Diacylglycerol lactone compound, process for producing the same, and immunostimulant containing the same as active ingredient
阅读说明：本技术 二酰基甘油内酯化合物、其制备方法以及含有其作为有效成分的免疫刺激剂 (Diacylglycerol lactone compound, process for producing the same, and immunostimulant containing the same as active ingredient ) 是由 孙起荣 金载和 尹善瑛 刘畅炫 郑珍善 于 2019-03-08 设计创作，主要内容包括：公开了一种促进嗜中性粒细胞迁移以增强免疫力和抑制感染的新型二酰基甘油内酯化合物、其制备方法以及包含其作为活性成分的免疫刺激剂。本说明书中的二酰基甘油内酯化合物由化学式1表示。在化学式1中,R1和R2独立地是2至30个碳原子的脂肪酸残基。(Disclosed are a novel diacylglycerol lactone compound that promotes neutrophil migration to enhance immunity and suppress infection, a method for preparing the same, and an immunostimulant comprising the same as an active ingredient. The diacylglycerol lactone compound in the present specification is represented by chemical formula 1. In chemical formula 1, R1 and R2 are independently a fatty acid residue of 2 to 30 carbon atoms.)
1. A diacylglycerol lactone compound represented by the following chemical formula 1,
[ chemical formula 1]
In chemical formula 1, R1 and R2 are independently a fatty acid residue of 2 to 30 carbon atoms.
2. The diacylglycerol lactone compound of claim 1, wherein R1 and R2 are independently selected from: acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, 2-methylbutyryl, cyclopropanecarbonyl, cyclohexanecarbonyl, hexanoyl, heptanoyl, nonanoyl, dodecanoyl, myristoyl, palmitoyl, linoleoyl, oleoyl, linoleoyl, eicosanoyl and arachidonyl.
3. The diacylglycerol lactone compound of claim 1, wherein the diacylglycerol lactone compound of chemical formula 1 is a compound represented by the following chemical formula 2a or chemical formula 2b,
[ chemical formula 2a ]
[ chemical formula 2b ]
4. A method of preparing a diacylglycerol lactone comprising the steps of:
as shown in the following reaction 1, a compound represented by the formula A is reacted with CH2＝CH-CH2MgX (X is a halogen atom) to produce a compound represented by the formula B,
[ reaction 1]
In reaction 1, P1And P2Is a protecting group;
hydroxylating the double bond of the compound of formula B to obtain a compound represented by formula C as shown in the following reaction 2,
[ reaction 2]
In reaction 2, P1And P2Is a protecting group;
as shown in the following reaction 3, a lactone cyclization reaction is performed on the compound of chemical formula C to obtain a compound represented by chemical formula D,
[ reaction 3]
In reaction 3, P1And P2Is a protecting group; and
the compound of chemical formula D is subjected to deprotection reaction and esterification reaction, as shown in the following reaction 4, to obtain a compound represented by chemical formula 1,
[ reaction 4]
In reaction 4, P1And P2Is a protecting group, R1 and R2 are independently fatty acid residues of 2 to 30 carbon atoms.
5. An immunostimulant comprising a diacylglycerol lactone compound represented by the following chemical formula 1 as an effective ingredient,
[ chemical formula 1]
In chemical formula 1, R1 and R2 are independently a fatty acid residue of 2 to 30 carbon atoms.
6. The immunostimulant according to claim 5, wherein the diacylglycerol lactone compound increases IL-8 cytokine expression.
7. The immunostimulant according to claim 5, wherein the diacylglycerol lactone compound promotes migration of neutrophils from blood to cells.
8. The immunostimulant according to claim 5, wherein the diacylglycerol lactone compound prevents or treats an immune disease selected from bacterial or viral infection, acute and chronic inflammatory lung disease, pneumonia and sepsis.
9. The immunostimulant according to claim 5, wherein the amount of the diacylglycerol lactone compound is from 0.0001 to 100.0 wt%.
10. A health functional food composition for enhancing immunity comprises diacylglycerol lactone compound represented by the following chemical formula 1 as an effective ingredient,
[ chemical formula 1]
In chemical formula 1, R1 and R2 are independently a fatty acid residue of 2 to 30 carbon atoms.
11. A method of enhancing immunity, the method comprising administering to a non-human subject an immunostimulant according to any one of claims 5 to 9.
Technical Field
The present invention relates to diacylglycerolactone compounds, and more particularly, to novel diacylglycerolactone compounds for promoting neutrophil migration to enhance immunity and suppress infection, a method for preparing the same, and an immunostimulant comprising the same as an active ingredient.
Background
Immunization is a self-defense reaction that physiologically recognizes, eliminates and/or metabolizes exogenous and endogenous foreign substances within the body. Immune responses can be divided into innate immunity, which is the initial immune response, and adaptive immunity, which is the late immune response. In the initial immune response, the host is protected by the inhibition of foreign substances (pathogens) by the activity of macrophages and natural killer cells (NK cells). At this time, macrophages engulf foreign substances and produce and secrete TNF- α as an active marker. NK cells destroy pathogen-infected cells by producing and secreting perforin, an active marker. Subsequently, cytotoxic T lymphocytes, helper T lymphocytes and B lymphocytes associated with acquired immunity are activated to kill infected cells or produce antibodies, thereby protecting the host. Cytotoxic T lymphocytes kill pathogen-infected cells by producing and secreting large numbers of perforin-like NK cells. B lymphocytes, either helper or independent, protect the host by producing antibodies. Inflammatory cytokines, such as IL-6, IL-8, TNF- α, are agents that mediate immune responses and are known to be particularly involved in the initial immune response.
Generally, in the case of immunodeficiency, resistance to infection is reduced, patients with insufficient antibody production cannot protect themselves from bacterial infection, and phagocytic capacity of neutrophils is also reduced. In addition, in this case, since activation of the complement system is also inhibited, leukocyte migration factors and the like are not produced, so that inflammation increases and viremia occurs, resulting in spread of the virus to the central nervous system or elsewhere. In the case of cancer patients, not only cancer cells but also normal cells are affected during chemotherapy or radiation therapy, and the immunity of the patients may be rapidly reduced due to side effects.
Immunostimulants are drugs for treating congenital and acquired immunodeficiency, and immunoglobulin, Interferon (INF), etc. are typically used as immunostimulants. Wherein the preparation obtained by concentrating and purifying IgG which is one of human immunoglobulin is used for preventing and treating measles, varicella, hepatitis B, mumps, etc. However, the preparation has disadvantages of pain at the injection site and lowering blood pressure. Interferon has been found to be an antiviral factor, but its cell proliferation inhibitory action, immunoregulatory action, etc. have been found thereafter, and it is used as an antiviral agent and an antitumor agent. However, its β type has side effects such as fever, boredom, anorexia, local pain upon injection, and alopecia, while its α type may cause side effects such as leukopenia due to temporary suppression of bone marrow function.
Disclosure of Invention
The invention aims to provide a novel diacylglycerol lactone compound with an immune enhancement effect and a preparation method thereof.
It is another object of the present invention to provide a diacylglycerol lactone compound for enhancing immunity and suppressing infection by increasing the expression of IL-8(CXCL8) and promoting neutrophil migration, and an immunostimulant comprising the diacylglycerol lactone compound as an active ingredient.
In some embodiments for achieving the above objects, the present invention provides a diacylglycerol lactone compound represented by the following
[ chemical formula 1]
In
In addition, the present invention provides an immunostimulant and health functional food composition for immune enhancement comprising the diacylglycerol lactone compound represented by
The diacylglycerol lactone compound of the present invention is a novel compound having an immunopotentiating effect. It increases the expression of IL-8(CXCL8) and promotes neutrophil migration, thereby enhancing immunity and inhibiting infection.
Drawings
FIG. 1 is a graph showing that CXCL8(IL-8) expression is increased in cells due to diacylglycerol lactone compounds according to the present invention.
FIG. 2 is a graph showing the bacterial infection inhibitory effect of diacylglycerol lactone compounds according to the present invention.
Fig. 3 is a photograph showing the results of a bacterial infection inhibition experiment of the diacylglycerol lactone compound according to the present invention.
Detailed Description
Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.
The present invention provides a novel diacylglycerol lactone compound represented by the following
[ chemical formula 1]
In
Preferred examples of the diacylglycerol lactone compound represented by
[ chemical formula 2a ]
[ chemical formula 2b ]
The diacylglycerol lactone compound represented by
[ reaction 1]
[ reaction 2]
[ reaction 3]
[ reaction 4]
In the esterification reaction of
The diacylglycerol lactone compound represented by
The diacylglycerol lactone compounds of the invention increase the expression of CXCL8(IL-8) in cells, promote neutrophil migration, and inhibit bacterial infection of bronchial fungi in animal models. Neutrophils Usually up to 10 per day in normal humans11And, mature from the bone marrow, then circulate in blood vessels for about 8 hours, infiltrate the tissue, survive for several days, and then die or disappear. When a bacterial infection occurs, the immune cells that primarily remove the bacteria are neutrophils. Vascular endothelial cells are activated by chemokines (CXCL8 or CXCL2, etc.) and various inflammatory factors secreted from damaged tissues in a region infected with bacteria, thereby circulating neutrophils in blood vessels to migrate to tissues and remove bacteria present in the infected tissues. In the experimental examples of the present invention, it was confirmed that the diacylglycerol lactone compound of
The diacylglycerol lactone compound of the present invention can be used as an immunostimulant alone, without being mixed with other substances, or can be used in the form of a pharmaceutical composition containing the diacylglycerol lactone compound as an active ingredient. When the diacylglycerol lactone compounds of the present invention are used in pharmaceutical compositions, conventional pharmaceutically acceptable carriers, excipients, or diluents can be included therein. The amount of the diacylglycerol lactone compound in the pharmaceutical composition may be widely varied without particular limitation, and is specifically 0.0001 to 100.0% by weight, preferably 0.001 to 95.0% by weight, more preferably 0.01 to 50% by weight, for example, 1 to 20% by weight, relative to the total amount of the composition.
The pharmaceutical composition may be formulated into any one selected from the group consisting of: tablets, boluses, powders, granules, capsules, suspensions, liquid solutions (liquidsolids), emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilizates, suppositories, and the like; and may be formulated in various forms for oral or parenteral administration. In formulating the composition, conventional excipients or diluents such as fillers, extenders, binders, wetting agents, disintegrants and surfactants may be used. Solid preparations for oral administration include tablets, boluses, powders, granules, capsules, and the like, and such solid preparations may be prepared by mixing one or more components with at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin, and the like. In addition to excipients, lubricating agents such as magnesium stearate and talc may be used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups and the like, and may include conventional diluents such as water and liquid paraffin, or may include various excipients such as wetting agents, sweeteners, flavoring agents and preservatives. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, suppositories, and the like, and solvents for solutions such as non-aqueous solutions, suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. The base material of suppositories may include witepsol, polyethylene glycol (macrogol), tween 61, cocoa butter, laurate and glycerogelatin.
