KR102688016B1 - Pharmaceutical composition for preventing or treating brain tumor comprising acetylenic acid analogue from the leaves of dendropanax morbiferus - Google Patents

Abstract

The present invention relates to a composition for preventing or treating brain tumors comprising an acetylenic acid derivative isolated from Hwangchil tree leaves as an active ingredient. The acetylenic acid derivative isolated from Hwangchil tree leaves according to the present invention exhibits brain tumor stem cell inhibitory activity, As a natural extract ingredient, it has relatively low side effects and toxicity rates and can effectively prevent and treat brain tumors in a bio-friendly manner.

Description

Pharmaceutical composition for preventing or treating brain tumors comprising acetylenic acid derivatives isolated from Hwangchil tree leaves as an active ingredient

The present invention relates to a composition for preventing or treating brain tumors comprising an acetylenic acid derivative isolated from Hwangchil tree leaves as an active ingredient.

A brain tumor refers to any tumor that occurs within the skull and occurs in brain tissue or the membrane tissue surrounding it. Representative examples include glioma, meningioma, and pituitary tumor, of which glioma originating from glial cells has the highest incidence rate. Glioma can be classified into malignant grades considering histological characteristics such as cell necrosis, degree of vascular endothelial cell proliferation, nuclear atypia, and mitosis. The highest grade 4, malignant glioma, is called glioblastoma multiforme (GBM). It is said. It accounts for 12-15% of all brain tumors, and the prognosis is considerably worse than other tumors, with the average survival time after diagnosis being only about 14 months.

Malignant glioblastoma is a representative incurable solid tumor, and treatment methods such as surgery, radiation therapy, and anticancer drug therapy have been attempted, but it is known that the patient survival rate is not significantly improved. In the case of radiation therapy, the side effects that accompany radiation therapy, such as neurotoxicity and dementia, are pointed out as problems, and anticancer drug therapy (chemotherapy) is receiving attention. However, in the case of drug therapy, there is a special barrier called the 'blood-brain barrier'. There are many drugs that cannot be delivered to the brain because they are blocked by the protective system, and are very limited due to cancer's resistance to anticancer drugs. Recently, as tumor stem cells, which exist in small numbers in tumor tissues, have become known as cells involved in anticancer drug resistance, interest has been focused on developing anticancer drugs targeting them.

Therefore, in order to treat brain tumors, there is an urgent need to develop a treatment targeting brain tumor stem cells from natural materials that complements the shortcomings of existing chemotherapy drugs and has fewer side effects.

Dendropanax morbiferus is a perennial evergreen tree belonging to the Araliaceae family and is mainly distributed in the southern coastal area of Korea. In Korea, the leaves and stem parts can be used for food. It is reported that Hwangchil tree leaves exert physiological activities such as antioxidant, anti-inflammatory, anti-obesity and anti-cancer activities. Hwangchil tree is reported to be effective in treating breast and lung cancer (Park Soyi et al., Korean J. Food Preserv., 25, 471~481, 2018), and although the presence of diacetylene as an active ingredient has been revealed, Hwangchil tree Research on the ingredients contained in leaves is insufficient.

Republic of Korea Patent Registration No. 10-1454359 Republic of Korea Patent Registration No. 10-2031569

The present inventors completed the present invention by purifying and separating compounds contained in Hwangchil tree leaves through numerous studies.

Therefore, the purpose of the present invention is to not only confirm that Hwangchil tree leaves contain various bioactive substances, but also to provide a compound with a new function in killing brain tumor stem cells.

Another object of the present invention is to provide a pharmaceutical composition, food composition, and health functional food composition for treating brain tumors by killing brain tumor stem cells using an acetylenic acid compound isolated from the leaves of Hwangchil tree and the structure of which has been determined.

The object of the present invention is not limited to the object mentioned above, and even if not explicitly mentioned, the object of the invention that can be recognized by a person of ordinary skill in the art from the description of the detailed description of the invention described later may also naturally be included. .

According to one embodiment of the present invention, there is provided a pharmaceutical composition for preventing and treating brain tumors comprising an acetylenic acid derivative represented by the following [Chemical Formula 1] or a pharmaceutically acceptable salt or solvate thereof as an active ingredient;

[Formula 1]

.

The acetylenic acid derivative is characterized in that it is isolated from Hwangchil tree leaves.

The acetylenic acid derivative isolated from Hwangchil tree leaves, which is the active ingredient of the present invention, and represented by [Formula 1] has the same structural formula as [Formula 1], 16-acetyl (9 Z, 16 S )-16-hydroxyocta It was named -10,17-dien-12,14-diynoic acid [16-acetyl-(9 Z, 16 S )-16-hydroxyoctadeca-10,17-dien-12,14-diynoic acid].

The compound of [Formula 1] according to the present invention can preferably be obtained by extraction and purification from Hwangchil tree leaves, and can be obtained through organic synthesis.

