KR100367115B1 - Novel compounds showing apoptosis-inhibitive effect purified from Isodon japonicus and a method for preparing the said compounds - Google Patents

Abstract

The present invention relates to a novel compound and a method for preparing the same, and more particularly, the present invention is prepared from Isodon japonicus using methanol and ethylacetate extraction and chromatography. The novel compounds of the invention exhibit excellent apoptosis inhibitory effects and can be usefully used as apoptosis inhibitors.

Description

Novel compounds showing apoptosis-inhibitive effect purified from Isodon japonicus and a method for preparing the said compounds}

The present invention relates to a novel compound and a method for preparing the same, and more particularly, methanol and ethyl acetate (ethylacetate) extraction and chromatograph (chro-matography) is produced from the pool and exhibits apoptosis inhibitory effect The present invention relates to a novel compound and a preparation method thereof.

Apoptosis or programmed cell death (PCD) is the basic intracellular process for spontaneous self-elimination of damaged or unwanted cells in a wide range of biological systems. Apoptosis is an essential mechanism for organ development, tissue remodeling, maintenance of intracellular homeostasis and removal of abnormal and damaged cells because the contents of cells that have died through the process of apoptosis are not extracellularly released and do not damage other cells.

As mentioned above, the apoptosis process, which plays an important role in living organisms, must be precisely regulated according to a predetermined program. If apoptosis is inhibited, diseases such as cancer, autoimmune disease and viral infectious diseases are induced. (Kerr et al ., Br. J. Cancer , 26, 239-257, 1972), Inappropriately increasing apoptosis can lead to liver failure caused by toxic substances such as acquired immunodeficiency, various neurodegenerative diseases, ischemic diseases (strokes) and alcohols. Diseases such as diseases are induced.

Early cells undergoing apoptosis develop chromatin aggregation, cytoplasm, and the like, which are induced during late stages of damage such as cell junctions, blebbing of cell membranes, and cell shrinkage. Nuclear enrichment (cytoplasm and nuclei condensation), loss of mitochondrial membrane potential, changes in plasma membrane composition and apoptotic body formation are induced and undergo morphological and biochemical changes throughout. DNA fragmentation in the form of oligonucleosomes, the final result of apoptosis, is a typical biochemical feature of cells undergoing apoptosis (Green DR, and Reed, JC, Science , 281, 1309-1312, 1998).

Many apoptosis-related studies have now identified key players and mechanisms involved in apoptosis in a variety of organisms. Representatively, in mammalian cells, caspase, a type of cellular cysteine protease, is known to play an important role in regulating apoptosis. The evolutionary phylogenetic tree can be classified into three types centered on ICE (interlukin 1 converting enzyme), CPP32, and ICH-1.

Caspases may be receptor-mediated signal transduction, depletion of growth factors, oxidative stress, DNA damage and cell-cells. ) Or activated by a variety of stimuli, including disruption of cell-matrix interactions, poly (ADP-ribose) polymerase (RARP), lamine, Induces apoptosis formation by breaking down cellular proteins such as cytokeratins and caspase-activated DNase inhibitors (ICAD) (Cryns, V., and Yuan, J., Genes Dev ., 12, 1551-1570, 1998).

Active studies of apoptosis have shown that in mammals, at least 14 different classes of caspases have so far been peptide substrates after aspartic acid residues, which play a pivotal role in the initiation and execution of apoptosis. Has been reported to induce apoptosis by cutting (Nunez et al ., Oncogene, 17, 3237-3245, 1998).

Accordingly, the present inventors have tried to search for compounds capable of modulating apoptosis. As a result, we have isolated novel compounds exhibiting apoptosis inhibitory effect from the cultivars, and have identified their physicochemical properties and said compounds have excellent apoptosis in apoptosis-induced cell lines. The present invention was completed by confirming the inhibitory effect.

It is an object of the present invention to provide novel compounds and salts thereof which exhibit excellent apoptosis inhibitory effects as apoptosis inhibitors.

It is also an object of the present invention to provide a production method for extracting and separating and purifying the compound from the parakeet.

It is also an object of the present invention to provide a pharmaceutical composition for apoptosis inhibitors containing the compound and / or pharmaceutically acceptable salts thereof as an active ingredient.

