阅读说明：本技术 从鹿茸分离的新颖化合物及其药物用途 (Novel compound isolated from velvet antler and pharmaceutical use thereof ) 是由 具永三 孙基男 于 2019-07-15 设计创作，主要内容包括：本发明提供一种将选自由化学式1至4表示的化合物中的一种以上作为有效成分的预防或治疗炎性疾病、白细胞减少症或中性粒细胞减少症的药物组合物以及其的合成方法。(The present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, leukopenia or neutropenia comprising at least one compound selected from the group consisting of compounds represented by chemical formulas 1 to 4 as an active ingredient, and a method for synthesizing the same.)

1. A pharmaceutical composition for preventing or treating inflammatory diseases, leukopenia or neutropenia, wherein at least one selected from the group consisting of the compounds represented by the following chemical formulae 1 to 4 is used as an active ingredient.

[ chemical formula 1]

[ chemical formula 2]

[ chemical formula 3]

[ chemical formula 4]

2. The pharmaceutical composition for preventing or treating inflammatory diseases, leukopenia or neutropenia according to claim 1, wherein the effective ingredient is a mixture of the compound represented by chemical formula 1 and the compound represented by chemical formula 2.

3. The pharmaceutical composition for preventing or treating inflammatory diseases, leukopenia or neutropenia according to claim 1, wherein the effective ingredient is a mixture of the compound represented by chemical formula 3 and the compound represented by chemical formula 4.

4. The pharmaceutical composition for preventing or treating inflammatory diseases, leukopenia or neutropenia according to claim 1, wherein the effective ingredient inhibits the production of inflammatory cytokines.

5. The pharmaceutical composition for preventing or treating inflammatory diseases, leukopenia or neutropenia according to claim 1, wherein the inflammatory diseases are selected from the group consisting of atopic dermatitis, edema, dermatitis, allergy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, pharyngolaryngitis, tonsillitis, pneumonia, gastritis, colitis, gout, ankylosing spondylitis, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, scapulohumeral periarthritis, myositis, hepatitis, cystitis, nephritis.

6. The pharmaceutical composition for preventing or treating inflammatory diseases, leukopenia or neutropenia according to claim 1 wherein the neutropenia disease is neutropenia induced by an anticancer chemotherapy drug.

7. A health functional food for preventing or improving inflammatory diseases, wherein compounds represented by the following chemical formulae 1 to 4 are used as an active ingredient.

[ chemical formula 1]

[ chemical formula 2]

[ chemical formula 3]

[ chemical formula 4]

8. A method for synthesizing 1-palmitoyl-2-conjugated linoleoyl-3-acetyl glycerol comprises the following steps:

(a) a step of reacting palmitic acid with a compound represented by the following chemical formula 5 under alkaline conditions to synthesize 1-palmitoyl glycerol;

(b) a step of reacting the 1-palmitoyl glycerol with acetyl halide under an alkaline condition to synthesize 1-palmitoyl-3-acetyl glycerol; and

(c) a step of reacting 1-palmitoyl-3-acetylglycerol with conjugated linoleic acid under an alkaline condition.

[ chemical formula 5]

(in the formula, R₁、R₂Are each independently C₁To C₃Alkyl group of (1). )

9. The method for synthesizing 1-palmitoyl-2-conjugated linoleoyl-3-acetyl glycerol according to claim 8, wherein, in the step (a), palmitic acid is activated by adding pivaloyl halide, or

In said step (c) the conjugated linoleic acid is activated by adding pivaloyl halide.

(the halide is Cl, Br or l.)

10. The method of synthesizing 1-palmitoyl-2-conjugated linoleoyl-3-acetyl glycerol according to claim 8, wherein the conjugated linoleic acid is a mixture of conjugated linoleic acid (cis-9, trans-11) and conjugated linoleic acid (trans-10, cis-12).

11. A method for synthesizing 1-conjugated linoleoyl-2-palmitoyl-3-acetyl glycerol comprises the following steps:

(a) a step of reacting conjugated linoleic acid with a compound represented by the following chemical formula 5 under basic conditions to synthesize 1-conjugated linoleoyl-glycerol;

(b) a step of reacting the 1-conjugated linoleoyl-glycerol with an acetyl halide under basic conditions to synthesize 1-conjugated linoleoyl-3-acetylglycerol; and

(c) a step of reacting the 1-conjugated linoleoyl-3-acetyl glycerol and palmitic acid under basic conditions.

[ chemical formula 5]

(in the formula, R₁、R₂Are each independently C₁To C₃Alkyl group of (1). )

12. The method for synthesizing 1-conjugated linoleoyl-2-palmitoyl-3-acetyl glycerol according to claim 11, wherein, in the step (a), the conjugated linoleic acid is activated by adding pivaloyl halide, or

Activating palmitic acid in said step (c) by adding pivaloyl halide.

(the halide is Cl, Br or l.)

13. The method of synthesizing 1-conjugated linoleoyl-2-palmitoyl-3-acetyl glycerol according to claim 11, wherein the conjugated linoleic acid is a mixture of conjugated linoleic acid (cis-9, trans-11) and conjugated linoleic acid (trans-10, cis-12).

Technical Field

The present invention relates to a novel compound separated from velvet antler and its pharmaceutical use, and also relates to a chemical synthesis method of the novel compound.

Background

Cornu Cervi Pantotrichum (Cervi Parvum Cornu) is prepared by cutting and drying uncured Cornu Cervi Pantotrichum belonging to Cervus and Cervus (Cervus) such as Cervus nippon, Cervus elaphus (Cervus elaphus), or Cervus canadensis (Cervus canadensis).