In the present invention, “prevention” includes all actions that inhibit or delay the onset of a brain tumor by administering the composition of the present invention, and “treatment” includes all actions that improve or beneficially change symptoms caused by a brain tumor by the composition of the present invention. Includes.

The acetylenic acid derivative of [Formula 1] can be used in the form of a pharmaceutically acceptable salt, exhibits the same efficacy, and includes salts, hydrates, and dissolved products prepared by conventional methods.

In the present invention, “pharmaceutically acceptable salt” means a salt formed with any inorganic acid or organic acid or base that does not cause serious irritation to the administered organism and does not impair the biological activity and physical properties of the compound. As the salt, salts commonly used in the art, such as acid addition salts formed with pharmaceutically acceptable free acids, can be used without limitation.

Acid addition salts can be prepared by conventional methods, for example, by dissolving the compound in an excess of aqueous acid solution and precipitating the salt using a water-miscible organic solvent, such as methanol, ethanol, acetone, or acetonitrile. Equimolar amounts of the compound and an acid or alcohol (e.g., glycol monomethyl ether) in water can be heated, and the mixture can then be evaporated to dryness, or the precipitated salt can be suction filtered.

For example, pharmaceutically acceptable salts may include sodium, calcium, and potassium salts of hydroxy groups, and other pharmaceutically acceptable salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, and hydrogen phosphate. , dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, benzylsulfonate (besylate), methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts, etc. and can be produced through salt production methods known in the art.

The pharmaceutical composition containing the compound represented by [Formula 1] or a pharmaceutically acceptable salt, solvate, or ester thereof of the present invention may be prepared using an appropriate carrier, excipient, or diluent commonly used in the preparation of pharmaceutical compositions. may additionally be included.

In the present invention, “pharmaceutically acceptable carrier” includes a carrier or diluent that does not irritate living organisms and does not inhibit the biological activity and properties of the injected compound. The type of carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable can be used. Non-limiting examples of the carrier include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, etc. These may be used alone or in combination of two or more types. Additionally, if necessary, other common additives such as antioxidants, buffers, and/or bacteriostatic agents may be added and used.

The pharmaceutical composition is any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, freeze-dried preparations, and suppositories. It may have a variety of oral or parenteral dosage forms. When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc. These solid preparations contain one or more compounds and at least one excipient, such as starch, calcium carbonate, sucrose, or lactose. It can be prepared by mixing (lactose), gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate, talc, etc. may also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is.

Additionally, preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.

According to another embodiment of the present invention, there is provided a health functional food for preventing and improving brain tumors containing the acetylenic acid derivative represented by [Formula 1] or a pharmaceutically acceptable salt or solvate thereof as an active ingredient.

When using the composition of the present invention in a health functional food, the composition can be added as is or used together with other health functional foods or health functional food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use. In general, when producing a food or beverage, the composition of the present invention can be added in an amount of preferably 15 parts by weight or less, more preferably 10 parts by weight or less, based on the raw materials. However, in the case of long-term intake for the purpose of health control and hygiene, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient can be used in an amount above the above range.

There is no particular limitation on the type of health functional food that can contain the composition of the present invention, and specific examples include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, and ice cream. There are dairy products, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, etc., and can include all health functional foods in the conventional sense, and can include foods used as feed for animals.

In addition, when the health functional food composition of the present invention is used in the form of a beverage, it may contain various sweeteners, flavoring agents, or natural carbohydrates as additional ingredients like ordinary beverages.

In the present invention, “brain tumor” refers to a tumor that occurs in the brain and central nervous system and can be used interchangeably with “brain cancer.” The brain tumor may be a primary brain tumor or a metastatic brain tumor.

The brain tumors include anaplastic astrocytomas, glioblastomas, meningiomas, pituitary tumors, schwannomas, CNS lymphoma, and oligodendrogliomas. , ependymomas, low-grade astrocytomas, medulloblastomas, astrocytic tumors, pilocytic astrocytoma, diffuse astrocytomas, Pleomorphic xanthoastrocytomas, subependymal giant cell astrocytomas, anaplastic oligodendrogliomas, oligoastrocytomas, anaplastic oligoastrocytomas ), myxopapillary ependymomas, subependymomas, ependymomas, anaplastic ependymomas, astroblastomas, notochord of the third ventricle Chordoid gliomas of the third ventricle, gliomatosis cerebris, glangliocytomas, desmoplastic infantile astrocytomas, desmoplastic infantile gangliogliomas ), dysembryoplastic neuroepithelial tumors, central neurocytomas, cerebellar liponeurocytomas, paragangliomas, ependymoblastomas, supratentorial primitive neuroectodermal Tumors (supratentorial primitive neuroectodermal tumors), choroids plexus papilloma, pinocytomas, pineoblastomas, pineal parenchymal tumors of intermediate differentiation, perivascular Hemangiopericytomas, tumors of the sellar region, craniopharyngioma, capillary hemangioblastoma, and primary CNS lymphoma.

Preferably, the brain tumor may be glioblastoma.