The compositions of the present invention may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" is used to refer to an amount of a treatment for a disease sufficient to achieve a reasonable benefit/risk ratio applicable to the desired result in medical treatment. A "pharmaceutically effective amount" may be determined according to: the species, age, sex, severity and type of the disease, activity of the drug, sensitivity to the drug, time of administration, route of administration, rate of excretion, duration of treatment, factors including concomitant drugs, and other factors well known in the medical arts. The compositions of the present invention may be administered alone or sequentially or simultaneously with other therapeutic agents. The composition of the invention may be administered one or more times. In view of all the above factors, it is important to administer an amount that can achieve the maximum effect in the minimum amount without side effects, which can be easily determined by the person skilled in the art. The preferred amount of the composition of the present invention may vary depending on the condition and body weight of the patient, the severity of the disease, the type of formulation of the drug, the route of administration and the course of treatment. An appropriate total amount administered every 1 day may be determined by a physician, and is typically from about 0.001mg/kg to about 1000mg/kg, preferably from about 0.05mg/kg to 200mg/kg, more preferably from about 0.1mg/kg to about 100mg/kg once daily, or may be administered in divided doses several times per day. The compound or composition may be applied to any subject without particular limitation for the purpose of preventing immune reduction, enhancing immunity, or treating immune diseases, as long as it is an individual. For example, the composition of the present invention can be administered not only to humans but also to non-human animals (particularly mammals) such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cows, sheep, pigs, goats, and birds and fish. The composition of the present invention can be administered by conventional various methods, for example, by oral or rectal administration, or by intravenous (i.v.), intramuscular (i.m.), subcutaneous (s.c.), epidural (intradural) or cerebrovascular injection.
In some embodiments, the present invention provides a health functional food composition for enhancing immunity, comprising the diacylglycerol lactone compound of
The nutraceutical composition may consist solely or essentially of the pure compounds of the present invention or may comprise the compounds of the present invention as well as other conventional ingredients of nutraceutical foods. The amount of the active ingredient in the health food composition may be appropriately determined depending on the intended use. Generally, when the compound of the present invention is contained in a food or beverage, the amount of the composition of the present invention is preferably less than 15% by weight, more preferably less than 10% by weight, relative to the total amount of raw materials. The amount may be less than the above range in long-term use for the purpose of health management and hygiene. The amount of the active ingredient is more than the above range since there is no problem in safety.
The foods to which the compound of the present invention can be added are not limited and include various foods such as meat, sausage, bread, chocolate, candy, snack, pizza, noodles, gums, daily products such as ice cream, soup, beverage, tea, drink, alcoholic drink, vitamin complex and any health functional food, and also include foods used as animal feeds. When the health functional food composition of the present invention is used in a beverage product, the beverage product may include a sweetener, a flavoring agent, or natural carbohydrates. Examples of natural carbohydrates include: monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol, and erythritol. The amount of carbohydrates in the beverage composition may vary widely without particular limitation, and is preferably 0.01g to 0.04g, more preferably 0.02g to 0.03g per 100ml of beverage. Examples of sweeteners include natural sweeteners such as thaumatin and stevia extracts, and artificial sweeteners such as saccharin and aspartame. In addition to the above, the health functional food composition of the present invention may include various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH control agents, stabilizers, preservatives, glycerin, alcohol, carbonation used in carbonated beverages, and the like. Also, the health functional food composition of the present invention may include fruits such as fruits used for preparing natural fruit juice and fruit juice beverages as well as vegetable beverages.
In some embodiments, the present disclosure provides a method for enhancing immunity or preventing or treating an immune-related disease comprising administering the pharmaceutical composition to a patient in need thereof. The term "patient in need thereof" includes any animal, including humans, that has or can develop an immune-related disease. Immune-related diseases can be treated or prevented by administering to a patient in need thereof an effective amount of a pharmaceutical composition containing a compound of the present invention or containing a compound of the present invention and pharmaceutically acceptable salts thereof. The term "administering" means introducing a pharmaceutical composition of the invention to a patient in need thereof by any suitable method. The composition of the present disclosure can be administered by a conventional variety of methods, for example, by oral or parenteral administration, as long as the target tissue can be reached. In some embodiments, the methods of the present disclosure comprise administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a diacylglycerol lactone compound of formula I. A suitable total amount administered every 1 day may be determined by a physician and is generally from about 0.001mg/kg to about 1000mg/kg, preferably from about 0.05mg/kg to 200mg/kg, more preferably from about 0.1mg/kg to about 100 mg/kg. The total daily amount administered may be administered once daily or in divided doses several times daily. However, the specific therapeutically effective amount of the pharmaceutical composition administered to a particular patient may vary depending on the following factors: the type and extent of the response to be achieved in the treatment, the particular composition (including whether or not the composition contains another agent), the age, weight, general health, sex, diet of the patient, time of administration, route of administration, composition ratio, course of treatment, other drugs used together in the treatment, and a variety of factors well known in the medical arts.
Embodiments for carrying out the invention
Hereinafter, the present invention is described in more detail by embodiments. The following examples are only for aiding understanding of the present invention, and the present invention is not limited by the following examples.
[ example 1]Synthesis of diacylglycerolactone Compound (EC-A129)
A. As shown in the following reaction 1a, 50g (402.77mmol) of 4-methoxyphenol were dissolved in 1500ml of acetone. Then 278g (2013.8mmol) of K were added2CO3And stirred at room temperature for 30 minutes. 126ml (1611.1mmol) of epichlorohydrin are added to the 4-methoxyphenol solution, the temperature is raised to 60 ℃ to 65 ℃ and refluxing is carried out for 72 hours. The reaction was confirmed by TLC (ethyl acetate (EA): hexane (Hex): 1: 9). When the reaction was completed, the reaction solution was filtered through a celite filter, and the filtrate was concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 10 (volume ratio) mixture) to obtain 68g of the objective compound (yield: 97.3%).
[ reaction 1a ]
B. As shown in reaction 1b below, 0.73ml (7.0755mmol) of benzyl alcohol was dissolved in 4ml of Dimethylformamide (DMF). 283mg (7.0755mmol) of 60% -NaH was added slowly and stirred at room temperature for 30 min. 850mg (4.717mmol) of the product of reaction 1a (SM) are dissolved in 3ml of Dimethylformamide (DMF). The dissolved solution was slowly added dropwise to the reaction solution, followed by stirring at 80 ℃ for 3 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction is complete, H is 2O was added to the reaction solution to stop the reaction, and Ethyl Acetate (EA)/H was used2And (4) extracting. Thereafter, the organic layer was washed 3 times with purified water and MgSO4Water was removed and then concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 4 (volume ratio) mixture) to obtain 1.07g of the objective compound (yield: 78.3%).
[ reaction 1b ]
C. As shown in reaction 1c below, 52.77g (297.79mmol) pyridinium chlorochromate (PCC) and 52.77g diatomaceous earth (celite) were added to 233.5ml Methylene Chloride (MC) and stirred. 23.55g (81.67mmol) of the product (SM) of reaction 1b were dissolved in 81.2ml of dichloromethane (MC) and slowly added dropwise. It was stirred at room temperature for 24 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, it was filtered through a celite filter, and the filtrate was concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): dichloromethane (MC): hexane (Hex) ═ 1: 1: 4 (volume ratio) mixture) to obtain 14.2g of the objective compound (yield: 60.7%).
[ reaction 1c ]
D. 370mg (1.29mmol) of the product (SM) of reaction 1c are dissolved in 1.3ml of Tetrahydrofuran (THF) as shown in reaction 1d below. Then, the user can use the device to perform the operation,under nitrogen (N) 2) After bubbling, it was cooled to 0 ℃. 1.94ml (3.877mmol) of allylmagnesium chloride (2M in THF) were slowly added dropwise and stirred at room temperature for 2 h. The reaction was confirmed by TLC (EA: Hex 1: 4). When the reaction was completed, a dilute aqueous hydrochloric acid solution was added to the reaction solution to stop the reaction. Then, it was treated with Ethyl Acetate (EA)/H2O extraction, then extraction with magnesium sulfate (MgSO)4) Removing water, and concentrating. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 7 (volume ratio) mixture) to obtain 250mg of the objective compound (yield: 59%).
[ reaction 1d ]
E. As shown in reaction 1e below, 6.5g (19.793mmol) of the product (SM) from reaction 1d was dissolved in 130ml of Tetrahydrofuran (THF). Then, under nitrogen (N)2) After bubbling, it was cooled to-78 ℃. 16ml (31.977mmol) of borane dimethylsulfide solution (2M, BH in THF) are slowly added dropwise3Me2S) and stirred at the same temperature overnight. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was completed, methanol (MeOH) was added to the reaction solution to stop the reaction, and then concentrated. 42.6g (197.93mmol) of pyridinium chlorochromate (PCC) and 42.6g of diatomaceous earth were added to 32.5ml of Methylene Chloride (MC) and stirred. A solution of concentrated reactant SM2 dissolved in the appropriate amount of dichloromethane (MC) was added and stirred at room temperature for 5 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, it was filtered through a celite filter, and the filtrate was concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 4 (volume ratio) mixture) to obtain 4.56g of the objective compound (yield: 67.3%).
[ reaction 1e ]
F. As shown in reaction 1f below, 2.94g (8.59mmol) of the product of reaction 1e (SM)) Dissolved in 70ml of a mixed solution (acetonitrile (ACN)/H)2O ═ 8: 2 (volume ratio) mixture). Then, it was cooled to 0 ℃. 14.2g (25.76mmol) of Cerium Ammonium Nitrate (CAN) were added at the same temperature. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, saturated sodium bicarbonate solution (saturated NaHCO)3Aqueous solution) was added to the reaction solution to stop the reaction. The reaction mixture was warmed to room temperature and extracted with Ethyl Acetate (EA)/H2And (4) extracting. With magnesium sulfate (MgSO)4) After removal of water, it was concentrated. 2.8g of the objective compound was obtained (yield: 137.9%).