Conventionally, velvet antler is a traditional Chinese medicine widely used as a representative tonic, and is known to have various effects such as a strengthening effect, a blood enriching effect, an essence strengthening effect, an analgesic effect, a hematopoietic effect, a growth and development promoting effect, a cardiac insufficiency treating effect and an hyperfunction effect, fatigue recovery, physical activity enhancement, kidney diuresis function enhancement and the like, according to the eastern medicine and the like.

Velvet antler is known to contain various free amino acids and polysaccharides, glycosaminoglycans (GAG), hyaluronic acid, keratin, sialic acid, cholesterol, fatty acids, phospholipids, inorganic components, and the like.

The application method of cornu Cervi Pantotrichum comprises extracting cornu Cervi Pantotrichum and various herbal materials with hot water, and administering the filtrate; or pulverizing the above materials with herbal materials to obtain powder, and then taking the powder in the form of pill, however, various studies have been made to extract or separate physiologically active ingredients from velvet antler by solvent extraction and fractionation.

Velvet antler (sika deer) was extracted with chloroform in korean patent laid-open No. 1999 0044781 and the chloroform extract was fractionated using silica gel column chromatography to isolate five monoacetyldiacylglycerol compounds, in which hematopoietic stem cell and platelet precursor cell proliferation promoting activities of 1-palmitoyl-2-linoleoyl-3-acetylglycerol (PLAG, hereinafter, referred to as "PLA glycerol") represented by the following chemical formula were disclosed, and subsequently, regarding the study of PLA glycerol, korean patent laid-open No. 2006 0047447 discloses an immunomodulator and an anticancer agent, korean patent laid-open No. 2015 0021464 discloses a composition for inhibiting blood cancer or cancer metastasis, korean patent laid-open No. 2015 0021465 discloses a composition for preventing or treating rheumatoid arthritis, korean patent laid-open No. 2017 0005484 discloses a composition for treating leukopenia and platelet-reduction A composition for treating hypofunction.

1-Palmitoyl-2-linoleoyl-3-acetyl glycerol(PLA glycerol)

Further, Korean patent publication No. 2000-0059468 discloses a structure of 4 phospholipid-based novel compounds including compounds of the following chemical formula and antifungal activity thereof by extracting velvet antler (Cervus nippon) with ethanol and fractionating the ethanol extract using silica gel column chromatography.

As described above, velvet antler has various pharmacological activities and its components are very diverse, and thus, there is a need for continuous research on pharmacologically active components that have yet to be reported.

Disclosure of Invention

An object of the present invention is to provide a composition for preventing or treating inflammatory diseases, a composition for treating leukopenia or neutropenia, which can stably mass-produce a novel compound having anti-inflammatory physiological activity from velvet antler by isolating the compound from velvet antler using a solvent extraction and fractionation method and performing chemical synthesis.

The present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, which analyzes an active ingredient isolated from velvet antler and uses one or more selected from the group consisting of compounds represented by the following chemical formulas 1 to 4 as the active ingredient.

In another aspect, the present invention provides a composition for preventing or treating leukopenia or neutropenia, wherein at least one selected from the group consisting of the compounds represented by the following chemical formulae 1 to 4 is used as an active ingredient.

[ chemical formula 1]

[ chemical formula 2]

[ chemical formula 3]

[ chemical formula 4]

The compound represented by chemical formula 1 and the compound represented by chemical formula 2 are isomers with each other, and the compound represented by chemical formula 3 and the compound represented by chemical formula 4 are isomers with each other.

The compound according to the present invention may be used for inflammatory diseases, which are inflammatory diseases developed by inflammatory cytokines, for example, selected from the group consisting of atopic dermatitis, edema, dermatitis, allergy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, pharyngolaryngitis, tonsillitis, pneumonia, gastritis, colitis, gout, ankylosing spondylitis, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, scrap arthritis, myositis, hepatitis, cystitis, nephritis.

The compound according to the present invention can be used for preventing or treating neutropenia (CIN) induced by anticancer chemotherapy drugs.

In addition, the invention provides a method for synthesizing 1-palmitoyl-2-conjugated linoleoyl-3-acetyl glycerol (PCA glycerol), which comprises the following steps:

(a) a step of reacting palmitic acid with a compound represented by the following chemical formula 5 under alkaline conditions to synthesize 1-palmitoyl glycerol;

(b) a step of reacting the 1-palmitoyl glycerol with acetyl halide under an alkaline condition to synthesize 1-palmitoyl-3-acetyl glycerol; and

(c) a step of reacting the 1-palmitoyl-3-acetyl glycerol with Conjugated linoleic acid (Conjugated linoleic acid) under alkaline conditions.

In addition, the present invention provides a method for synthesizing 1-conjugated linoleoyl-2-palmitoyl-3-acetylglycerol (CPA glycerol), comprising:

(a) a step of reacting the Conjugated linoleic acid (Conjugated linoleic acid) with a compound represented by the following chemical formula 5 under basic conditions to synthesize 1-Conjugated linoleoyl-glycerol;

(b) a step of reacting the 1-conjugated linoleoyl-glycerol with an acetyl halide under basic conditions to synthesize 1-conjugated linoleoyl-3-acetylglycerol; and

(c) a step of reacting the 1-conjugated linoleoyl-3-acetyl glycerol and palmitic acid under basic conditions.

The conjugated linoleic acid used for the synthesis may be conjugated linoleic acid (cis-9, trans-11) and conjugated linoleic acid (trans-10, cis-12) alone or in a mixture.