The subtype of glioblastoma may be a proneural subtype, mesenchymal subtype, classical subtype, or neural subtype.

According to another embodiment of the present invention, a method for producing an acetylenic acid derivative of [Formula 1] is provided, which method includes obtaining a Hwangchil tree leaf extract; Concentrating the Hwangchil tree leaf extract under reduced pressure; It may include obtaining an ethyl acetate fraction through solvent fractionation of the concentrate and purifying the ethyl acetate fraction by MPLC using ODS column chromatography.

An example of the method for producing an acetylenic acid derivative of [Formula 1] according to the present invention is as follows. Hwangchil tree leaves are extracted by adding methanol at room temperature. The methanol extract is concentrated under reduced pressure at 45°C using a vacuum concentrator. The obtained concentrate is fractionated using hexane, chloroform, ethyl acetate, and saturated butanol. The ethyne acetate fraction was injected into ODS-MPLC and then eluted using a mixed solvent of water (H ₂ O) and acetone nitrile (MeCN) to obtain 29 fractions. Among the above fractions, fraction 21 was purified using ODS-MPLC.

The step of obtaining the Hwangchil tree leaf extract can be performed by pulverizing the Hwangchil tree leaves and then extracting them with a lower alcohol having 1 to 4 carbon atoms, wherein the lower alcohol is methanol.

According to the present invention constructed as described above, the acetylenic acid derivative of [Formula 1] isolated from the leaves of Hwangchil tree according to the present invention exhibits brain tumor cell inhibitory activity and, as a natural product extract, has a relatively low side effect and toxicity rate and is biocompatible. Brain tumors can be effectively prevented and treated.

Figure 1 shows compound 1 isolated from Hwangchil tree leaves, ( E )-9,16-dihydroxyocta-10,17-diene-12,14-diinoic acid [( E )-9,16-dihydroxyoctadeca-10 , 17-dien-12,14-diynoic acid] shows ^{the 1} H- ¹ H-COZY correlation (bold line), gHMBC correlation (arrow), and structural formula.
Figure 2 shows compound 2 isolated from Hwangchil tree leaves, (9 Z, 16 S )-16-hydroxyocta-10,17-diene-12,14-diinoic acid [(9 Z, 16 S )-16- ^{The 1} H- ¹ H-COZY correlation (thick line), gHMBC correlation (arrow), and structural formula of [hydroxyoctadeca-10,17-dien-12,14-diynoic acid] are shown.
Figure 3 shows compound 3 isolated from Hwangchil tree leaves, methyl (9 Z, 16 S ) -16-hydroxyocta-10,17-diene-12,14-diinoate [methyl- (9 Z, 16 S ) -16-hydroxyoctadeca-10,17-dien-12,14-diynoate].
Figure 4 shows compound 4 isolated from Hwangchil tree leaves, 16-acetyl (9 Z, 16 S ) -16-hydroxyocta-10,17-diene-12,14-diinoic acid [16-acetyl- (9 Z , 16 S )-16-hydroxyoctadeca-10,17-dien-12,14-diynoic acid] shows ^{the 1} H- ¹ H-COZY correlation (thick line), gHMBC correlation (arrow), and structural formula.
Figure 5 shows the results showing the effect of compounds 1 to 4 isolated from Hwangchil tree leaves on reducing the survival rate of brain tumor stem cells.
Figure 6 shows compound 4 isolated from Hwangchil tree leaves, 16-acetyl (9 Z, 16 S ) -16-hydroxyocta-10,17-diene-12,14-diinoic acid [16-acetyl- (9 Z , 16 S )-16-hydroxy octadeca-10,17-dien-12,14-diynoic acid] shows the effect of killing brain tumor stem cells by apoptosis.
Figure 7 shows compound 4 isolated from Hwangchil tree leaves, 16-acetyl (9 Z, 16 S ) -16-hydroxyocta-10,17-diene-12,14-diinoic acid [16-acetyl- (9 Z , 16 S )-16-hydroxy octadeca-10,17-dien-12,14-diynoic acid] shows the effect of killing brain tumor stem cells by apoptosis.

Hereinafter, the present invention will be described in detail through examples. However, the following examples are for illustrating the present invention, and the scope of the present invention is not limited by the following examples.

Example 1. Isolation of acetylenic acid derivatives from Hwangchil tree leaves

1. Extraction and solvent fractionation

Hwangchil tree leaves were used as dried and sold in Boseong-gun, Jeollanam-do. 1.5 kg of dried Hwangchil tree was pulverized, then 15 L of methanol was added and extracted at room temperature for 24 hours. Whatman Filter Paper No. After filtration, 6 L of methanol was added to the obtained residue, and the mixture was extracted and filtered again in the same manner. The obtained extraction filtrate was concentrated under reduced pressure at 45°C using a vacuum concentrator (A-3S, Eyela, Tokyo, Japan) equipped with an aspirator (CCA-1110, Eyela, Tokyo, Japan). The obtained concentrate was sequentially subjected to solvent fractionation with 1 L each of hexane, chloroform, ethyl acetate, and saturated butanol three times.