[ reaction 1f ]
G. 5.88g (22.945mmol) palmitic acid and 2.88ml (22.945mmol) pivaloyl chloride were added to 58.8ml dichloromethane (MC) and cooled to 0 ℃ as shown in the reaction 1g below. While maintaining the same temperature, 7.4ml (52.95mmol) of Triethylamine (TEA) was slowly dropped, followed by stirring at the same temperature for 30 minutes. 1.47g (17.65mmol) of the product of reaction 1f (SM) are added, and 216mg (1.765mmol) of 4-Dimethylaminopyridine (DMAP) are added, after which the temperature is raised to room temperature. Then, it was stirred at the same temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was completed, the reaction solution was extracted with aqueous potassium hydroxide solution (KOH solution)/dichloromethane (MC), and extracted with hydrochloric acid solution (HCl solution)/dichloromethane (MC), and then concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 4: 5 (volume ratio) mixture) to obtain 1.9g of the objective compound (yield: 22.6%).
Alternatively, SM (2.8g, 11.85mmol, 1 eq)/palmitic acid (1.3 eq)/N, N' -dicyclohexylcarbodiimide (DCC, 1.3 eq)/DMAP (0.1 eq)/MC (10 times palmitic acid) was reacted. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, the solvent was concentrated. Then, it was slurried with an appropriate amount of hexane, filtered, and concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 7 (volume ratio) mixture) to obtain 3.66g of the objective compound (yield: 65%).
[ reaction 1g ]
H. 3.66g (7.71mmol) of product (SM) reacted to 1g was added to 38ml of dichloromethane (MC) as shown in reaction 1h below. Then, under nitrogen (N)2) After bubbling, it was cooled to-78 ℃. 23ml (23.11mmol) of boron trichloride (1M in MC, BCl) are slowly added dropwise while maintaining the same temperature3) And then stirred at the same temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 1). When the reaction was complete, saturated sodium bicarbonate solution (saturated NaHCO)3Aqueous solution) was added to the reaction solution to stop the reaction. In the presence of Methylene Chloride (MC)/H2After O extraction, it was concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 2 (volume ratio) mixture) to obtain 2.68g of the objective compound (yield: 90%).
[ reaction 1h ]
I. As shown in the following reaction 1i, 100g (0.26mmol) of the product (SM) reacted for 1h, 72.5mL (0.52mmol) of Triethylamine (TEA), 3.17mg (0.026mmol) of 4-Dimethylaminopyridine (DMAP) were added to 10mL of dichloromethane (MC) and stirred at room temperature for 30 minutes. 24mL (0.338mmol) of acetyl chloride was slowly added dropwise and stirred at the same temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was completed, the reaction solution was extracted with aqueous potassium hydroxide solution (KOH solution)/dichloromethane (MC), and extracted with hydrochloric acid solution (HCl solution)/dichloromethane (MC), and then concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 3 (volume ratio) mixture) to obtain 87.4mg of the objective compound (yield: 78.8%).
[ reaction 1i ]
EXAMPLE 2 Synthesis of diacylglycerolactone Compound (EC-A51)
A. 350ml of pyridine, 10g (111.01mmol) of dihydroxyacetone and 3.53g (28.86mmol) of 4-Dimethylaminopyridine (DMAP) were dissolved as shown in the following reaction 2 a. Then, under nitrogen (N)2) After bubbling, it was cooled to 0 ℃. While maintaining the same temperature, 30.51g (111.01mmol) of tert-butyl (chloro) diphenylsilane (TBDPSCl) were slowly added dropwise. The reaction was stirred at the same temperature for 15 minutes and heated to 25 ℃ to 30 ℃. It was stirred for 16 hours. The reaction was confirmed by TLC (PE: EA: 10: 1). When the reaction was complete, it was extracted 4 times with Ethyl Acetate (EA)/purified water. The organic layer was washed with 1M hydrochloric acid and brine solution in this order, and the organic layer was washed with sodium sulfate (Na) 2SO4) Dehydrating and concentrating. The concentrate was purified by flash column (eluent: Petroleum Ether (PE): Ethyl Acetate (EA): 50: 1 (volume ratio) mixture) to obtain 7g of the objective compound (yield: 19.2%).
[ reaction 2a ]
B. As shown in reaction 2b below, 361.21mg (4.57mmol) of pyridine, 300mg (0.9133mmol) of the product of reaction 2a (SM) and 16.74mg (0.13699mmol) of 4-methylaminopyridine (DMAP) were dissolved in 10ml of dichloromethane (MC). Then, under nitrogen (N)2) After bubbling, it was cooled to 0 ℃. 78.86mg (1mmol) of acetyl chloride were slowly added dropwise while maintaining the same temperature. The reaction was stirred at the same temperature for 5 minutes and heated to 25 ℃ to 30 ℃. Stirred for 4 hours. The reaction was confirmed by TLC (PE: EA 5: 1). When the reaction was complete, ice water was added to the reaction and stirred for 5 minutes. Extracted 4 times with Ethyl Acetate (EA)/purified water. The organic layer was washed with 1M hydrochloric acid and brine solution in this order, and the organic layer was washed with sodium sulfate (Na)2SO4) Dehydrating and concentrating. The concentrate was purified by flash column (eluent: Petroleum Ether (PE): Ethyl Acetate (EA): 50: 1 (volume ratio) mixture) to give 200mg of the target compound (yield:59.1％)。
[ reaction 2b ]
C. As shown in reaction 2c below, 250mg (0.6748mmol) of the product (SM) of reaction 2b was dissolved in 2ml of Tetrahydrofuran (THF). Then, with nitrogen (N) 2) After bubbling, it was cooled to-70 ℃. 337.3mL (0.6748mmol) of allylmagnesium chloride (2M in THF) were slowly added dropwise while maintaining the same temperature. The reaction was stirred at the same temperature for 15 minutes and heated to 25 ℃ to 30 ℃. It was stirred for 3 hours. The reaction was confirmed by TLC (PE: EA: 10: 1). When the reaction was complete, ice water was added to the reaction and stirred for 5 minutes. Extracted 4 times with Ethyl Acetate (EA)/purified water. The organic layer was washed with 1M hydrochloric acid and brine solution in this order, and the organic layer was washed with sodium sulfate (Na)2SO4) Dehydrating and concentrating. The concentrate was purified by flash column (eluent: Petroleum Ether (PE): Ethyl Acetate (EA): 50: 1 (volume ratio) mixture) to obtain 100mg of the objective compound (yield: 28.74%).
[ reaction 2c ]
D. As shown in reaction 2d below, 2g (4.85mmol) of the product (SM) of reaction 2c was dissolved in 50ml of Tetrahydrofuran (THF). Then, under nitrogen (N)2) After bubbling, it was cooled to-78 ℃. 970mL (9.7mmol) of borane dimethylsulfide (10M in THF) were slowly added dropwise while maintaining the same temperature. The reaction was stirred at the same temperature for 30 minutes and heated to 25 ℃ to 35 ℃. It was stirred for 15.5 hours. The reaction was confirmed by TLC (PE: EA: 10: 1). When the reaction was completed, 80ml of methanol was added to stop the reaction. The organic layer was concentrated to obtain 2.5g of the objective compound. The reaction product was used directly in the next reaction.
[ reaction 2d ]
E. As shown in reaction 2e below, 110mg (0.25545mmol) of the product (SM) from reaction 2d was dissolved in 10ml dichloromethane (MC). Then, under nitrogen (N)2) After bubbling, it was heated to 25 ℃ to 30 ℃. 550.65mg (2.55mmol) pyridinium chlorochromate (PCC) were added to one portion while maintaining the same temperature. The reaction solution was stirred at the same temperature for 36 hours. The reaction was confirmed by TLC (PE: EA: 2: 1). When the reaction was complete, it was extracted 4 times with dichloromethane (MC)/purified water. The organic layer was washed with a saturated saline solution, and the organic layer was washed with sodium sulfate (Na)2SO4) Dehydrating and concentrating. The concentrate was purified by flash column (eluent: Petroleum Ether (PE): Ethyl Acetate (EA): 5: 1 (volume ratio) mixture) to obtain 60mg of the objective compound (yield: 44.05%).
[ reaction 2e ]
F. As shown in reaction 2f below, 200mg (0.46885mmol) of the product (SM) of reaction 2e was dissolved in 2ml of Tetrahydrofuran (THF). Then, under nitrogen (N)2) After bubbling, it was heated to 25 ℃ to 30 ℃. While maintaining the same temperature, 609.51mL (1.3 equiv.) of tetrabutylammonium fluoride (1M in THF, TBAF) were added to one portion. The reaction solution was stirred at the same temperature for 2 hours. The reaction was confirmed by TLC (PE: EA: 10: 1). When the reaction was complete, the reaction solution was filtered and concentrated. The concentrate was purified by flash column (eluent: Petroleum Ether (PE): Ethyl Acetate (EA): 10: 1 (volume ratio) mixture) to obtain 60mg of the objective compound (yield: 54.4%).
[ reaction 2f ]
G. 19.96mg (0.10608mmol) of the product of reaction 2f (SM) and 35mg (0.1248mmol) of linoleic acid are dissolved in 2ml of dichloromethane (MC) as shown in reaction 2g below. Then, under nitrogen (N)2) After bubbling, it was heatedTo 25 ℃ to 35 ℃. 30.9mg (0.14976mmol) of N, N' -Dicyclohexylcarbodiimide (DCC) and 3.05mg (0.02496mmol) of 4-Dimethylaminopyridine (DMAP) are added to one portion while maintaining the same temperature. The reaction was stirred at the same temperature for 16 hours. The reaction was confirmed by TLC (PE: EA: 10: 1). When the reaction was completed, ice water was added to the reaction solution and stirred for 5 minutes. Extracted 4 times with Ethyl Acetate (EA)/purified water. The organic layer was washed with brine solution. Organic layer with sodium sulfate (Na)2SO4) Dehydrating and concentrating. The concentrate was purified by flash column (eluent: Petroleum Ether (PE): Ethyl Acetate (EA): 10: 1 (volume ratio) mixture) to obtain 20mg of the objective compound (yield: 33.79%).
[ reaction 2g ]
[ example 3]Synthesis of diacylglycerol lactone Compound (EC-A52)
A. As shown in reaction 3a below, 350ml of pyridine, 10g (111.01mmol) of dihydroxyacetone and 3.53g (28.86mmol) of 4-Dimethylaminopyridine (DMAP) were dissolved. Then, under nitrogen (N) 2) After bubbling, it was cooled to 0 ℃. While maintaining the same temperature, 30.51g (111.01mmol) of tert-butyl (chloro) diphenylsilane (TBDPSCl) were slowly added dropwise. The reaction solution was stirred at the same temperature for 15 minutes and heated to 25 ℃ to 30 ℃. It was stirred for 16 hours. The reaction was confirmed by TLC (PE: EA: 10: 1). When the reaction was complete, it was extracted 4 times with Ethyl Acetate (EA)/purified water. The organic layer was washed with 1M hydrochloric acid and brine solution in this order, and the organic layer was washed with sodium sulfate (Na)2SO4) Dehydrating and concentrating. The concentrate was purified by flash column (eluent: Petroleum Ether (PE): Ethyl Acetate (EA): 50: 1 (volume ratio) mixture) to obtain 7g of the objective compound (yield: 19.2%).