1 shows a hydrogen nuclear magnetic resonance ( ¹ H-NMR) spectrum of the novel compound represented by Formula 1,

Figure 2 shows the carbon nuclear magnetic resonance ( ¹³ C-NMR) spectrum of the novel compound represented by Formula 1,

Figure 3 shows the hydrogen nuclear magnetic resonance spectrum of the novel compound represented by the formula (2),

Figure 4 shows the carbon nuclear magnetic resonance spectrum of the novel compound represented by the formula (2).

In order to achieve the above object, the present invention provides a novel compound represented by the formula (1) and formula (2) and salts thereof.

<Formula 1>

<Formula 2>

Physicochemical properties of the compounds of the present invention represented by Chemical Formulas 1 and 2 by using methods such as electron spray ionization mass spectrometer (ESI-MS), high resolution electron spray ionization mass spectrometer (HRESI-MS), and nuclear magnetic resonance spectra As a result of analysis, the compound of the present invention was confirmed to have the following physicochemical properties.

1) Compound of Formula 1

i) Material Properties: Powder ii) Molecular Weight: 343

iii) Molecular formula: C ₁₉ H ₂₁ NO ₅ iv) Mass spectrometry (M + H): 344 ( m / z )

2) a compound of formula 2

i) Material Properties: Powder ii) Molecular Weight: 374

iii) Molecular formula: C ₁₉ H ₁₈ NO ₈ iv) Mass spectrometry (M + H): 375 ( m / z )

In addition, hydrogen nuclear magnetic resonance spectra and carbon nuclear magnetic resonance spectra were measured using a methanol (CD ₃ OD) as a solvent, the compounds of formulas (1) and (2) were found to be new compounds that have not been reported to date. (See FIGS. 1 , 2 , 3 and 4 ).

The compounds of the present invention represented by the formulas (1) and (2) can be used in the form of pharmaceutically acceptable salts, and acid addition salts formed by pharmaceutically acceptable free acids are useful as salts. Compounds of formulas (1) and (2) can form pharmaceutically acceptable acid addition salts according to methods conventional in the art. Free acid and inorganic acid may be used as the free acid, and hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, and fumaric acid may be used as the organic acid. (fumaric acid), formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galluxuronic acid, embonic acid, glutamic acid or aspartic acid Can be used.

The present invention also provides a method for extracting, separating and purifying compounds of formulas (1) and (2) from a mill.

The manufacturing method of the present invention

1) extract the methanol extract with methanol, concentrated under reduced pressure and extracted with ethyl acetate to obtain an ethyl acetate extract (step 1);

2) concentrating the ethyl acetate extract, dissolving it in a small amount of mixed solvent, and performing chromatography to concentrate the active fraction to obtain a concentrate (step 2);

3) dissolving the concentrate in methanol and performing chromatography to collect an active fraction (step 3); And

4) The active fraction is separated by chromatography, the solvent is removed with a reduced pressure dryer, and freeze-dried to obtain a target compound (step 4).

Specifically, the step 1 is cut, the extract was extracted with 100% methanol for 48 hours and concentrated under reduced pressure after extraction using ethyl acetate.

Specifically, step 2 is described, the concentrated ethyl acetate extract is dissolved in a mixed solvent of chloroform and methanol (100: 1), and then a mixed solvent of chloroform and methanol (100: 1, 50: 1 and 10: 1) is added. The active fraction is concentrated by silica gel column adsorption chromatography used as the elution solvent.

Specifically, step 3 is described in detail. After dissolving the concentrated active fraction in methanol, the active fractions are collected by performing Sephadex LH-20 gel column chromatography.

Specifically, step 4 is described, high pressure liquid chromatography using a C18 column and an elution solvent of water-acetonitrile (20% acetonitrile to 100% acetonitrile for 30 minutes). The active fraction was separated to obtain the compound of formula 1 from the active fraction of the holding time of 21 minutes and the compound of formula 2 from the active fraction of the holding time of 22 minutes.

Finally, the present invention provides a pharmaceutical composition for apoptosis inhibitors containing as an active ingredient a compound of Formula 1 and Formula 2 and / or a pharmaceutically acceptable salt thereof.