Effects of the invention

It was confirmed that the novel compounds of examples 1 and 2 according to the present invention significantly inhibited the production of IL-6, IL-1 β and TNF-a, which are representative inflammatory cytokines, and also significantly inhibited neutropenia caused by an anticancer agent, compared to the control group, and that the effects of inhibiting the production of cytokines and inhibiting neutropenia are also significant, compared to PLA glycerol of comparative example. Therefore, it can be effectively used as a composition for preventing, ameliorating or treating inflammatory diseases according to the present invention and shows a significant inhibitory effect on neutropenia (CIN) induced by an anticancer chemotherapy drug, and thus, can be used as a prophylactic or therapeutic agent thereof.

Drawings

Fig. 1 is a flowchart illustrating an extraction method from velvet antler using 3 organic solvents according to the present invention.

Fig. 2 is a flowchart illustrating a fractionation method of an extract C2 extracted from velvet antler according to the present invention.

FIG. 3 is mass analysis data of fraction C2-2-E-a-Ms according to the present invention.

FIG. 4 is the LC data for fraction C2-2-E-a-Ms according to the present invention.

FIG. 5(A) is the UV analysis data of fraction C2-2-E-a-Ms according to the present invention, (B) is the UV analysis data of PLA glycerin of comparative example, (C) is the UV analysis data of conjugated linoleic acid (conjugated linoleic acid), and (D) is the UV analysis data of linoleic acid.

Fig. 6 is a graph showing the results of measuring cell viability (cell viability) by MTT method (MTT assay) of the novel compound according to examples 1 and 2 of the present invention.

FIG. 7 is a graph showing the IL-6 production inhibitory effect of the novel compounds of examples 1 and 2 of the present invention.

FIG. 8 is a graph showing the inhibitory effect on IL-1. beta. production of the novel compounds of examples 1 and 2 of the present invention.

FIG. 9 is a graph showing the TNF- α inhibitory effect of the novel compounds of examples 1 and 2 of the present invention.

Fig. 10 is a graph showing the NO formation inhibitory effect of the novel compounds of examples 1 and 2 of the present invention.

Fig. 11 is a graph showing the inhibitory effect of the novel compounds of examples 1 and 2 of the present invention on neutropenia induced by anticancer agent (gemcitabine).

Fig. 12 is a graph showing the inhibitory effect of the novel compounds of examples 1 and 2 of the present invention on neutropenia induced by an anticancer agent (tamoxifen).

Detailed Description

In order to develop a pharmaceutical composition derived from velvet antler for the prevention or treatment of inflammatory diseases, the present inventors analyzed the solvent extraction method and fractionation method of the prior art from various angles, isolated a novel compound having excellent physiological activity on inflammatory diseases by the following method, and confirmed that the novel compound has a significant inhibitory effect on neutropenia (CIN) induced by anticancer chemotherapy drugs, thereby developing a novel synthetic method of the compound to complete the present invention.

In order to extract physiologically active ingredients from velvet antler, velvet antler (sika deer) was sequentially extracted using 3 organic solvents of hexane, chloroform and 70% ethanol as shown in fig. 1, and then, novel compounds having excellent anti-inflammatory activity were isolated by performing silica gel column chromatography and thin film liquid chromatography as shown in fig. 2.

By extraction with organic solvents

2kg of a commercially available Cervus nippon (Cervus nippon) was pulverized, put into a beaker, and 9L of Hexane (n-Hexane) was added as a primary extraction solvent, and then, after 2 times of extraction by heating at a temperature of 80 ℃ for 2 hours, filtration was carried out to obtain a Hexane extract, and then, the Hexane extract was distilled and dried under reduced pressure to obtain 30.3g of extract C1.

After extraction of hexane, 9L of chloroform (CHCl) was added to the residue as a secondary extraction solvent₃) Then, the mixture was heated at 80 ℃ for 2 hours and extracted 2 times, and after obtaining a chloroform extract by filtration, the chloroform extract was distilled and dried under reduced pressure to obtain 17.9g of extract C2.

After extracting chloroform, 9L of 70% ethanol was added to the residue as a solvent for three times, followed by extraction 2 times by heating at 80 ℃ for 5 hours, filtration to obtain an ethanol extract, and distillation and drying of the ethanol extract under reduced pressure to obtain 89.2g of extract C3.

As a result of anti-inflammatory activity experiments on each of the extracts C1, C2, and C3 obtained as described above, it was confirmed that the anti-inflammatory activity effect was high in the extract C2, and the physiologically active ingredients were fractionated using silica gel column chromatography using the extract C2 as a target.

Silica gel column chromatography

Addition of CHCl to the powdered silica gel₃The mixed solution of/MeOH (500: 1, v/v) was swollen and then packed into an open column. 17.9g of the obtained extract C2 were dissolved in CHCl₃/MeOH(500: 1, v/v) and is suitable for use on a column packed with silica gel. Mixing CHCl₃the/MeOH (500: 2, v/v) was passed into the column as eluent to give elution fractions per 50ml, followed by TLC (elution solvent: CHCl)₃300: 1) and, after development with iodine (I), separated according to Rf value, 7 main fractions were isolated. The separated fractions were distilled under reduced pressure and dried to obtain fractions C2-1 to C2-7. As a result of the anti-inflammatory activity test on the 7 fractions, it was confirmed that the anti-inflammatory activity was high in the fraction C2-2, and thus the physiologically active ingredients were fractionated again using silica gel column chromatography with the fraction C2-2 as the subject.