[ reaction 3a ]
B. As in the following reaction 3b6g (76.1mmol) of pyridine, 5g (15.22mmol) of the product (SM) of reaction 3a and 185.94mg (1.52mmol) of 4-Dimethylaminopyridine (DMAP) are dissolved in 150ml of dichloromethane (MC). Then, under nitrogen (N)2) After bubbling, it was heated to 25 ℃ to 35 ℃. While maintaining the same temperature, 4.18g (15.22mmol) of palmitoyl chloride was slowly added dropwise. The reaction solution was stirred at the same temperature for 5 minutes and heated to 25 to 35 ℃. It was stirred for 16 hours. The reaction was confirmed by TLC (PE: EA: 10: 1). When the reaction is complete, an aqueous ammonium chloride solution (NH) is added 4Cl solution) was added to the reaction solution to stop the reaction. Then, it was extracted 3 times with Ethyl Acetate (EA)/purified water. The organic layer was washed with brine solution. Organic layer with sodium sulfate (Na)2SO4) Dehydrating and concentrating. The concentrate was purified by flash column (eluent: Petroleum Ether (PE): Ethyl Acetate (EA): 50: 1 (volume ratio) mixture) to obtain 4g of the objective compound (yield: 41.72%).
[ reaction 3b ]
C. As shown in reaction 3c below, 3.15g (5.55mmol) of the product (SM) of reaction 3b was dissolved in 50ml of Tetrahydrofuran (THF). Then, under nitrogen (N)2) After bubbling, it was cooled to 0 ℃. While maintaining the same temperature, 1.61g (11.1mmol) of allyl magnesium bromide was slowly added dropwise. The reaction solution was stirred at the same temperature for 15 minutes and heated to 25 ℃ to 30 ℃. It was stirred for 16 hours. The reaction was confirmed by TLC (PE: EA: 10: 1). When the reaction was complete, it was extracted 3 times with Ethyl Acetate (EA)/purified water. The organic layer was washed with brine solution. Organic layer with sodium sulfate (Na)2SO4) Dehydrating and concentrating. The concentrate was purified by flash column (eluent: Petroleum Ether (PE): Ethyl Acetate (EA): 20: 1 (volume ratio) mixture) to obtain 1.5g of the objective compound (yield: 44.32%).
[ reaction 3c ]
D. As shown in reaction 3d below, 1.2g (2.91mmol) of the product (SM) of reaction 3c was dissolved in 30ml of Tetrahydrofuran (THF). Then, under nitrogen (N)2) After bubbling, it was cooled to-78 ℃. 662.87mg (8.73mmol) of borane dimethylsulfide are slowly added dropwise while maintaining the same temperature. The reaction solution was stirred at the same temperature for 1 hour and heated to 25 to 35 ℃. Stirred for 15 hours. The reaction was confirmed by TLC (PE: EA: 10: 1). When the reaction was completed, 80ml of methanol was added to the reaction solution to stop the reaction. The organic layer was concentrated to obtain 1.3g of the objective compound. The reaction product was used directly in the next reaction.
[ reaction 3d ]
E. As shown in reaction 3e below, 150mg (0.23924mmol) of the product (SM) from reaction 3d was dissolved in 15ml dichloromethane (MC). Then, with nitrogen (N)2) After bubbling, it was heated to 25 ℃ to 30 ℃. 361mg (1.67mmol) of pyridinium chlorochromate (PCC) are added to one portion while maintaining the same temperature. The reaction solution was stirred at the same temperature for 48 hours. The reaction was confirmed by TLC (PE: EA 5: 1). When the reaction was completed, the reaction product was concentrated to obtain 150mg of the objective compound.
[ reaction 3e ]
F. As shown in reaction 3f below, 1.5g (2.41mmol) of the product (SM) of reaction 3e was dissolved in 5ml of Tetrahydrofuran (THF). Then, under nitrogen (N) 2) After bubbling, it was heated to 25 ℃ to 35 ℃. While maintaining the same temperature, 3.13mL (1.3 equiv.) of tetrabutylammonium fluoride (1M in THF, TBAF) was added to one portion. The reaction solution was stirred at the same temperature for 2 hours. The reaction was confirmed by TLC (PE: EA 5: 1). When the reaction was complete, it was extracted 3 times with Ethyl Acetate (EA)/purified water. The organic layer was washed with brine solution, and the organic layer was washed with sodium sulfate (Na)2SO4) Dehydrating and concentrating. The concentrate was purified by flash column (eluent: Petroleum Ether (PE): Ethyl Acetate (EA): 5: 1 (volume ratio) mixture) to obtain 250mg of the objective compound (yield: 24.28%).
[ reaction 3f ]
G. As shown in reaction 3g below, 250mg (0.65011mmol) of the product of reaction 3f (SM) and 182.32mg (0.65011mmol) linoleic acid were dissolved in 5ml dichloromethane (MC). Then, under nitrogen (N)2) After bubbling, it was heated to 25 ℃ to 35 ℃. 160.96mg (0.78013mmol) of N, N' -Dicyclohexylcarbodiimide (DCC) and 7.94mg (0.06501mmol) of 4-Dimethylaminopyridine (DMAP) are added to one portion while maintaining the same temperature. The reaction solution was stirred at the same temperature for 16 hours. The reaction was confirmed by TLC (PE: EA 5: 1). When the reaction was complete, the reaction solution was filtered and concentrated. The concentrate was purified by flash column (eluent: Petroleum Ether (PE): Ethyl Acetate (EA): 5: 1 (volume ratio) mixture) to obtain 100mg of the objective compound (yield: 22.59%).
[ reaction 3g ]
[ example 4 ]]Synthesis of diacylglycerolactone Compound (EC-A115)
A. As shown in the following reaction 4a, 65mg (0.275mmol) of the product (SM) of reaction 1f, 77mL (0.55mmol) of Triethylamine (TEA) and 3.36mg (0.0275mmol) of 4-Dimethylaminopyridine (DMAP) were added to 2mL of dichloromethane (MC), stirred and then stirred at room temperature for 30 minutes. Propionyl chloride 31.2mL (0.358mmol) was slowly added dropwise to the reaction solution, and stirred at room temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). After completion of the reaction, extraction was performed with 0.1N aqueous hydrochloric acid (c-HCl solution)/dichloromethane (MC). The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 3 (volume ratio) mixture) to obtain 51.2mg of the objective compound (yield: 63.7%).
[ reaction 4a ]
B. 51.2mg (0.175mmol) of the product (SM) of reaction 4a was dissolved in 1ml of dichloromethane (MC) as shown in reaction 4b below. Then, under nitrogen (N)2) After bubbling, it was cooled to-78 ℃. 0.53ml (0.525mmol) of boron trichloride (1M in MC, BCl)3) Slowly added dropwise to the coolant and stirred at the same temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, a saturated sodium bicarbonate solution (saturated NaHCO) was added to the reaction solution 3Solution) to stop the reaction and the temperature was raised to room temperature. Extracted with dichloromethane (MC) and concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 2: 1 (volume ratio) mixture) to obtain 25.6mg of the objective compound (yield: 72.3%).
[ reaction 4b ]
C. As shown in reaction 4c below, 35.4mg (0.126mmol) linoleic acid and 15.2mL (0.1236mmol) pivaloyl chloride were added to 1mL Methylene Chloride (MC) and cooled to 0 deg.C to 5 deg.C. 34.5mL (0.2472mmol) of Triethylamine (TEA) were slowly added dropwise and stirred at the same temperature for 30 min. 25mg (0.1236mmol) of the product of reaction 4b (SM) and 1.5mg (0.0124mmol) of 4-Dimethylaminopyridine (DMAP) are added and the mixture is stirred at room temperature overnight. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, it was extracted with 0.1N potassium hydroxide solution (KOH solution)/dichloromethane (MC). Then, the extract was extracted with 0.07N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), followed by magnesium sulfate (MgSO)4) Removing water, and concentrating. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 3.5 (volume ratio) mixture) to obtain 29.9mg of the objective compound (yield: 52%).
[ reaction 4c ]
[ example 5]Synthesis of diacylglycerolactone Compound (EC-A116)
A. As shown in reaction 5a below, 150mg (0.635mmol) of the product (SM) of reaction 1f and 0.18ml (1.27mmol) of Triethylamine (TEA) were added to 6.35ml of dichloromethane (MC) and stirred at room temperature for 30 minutes. 86.2mL (0.825mmol) of butyryl chloride was slowly added dropwise to the reaction solution, and stirred at room temperature for 3 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). After completion of the reaction, it was extracted with 0.1N aqueous hydrochloric acid (c-HCl solution)/dichloromethane (MC), and concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 3.5 (volume ratio) mixture) to obtain 106mg of the objective compound (yield: 54.5%).
[ reaction 5a ]
B. 106mg (0.346mmol) of the product (SM) of reaction 5a was dissolved in 1ml of dichloromethane (MC) as shown in reaction 5b below. Then, under nitrogen (N)2) After bubbling, it was cooled to-78 ℃. Slowly dropwise addition of 1ml (1.038mmol) of boron trichloride (1M in MC, BCl)3) And stirred at the same temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 1). When the reaction was complete, saturated sodium bicarbonate solution (saturated NaHCO)3Aqueous solution) was added to the reaction solution to stop the reaction, and it was heated to room temperature. Extraction was performed with dichloromethane (MC). The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1 (volume ratio) mixture) to obtain 52.8mg of the objective compound (yield: 70.5%).
[ reaction 5b ]
C. As shown in the following reaction 5c, 70mg (0.249mmol) of methylene was addedOleic acid and 30mL (0.24436mmol) of pivaloyl chloride were added to 1mL of dichloromethane (MC) and cooled to 0 deg.C to 5 deg.C. 68mL (0.488mmol) of Triethylamine (TEA) were slowly added dropwise and stirred at the same temperature for 30 minutes. 52.8mg (0.244mmol) of the product (SM) of reaction 5b and 3mg (0.0244mmol) of 4-Dimethylaminopyridine (DMAP) were added to the reaction solution at the same temperature and stirred at room temperature overnight. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, it was extracted with 0.1N potassium hydroxide solution (KOH solution)/dichloromethane (MC). The extract was extracted with 0.07N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), followed by magnesium sulfate (MgSO)4) Removing water, and concentrating. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 3.5 (volume ratio) mixture) to obtain 78.4mg of the objective compound (yield: 67%).
