuclear receptor reporter assay kit, the value of Vmax increased to 166% as an average value when the sample 1 was added as compared with the solvent control group. In sample 2, it increased to 288%. On the other hand, when NGF was added, it was 107%. Since Vmax shows the maximum binding ability between estrogen and estrogen receptor, Specimen 1 and Specimen 2 showed better binding to estrogen receptor than NGF. This means that the reactivity with the estrogen receptor was enhanced, which is the activation of the estrogen receptor. Furthermore, the combined use of Specimen 2 and NGF resulted in 566%, and the synergistic effect of the combined use with NGF was observed.

On the other hand, the km value indicating the binding concentration of the estrogen receptor to estrogen decreased to 80% as an average value when the sample 1 was added as compared with the solvent control group. In sample 2, it decreased to 69%. On the other hand, it was 99% with the addition of NGF. Since the Km value shows the affinity of the binding between estrogen and the estrogen receptor, the sample 1 and the sample 2 showed the superior affinity with the estrogen receptor at a lower concentration than that of NGF. This means that the reactivity with the estrogen receptor was enhanced, which is the activation of the estrogen receptor. Furthermore, the combined use of Specimen 2 and NGF resulted in 59%, and the synergistic effect of the combined use with NGF was observed.

Furthermore, the binding property of Specimen 1 and Specimen 2 was reduced by treating the estrogen at a high concentration. This reduction indicates that the activating action is antagonistic activation.

The mRNA expression level (number of copies) of the estrogen receptor in the above cells was 17 copies in the solvent control group, 48 copies in the sample 1 treatment group, 122 copies in the sample 2 treatment group, and 29 copies in the NGF treatment group. .. In addition, the combined use of Specimen 2 and NGF resulted in 552 copies, and a synergistic effect with NGF was observed.

The mRNA expression level of the estrogen receptor was remarkable in Specimen 1 and Specimen 2, and was superior to NGF. This showed the mRNA-inducing action of the estrogen receptor by Specimen 1 and Specimen 2. In addition, a synergistic effect was observed when used in combination with NGF.

The quinoline derivative obtained in the present invention exhibits an estrogen receptor activating action and activates various cell functions such as skin cells and nerve cells. As a result, the QOL of the people can be improved, the healthy working population can be increased, and medical expenses can be reduced.

The quinoline derivative obtained by the present invention increases skin cells, contributes to the improvement of the skin of those suffering from skin troubles such as wrinkles and tarmi as cosmetics, and contributes to the development of the cosmetics industry.

The quinoline derivative obtained by the present invention increases nerve cells and can be used for the treatment of dementia and Alzheimer's disease, and also proliferates osteoblasts and contributes to the maintenance of national health.

Claims (1)

A quinoline derivative exhibiting an estrogen receptor activating action represented by the following formula (1).