2. Separation of ingredients

Medium pressure liquid chromatography (MPLC; Isolera One, Biotage) coupled to an octadecylsilane column (SNAP Ultra C18 120 g, Biotage, Uppsala, Sweden) was used on 13.7796 g of the ethyl acetate fraction obtained by solvent fractionation of the methanol extract of Hwangchil tree. The components were separated and purified.

The ethyl acetate fraction was injected into ODS-MPLC and then eluted and fractionated using a mixed solvent of water (H ₂ O) and acetone nitrile (MeCN). That is, H ₂ O/MeCN = 9:1 (v/v) was maintained for the first 30 minutes, and then eluted using a grdient method to maintain H ₂ O/MeCN = 0:10 (v/v) for the next 130 minutes. At this time, the flow rate was 50 mL/min, and components were detected at 210 nm and 254 nm. The ethyl acetate fraction was fractionated into 29 fractions (D1 to D29) through ODS-MPLC.

D13, D19, and D21 obtained by ODS-MPLC of the ethyl acetate fraction were purified again using MPLC equipped with an ODS column (SNAP Ultra C18 30 g, Biotage). At this time, the flow rate was 25 mL/min, and components were detected at 210 nm and 254 nm. By ODS-MPLC of the ethyl acetate fraction, D13a (Compound 1) was separated from D13, D19a (Compound 2) and D19a (Compound 3) were separated from D19, and D21c (Compound 4) was separated from D21.

Example 2. Structural analysis of compounds

1. Compound 1

The structure of Compound 1 obtained in Example 1 was analyzed as follows, and the analysis results are shown in Table 1 and Figure 1.

In the HR-ESI-MS (positive) spectrum of compound 1, the sodiated molecular ion peak, m/z 327.1569 [M+Na] ^+, was observed, and it can be seen that the molecular formula of this compound is C ₁₈ H ₂₄ O ₄ (molecular weight 304). there was. And in the ¹ H-NMR (500 MHz, CD ₃ OD) spectrum, five types of olefinic double bond proton signals [δ 6.33 (1H, dd, J = 16.0, 5.5 Hz, H-10), 5.77 (1H, ddd, J = 16.0, 1.5, 1.0 Hz, H-11), 5.93 (1H, ddd, J = 17.0, 10.0, 5.5 Hz, H-17), 5.41 (1H, dt, J = 17.0, 1.5 Hz, H-18a ) and 5.19 (1H, dt, J = 10.0, 1.5 Hz, H-18b)] and two oxygenated proton signals [δ 4.92 (1H, d, J = 5.5 Hz, H-16) and 4.12 (1H, dt, J = 6.0, 5.5 Hz, H-9)] was observed. Additionally, 16H methylene proton singals [δ 1.35-2.28 (H-2~H-8) were observed. In particular, the coupling contant values ( J ) of δ 6.33 (H-10) and 5.77 (H-11), which show proton-proton correlation in the ¹ H- ¹ H COZY spectrum, are 16.0 Hz, and one type of olefine double bond is trans I could see that it was. ¹³ In the C-NMR spectrum, one carbonyl carbon signal [δ 177.9 (C-1)] and four quaternary carbon signals [δ 78.1 (C-12), 74.2 (C-13), 79.7 (C-) A total of 18 types of carbon signals were observed, including [14) and 82.4 (C-15)].

From the above ESI-MS and 1D-NMR results, compound 1 was suggested to be octadecadiendiynoic acid having two types of hydroxyl groups.

For a more accurate structural analysis of Compound 1, 2D-NMR analyzes such as gHSQC, ¹ H- ¹ H-COZY, and gHMBC were performed. In the ¹ H- ¹ H-COZY results, a series of proton-proton correlations between H-1 to H-11 and H-16 to H-18 were observed. In particular, in the gHMBC spectrum there is a correlation between δ 1.59-1.62 (H-3) and 177.9 (C-1), and δ 6.33 (H-10) and 78.1 (C-12), δ 5.77 (H-11). Correlations were observed between δ 4.92 (H-16) and 70.7 (C-14) and δ 5.93 (H-17) and 82.4 (C-15).

From Table 1 below, where the instrumental analysis results are presented, Compound 1 is ( E )-9,16-dihydroxyocta-10,17-diene-12,14-diinoic acid [( E )-9,16-dihydroxyoctadeca- 10,17-dien-12,14-diynoic acid].

Table 1 below shows ¹ H- (600 MHz) and ¹³ C-NMR (150 MHz) of ( E )-9,6-dihydroxyocta-10,17-diene-12,14-diinoic acid (Compound 1) ) data (CD ₃ OD). Additionally , the ¹ H- ¹ H-COZY correlation (bold line), gHMBC correlation (arrow) and The structural formula is as shown in Figure 1.