[ reaction 5c ]
[ example 6]Synthesis of diacylglycerolactone Compound (EC-A117)
A. As shown in reaction 6a below, 150mg (0.635mmol) of the product (SM) of reaction 1f and 0.18ml (1.27mmol) of Triethylamine (TEA) were added to 2ml of dichloromethane (MC) and stirred. Then stirred at room temperature for 30 minutes. 0.1ml (0.825mmol) of valeryl chloride was slowly added dropwise to the reaction solution and stirred at room temperature for 3 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was completed, it was extracted with 0.1N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), and then concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 3.5 (volume ratio) mixture) to obtain 89.1mg of the objective compound (yield: 43.8%).
[ reaction 6a ]
B. 89.1mg (0.278mmol) of the product (SM) of reaction 6a was dissolved in 1.4ml of dichloromethane (MC) as shown in reaction 6b below. Then, under nitrogenQi (N)2) After bubbling, it was cooled to-78 ℃. 0.83ml (0.834mmol) of boron trichloride (1M in MC, BCl) are slowly added dropwise3). The reaction solution was stirred at the same temperature for 1 hour. The reaction was confirmed by TLC (EA: Hex 1: 1). When the reaction was complete, saturated sodium bicarbonate solution (saturated NaHCO)3Aqueous solution) was added to the reaction solution to stop the reaction, and it was heated to room temperature. Extracted with dichloromethane (MC) and then concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1 (volume ratio) mixture) to obtain 46.8mg of the objective compound (yield: 73.1%).
[ reaction 6b ]
C. As shown in reaction 6c below, 58.1mg (0.207mmol) linoleic acid and 25mL (0.203mmol) pivaloyl chloride were added to 1mL Methylene Chloride (MC) and cooled to 0 deg.C to 5 deg.C. 57mL (0.406mmol) of Triethylamine (TEA) was slowly added dropwise and stirred at the same temperature for 30 minutes. 46.8mg (0.203mmol) of the product of reaction 6b (SM) and 2.5mg (0.0203mmol) of 4-Dimethylaminopyridine (DMAP) were added at the same temperature and stirred at room temperature overnight. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, it was extracted with 0.1N potassium hydroxide solution (KOH solution)/dichloromethane (MC). The extract was extracted with 0.07N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), followed by magnesium sulfate (MgSO) 4) Removing water, and concentrating. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 3.5 (volume ratio) mixture) to obtain 42.5mg of the objective compound (yield: 42.5%).
[ reaction 6c ]
[ example 7]Synthesis of diacylglycerolactone Compound (EC-A118)
A. As shown in reaction 7a below, 150mg (0.635mmol) of the product (SM) of reaction 1f, 0.18ml (1.27mmol) of Triethylamine (TEA) and 7.8mg (0.063mmol) of 4-Dimethylaminopyridine (DMAP) were added to 2ml of dichloromethane (MC) and stirred. Then, the mixture was stirred at room temperature for 30 minutes. 0.113mL (0.825mmol) of hexanoyl chloride was slowly added dropwise. The reaction solution was stirred at room temperature for 3 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was completed, it was extracted with 0.1N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), and then concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 3.5 (volume ratio) mixture) to give 135.4mg of the product (yield: 63.7%).
[ reaction 7a ]
B. 135.4mg (0.405mmol) of the product (SM) of reaction 7a are dissolved in 2ml of dichloromethane (MC) as shown in reaction 7b below. Then, under nitrogen (N)2) After bubbling, it was cooled to-78 ℃. 1.2ml (1.21mmol) of boron trichloride (1M in MC, BCl) are slowly added dropwise 3). The reaction solution was stirred at the same temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 1). When the reaction was complete, saturated sodium bicarbonate solution (saturated NaHCO)3Aqueous solution) was added to the reaction solution to stop the reaction, and it was heated to room temperature. Extracted with dichloromethane (MC) and concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1 (volume ratio) mixture) to give 70.5mg of the product (yield: 71.3%).
[ reaction 7b ]
C. As shown in reaction 7c below, 83mg (0.294mmol) linoleic acid and 35.5mL (0.2886mmol) pivaloyl chloride were added to 1mL Methylene Chloride (MC). Then, it was cooled to 0 ℃ to 5 ℃. 80mL (0.5772mmol) of Triethylamine (TEA) was slowly added dropwise and stirred at the same temperature for 30 minutes. 70.5mg (0.2886mmol) of the product of reaction 7b (SM) and 4.3mg (0.035mmol) of 4-Dimethylaminopyridine (DMAP) are added at the same temperature and the mixture is cooled to room temperatureStirring was continued overnight. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, it was extracted with 0.1N potassium hydroxide solution (KOH solution)/dichloromethane (MC). The extract was extracted with 0.07N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), followed by magnesium sulfate (MgSO) 4) Removing water, and concentrating. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 4 (volume ratio) mixture) to obtain 20mg of the objective compound (yield: 13.7%).
[ reaction 7c ]
[ example 8]Synthesis of diacylglycerolactone Compound (EC-A119)
A. As shown in reaction 8a below, 150mg (0.635mmol) of the product (SM) of reaction 1f, 0.18ml (1.27mmol) of Triethylamine (TEA) and 7.8mg (0.063mmol) of 4-Dimethylaminopyridine (DMAP) were added to 3ml of dichloromethane (MC) and stirred. Then, the mixture was stirred at room temperature for 30 minutes. 0.13ml (0.825mmol) of heptanoyl chloride was slowly added dropwise and stirred at room temperature for 3 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was completed, it was extracted with 0.1N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), and then concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 3.5 (volume ratio) mixture) to give 126.1mg of the product (yield: 57%).
[ reaction 8a ]
B. As shown in reaction 8b below, 126mg (0.362mmol) of the product (SM) of reaction 8a was dissolved in 2ml of dichloromethane (MC). Then, under nitrogen (N)2) After bubbling, it was cooled to-78 ℃. 1.1ml (1.08mmol) of boron trichloride (1M in MC, BCl) are slowly added dropwise 3). The reaction solution was stirred at the same temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, it was extracted with 0.1N aqueous hydrochloric acid (c-HCl solution)/dichloromethane (MC) and concentrated. Will be provided withThe concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1 (volume ratio) mixture) to give 77.8mg of the product (yield: 83.2%).
[ reaction 8b ]
C. As shown in reaction 8c below, 86.2mg (0.3072mmol) linoleic acid and 37mL (0.3012mmol) pivaloyl chloride were added to 1mL dichloromethane (MC). Then, it was cooled to 0 ℃ to 5 ℃. 84mL (0.6024mmol) of Triethylamine (TEA) were slowly added dropwise and stirred at the same temperature for 30 minutes. 77.8mg (0.3012mmol) of the product of reaction 8b (SM) and 3.7mg (0.0301mmol) of 4-Dimethylaminopyridine (DMAP) are added at the same temperature and stirred at room temperature overnight. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, it was extracted with 0.1N potassium hydroxide solution (KOH solution)/dichloromethane (MC). The extract was extracted with 0.07N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), followed by magnesium sulfate (MgSO)4) Removing water, and concentrating. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 3.5 (volume ratio) mixture) to obtain 75.1mg of the objective compound (yield: 47.9%).
[ reaction 8c ]
[ example 9]Synthesis of diacylglycerolactone Compound (EC-A120)
A. As shown in reaction 9a below, 150mg (0.635mmol) of the product (SM) of reaction 1f, 0.18ml (1.27mmol) of Triethylamine (TEA) and 7.8mg (0.063mmol) of 4-Dimethylaminopyridine (DMAP) were added to 2ml of dichloromethane (MC) and stirred. Then, it was stirred at room temperature for 30 minutes. Slowly 0.16ml (0.825mmol) of nonanoyl chloride are added dropwise. The reaction solution was stirred at room temperature for 3 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was completed, it was extracted with 0.1N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), and then concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 3.5 (volume ratio) mixture) to obtain 154mg of the objective compound (yield: 64%).
[ reaction 9a ]
B. As shown in reaction 9b below, 154mg (0.41mmol) of the product (SM) of reaction 9a was dissolved in 2ml of dichloromethane (MC). Then, under nitrogen (N)2) After bubbling, it was cooled to-78 ℃. 1.21ml (1.227mmol) of boron trichloride (1M in MC, BCl) are slowly added dropwise3). The reaction solution was stirred at the same temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was completed, it was extracted with 0.1N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), and then concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1 (volume ratio) mixture) to obtain 99.7mg of the objective compound (yield: 85%).
[ reaction 9b ]
C. As shown in reaction 9c below, 100mg (0.355mmol) linoleic acid and 43mL (0.355mmol) pivaloyl chloride are added to 1mL Methylene Chloride (MC). Then, it was cooled to 0 ℃ to 5 ℃. 97mL (0.696mmol) of Triethylamine (TEA) are slowly added dropwise. The reaction solution was stirred at the same temperature for 30 minutes. 99.7mg (0.348mmol) of the product (SM) of reaction 9b and 4.3mg (0.035mmol) of 4-Dimethylaminopyridine (DMAP) were added at the same temperature and stirred at room temperature overnight. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, it was extracted with 0.1N potassium hydroxide solution (KOH solution)/dichloromethane (MC). The extract was extracted with 0.07N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), followed by magnesium sulfate (MgSO)4) Removing water, and concentrating. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 3.5 (volume ratio) mixture) to obtain 87.1mg of the objective compound (yield: 45.6%).
[ reaction 9c ]
[ example 10]Synthesis of diacylglycerolactone Compound (EC-A121)
A. As shown in reaction 10a below, 165.264mg (0.825mmol) of lauric acid and 43mL (0.825mmol) of pivaloyl chloride were added to 2mL of dichloromethane (MC). Then, it was cooled to 0 ℃. 0.27ml (3mmol) Triethylamine (TEA) was slowly added dropwise. The reaction solution was stirred at the same temperature for 30 minutes. 150mg (0.635mmol) of the product (SM) of reaction 1f and 7.8mg (0.0635mmol) of 4-Dimethylaminopyridine (DMAP) are added at the same temperature and stirred at room temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, it was extracted with 0.1N potassium hydroxide solution (KOH solution)/dichloromethane (MC). The extract was extracted with 0.07N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), followed by magnesium sulfate (MgSO) 4) Removing water, and concentrating. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 4 (volume ratio) mixture) to obtain 159.1mg of the objective compound (yield: 59.8%).
[ reaction 10a ]
B. 159mg (0.3798mmol) of the product (SM) of reaction 10a was dissolved in 1.9ml of dichloromethane (MC) as shown in reaction 10b below. Then, under nitrogen (N)2) After bubbling, it was cooled to-78 ℃. 1.14ml (1.14mmol) of boron trichloride (1M in MC, BCl) are slowly added dropwise3). The reaction solution was stirred at the same temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, it was concentrated with 0.1N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC). 113.6mg of the objective compound was obtained (yield: 91.1%).