The pharmaceutical composition contains a compound represented by Formula 1 and Formula 2 and / or a pharmaceutically acceptable salt thereof as an active ingredient exhibiting an apoptosis inhibitory effect, thereby inhibiting abnormally-induced apoptosis mechanisms, thereby resulting in increased apoptosis. It can be usefully used to prevent or prevent immunodeficiency, various neurodegenerative diseases, ischemic diseases (strokes) and liver diseases caused by toxic substances such as alcohol.

The compounds of formulas (1) and (2) may be administered orally or parenterally, for example, intravenously, subcutaneously, intraperitoneally or topically, during clinical administration and may be used in the form of general pharmaceutical formulations.

The pharmaceutical compositions of the present invention may be formulated for oral administration such as tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs formulated as elixirs. Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin for preparation in formulations such as tablets and capsules; Excipients such as dicalcium phosphate; Disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. Capsules contain liquid carriers, such as fatty oils, in addition to the substances mentioned above.

In addition, the pharmaceutical composition of the present invention can be administered parenterally, and parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection. To formulate into parenteral dosage forms, the compounds of Formula 1 and Formula 2 are mixed in water with stabilizers or buffers to prepare solutions or suspensions, which are formulated in unit dosage forms of ampoules or vials.

The effective dose of the compound represented by the formula (1) is generally 1 to 50 mg / day, preferably 5 to 20 mg / day, per kg of adult patient, and 1 day at regular time intervals according to the judgment of the doctor or pharmacist. It may be administered several times, preferably two to three times daily.

As a result of the acute toxicity test on the compound of Formula 1, MLD (mouse lethal dosage) value was very stable above 500 mg / kg.

In addition, the effective dose of the compound represented by Formula 2 is generally 1 to 50 mg / day, preferably 5 to 20 mg / day, per kg of adult patient, at regular time intervals according to the judgment of the doctor or pharmacist. It may be administered several times a day, preferably dividedly two to three times a day.

As a result of acute toxicity test on the compound of Formula 2, MLD (mouse lethal dosage) value was very stable at 500 mg / kg or more.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Isolation and Purification of Apoptosis Inhibitors

In order to search for compounds capable of modulating apoptosis mechanisms, we isolated and purified novel compounds from the pool using extraction and chromatography.

First, 3 kg of the plant extracts from Yasan around Gyeongnam Jinju were cut into 5 cm sizes, extracted with 100% methanol for 48 hours, concentrated under reduced pressure, and extracted with ethyl acetate. The ethyl acetate layer was concentrated and dissolved in a small amount of a mixed solvent of chloroform and methanol (100: 1), followed by silica gel column chromatography (20-230 mes, Merck) to concentrate the active fraction. The concentrated material was dissolved in 2 ml of methanol, and then Sephadex LH gel column chromatography was performed to collect the active fractions. High pressure liquid chromatography (column: C18, flow rate: 1.5 mL / min, 220 nm detection) using the active fraction as an elution solvent for water-acetonitrile (20% acetonitrile to 100% acetonitrile for 30 minutes) After separating the active fraction of the holding time 21 minutes and removing the solvent with a vacuum dryer, the residue obtained by freeze drying to obtain 12 mg of the compound of formula 1, the active fraction of the holding time of 22 minutes was separated and dried under reduced pressure The solvent was removed, and the obtained residue was freeze-dried to obtain 6 mg of the compound of formula (2).

<Compound of Formula 1>

¹³ C NMR (CD ₃ OD) 47.0, 56.4, 56.8, 82.3, 111.6, 116.3, 116.5, 118.7, 123.3, 128.3, 129.2, 131.5, 142.2, 149.3, 149.9, 158.5, 169.2

<Compound of Formula 2>

¹³ C NMR (CD ₃ OD) 37.9, 52.7, 74.7, 114.2, 115.3, 116.3, 116.5, 117.5, 121.8, 123.2, 127.6, 123.2, 127.6, 128.8, 145.4, 146.2, 146.8, 147.9, 149.8, 168.3, 172.1

Example 2 Physicochemical Characterization of Example 1 Compound

In order to analyze the physicochemical properties of the compounds represented by Formula 1 and Formula 2 obtained in Example 1, the inventors of the present invention, ESI-MS (electrospray ionization mass spectrometry, Fisons VG Quattro 400 mass spectrometer, USA), HRESI-MS ( High resolution electrospray ionization mass spectrometry, Fisons VG Quattro 400 mass spectrometer, USA) and hydrogen and carbon nuclear magnetic resonance spectra were used. In the NMR experiment, each sample was dissolved in methanol (CD3OD) solvent and measured in a 5 mm NMR tube. The chemical shift was measured based on the peak of each solvent or the peak of TMS (tetramethylsilane). As a result of analyzing the property, molecular weight, molecular formula and mass of the compound, it was confirmed that the novel compound of the present invention had the following physical and chemical properties.