1.8g of the fraction C2-2 were taken. To 20g of powdery silica gel, 50ml of a mixed solution of hexane (n-Hx)/Ethyl Acetate (EA) (50: 1) was added and allowed to swell, and then the column was packed. 1.8g of fraction C2-2 were dissolved in a minimum amount of eluent, n-Hx/EA (50: 1, v/v) and applied to a silica gel column. The eluate was passed through the silica gel column to obtain elution fractions of 15ml each, eluted by TLC (elution solvent: n-Hx/EA: 50: 1, v/v), developed with iodine, and then separated by Rf to separate 6 major fractions. The separated fractions were distilled under reduced pressure and dried to obtain fractions C2-2-A through C2-2-F. As a result of the anti-inflammatory activity test on the 6 fractions, it was confirmed that the anti-inflammatory activity was high in the fraction C2-2-E, and thus the physiologically active ingredients were fractionated again using silica gel column chromatography with the fraction C2-2-E as the subject.

212mg of the fraction C2-2-E were taken. To 3.5g of powdery silica gel, 10ml of a mixed solution of n-Hx/EA/AcOH (20: 1: 0.5, v/v/v) was added and allowed to swell, and then the mixture was packed into a column. 212mg of fraction C2-2-E were dissolved in the eluent n-Hx/EA/AcOH (20: 1: 0.5, v/v/v) and applied to a silica gel column. The eluate was passed through the silica gel column to obtain elution fractions of 10ml each, eluted by TLC (elution solvent: n-Hx/EA/AcOH ═ 20: 1: 0.5, v/v/v), developed with iodine, and then separated into 2 major fractions by Rf. The separated fractions were distilled under reduced pressure and dried to obtain fractions C2-2-E-a and C2-2-E-b. As a result of the anti-inflammatory activity test conducted on the 2 fractions, it was confirmed that the anti-inflammatory activity was high in the fraction C2-2-E-a, and thus the physiologically active ingredients were fractionated again using silica gel column chromatography using the fraction C2-2-E-a as a subject.

137mg of the fraction C2-2-E-a was taken. 5ml of methanol was added to the C2-2-E-a and mixed, separated into a methanol-soluble fraction C2-2-E-a-Ms and a methanol-insoluble fraction C2-2-E-a-Mi, and each fraction was distilled under reduced pressure and dried to obtain 63mg of a fraction C2-2-E-a-Ms. As a result of the anti-inflammatory activity test conducted on the 2 fractions, it was confirmed that the fraction C2-2-E-a-Ms had a high anti-inflammatory activity.

The fractions were evaluated for anti-inflammatory inhibition by the method of experimental example 2 described later, and the results are shown in table 1 below.

[ TABLE 1]

As is clear from Table 1, the production of inflammatory cytokines IL-6, IL-1. beta. and TNF-. alpha.was inhibited as the fraction was smaller, and in order to confirm the effective components of fraction C2-2-E-a-Ms having the highest inflammation inhibitory activity among the fractions, a MALDI-TOF/TOF mass spectrometer (Bruker Ultraflexreeme) was used^TMGermany), Liquid Chromatography (LC) and UV analyzer.

FIG. 3 is the mass analysis data of fraction C2-2-E-a-Ms, FIG. 4 is the LC data of fraction C2-2-E-a-Ms, FIG. 5(A) is the UV analysis data of fraction C2-2-E-a-Ms according to the invention, FIG. 5(B) is the UV analysis data of PLA glycerol of comparative example, FIG. 6(C) is the UV analysis data of conjugated linoleic acid (D) is the UV analysis data of linoleic acid.

As a result of analyzing the quality of the effective components of the fraction C2-2-E-a-Ms of the present invention, the quality was the same as that of the known PLA glycerol isolated from velvet antler (Compound KJ-3 of Korean patent laid-open No. 1999-0044781), but the result of UV analysis showed a completely different UV analysis pattern from that of PLA glycerol. The comparison of the UV patterns of Conjugated Linoleic acid (Conjugated Linoleic acid) and Linoleic acid (Linoleic acid) in FIG. 5(C) confirms that the compound of fraction C2-2-E-a-Ms of the present invention has a novel structure of Conjugated linoleoyl group (Conjugated Linoleic acid).

It was confirmed in the present invention that the anti-inflammatory physiologically active ingredients according to the present invention are novel compounds represented by the following chemical formulae 1 to 4 in which acetyl (acetyl), palmitoyl (palmitoyl), Conjugated linoleoyl (Conjugated linoleoyl) are bonded to a glycerol structure.

[ chemical formula 1]

[ chemical formula 2]

[ chemical formula 3]

[ chemical formula 4]

Chemical Synthesis of PCA Glycerol represented by the above-mentioned chemical formula 1 and chemical formula 2

The compounds of chemical formula 1 and chemical formula 2 (PCA glycerol) according to the present invention can be prepared in high yield by the following reaction formula 1.

[ reaction formula 1]

The PCA glycerol according to the invention comprises:

(a) reacting palmitic acid with a compound represented by the following chemical formula 5 under alkaline conditions to produce a palmitic acid

(a) Reacting palmitic acid with a compound represented by the following chemical formula 5 under alkaline conditions to synthesize 1-palmitoyl glycerol;

(b) a step of reacting the 1-palmitoyl glycerol with acetyl halide under an alkaline condition to synthesize 1-palmitoyl-3-acetyl glycerol; and

(c) a step of reacting the 1-palmitoyl-3-acetyl glycerol with Conjugated linoleic acid (Conjugated linoleic acid) under alkaline conditions.