[ reaction 10b ]
C. As shown in reaction 10c below, 99mg (0.3528mmol) linoleic acid and 42.56mL (0.3459mmol) pivaloyl chloride were added to 1mL Methylene Chloride (MC). Then, it was cooled to 0 ℃. 0.15ml (1.0377mmol) Triethylamine (TEA) are slowly added dropwise. The reaction solution was stirred at the same temperature for 30 minutes. 113.6mg (0.3459mmol) of the product of reaction 10b (SM) and 4mg (0.0346mmol) of 4-Dimethylaminopyridine (DMAP) were added at the same temperature and stirred at room temperature overnight. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, it was extracted with 0.1N potassium hydroxide solution (KOH solution)/dichloromethane (MC). The extract was extracted with 0.07N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), followed by magnesium sulfate (MgSO) 4) Removing water, and concentrating. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 5 (volume ratio) mixture) to obtain 128mg of the objective compound (yield: 62.6%).
[ reaction 10c ]
[ example 11]Synthesis of diacylglycerol lactone Compound (EC-A122)
A. As shown in reaction 11a below, 188.405mg (0.825mmol) myristic acid and 43mL (0.825mmol) pivaloyl chloride were added to 2mL dichloromethane (MC). Then, it was cooled to 0 ℃. 0.27ml (3mmol) Triethylamine (TEA) was slowly added dropwise. The reaction solution was stirred at the same temperature for 30 minutes. 150mg (0.635mmol) of the product (SM) of reaction 1f and 7.8mg (0.0635mmol) of 4-Dimethylaminopyridine (DMAP) are added at the same temperature and stirred at room temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, it was extracted with 0.1N potassium hydroxide solution (KOH solution)/dichloromethane (MC). The extract was extracted with 0.07N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), followed by magnesium sulfate (MgSO)4) Removing water, and concentrating. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 4 (volume ratio) mixture) to obtain 190.7mg of the objective compound (yield: 67.2%).
[ reaction 11a ]
B. 190.7mg (0.427mmol) of the product (SM) of reaction 10a was dissolved in 2ml of dichloromethane (MC) as shown in reaction 11b below. Then, under nitrogen (N)2) After bubbling, it was cooled to-78 ℃. 1.28ml (1.28mmol) of boron trichloride (1M in MC, BCl) are slowly added dropwise3). The reaction solution was stirred at the same temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was completed, it was extracted with 0.1N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), and then concentrated. 111.2mg of the objective compound was obtained (yield: 73%).
[ reaction 11b ]
C. As shown in reaction 11c below, 89mg (0.3176mmol) linoleic acid and 38mL (0.3012mmol) pivaloyl chloride were added to 1mL Methylene Chloride (MC). Then, it was cooled to 0 ℃. 94.5mL (0.9341mmol) of Triethylamine (TEA) were slowly added dropwise. The reaction solution was stirred at the same temperature for 30 minutes. 111mg (0.3113mmol) of the product of reaction 10b (SM) and 3.8mg (0.031mmol) of 4-Dimethylaminopyridine (DMAP) are added at the same temperature and stirred at room temperature overnight. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, it was extracted with 0.1N potassium hydroxide solution (KOH solution)/dichloromethane (MC). The extract was extracted with 0.07N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), followed by magnesium sulfate (MgSO) 4) Removing water, and concentrating. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 5 (volume ratio) mixture) to obtain 104.5mg of the objective compound (yield: 54.2%).
[ reaction 11c ]
[ example 12]Diacylglycerol lactone compound (EC-A)123) Synthesis of (2)
A. As shown in reaction 12a below, 257.84mg (0.825mmol) of arachidonic acid and 43mL (0.825mmol) of pivaloyl chloride were added to 2mL of dichloromethane (MC). Then, it was cooled to 0 ℃. 0.27ml (3mmol) Triethylamine (TEA) was slowly added dropwise. The reaction solution was stirred at the same temperature for 30 minutes. 150mg (0.635mmol) of the product (SM) of reaction 1f and 7.8mg (0.0635mmol) of 4-Dimethylaminopyridine (DMAP) are added at the same temperature and stirred at room temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, it was extracted with 0.1N potassium hydroxide solution (KOH solution)/dichloromethane (MC). The extract was extracted with 0.07N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), followed by magnesium sulfate (MgSO)4) Removing water and concentrating. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 4 (volume ratio) mixture) to obtain 217.6mg of the objective compound (yield: 64.6%).
[ reaction 12a ]
B. As shown in reaction 12b below, 217.6mg (0.498mmol) of the product (SM) of reaction 12a was dissolved in 2ml of dichloromethane (MC). Then, under nitrogen (N)2) After bubbling, it was cooled to-78 ℃. 1.23ml (1.23mmol) of boron trichloride (1M in MC, BCl) are slowly added dropwise3). The reaction solution was stirred at the same temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was completed, it was extracted with 0.1N aqueous hydrochloric acid (c-HCl solution)/dichloromethane (MC), and then concentrated. 153mg of the objective compound was obtained (yield: 84.7%).
[ reaction 12b ]
C. As shown in reaction 12c below, 99.3mg (0.3542mmol) linoleic acid and 42.7mL (0.3472mmol) pivaloyl chloride were added to 1mL Methylene Chloride (MC). Then, it was cooled to 0 ℃. 0.15ml (1.0416mmol) Triethylamine (TEA) are slowly added dropwise. Will be provided withThe reaction solution was stirred at the same temperature for 30 minutes. 153mg (0.3472mmol) of the product of reaction 12b (SM) and 4.2mg (0.035mmol) of 4-Dimethylaminopyridine (DMAP) are added at the same temperature and stirred at room temperature overnight. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, however, it was extracted with 0.1N potassium hydroxide solution (KOH solution)/dichloromethane (MC). The extract was extracted with 0.07N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), followed by magnesium sulfate (MgSO) 4) Removing water, and concentrating. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 5 (volume ratio) mixture) to obtain 113.2mg of the objective compound (yield: 46.3%).
[ reaction 12c ]
[ example 13]Synthesis of diacylglycerolactone Compound (EC-A124)
A. As shown in reaction 13a below, 150mg (0.635mmol) of the product (SM) of reaction 1f was dissolved in 2ml of dichloromethane (MC). Then, 0.18ml of Triethylamine (TEA) and 7.8mg of 4-Dimethylaminopyridine (DMAP) were added and stirred. Then, the mixture was stirred at room temperature for 30 minutes. 87mL (0.825mmol) of isobutyryl chloride was slowly added dropwise to the reaction solution, and stirred at room temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was completed, it was extracted with 0.1N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), and then concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 4 (volume ratio) mixture) to give 111.7mg of the product (yield: 57.4%).
[ reaction 13a ]
B. As shown in reaction 13b below, 111.7mg (0.3646mmol) of the product (SM) of reaction 13a was dissolved in 2ml of dichloromethane (MC). Then, under nitrogen (N)2) After bubbling, it was cooled to-78 ℃. 1.1ml (1.09mmol) of boron trichloride (1M in MC, BCl) are slowly added dropwise 3). The reaction solution was stirred at the same temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 1). When the reaction was complete, it was extracted with 0.1N aqueous hydrochloric acid (c-HCl solution)/dichloromethane (MC) and concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1 (volume ratio) mixture) to give 61.7mg of the product (yield: 78.3%).
[ reaction 13b ]
C. As shown in reaction 13c below, 81.6mg (0.291mmol) linoleic acid and 37mL (0.3012mmol) pivaloyl chloride were added to 1mL dichloromethane (MC). Then, it was cooled to 0 ℃. 0.12ml (0.8559mmol) Triethylamine (TEA) was slowly added dropwise and stirred at the same temperature for 30 minutes. 61.7mg (0.2853mmol) of the product (SM) of reaction 13b and 3.5mg (0.029mmol) of 4-Dimethylaminopyridine (DMAP) were added at the same temperature and stirred at room temperature overnight. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, it was extracted with 0.1N potassium hydroxide solution (KOH solution)/dichloromethane (MC). The extract was extracted with 0.07N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), followed by magnesium sulfate (MgSO)4) Removing water, and concentrating. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 5 (volume ratio) mixture) to obtain 110.91mg of the objective compound (yield: 81.2%).
[ reaction 13c ]
[ example 14]Synthesis of diacylglycerolactone Compound (EC-A125)
A. As shown in reaction 14a below, 150mg (0.635mmol) of the product (SM) of reaction 1f was dissolved in 2ml of dichloromethane (MC). Then, 0.18ml (1.27mmol) of Triethylamine (TEA) and 7.8mg (0.064mmol) of 4-Dimethylaminopyridine (DMAP) were added and stirred. Then, the mixture was stirred at room temperature for 30 minutes. 0.1ml (0.825mmol) of pivaloyl chloride are slowly added dropwise and stirred at room temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex. 1: 1.5). When the reaction was completed, it was extracted with 0.1N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), and then concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 4 (volume ratio) mixture) to give 117.5mg of the product (yield: 57.7%).
[ reaction 14a ]
B. 117mg (0.3652mmol) of the product (SM) of reaction 14a was dissolved in 1.83ml of dichloromethane (MC) as shown in reaction 14b below. Then, under nitrogen (N)2) After bubbling, it was cooled to-78 ℃. 1.1ml (1.09mmol) of boron trichloride (1M in MC, BCl) are slowly added dropwise3). The reaction solution was stirred at the same temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was completed, it was extracted with 0.1N aqueous hydrochloric acid (c-HCl solution)/dichloromethane (MC), and then concentrated. This gave 73.4mg of the titled compound (yield: 87.3%).
[ reaction 14b ]
C. As shown in reaction 14c below, 91.2mg (0.3251mmol) linoleic acid and 40mL (0.31877mmol) pivaloyl chloride were added to 1mL Methylene Chloride (MC). Then, it was cooled to 0 ℃. 0.13ml (0.9563mmol) Triethylamine (TEA) was slowly added dropwise. It was stirred at the same temperature for 30 minutes. 73.4mg (0.31877mmol) of the product (SM) from reaction 14b and 4mg (0.032mmol) of 4-Dimethylaminopyridine (DMAP) were added at the same temperature and stirred at room temperature overnight. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, it was extracted with 0.1N potassium hydroxide solution (KOH solution)/dichloromethane (MC). The extract was extracted with 0.07N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), followed by magnesium sulfate (MgSO)4) Removing water, and concentrating. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 5 (volume ratio) mixture)) To obtain 73.6mg of the objective compound (yield: 46.9%).