As a result, the material property of the compound of Formula 1 was powder, the molecular weight was 343, the molecular formula was confirmed as C ₁₉ H ₂₁ NO ₅ , and the mass spectrometry (M + H) of the compound was 344 ( m / z ). Hydrogen nuclear magnetic resonance spectra and carbon nuclear magnetic resonance spectra of methanol (CD ₃ OD) with solvent as a result of physicochemical properties showed that the compounds of the present invention represented by the general formula (1) have not been reported until now. It was found to be a novel compound that represents ( FIGS. 1 and 2 ).

In addition, the material property of the compound of Formula 2 was powder, the molecular weight was 374, the molecular formula was confirmed as C ₁₉ H ₁₈ NO 8, The mass spectrometry (M + H) of the compound was 375 ( m / z ). Hydrogen nuclear magnetic resonance spectra and carbon nuclear magnetic resonance spectra of methanol (CD ₃ OD) as a solvent, together with the above physicochemical properties, showed that the compound of the present invention represented by Formula 2 also has not been reported until now. It was found to be a novel compound that produces an effect ( FIGS. 3 and 4 ).

Experimental Example 1 Measurement of Apoptosis Inhibitory Effect of the Novel Compound of Example 1

In order to measure the apoptosis inhibitory effect of the compounds represented by Formula 1 and Formula 2 obtained in Example 1, the present inventors performed the following in vitro experiment using U937 cells, a human blood cancer cell line.

U937 cells were incubated in 96-well microplates until 5 X 10 ⁶ cells / well, and then 10 µg / ml of apoptotic inducer etoposide and 1 to 100 µg / Treatment with the U937 cells at a concentration of ml and incubated for 5 hours at 37 ℃ in 5% CO ₂ -95% air conditions to compare the apoptosis inhibitory effect. At this time, cells treated with only etoposide were used as the positive control group, and cells not treated with both etoposide and the compound of the present invention were used as the negative control group. After 5 hours, the shape of U937 cells was observed under a microscope to determine apoptosis. The cells obtained by centrifugation were subjected to DEVD-AFC (Z-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin). ) Was used to measure the kease phase-3 activity. Determination of the apoptosis inhibitory effect exhibited by the compounds of the present invention is based on the kease phase-3 activity of the positive control (only treated with etoposide) and the keasephase-3 activity of the negative control (untreated with etoposide and compounds). It is shown in Table 1 below.

Table 1 Comparison of apoptosis inhibitory effect in U937 cell line

compound Inhibition Rate (IC ₅₀ , Μg / ml) Compound of Formula 1 15.0 Compound of formula (2) 20.0 Pyrrolidine dithiocarbonate (PDTC) 15.0

As shown in Table 1, the inhibitory activity concentration of the compound represented by the formula (1) on the apoptosis of etoposide-induced U937 cells (IC ₅₀ , the concentration required to exhibit an inhibitory effect of 50%) is 15 µg / ml , The inhibitory activity concentration of the compound represented by the formula (2) is 20 ㎍ / ㎖ and 15.0 ㎍ / ㎖ inhibitory activity concentration of the standard comparative compound PDTC (pyrrolidine dithiocarbonate), the compound of formula 1 of the present invention Almost the same apoptosis inhibitory effect as PDTC, the compound of formula 2 was found to have a better apoptosis inhibitory effect than the standard comparator. From the above results, it was confirmed that the compounds of Formula 1 and Formula 2 of the present invention are novel compounds derived from natural products that can inhibit apoptosis in vitro.

Experimental Example 2 Oral Acute Toxicity in Rats

Acute toxicity test was performed using 6-week-old SPF SD rats. Two animals per group were suspended orally at a dose of 1 g / kg / 1 ml by suspending the compounds of Formula 1 and Formula 2 in 0.5% methylcellulose solution, respectively. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities. As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxicity change was observed in weight change, blood test, blood biochemistry test, autopsy findings, etc. As a result, all of the tested compounds did not show toxic changes up to 500 mg / kg in rats, and the minimum lethal dose (LD ₅₀ ) was determined to be a safe substance of 500 mg / kg or more.