In the step (a), the compound of chemical formula 5 is a glycerol derivative protected by a 1, 3-diol compound. This reaction can be performed under basic conditions such as triethylamine (triethylamine), and in order to maximize the reactivity of palmitic acid, Pivaloyl halide (Pivaloyl halide) can be added to perform an acid anhydride reaction (anhydride reaction). After the reaction, deprotection using hydrochloric acid or the like synthesizes 1-palmitoyl glycerol. The equivalent (eq.) of palmitic acid and the compound of chemical formula 5 is preferably 0.9: 1.1 to 1.1: 0.9, but is not limited thereto.

In the step (b), the equivalent weight of 1-palmitoyl glycerol and acetyl halide is preferably 1: 1 to 1: 1.6, but is not limited thereto.

In the step (c), in order to maximize the reactivity of the conjugated linoleic acid, Pivaloyl halide (Pivaloyl halide) may be added to proceed through an acid anhydride reaction (anhydride reaction). The equivalent (eq.) of 1-palmitoyl-3-acetylglycerol and conjugated linoleic acid is preferably 0.9: 1.1 to 1.1: 0.9, but is not limited thereto.

In addition, the compounds of chemical formulae 3, 4 (CPA glycerol) according to the present invention can be prepared in high yield by the following reaction formula 2.

[ reaction formula 2]

CPA glycerol according to the present invention comprises:

(a) a step of reacting Conjugated linoleic acid (Conjugated linoleic acid) with a compound represented by the following chemical formula 5 under basic conditions to synthesize 1-Conjugated linoleoyl glycerol;

(b) a step of reacting the 1-conjugated linoleoyl glycerol with acetyl halide under basic conditions to synthesize 1-conjugated linoleoyl-3-acetyl glycerol; and

(c) a step of reacting the 1-conjugated linoleoyl-3-acetyl glycerol with palmitic acid under basic conditions to synthesize 1-conjugated linoleoyl-2-palmitic acid 3-acetyl glycerol (CPA glycerol). Pivaloyl halide (Pivaloyl halide) may be added for the acid anhydride reaction in the steps (a), (c).

The following examples illustrate the synthesis of the novel compounds of the present invention.

Example 1: preparation of 1-Palmitoyl-2-conjugated linoleoyl-3-acetyl glycerol (PCA glycerol) (1-palmityl-2-conjugated-linoleoyl-3-acetyl glycerol (PCA glycerol))

(1) Preparation of 1-palmitoyl glycerol (1-palmitoyl glycerol)

To 200ml of Methylene chloride (methyl chloride, MC) were added 20.0g of Palmitic acid (palmitc acid) and 17.0g of triethylamine (triethylamine) and cooled to 0 ℃, then 10.0g of pivaloyl chloride was slowly added dropwise and stirred for 2 hours, then 10.8g of acetone glycerol (Solketal) and 0.1g of glycerol 4-Dimethylaminopyridine (4-Dimethylaminopyridine) were added and stirred at room temperature for 12 hours, and 200ml of purified water was added for layer separation and then the organic layer was concentrated in vacuo. To the concentrated mother liquor were added 200ml of methanol and 20ml of hydrochloric acid, and after stirring at room temperature for 10 hours, 300ml of n-hexane (n-hexane) and 300ml of purified water were added, and after stirring for 3 hours, filtration and vacuum drying were carried out to obtain 21.9g of the objective compound (yield: 85.0%).

(2) Preparation of 1-Palmitoyl-3-acetylglycerol (1-palmityl-3-acetyl glycerol)

To 200ml of Methylene Chloride (MC), 20 was added0g of the obtained 1-palmitoyl glycerol, 33.5g of Pyridine (Pyridine), and 0.2g of 4-Dimethylaminopyridine (4-diamylamine) were stirred for 1 hour, and then 7.1g of Acetyl chloride (Acetyl chloride) was added dropwise thereto and stirred for 5 hours. Adding 200ml purified water, neutralizing with dilute hydrochloric acid, separating layers, and collecting organic layer with MgSO₄After dehydration, the reaction mixture was concentrated in vacuo, 100ml of n-hexane was added, the mixture was crystallized at a temperature of 10 ℃ or lower, filtered, and dried in vacuo to obtain 18.5g of the objective compound (yield: 82.0%).

(3) Preparation of 1-Palmitoyl-2-conjugated linoleoyl-3-acetyl glycerol (PCA glycerol) (1-Palmitoyl-2-C-linoleoyl-3-acetyl glycerol (PCA glycerol))

To 180ml of n-hexane, 14.0g of conjugated linoleic acid (cis-9, trans-11/trans-10, cis-12) and 10.8g of triethylamine (triethylamine) were added, cooled to 0 ℃ and 7.0g of pivaloyl chloride was slowly added dropwise. After completion of the dropwise addition, stirring was performed at the same temperature for 1 hour, and then 18.0g of 1-palmitoyl-3-acetylglycerin and 1.0g of 4-dimethylaminopyridine were added, followed by stirring at room temperature for 10 hours. Adding 180ml purified water, separating the layers, and separating the organic layer with MgSO₄After dehydration, the reaction mixture was concentrated in vacuo and purified with a silica gel column (eluent: n-Hx: EA: 20: 1, v/v) to obtain 26.1g of the objective compound (yield: 85%).