[ reaction 14c ]
[ example 15]Synthesis of diacylglycerolactone Compounds (EC-A126)
A. As shown in reaction 15a below, 150mg (0.635mmol) of the product (SM) of reaction 1f was dissolved in 2ml of dichloromethane (MC). Then, 0.18ml (1.27mmol) of Triethylamine (TEA) and 7.8mg (0.064mmol) of 4-Dimethylaminopyridine (DMAP) were added and stirred. It was stirred at 0 ℃ for 30 minutes. 71mL (0.825mmol) of 2-methylbutyryl chloride was added to the reaction solution, and stirred at room temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was completed, it was extracted with 0.1N aqueous hydrochloric acid (c-HCl solution)/dichloromethane (MC), and then concentrated. 102.4mg of the objective compound was obtained (yield: 50.3%).
[ reaction 15a ]
B. As shown in reaction 15b below, 102mg (0.31842mmol) of the product (SM) of reaction 15a was dissolved in 1.6ml dichloromethane (MC). Then, under nitrogen (N)2) After bubbling, it was cooled to-78 ℃. Slowly dropwise addition of 1.0ml (0.955mmol) of boron trichloride (1M in MC, BCl)3A solution). The reaction solution was stirred at the same temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was completed, it was extracted with 0.1N aqueous hydrochloric acid (c-HCl solution)/dichloromethane (MC), and then concentrated. 58.5mg of the objective compound was obtained (yield: 79.8%).
[ reaction 15b ]
C. As shown in reaction 15c below, 72.7mg (0.259mmol) linoleic acid and 31mL (0.254mmol) pivaloyl chloride were added to 1mL dichloromethane (MC).Then, it was cooled to 0 ℃. 0.11ml (0.762mmol) Triethylamine (TEA) are slowly added dropwise. It was stirred at the same temperature for 30 minutes. 58.5mg (0.254mmol) of the product (SM) of reaction 15b and 3.1mg (0.0254mmol) of 4-Dimethylaminopyridine (DMAP) were added at the same temperature and stirred at room temperature overnight. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, it was extracted with 0.1N potassium hydroxide solution (KOH solution)/dichloromethane (MC). The extract was extracted with 0.07N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), followed by magnesium sulfate (MgSO) 4) Removing water, and concentrating. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 5 (volume ratio) mixture) to obtain 64.7mg of the objective compound (yield: 51.7%).
[ reaction 15c ]
[ example 16]Synthesis of diacylglycerolactone Compound (EC-A127)
A. As shown in reaction 16a below, 150mg (0.635mmol) of the product (SM) of reaction 1f was dissolved in 2ml of dichloromethane (MC). Then, 0.18ml (1.27mmol) of Triethylamine (TEA) and 7.8mg (0.064mmol) of 4-Dimethylaminopyridine (DMAP) were added and stirred. Then, it was stirred at 0 ℃ for 30 minutes. To the reaction solution was added 75mL (0.825mmol) of cyclopropanecarbonyl chloride, and the mixture was stirred at room temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was completed, it was extracted with 0.1N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), and then concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 3.5 (volume ratio) mixture) to give 123.3mg of the product (yield: 63.8%).
[ reaction 16a ]
B. As shown in reaction 15b below, 123.3mg (0.405mmol) of the product (SM) of reaction 15a was dissolved in 1.4ml of dichloromethane (MC). Then, under nitrogen (N) 2) After bubbling, it was cooled to-78 ℃. 1.2ml (1.215mmol) of boron trichloride (1M in MC, BCl) are slowly added dropwise3). The reaction solution was stirred at the same temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, it was extracted with 0.1N aqueous hydrochloric acid (c-HCl solution)/dichloromethane (MC) and concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1 (volume ratio) mixture) to give 50mg of the product (yield: 57.6%).
[ reaction 16b ]
C. As shown in reaction 16c below, 66.76mg (0.238mmol) linoleic acid and 29mL (0.2334mmol) pivaloyl chloride were added to 1mL Methylene Chloride (MC). Then, it was cooled to 0 ℃. 0.1ml (0.7002mmol) Triethylamine (TEA) was slowly added dropwise. The reaction solution was stirred at the same temperature for 30 minutes. 50mg (0.2334mmol) of the product (SM) from reaction 16b and 2.85mg (0.023mmol) of 4-Dimethylaminopyridine (DMAP) are added at the same temperature and stirred at room temperature overnight. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, it was extracted with 0.1N potassium hydroxide solution (KOH solution)/dichloromethane (MC). The extract was extracted with 0.07N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), followed by magnesium sulfate (MgSO) 4) Removing water, and concentrating. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 4 (volume ratio) mixture) to obtain 29.70mg of the objective compound (yield: 62.3%).
[ reaction 16c ]
[ example 17]Synthesis of diacylglycerolactone Compounds (EC-A128)
A. As shown in reaction 17a below, 150mg (0.635mmol) of the product (SM) of reaction 1f was dissolved in 2ml of dichloromethane (MC). Then, 0.18ml (1.27mmol) of Triethylamine (TEA) and 7.8mg (0.064mmol) of 4-Dimethylaminopyridine (DMAP) were added and stirred. Then, it was stirred at 0 ℃ for 30 minutes. 0.11ml (0.825mmol) of cyclohexanecarbonyl chloride was added to the reaction solution, and stirred at room temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was completed, it was extracted with 0.1N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), and then concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 3.5 (volume ratio) mixture) to give 140.9mg of the product (yield: 64%).
[ reaction 17a ]
B. As shown in reaction 17b below, 140.9mg (0.4067mmol) of the product (SM) of reaction 17a was dissolved in 1.4ml of dichloromethane (MC). Then, under nitrogen (N) 2) After bubbling, it was cooled to-78 ℃. 1.2ml (1.22mmol) of boron trichloride (1M in MC, BCl) are slowly added dropwise3). The reaction solution was stirred at the same temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was completed, it was extracted with 0.1N aqueous hydrochloric acid (c-HCl solution)/dichloromethane (MC), and then concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1 (volume ratio) mixture) to give 95.1mg of the product (yield: 91.2%).
[ reaction 17b ]
C. 106.1mg (0.378mmol) of linoleic acid and 45.6mL (0.371mmol) of pivaloyl chloride are added to 1mL of dichloromethane (MC) as shown in reaction 17c below. Then, it was cooled to 0 ℃. 0.15ml (1.113mmol) Triethylamine (TEA) are slowly added dropwise. The reaction solution was stirred at the same temperature for 30 minutes. 95.1mg (0.371mmol) of the product (SM) of reaction 17b and 4.5mg (0.037mmol) of 4-Dimethylaminopyridine (DMAP) were added at the same temperature and stirred at room temperature overnight. The reaction was confirmed by TLC (EA: Hex 1: 2). After completion of the reaction, extraction was performed with 0.1N potassium hydroxide solution (KOH solution)/dichloromethane (MC)And (6) taking. The extract was extracted with 0.07N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), followed by magnesium sulfate (MgSO) 4) Removing water, and concentrating. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 4 (volume ratio) mixture) to obtain 126.4mg of the objective compound (yield: 65.7%).
[ reaction 17c ]
EXAMPLE 18]Synthesis of diacylglycerolactone Compound (EC-A130)
A. As shown in reaction 18a below, 5.88g (22.945mmol) palmitic acid and 2.88ml (22.945mmol) pivaloyl chloride were added to 1ml dichloromethane (MC). Then, it was cooled to 0 ℃. 7.4ml (52.95mmol) Triethylamine (TEA) were slowly added dropwise. The reaction solution was stirred at the same temperature for 30 minutes. 1.47g (17.65mmol) of the product of reaction 1f (SM) and 216mg (1.765mmol) of 4-Dimethylaminopyridine (DMAP) were added at the same temperature and stirred at room temperature overnight. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, it was extracted with 0.1N potassium hydroxide solution (KOH solution)/dichloromethane (MC). The extract was extracted with 0.07N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), followed by magnesium sulfate (MgSO)4) Removing water and concentrating. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 4.5 (volume ratio) mixture) to obtain 1.9g of the objective compound (yield: 22.6%).
[ reaction 18a ]
B. As shown in reaction 18b below, 1.9g (4.003mmol) of the product (SM) of reaction 18a was dissolved in 20ml of dichloromethane (MC). Then, under nitrogen (N)2) After bubbling, it was cooled to-78 ℃. 12ml (12.01mmol) of boron trichloride (1M in MC, BCl) are slowly added dropwise3A solution). The reaction solution was stirred at the same temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 1). When it is reversedWhen this was done, it was extracted with 0.1N aqueous hydrochloric acid (c-HCl solution)/dichloromethane (MC) and then concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 1.5 (volume ratio) mixture) to obtain 1.287g of the target compound (yield: 83.6%).
[ reaction 18b ]
C. As shown in reaction 18c below, 100mg (0.26mmol) of the product (SM) of reaction 18b was dissolved in 1ml of dichloromethane (MC). Then, 72.5mL (0.52mmol) of Triethylamine (TEA) and 3.17mg (0.026mmol) of 4-Dimethylaminopyridine (DMAP) were added and stirred. It was then stirred at 0 ℃ for 30 minutes. 35.3mL (0.338mmol) of butyryl chloride was added to the reaction solution. It was stirred at room temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was completed, it was extracted with 0.1N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), and then concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 3 (volume ratio) mixture) to obtain 86.8mg of the objective compound (yield: 73.4%).
[ reaction 18c ]
[ example 19]Synthesis of diacylglycerolactone Compound (EC-A131)
As shown in reaction 19a below, 100mg (0.26mmol) of the product (SM) of reaction 18b was dissolved in 1ml of dichloromethane (MC). Then, 72.5mL (0.52mmol) of Triethylamine (TEA) and 3.17mg (0.026mmol) of 4-Dimethylaminopyridine (DMAP) were added and stirred. Then, it was stirred at 0 ℃ for 30 minutes. 36mL (0.338mmol) of isobutyryl chloride was added to the reaction solution, and stirred at room temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was completed, it was extracted with 0.1N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), and then concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 4 (volume ratio) mixture) to give 86.8mg of the product (yield: 73.4%).
[ reaction 19a ]
Example 20]Synthesis of diacylglycerolactone Compound (EC-A132)
As shown in reaction 20a below, 100mg (0.26mmol) of the product (SM) of reaction 18b was dissolved in 1ml of dichloromethane (MC). Then, 72.5mL (0.52mmol) of Triethylamine (TEA) and 3.17mg (0.026mmol) of 4-Dimethylaminopyridine (DMAP) were added and stirred. Then, it was stirred at 0 ℃ for 30 minutes. To the reaction solution was added 41.6mL (0.338mmol) of pivaloyl chloride, and the mixture was stirred at room temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was completed, it was extracted with 0.1N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), and then concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 4 (volume ratio) mixture) to give 84.9mg of the product (yield: 69.7%).