Position δ _H ( Int. , Multi. , J in Hz) _δC One - 177.87 2 2.28 (2H, t, 7.5) 35.12 3 1.59-1.62 (2H, m) 26.21 4 1.35 (6H, m) ^{a 30.30a} 5 ^30.46a 6 ^30.60a 7 1.40-1.43 (2H, m) 26.52 8 1.48-1.51 (2H, m) 37.93 9 4.12 (2H, dt, 6.0, 5.5) 72.56 10 6.33 (1H, dd, 16.0, 5.5) 152.00 11 5.77 (1H, ddd, 16.0,1.5,1.0) 108.49 12 - 78.14 13 - 74.18 14 - 70.72 15 - 82.35 16 4.92 (1H, d, 5.5) 64.12 17 5.93 (1H, ddd, 17.0 10.0, 5.5) 138.23 18a
18b 5.41 (1H, dt, 17.0, 1.5)
5.19 (1H, dt, 10.0, 1.5) 116.73 ^a C-4~C-6 signal values can be changed.

2. Compound 2

The structure of Compound 2 obtained in Example 1 was analyzed as follows, and the analysis results are shown in Table 2 and Figure 2.

In the HR-ESI-MS (positive) spectrum of compound 2, a molecular weight peak of m/z 311.1621 [M+Na] ⁺ was observed, and the molecular formula of this compound was interpreted as C ₁₈ H ₂₄ O ₃ (molecular weight 288). The ¹ H- (500 MHz) and ¹³ C-NMR (125 MHz) spectra of compound 2 were similar to those of compound 1, but instead of one oxygenated proton and carbon signal, one methylene proton signal [δ 3.06 (2H, br. d, J = 7.0 Hz, H-11)] and carbon signal [δ 18.23 (C-11)] were observed. In addition, one cis -type olefinic double bond proton signal observed in the ¹ H- and ¹³ C-NMR spectra of compound 2 [δ 5.51 (1H, dd, J = 10.5, 7.0 Hz, H-10) and 5.37-5.40 (1H, ddd, J = 10.5, 7.0 Hz, H-11)] and carbon signals [δ 133.92 (C-10) and 123.65 (C-11)] showed differences from those of compound 1. ¹ H- and ¹³ C-NMR spectra of compound 2 (9 Z, 16 S )-16-hydroxyocta-10,17-diene reported in a previous paper (Kim et al., Food Bioscience, 100878, 2021) It was consistent with those of -12,14-diynoic acid [(9 Z, 16 S )-16-hydroxyoctadeca-10,17-dien-12,14-diynoic acid].

From Table 2 below, where the instrumental analysis results are presented, Compound 2 is (9 Z, 16 S )-16-hydroxyocta-10,17-diene-12,14-diinoic acid [(9 Z, 16 S )-16 -hydroxyoctadeca-10,17-dien-12,14-diynoic acid] (Compound 2).

Table 2 below shows ^{the 1} H- (500 MHz) and ¹³ C-NMR (125 MHz) data (CD ₃ OD).

Position δ _H ( Int. , Multi. , J in Hz) _δC One - 177.87 2 2.28 (1H, t, 7.5) 35.12 3 1.59-1.62 (2H, m) 26.21 4 1.34 (8H, m) ^30.39a 5 ^30.34a 6 ^30.39a 7 ^30.41a 8 1.34-1.41 (2H, m) 28.16 9 5.51 (1H, dt, 10.5, 7.0) 133.92 10 5.37-5.40 (1H, dd, 10.5, 7.0) 123.65 11 3.06 (1H, br. d, 7.0) 18.23 12 - 80.47 13 - 71.19 14 - 65.14 15 - 76.19 16 4.85 (1H, d, 5.5) 63.97 17 5.90 (1H, ddd, 17.0 10.0, 5.5) 138.35 18a
18b 5.39 (1H, br. d, 17.0)
5.18 (1H, dt, 10.5, 1.5) 116.63 ^a C-4~C-6 signal values can be changed.

3. Compound 3

The structure of Compound 3 obtained in Example 1 was analyzed as follows, and the analysis results are shown in Table 3 and Figure 3.

In the HR-ESI-MS (positive) spectrum of compound 3, a molecular weight peak of m/z 325.1778 [M+Na] ⁺ was observed, and the molecular formula of this compound was interpreted as C ₁₉ H ₂₆ O ₃ (molecular weight 302). The ¹ H- (500 MHz) and ¹³ C-NMR (125 MHz) spectra of compound 3 were very similar to those of compound 2, but one methoxy proton signal [δ 3.65 (3H, s)] and a carbon signal (δ 51.12) was additionally observed. From the MS and 1D-NMR analysis results, Compound 3 has a methyl group bonded to (9 Z, 16 S )-16-hydroxyocta-10,17-diene-12,14-diinoic acid, which is the structural formula of Compound 2. It was suggested as a compound.