Example 2: preparation of 1-Conjugated linoleoyl-2-palmitoyl-3-acetyl glycerol (CPA glycerol) (1-Conjugated-linoleoyl-2-palmitoyl-3-acetyl glycerol)

(1) Preparation of 1-conjugated linoleoyl glycerol (1-C-linoleoyl glycerol)

To 200ml of n-Hexane (n-Hexane) were added 22.0g of conjugated linoleic acid (cis-9, trans-11/trans-10, cis-12) and 10.8g of triethylamine (triethylamine) and cooled to 0 ℃, then 7.0g of Pivaloyl chloride (Pivaloyl chloride) was slowly added dropwise at the same temperature and stirred for 1 hour, then 10.8g of acetone glycerol (Solketal) and 0.1g of 4-dimethylaminopyridine were added and stirred for 12 hours at room temperature, and after adding 200ml of purified water, layer separation was performed, and then, the organic layer was concentrated in vacuo. 200ml of methanol and 20ml of hydrochloric acid were added to the concentrated mother liquor, and the mixture was stirred at room temperature for 10 hours, then 300ml of n-hexane and 300ml of purified water were added thereto, and after stirring for 3 hours, filtration and vacuum drying were carried out to obtain 22.8g of the objective compound (yield: 82%)

(2) Preparation of 1-conjugated linoleoyl-3-acetyl glycerol (1-C-linoleoyl-3-acetyl glycerol)

To 200ml of Methylene Chloride (MC), 20.0g of 1-conjugated linoleoyl glycerol, 33.5g of pyridine (pyridine), and 0.2g of 4-Dimethylaminopyridine (4-dimethylamino pyridine) were added, and the mixture was stirred for 1 hour, followed by dropwise addition of 6.6g of Acetyl chloride (Acetyl chloride) and stirring for 5 hours. Adding 200ml purified water, neutralizing with dilute hydrochloric acid, separating layers, and collecting organic layer with MgSO₄After dehydration, the reaction mixture was concentrated in vacuo, 100ml of n-hexane was added, crystallization was carried out at a temperature of 10 ℃ or lower, and filtration and vacuum drying were carried out to obtain 17.7g of the objective compound (yield: 79.0%).

(3) Preparation of 1-conjugated linoleoyl-2-palmitoyl-3-acetylglycerol (CPA glycerol) (1-C-linoleoyl-2-palmitoyl-3-acetyl glycerol (CPA glycerol))

To 180ml of n-Hexane (n-Hexane) were added 12.2g of Palmitic acid (Palmitic acid) and 10.8g of triethylamine (triethylamine) and cooled to 0 ℃, and then 7.0g of Pivaloyl chloride (Pivaloyl chloride) was slowly added dropwise theretoAfter stirring at the same temperature for 1 hour, 18.0g of 1-conjugated linoleoyl-3-acetylglycerol (1-C-linoleoyl-3-acetyl glycerol) and 1.0g of 4-Dimethylaminopyridine (4-methylenediaminopyradine) were added, and then, stirring was performed at room temperature for 10 hours. Adding 180ml purified water, separating the layers, and then, using MgSO as organic layer₄The reaction mixture was dehydrated, concentrated in vacuo, and purified with a silica gel column (eluent: n-Hx: EA: 20: 1, v/v) to obtain 25.9g of the title compound (yield: 86%).

Comparative example: preparation of 1-Palmitoyl-2-linoleoyl-3-acetyl glycerol (PLA glycerol) (1-Palmitoyl-2-lineoyl-3-acetyl glycerol (PLA glycerol))

PLA glycerol was synthesized in the same manner as in example 1, in this case, Linoleic acid (Linoleic acid) (cis-9, cis-12) was used instead of conjugated Linoleic acid (cis-9, trans-11/trans-10, cis-12).

To 180ml of n-Hexane (n-Hexane) were added 14.0g of Linoleic acid (cis 9, cis 12) (Linoleic acid (cis-9, cis-12)) and 10.8g of triethylamine (triethylamine), and after cooling to 0 ℃, 7.0g of Pivaloyl chloride (Pivaloyl chloride) was slowly added dropwise. After completion of the dropwise addition, stirring was carried out at the same temperature for 1 hour, and then, after adding 18.0g of 1-Palmitoyl-3-acetylglycerol (1-palmityl-3-acetyl glycerol) and 1.0g of 4-Dimethylaminopyridine (4-methylenepyridodine), stirring was carried out at room temperature for 10 hours. Adding 180ml purified water, separating the layers, and collecting the organic layer with MgSO₄The reaction mixture was dehydrated, concentrated in vacuo, and purified with a silica gel column (eluent: n-Hx: EA: 20: 1, v/v) to obtain 24.9g of the title compound (yield: 81%).

The anti-inflammatory activity test of the compounds synthesized in examples 1 and 2 and comparative example was evaluated by the following animal test.

A. Laboratory animal

As experimental animals, 8-week-old C57BL/6 mice were kept in an environment of 22. + -. 2 ℃ temperature, 65. + -. 5% relative humidity and 12 hours light-dark cycle for 7 days and adapted to the laboratory environment before being used for experiments. The solid feed (three-nutrient feed) and water are sufficiently supplied.

B. Isolated culture of peritoneal macrophages

Mouse was intraperitoneally injected with HBSS to extract macrophages (macrophage), centrifuged at 3,000rpm for 5 minutes, and then 100 units/ml of penicillin/streptomycin (penicillin/streptomycin) was added to DMEM medium supplemented with 10% Fetal Bovine Serum (FBS), and peritoneal macrophages were isolated, at 37 ℃ and 5% CO₂Was used for the experiment after 24 hours of culture in the incubator of (1).