As confirmed above, the compounds of the present invention represented by the formula (1) and (2) exhibit excellent apoptosis inhibitory effect can be formulated into a pharmaceutical composition for apoptosis inhibitors.

Preparation Example 1 Manufacturing Method of Syrup

A syrup containing the compound of Formula 1 or Formula 2 of the present invention and a pharmaceutically acceptable salt thereof as an active ingredient of 2% (weight / volume) is prepared by the following method.

Acid addition salts, saccharin and sugars of the compound of formula 1 or formula 2 were dissolved in 80 g of warm water. After the solution was cooled, a solution consisting of glycerin, saccharin, spices, ethanol, sorbic acid and distilled water was prepared and mixed thereto. Water was added to this mixture to 100 ml. The addition salt can be replaced with other salts according to the examples.

The components of the syrup are as follows.

Compound of formula (1) or formula (2) .HCl salt ... 2 g

Saccharin 0.8 g

25.4 g of sugar

Glycerin ... 8.0 g

Spices ··················· 0.04 g

Ethanol 4.0 g

0.4 g of sorbic acid

Distilled water ·····················

Preparation Example 2 Preparation of Tablet

A tablet containing 15 mg of active ingredient is prepared by the following method.

250 g of the compound of the formula (1) or (Chemical Formula 2) hydrochloride were mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

The components of the tablet are as follows.

250 g of compounds of the formula (1) or (2)

Lactose ········ 175.9 g

Potato starch ········· 180 g

Colloidal silicic acid 32 g

10% gelatin solution

Potato starch · 160 g

Talc · 50 g

Magnesium stearate 5 g

Preparation Example 3 Manufacturing Method of Injection Solution

Injection solution containing 10 mg of the active ingredient was prepared by the following method.

1 g of a compound of the formula (1) or (2), hydrochloride, 0.6 g of sodium chloride, and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

The components of the injection solution are as follows.

Compound of formula (I) or formula (II)

Sodium Chloride ・ ・ ・ ・ 0.6 g

0.1 g of ascorbic acid

Distilled water ··················

As described above, the compounds of the present invention represented by the formula (1) and (2) is a novel compound derived from the pool, it is non-toxic and shows excellent apoptosis inhibitory effect can be useful as an apoptosis inhibitor.

Claims (6)

The novel compounds represented by the following formula (1) and salts thereof. Formula 1 The novel compounds represented by the following formula (2) and salts thereof. Formula 2 1) extract the methanol extract with methanol (methanol) and concentrated under reduced pressure and then extracted with ethyl acetate (ethylacetate) to obtain an ethyl acetate extract (step 1); 2) concentrating the ethyl acetate extract, dissolving it in a small amount of mixed solvent, performing chromatography (chromatography), and concentrating the active fraction to obtain a concentrate (step 2); 3) dissolving the concentrate in methanol and performing chromatography to collect an active fraction (step 3); And 4) The method of claim 1 and 2, wherein the active fraction is separated by chromatography, the solvent is removed with a reduced pressure dryer, and freeze-dried to obtain a target compound (step 4). The method of claim 3, wherein the chromatography of step 2 is silica gel column adsorption chromatography, and the chromatography of step 3 is sephadex LH-20 gel column chromatography. The chromatography of step 4 is characterized in that the high pressure liquid chromatography (high pressure liquid chromatography). From the group consisting of acquired immunodeficiency disease, neurodegenerative disease, ischemic disease including stroke and alcoholic liver disease caused by toxic substances, including the compound represented by Formula 1 of claim 1 and / or a pharmaceutically acceptable salt thereof as an active ingredient Pharmaceutical compositions for the prophylaxis or treatment of selected diseases. From the group consisting of acquired immunodeficiency syndrome, neurodegenerative diseases, ischemic diseases including stroke, and liver diseases caused by toxic substances such as alcohol, containing the compound represented by the formula (2) of claim 2 and / or a pharmaceutically acceptable salt thereof as an active ingredient Pharmaceutical compositions for the prophylaxis or treatment of selected diseases.