For a more accurate structural analysis of compound 3, 2D-NMR analyzes such as gHSQC, ¹ H- ¹ H-COZY, and gHMBC were performed. As a result, the basic skeleton of compound 3 was interpreted as (9 Z, 16 S )-16-hydroxyocta-10,17-diene-12,14-diinoic acid, which is the same as compound 2. In particular, a correlation between δ 3.65 (-OCH ₃ ) and 176.15 (C-1) was observed in the gHMBC spectrum.

From Table 3 below, which presents the instrumental analysis results, Compound 3 is methyl (9 Z, 16 S )-16-hydroxyocta-10,17-diene-12,14-dinoate [methyl-(9 Z, 16 S) )-16-hydroxyoctadeca-10,17-dien-12,14-diynoate].

Table 3 ^{below shows 1 H- (600 MHz) and 13} C - ^NMR ( 150 MHz) data (CD ₃ OD). Additionally, ^{the 1} H- ¹ H-COZY correlation of methyl (9 Z, 16 S )-16-hydroxyocta-10,17-diene-12,14-dinoate (bold line), gHMBC correlation (arrow) ) and the structural formula is as shown in Figure 3.

Position δ _H ( Int. , Multi. , J in Hz) _δC One - 176.15 2 2.32 (2H, t, 7.5) 34.94 3 1.59-1.62 (2H, m) 26.15 4 1.33 (6H, m) ^30.21a 5 ^30.29a 6 ^30.35a 7 1.36-1.40 (2H, m) 30.40 8 2.07 (2H, q, 7.0) 28.14 9 5.52 (1H, br dt, 10.5, 1.5) 133.92 10 5.36-5.40 (1H, dd, 10.5, 7.0) 123.70 11 3.06 (1H, br. d, 7.0) 18.22 12 - 80.46 13 - 71.17 14 - 65.13 15 - 76.22 16 4.84 (1H, d, 5.5) 63.99 17 5.90 (1H, ddd, 17.0 10.0, 5.5) 138.42 18a
18b 5.39 (1H, br. d, 17.0)
5.18 (1H, dt, 10.5, 1.5) 116.59 -OCH ₃ 3.65 (3H, s) 52.12 ^a C-4~C-6 signal values can be changed.

4. Compound 4

The structure of Compound 4 obtained in Example 1 was analyzed as follows, and the analysis results are shown in Table 4 and Figure 4.

From the HR-ESI-MS (positive) spectrum of compound 4, a molecular weight peak of m/z 353.1726 [M+Na] ⁺ was observed, and the molecular formula of this compound was found to be C ₂₀ H ₂₆ O ₄ (molecular weight 330). And the ¹ H- (500 MHz) and ¹³ C-NMR (125 MHz) spectra of compound 4 were very similar to those of compound 2, but the proton corresponding to the acetyl group [δ 2.07 (3H, s, H-2') ] and carbon signals [δ 171.11 (C-1') and 20.91 (C-2')] were observed.

For a more accurate structural analysis of compound 4, 2D-NMR analyzes such as gHSQC, ¹ H- ¹ H-COZY, and gHMBC were performed. As a result, the basic skeleton of compound 4 was interpreted as (9 Z, 16 S )-16-hydroxyocta-10,17-diene-12,14-diinoic acid, which is the same as compound 2. In particular, a correlation between δ 5.88 (H-16) and 176.15 (C-1) was observed in the gHMBC spectrum.

From Table 4 below, where the instrumental analysis results are presented, compound 4 is 16-acetyl (9 Z, 16 S )-16-hydroxyocta-10,17-diene-12,14-diinoic acid [16-acetyl-(9 Z, 16 S )-16-hydroxyoctadeca-10,17-dien-12,14-diynoic acid].

Table 4 below shows the ¹ H- (600 MHz) and ¹³ C- of 16-acetyl (9 Z, 16 S )-16-hydroxyocta-10,17-diene-12,14-diinoic acid (Compound 4). NMR (150 MHz) data (CD ₃ OD). In addition, ¹ H- ¹ H-COZY correlation of 16-acetyl (9 Z, 16 S )-16-hydroxyocta-10,17-diene-12,14-diinoic acid (bold line), gHMBC correlation (arrow) and the structural formula are as shown in Figure 4.

Position δ _H ( Int. , Multi. , J in Hz) _δC One - 177.74 2 2.28 (2H, t, 7.2) 35.06 3 1.58-1.63 (2H, m) 26.16 4 1.35 (8H, m) ^30.29a 5 ^30.34a 6 ^30.20a 7 1.37-1.42 (2H, m) 30.35 8 2.09 (2H, m) 28.16 9 5.33 (1H, dd, 11.4, 1.8) 134.14 10 5.53 (1H, dt, 11.4, 1.2) 123.30 11 3.07 (2H, br. d, 7.2) 18.24 12 - 81.56 13 - 72.64 14 - 64.73 15 - 70.02 16 5.88 (1H, d, 5.5) 65.82 17 5.91 (1H, ddd, 16.4, 10.0, 5.6) 133.92 18a
18b 5.51 (1H, dd, 16.4, 1.2)
5.32 (1H, dt, 9.6, 1.2) 119.74 One' - 171.11 2' 2.07 (3H, s) 20.91 ^a C-4~C-6 signal values can be changed.