Experimental example 1: evaluation of cytotoxicity by MTT method

Culturing the peritoneal macrophages at 3 × 10⁵Cells/well were plated per 100uL aliquot into 96-well plates and cultured overnight. After removal of the medium, the compounds of example 1(PCA glycerol) and example 2(CPA glycerol) were treated at concentrations (10. mu.g/ml, 100. mu.g/ml and 200. mu.g/ml, respectively) on peritoneal macrophages at 37 ℃ with 5% CO₂Was cultured in an incubator for 24 hours. After incubation, the medium was removed, MTT (5mg/mL) reagent was aliquoted per 40uL and incubated in CO₂After 4 hours of incubation in the incubator, the MTT reagent was removed, and the DMSO reagent was dispensed in an amount of 600uL, and then allowed to stand at room temperature for 30 minutes. Then, the absorbance (OD) was measured at 540nm with a microplate reader.

Cell viability (cellviality) according to the MTT method was measured for the compounds of examples 1 and 2 and is shown in table 2 below and the graph of fig. 6. As shown in Table 2 and FIG. 6, it can be seen that there was no significant difference in cell viability even at the treatment of 200. mu.g/ml at the maximum. Thus, the novel compounds according to the present invention were confirmed to be not cytotoxic.

[ TABLE 2]

Experimental example 2: anti-inflammatory Activity test (evaluation of production inhibition of IL-6, IL-1. beta. and TNF-. alpha.)

The anti-inflammatory activity was evaluated by measuring the secretion amounts of IL-6, IL-1. beta. and TNF-a secreted from mouse peritoneal macrophages induced by Lipopolysaccharides (LPS) of inflammatory response inducing factors using an ELISA test method (Millipore, USA).

To obtain cell culture medium, mouse peritoneal macrophages were adjusted to 3x10⁵Cells/ml, and inoculated into 96-well plates, and after 24 hours of culture, the compounds of example 1(PCA glycerol), example 2(CPA glycerol), and comparative example (PLA glycerol) were treated at concentrations (10. mu.g/ml, 100. mu.g/ml, 200. mu.g/ml, respectively), and LPS (1. mu.g/ml) was treated. The normal group was not treated, while the control group treated only LPS (1. mu.g/ml) in peritoneal macrophages. After 12 hours of culture, the supernatant was obtained by centrifugation. ELISA was performed on a microplate to isolate anti-mouse IL-6, IL-1. beta.3 and TNF-a as coating antibodies (capture antibodies). Then, washed with Phosphate Buffered Saline (PBST) containing 0.05% Tween 20, blocked with 10% FBS and washed with PBST, and the cell culture supernatant was aliquoted into wells and reacted at room temperature for 2 hours. After the reaction, the reaction mixture was washed with PBST, and then, a biotinylated anti-mouse IL-6, IL-1. beta. and TNF-. alpha.detection antibody (detection antibody) and a streptavidin-horseradish peroxidase conjugate (streptavidin-horse radish peroxidase conjugate) were separately diluted and reacted at room temperature for 1 hour. Then, the OPD solution was added thereto by washing with PBST and the reaction was carried out in the dark at room temperature for 30 minutes. By 2NH₂SO₄After the reaction was completed, the absorbance was measured at 450nm using a microplate reader.

The amounts of IL-6 produced by the compounds of examples 1 and 2 were measured, and are shown in Table 3 below and shown in the graph of FIG. 7. As shown in table 3 and fig. 7, it is understood that the compounds prepared in examples 1 and 2 significantly reduced the amount of IL-6 produced as compared to the control group, and had a significant IL-6 production inhibitory effect as compared to the comparative example.

[ TABLE 3]

The amounts of IL-1. beta. produced by the compounds of examples 1 and 2 were measured, and are shown in Table 4 below and shown in the graph of FIG. 8. As shown in table 4 and fig. 8, it is understood that the compounds prepared in examples 1 and 2 significantly reduced the amount of IL-1 β production as compared with the control group, and had a significant effect of inhibiting IL-1 β production as compared with the comparative example.

[ TABLE 4]

The amounts of TNF-. alpha.produced by the compounds of examples 1 and 2 were measured and shown in Table 5 below and in the graph of FIG. 9. As shown in Table 5 and FIG. 9, it was found that the compounds prepared in examples 1 and 2 significantly reduced the amount of TNF-a produced as compared with the control group, and had a significant effect of inhibiting the production of TNF- α as compared with the comparative example.

[ TABLE 5]

Experimental example 3: anti-inflammatory Activity test (evaluation of NO formation inhibition)

Suspending the cultured peritoneal macrophages in DMEM containing 10% FBS, and then performing cell culture at 5X 10⁵Cells/well were plated in 96-well plates at 37 ℃ with 5% CO₂After 24 hours of incubation in an incubator and exchange of fresh DMEM medium, the compounds of examples 1, 2 and comparative examples were treated at concentrations (10. mu.g/ml, 100. mu.g/ml and 200. mu.g/ml, respectively) on peritoneal macrophages, followed by LPS treatment (1. mu.g/ml) for 24 hours. After incubation, the supernatant was separated, centrifuged at 3000rpm for 5 minutes, and the separated supernatant was aliquoted onto a new microplate (microplate). The normal group was untreated, and the control group treated only LPS (1. mu.g/ml) in peritoneal macrophages.

Identical amounts of Griess reagent (1% sulfonamide), 0.1% naphthyl-ethylenediamine hydrochloride (naphthalene-ethylene diamine), 2% phosphoric acid (phosphoric acid) were treated and reacted at room temperature for 10 minutes, and after allowing the culture solution and Griess solution to react for 5 minutes, absorbance (OD) was measured at 540 nm.

Fig. 10 is a graph showing the NO production inhibitory effect of the novel compounds of examples 1 and 2 of the present invention.