[ reaction 20a ]
EXAMPLE 21 Synthesis of diacylglycerolactone Compound (EC-A133)
As shown in reaction 21a below, 100mg (0.26mmol) of the product (SM) of reaction 18b was dissolved in 1ml of dichloromethane (MC). Then, 0.1ml (0.78mmol) of Triethylamine (TEA) and 3.17mg (0.026mmol) of 4-Dimethylaminopyridine (DMAP) were added and stirred. Then, it was stirred at 0 ℃ for 30 minutes. To the reaction solution, 29mL (0.338mmol) of 2-methylbutyryl chloride was added, and the mixture was stirred at room temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was completed, it was extracted with 0.1N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), and then concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 4 (volume ratio) mixture) to give 87.6mg of the product (yield: 71.9%).
[ reaction 21a ]
EXAMPLE 22]Synthesis of diacylglycerolactone Compound (EC-A134)
As shown in reaction 22a below, 100mg (0.26mmol) of the product (SM) of reaction 18b was dissolved in 1ml of dichloromethane (MC). Then, 72.5mL (0.52mmol) of Triethylamine (TEA) and 3.17mg (0.026mmol) of 4-Dimethylaminopyridine (DMAP) were added and stirred. Then, it was stirred at 0 ℃ for 30 minutes. To the reaction solution was added 30.7mL (0.338mmol) of cyclopropanecarbonyl chloride, and the mixture was stirred at room temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was completed, it was extracted with 0.1N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), and then concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 4 (volume ratio) mixture) to give 89.3mg of the product (yield: 75.8%).
[ reaction 22a ]
[ example 23]Synthesis of diacylglycerolactone Compound (EC-A135)
As shown in reaction 23a below, 100mg (0.26mmol) of the product (SM) of reaction 18b was dissolved in 1ml of dichloromethane (MC). Then, 72.5mL (0.52mmol) of Triethylamine (TEA) and 3.17mg (0.026mmol) of 4-Dimethylaminopyridine (DMAP) were added and stirred. Then, it was stirred at 0 ℃ for 30 minutes. 45.2mL (0.338mmol) of cyclopropanecarbonyl chloride was added to the reaction solution, and stirred at room temperature for 2 hours. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was completed, it was extracted with 0.1N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), and then concentrated. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 4 (volume ratio) mixture) to give 88.1mg of the product (yield: 68.5%).
[ reaction 23a ]
EXAMPLE 24]Synthesis of diacylglycerolactone Compound (EC-A136)
As shown in reaction 24a below, 67.7mg (0.338mmol) of lauric acid and 42mL (0.338mmol) of pivaloyl chloride were added to 1mL of dichloromethane (MC). Then, it was cooled to 0 ℃. 0.1ml (0.78mmol) Triethylamine (TEA) was slowly added dropwise at the same temperature. It was stirred at the same temperature for 30 minutes. 100mg (0.26mmol) of the product (SM) of reaction 18b and 3.17mg (0.026mmol) of 4-Dimethylaminopyridine (DMAP) are added at the same temperature. It was stirred at room temperature overnight. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, it was extracted with 0.1N potassium hydroxide solution (KOH solution)/dichloromethane (MC). The extract was extracted with 0.07N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC), followed by magnesium sulfate (MgSO) 4) Removing water, and concentrating. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 4 (volume ratio) mixture) to obtain 103.6mg of the objective compound (yield: 70.3%).
[ reaction 24a ]
EXAMPLE 25]Synthesis of diacylglycerolactone Compound (EC-A137)
77.2mg (0.338mmol) of myristic acid and 42mL (0.338mmol) of pivaloyl chloride are added to 1mL of dichloromethane (MC) as shown in reaction 25a below. Then, it was cooled to 0 ℃. 0.1ml (0.78mmol) Triethylamine (TEA) was slowly added dropwise at the same temperature. It was stirred at the same temperature for 30 minutes. 100mg (0.26mmol) of the product (SM) of reaction 18b and 3.17mg (0.026mmol) of 4-Dimethylaminopyridine (DMAP) are added at the same temperature. It was stirred at room temperature overnight. The reaction was confirmed by TLC (EA: Hex 1: 2). When the reaction was complete, it was extracted with 0.1N potassium hydroxide solution (KOH solution)/dichloromethane (MC). Extracting the extractive solution with 0.07N hydrochloric acid solution (c-HCl solution)/dichloromethane (MC)Taking, and adding magnesium sulfate (MgSO)4) Removing water and concentrating. The concentrate was purified by flash column (eluent: Ethyl Acetate (EA): hexane (Hex): 1: 4 (volume ratio) mixture) to obtain 106.4mg of the objective compound (yield: 68.8%).
[ reaction 25a ]
[ Experimental examples 1-1]Evaluation of cytotoxicity of diacylglycerol lactone Compounds
RAW264.7 cells (cells of the mouse macrophage family) were plated at 1x 105The cells/ml were suspended in DMEM (Dulbecco's modified Eagle's Medium) supplemented with 10% fetal bovine serum, then seeded into 96-well plates in 100. mu.l, and cultured for 15 hours. Next, the culture broth was treated with diacylglycerol lactone compounds of the kind and concentration shown in Table 1 below, and then cultured for another 24 hours. According to the manual EZ-CYTOX (Daeillab _ EZ-1000) for measuring the number of living cells using WST, 10. mu.l of EZ-Cytox was added to each well and reacted for 2 hours from 30 minutes, and then the Optical Density (OD) at 450nm was measured. The cell viability was calculated according to the
[ equation 1]
[ TABLE 1]
As shown in table 1, according to the results of observing the cell viability of RAW264.7 cells depending on the diacylglycerol lactone compound of the present invention, it was confirmed that all compounds showed no cytotoxicity at a concentration of 100 μ g/ml.
[ Experimental example 2-1]Increased expression of CXCL8(IL-8) of diacylglycerol lactone compounds
THP-1 cells of the human macrophage family were plated at 1X 10 5The individual cells/ml were suspended in RPMI (Hyclone, Thermo Scientific) medium with 10% fetal bovine serum and incubated at 37 ℃ in 5% CO2Culturing in a humidified incubator. Culturing THP-1 cells at 1X 106One cell/ml was seeded into 12-well plates and stabilized for 30 minutes. Then, the culture broth was treated with diacylglycerol lactone compounds of the type shown in Table 2 below for 1 hour, followed by treatment with Gemcitabine (Gemcitabine, 2. mu.g/ml), which was a cell-stimulating agent, followed by further incubation for 24 hours. Thereafter, 1.5ml of culture supernatant was collected per well and centrifuged (at 3000rpm, 5 minutes) to recover the supernatant. The level of CXCL8(IL-8) in the recovered supernatant was determined according to the manual supplied by the human IL-8ELISA kit (BD Biosciences). The day before ELISA was performed, the IL-8 capture antibody was diluted in phosphate buffered saline, coated in microwells, and then stored overnight at 4 ℃. Each well was washed 3 times with wash buffer at room temperature and then blocked with 2% Bovine Serum Albumin (BSA) for 1 hour. After 3 washes with wash buffer, 100 μ l of sample was dispensed into each well and left for 2 hours at room temperature. The detection antibody, which was washed 3 times with the washing buffer and diluted, was dispensed into each well, allowed to react for 1 hour at room temperature, and left to stand for 1 hour at room temperature. Thereafter, a secondary HRP-labeled antibody (secondary HRP conjugated antibody) was reacted at room temperature for 30 minutes, washed 3 times with a washing buffer, then each well was treated with 50 μ l of a stop solution, and then the optical density was measured at 450nm with an ELISA microplate guide. The results of the expression increase rate are shown in the following table 2 and fig. 1.
[ TABLE 2 ]
According to table 2 and fig. 1, it was confirmed that when THP-1 cells were treated with gemcitabine, which is an anticancer drug, secretion of CXCL8(IL-8) chemokine was increased by about 5-fold as compared with the negative control group, and addition of diacylglycerol lactone compound increased CXCL8 chemokine secretion of THP-1 cells by at least 1.3-fold and up to 12-fold relative to the anticancer drug-treated group.
[ Experimental example 3-1]Animal model and sample administration of pulmonary infectious bacteria
To obtain a mouse model with bacterial lung infection, 12-week-old Balb/c male mice were purchased from Koatech Corporation (South Korea) and maintained in some pathogen-free facilities at moderate temperatures and light cycles. To obtain bacteria inducing lung infection, pseudomonas Aeruginosa K (aeroginosa K, PAK) of the genus pseudomonas (Psuedomonas) was incubated in LB broth or LB agar plate at 37 ℃ overnight, and then the culture broth was centrifuged at 13000x g for 2 minutes to obtain a bacterial pellet. Thereafter, the bacterial pellet was suspended in Phosphate Buffered Saline (PBS), and the optical density of the serial dilutions was measured and plated on agar plates, thereby obtaining a bacterial inoculum with Colony Forming Units (CFU). For use in the following experiments, at 1X 10 per 20. mu.l 5Concentration of CFU bacterial inoculum was prepared for infection.
[ Experimental example 4-1]Confirmation of CFU levels in P.aeruginosa-infected mice
The PAK bacteria inoculum prepared in Experimental example 3 (1X 10 per mouse in 20. mu.l PBS)5CFU) was administered to a total of eight 12-week-old Balb/c mice by nasal injection, in which diacylglycerol lactone compound (EC _ a129) was orally administered at 250mg/kg to four mice in the PAK-treated group and PBS was administered to the control group. After 4 hours, bronchoalveolar lavage fluid (BALF) samples were collected from the PAK treated group. Collected BALF samples were mixed with PBS at 1: 1000, and the diluted samples were plated on LB agar and incubated overnight at 37 ℃. CFU levels in BALF were confirmed by measuring the number of surviving bacteria by plate counting method, and the results thereof are shown in table 3 below, fig. 2 and fig. 3.
[ TABLE 3 ]
As shown in table 3 and fig. 2, it was confirmed that bacterial CFU in BALF rapidly increased 4 hours after PAK administration. On the other hand, when a129 compound, which significantly increases expression of neutrophil recruitment factor CXCL8, among diacylglycerolactone compounds was administered together with PAK, bacterial CFU in alveolar lavage fluid at 4 hours was significantly lower than that of PAK only group. As described in experimental example 3, pooled BALF from each mouse of each group was mixed with PBS at a ratio of 1: 1000 dilution, and 100 u l added to LB plate to temperature bacteria for 16 hours, then the bacterial count. Before measuring the bacterial count, LB plates were photographed, shown in figure 3, demonstrating that diacylglycerol lactone compounds promote bacterial removal early in infection in PAK infected mice.