Experiment example. Confirmation of apoptosis effect of brain tumor stem cells

1. Samples and cell culture

The brain tumor stem cells used in the following examples were brain tumor stem cell lines derived from brain tumor patients, GSC11 (glioblastoma, Joshua M. Shulman, Texas Children's Hospital), GSC23 (glioblastoma, Joshua M. Shulman, Texas Children's Hospital), and malignant brain tumor cell lines. U87MG (glioblastoma, ATCC) and normal astrocyte NHA (ATCC) were used. Each cell was cultured under brain tumor stem cell culture conditions: 1% antimicrobial agent (apenicillin/streptomycin, Lonza, Basel, Switzerland), 0.04% B27 (Invitrogen, MA, USA), epidermal growth factor (20 ng/mL; R&D) Systems, MN, USA) and fibroblast growth factor (basic broblast growth factor, 20 ng/mL; R&D Systems) culture medium (Hyclone, GE healthcare life sciences, IL, USA) at 37°C for 5 days. It was used by culturing under % CO ₂ conditions. U87 cells were cultured in 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin, and NHA were cultured in 10% FBS, 1% penicillin/streptomycin, and Astrocyte medium.

2. Evaluation of apoptosis of brain tumor stem cells of each compound

The present inventors performed the following experiment to determine whether compounds 1 to 4 isolated from the leaves of Hwangchil tree have an apoptotic effect on brain tumor stem cells.

1) Cell viability

For this purpose, brain tumor stem cell lines GSC11 and GSC23 and normal astrocytes NHA cells were each dispensed at 3,000 cells/well in a 96 well plate (SPL, Korea), and after 24 hours, the compounds of the present invention were added at different concentrations (0 , 6, 12, 25, 50 μM) and then cultured for 2 days. Afterwards, 20 μL of Alamarblue (invitrogen, MA, USA) analysis reagent was added per well and reacted for 4 hours, followed by SYNERGY HTX multi-mode reader (Bio-Tek Instruments Inc.) at a wavelength of 530 (Ex) ~ 590 (Em) nm. , VT, USA), the degree of cell death according to the compound treatment concentration for each cell line was measured by measuring fluorescence using a device. At this time, the measurement values were recorded as mean ± SEM.

2) Annex V/PI

Cell death analysis using Annexin V/PI staining was performed to confirm the effect of the compound in promoting apoptosis of brain tumor stem cells. For this purpose, the GSC11, GSC23, and NHA cells used in the above experiment were distributed in 60 mm dishes (SPL, Korea) at 800,000 cells/well. After culturing for 24 hours, the compound was treated at a concentration of 10 μM and cultured for 24 hours. After incubation, the cells were washed twice with PBS, and then the cells were detached using 0.25% trypsin-EDTA. The cells were precipitated by centrifugation at 800 rpm for 5 minutes, and washed once with cold PBS. Then, the cells were suspended in a 1.5 ml tube with 100 μl of 1X annexin-binding buffer (Thermo scientific, MA, USA). Then, 5 μl of staining agent FITC annexin V (Thermo scientific, MA, USA) and PI working solution (100 μg/mL) (Thermo scientific, MA, USA) were added to the cells and reacted at room temperature for 15 minutes, and then each cell was Fluorescence analysis was performed using a fluorescence-based cell sorter. At this time, the measured values were reported as mean ± SEM.

3) Optical Diffraction Tomography (ODT)

Optical Diffraction Tomography (ODT) is a quantitative phase imaging technique that uses the refractive index of a laser to pass through a thin, transparent sample. In order to image living and dead cells without additional staining (label-free) using this imaging technology, holotomography (HT-2S) (Tomocube, Inc.), which operates on the principle of quantitative phase imaging, was used. , Korea) was used.

GSC11 and GSC23 were treated with 50 μM of the above compound, cultured for 24 hours, and then imaged using holotomography together with the control group. Imaging was performed maintaining conditions of 5% CO ₂ and 37°C.

As shown in Figure 5, it was confirmed that Compounds 1 to 3 hardly reduced the survival rate of brain tumor stem cell lines GSC11, GSC23, and U87MG even when treated at a concentration of 50 μM. On the other hand, compound 4 was found to have the effect of reducing cell viability in a concentration-dependent manner.

As shown in [Table 5] below, when compound 4 was treated at a concentration of 25 μM, the survival rate of normal cells, NHA, was 96 ± 1%, and the survival rate of brain tumor stem cell lines GSC11, GSC23, and U87MG was 62 ± 1%. It was found to be 75±2 and 84±1%.