The production rates of Nitric Oxide (NO) of the compounds of examples 1 and 2 were measured, and are shown in table 6 below and shown in the graph of fig. 10. As shown in table 6 and fig. 10, it is understood that the compounds prepared in examples 1 and 2 have a significantly reduced NO production rate as compared with the control group, and also have a significant NO production inhibitory effect as compared with the comparative example.

[ TABLE 6 ]

The compound according to the present invention may be used for preventing or treating inflammatory diseases, which are inflammatory diseases caused by inflammatory cytokines, for example, selected from the group consisting of atopic dermatitis, edema, dermatitis, allergy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, pharyngolaryngitis, tonsillitis, pneumonia, gastritis, colitis, gout, ankylosing spondylitis, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, scapulohumeral periarthritis, myositis, hepatitis, cystitis, nephritis.

PLA glycerol in the comparative example is known to be a candidate for drug-induced neutropenia (CIN) of anticancer chemotherapy, and phase 2 clinical trials of FDA have recently been completed, reporting that neutropenia is inhibited by a different mechanism of action from that of the existing therapeutic agent, so-called G-CSF (filgrastim) or its derivative (pegfilgrastim).

Next, the inhibitory effects of the compounds of examples 1 and 2 of the present invention and the compounds of comparative examples on neutropenia induced by anticancer chemotherapeutic drugs were verified by mouse experiments.

Experimental example 4: mouse experiments for neutropenia induced by anticancer chemotherapy

Gemcitabine (Gemcitabine) 50mg/kg as an anticancer agent was injected into the abdominal cavity of mice daily for 3 consecutive weeks as a control group, and 500mg/kg of the compounds of example 1, example 2 and comparative example 1 was orally administered daily for 3 consecutive weeks, and the number of neutrophils in 1 μ l of blood was measured after 3 consecutive weeks using an automatic blood sample Analyzer (Auto Hematology Analyzer BC-5900, Mindray company), and the results thereof are shown in the following table 7 and fig. 11.

[ TABLE 7 ]

As shown in table 7 and fig. 11, it was confirmed that the number of neutrophils was reduced by about 45% in mice administered with gemcitabine, 14.5% in the compound of comparative example (PLA glycerin), about 2% in the compound of example 1(PCA glycerin), and about 6.7% in the compound of example 2(CPA glycerin), not only the control, but also the significant effect was exhibited as compared with the comparative example. 50mg/kg of tamoxifen used as an anticancer agent was injected into the abdominal cavity of C57BL/6 mice as a control group every day for 3 consecutive weeks, and 500mg/kg of the compounds of example 1, example 2, and comparative example 1 was orally administered every day for 3 consecutive weeks, after which the number of neutrophils in 1. mu.l of blood was measured using an automatic blood sample Analyzer (Auto Hematology Analyzer BC-5900, Mindray Co.) and the results thereof are shown in Table 8 below and FIG. 12.

TABLE 8

As shown in table 8 and fig. 12, it was found that the number of neutrophils was reduced by about 37% in the mice administered with tamoxifen, while the number of neutrophils was reduced by 12.9% in the compound of comparative example (PLA glycerin), by about 6.3% in the compound of example 1(PCA glycerin), and by about 8.2% in the compound of example 2(CPA glycerin), and significant effects were confirmed not only in the control group, but also in comparison with the comparative example. The compound of the present invention, in addition to the anticancer chemotherapeutic drugs, known anticancer chemotherapeutic drugs are effective as suppressive drugs for neutropenia caused by Cyclophosphamide (Cyclophosphamide), Imatinib (Imatinib), Lenalidomide (Lenalidomide), Bortezomib (Bortezomib), Pemetrexed (Pemetrexed), Methotrexate (Methotrexate), Paclitaxel (Paclitaxel), Etoposide (Etoposide), Topotecan (Topotecan), irinotecan (irinotecan), mechleretannine, Chlorambucil (chlomambucil), Melphalan (Melphalan), Carmustine (Carmustine, BCNU), Lomustine (CCNU), isocyclosfamide (Cyclophosphamide), Procarbazine (Procarbazine), carbapenem (Carboplatin), Carboplatin (Doxorubicin (d), Carboplatin (Doxorubicin), platinum (Doxorubicin), Carboplatin (d), platinum (Doxorubicin), platinum (Doxorubicin), Carboplatin (platinum (doxylamine), platinum (Doxorubicin), Carboplatin), platinum (Doxorubicin (platinum (Doxorubicin), Carboplatin), platinum (Doxorubicin, Carboplatin), Carboplatin (platinum (D), Carboplatin), platinum (platinum), platinum (platinum), platinum (platinum, 6-Mercaptopurine (6-Mercaptopurine), 6-Thioguanine (6-Thioguanine), idarubicin (idarubicin), Epirubicin (Epirubicin), Mitoxantrone (Mitoxantrone), Azathioprine (Azathioprine), 2-chlorodeoxyadenosine (2-Chloro deoxyadenosine), Hydroxyurea (Hydroxyurea), 5-Fluorouracil (5-Fluorouracil), cytarabine (cytarabine), Azacytidine (azatidine), Fludarabine phosphate (Fludarabine phosphate), Vincristine (Vincristine), Vinblastine (Vinblastine), Vinorelbine (vinorelbrexed), Docetaxel (Docetaxel), nano-albumin-bound paclitaxel (Nab-paclitaxel), Dasatinib (danitinib), etc.

[ Industrial Applicability ] of the invention

The present invention relates to a novel compound isolated from velvet antler and its pharmaceutical use, and also relates to a chemical synthesis method of the novel compound.