In addition, when compound 4 was treated at a concentration of 50 μM, the survival rate of normal cells, NHA, was 72 ± 1%, and the survival rates of brain tumor stem cell lines GSC11, GSC23, and U87MG were 40 ± 1, 60 ± 3, and 38 ± 3%. I could see that it was.

cell Cell viability (%) after treatment with compound 4 6 μM 12 μM 25 μM 50 μM NHA 97±2 95±2 96±1 72±1 GSC11 87±3 75±1 62±1 40±1 GSC23 89±3 85±1 75±2 60±3 U87MG 95±3 100±2 84±1 38±3

In addition, as can be seen in Figures 6 and 7, Annexin V/PI staining shows that when compound 4 is treated with brain tumor stem cells GSC11 and GSC23 at a concentration of 10 μM, this compound causes a killing effect by apoptosis on brain tumor stem cell lines. This was confirmed through holotomography analysis.

Therefore, through these results, the present inventors were able to confirm that Compound 4 has the ability to kill brain tumor stem cells, and that the compound can be used as a new brain tumor treatment targeting brain tumor stem cells.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.

Claims (10)

16-acetyl (9 Z, 16 S )-16-hydroxyocta-10,17-diene-12,14-diinoic acid [16-acetyl-(9 Z, 16 S )-16-hydroxyoctadeca-10,17 -dien-12,14-diynoic acid] A pharmaceutical composition for preventing and treating brain tumors comprising an acetylenic acid derivative or a pharmaceutically acceptable salt or solvate thereof as an active ingredient.
According to paragraph 1,
The acetylenic acid derivative is an acetylenic acid derivative, characterized in that it is isolated from Hwangchil tree leaves.
According to paragraph 1,
The brain tumors include anaplastic astrocytomas, glioblastomas, meningiomas, pituitary tumors, schwannomas, CNS lymphoma, and oligodendrogliomas. , ependymomas, low-grade astrocytomas, medulloblastomas, astrocytic tumors, pilocytic astrocytoma, diffuse astrocytomas, Pleomorphic xanthoastrocytomas, subependymal giant cell astrocytomas, anaplastic oligodendrogliomas, oligoastrocytomas, anaplastic oligoastrocytomas ), myxopapillary ependymomas, subependymomas, ependymomas, anaplastic ependymomas, astroblastomas, notochord of the third ventricle Chordoid gliomas of the third ventricle, gliomatosis cerebris, glangliocytomas, desmoplastic infantile astrocytomas, desmoplastic infantile gangliogliomas ), dysembryoplastic neuroepithelial tumors, central neurocytomas, cerebellar liponeurocytomas, paragangliomas, ependymoblastomas, supratentorial primitive neuroectodermal Tumors (supratentorial primitive neuroectodermal tumors), choroids plexus papilloma, pinocytomas, pineoblastomas, pineal parenchymal tumors of intermediate differentiation, perivascular characterized by being selected from the group consisting of hemangiopericytomas, tumors of the sellar region, craniopharyngioma, capillary hemangioblastoma, and primary CNS lymphoma. Pharmaceutical composition for preventing and treating brain tumors.
According to paragraph 1,
A pharmaceutical composition for preventing and treating brain tumors, wherein the brain tumor is glioblastoma.
16-acetyl (9 Z, 16 S )-16-hydroxyocta-10,17-diene-12,14-diinoic acid [16-acetyl-(9 Z, 16 S )-16-hydroxyoctadeca-10,17 -dien-12,14-diynoic acid] A health functional food for preventing and improving brain tumors containing an acetylenic acid derivative or a pharmaceutically acceptable salt or solvate thereof as an active ingredient.
According to clause 5,
The brain tumors include anaplastic astrocytomas, glioblastomas, meningiomas, pituitary tumors, schwannomas, CNS lymphoma, and oligodendrogliomas. , ependymomas, low-grade astrocytomas, medulloblastomas, astrocytic tumors, pilocytic astrocytoma, diffuse astrocytomas, Pleomorphic xanthoastrocytomas, subependymal giant cell astrocytomas, anaplastic oligodendrogliomas, oligoastrocytomas, anaplastic oligoastrocytomas ), myxopapillary ependymomas, subependymomas, ependymomas, anaplastic ependymomas, astroblastomas, notochord of the third ventricle Chordoid gliomas of the third ventricle, gliomatosis cerebris, glangliocytomas, desmoplastic infantile astrocytomas, desmoplastic infantile gangliogliomas ), dysembryoplastic neuroepithelial tumors, central neurocytomas, cerebellar liponeurocytomas, paragangliomas, ependymoblastomas, supratentorial primitive neuroectodermal Tumors (supratentorial primitive neuroectodermal tumors), choroids plexus papilloma, pinocytomas, pineoblastomas, pineal parenchymal tumors of intermediate differentiation, perivascular characterized by being selected from the group consisting of hemangiopericytomas, tumors of the sellar region, craniopharyngioma, capillary hemangioblastoma, and primary CNS lymphoma. Health functional food for preventing and improving brain tumors.
According to clause 5,
A health functional food for preventing and improving brain tumors, wherein the brain tumor is glioblastoma.
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