JP2008044940A - Novel compound isolated from fruit body of Antrodiacamphorata and pharmaceutical composition thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel diterpene compound separated from a fruiting body of Antrodia camphorata, and a pharmaceutical composition containing the compound. <P>SOLUTION: This compound is compound 1 having a structural formula, or 3β,19-hydroxylabda-8(17), 11E-diene-16, 15-olide and its pharmaceutically acceptable derivatives. This pharmaceutical composition contains the compounds and is used for the protection of nerve cells. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention relates to a novel diterpene compound isolated from the fruit body of Antrodia camphorata, its isolation method, and its use related to the protection of brain neurons.

Anthracite is also known as cow beetle or beef bowl, and is a perennial gonococcus belonging to the order Hidanasida, Sarnococcidae, a Taiwan-specific fungus with the scientific name (Antrodia camphorata), and a cattle that grows only in Taiwan It parasitizes the coconut trees. Since it is unique to Taiwan and is a valuable medicinal fungus, it has a high commercial value and is attracting a great deal of interest as a research subject, and is currently the most expensive natural fungus in Taiwan.

Anthracnose grows only in the mountainous area of Taiwan, between 450 and 2000 meters above sea level, especially on the rotten inner wall of the trunk of beef tree over 100 years old and the wet surface of dead beef tree. The environment in which turfgrass grows is a dark, moist and slightly cold area above the sea level, and because of its slow growth rate, it takes a considerable amount of time to form fruit bodies. Natural turfgrass grows on beef vines with a long age and forms fruiting bodies inside the hollow trunk. The shape of the fruiting body changes variously, such as a plate shape, a bell shape, a horseshoe shape, and a tower shape. It is bright red when formed, and gradually grows white, light pink, and ocher as it grows.

In the private sector, Anthracnose has been considered to have detoxification, anti-cancer, alcohol, and anti-inflammatory effects, but it has not been demonstrated with scientific evidence. The National Science Committee (Agricultural Authority of Taiwan), the National Agriculture Committee, and the Health Food Program of the Sanitation Department have so far studied the effects of turfgrass on tumors, cholesterol and blood sugar. In recent biological research, it has been clarified that the fruit body of Antrodia camphor has immunomodulating, antioxidant, and liver protecting effects (Hsiao, G. et al., JRJ Agric. Food Chem. 2003, 51, 3302-3308). Furthermore, the cultured mycelium may have anti-inflammatory activity, vascular relaxation activity, cytotoxicity against several types of tumor cells, action to protect oxidative damage of erythrocytes of healthy subjects, anti-hepatitis B virus activity, etc. (Liu, JJ et al., Toxicol. Appl. Pharmacol. 2004, 201, 186-193, Hseu, YC et al., Life Sci. 2002, 71, 469-482, Lee, IH et al., FEMS Microbiol
Lett. 2002, 209, 63-67).

The composition of Anthracis is complex, including many polysaccharides, triterpene compounds, SOD (super oxide dismutase, adenosine, ultra-low molecular protein, vitamins, trace elements, nucleic acids, solid alcohols, blood pressure stabilizers, etc.) Contains bioactive substances. Chemical research on the mycelium culture of Antrodia camphorata has only been reported by the group of Nakamura et al., And it is clear that five of these have cytotoxic succinic acid derivatives. (Nakamura.N. Et al., J.Nat.Prod. 2004, 67, 46-48). In research on the fruit bodies of Antrodia camphorata, it has been reported that fruit bodies are composed of multiple types including fatty acids, lignans, phenyl group derivatives, sesquiterpenes, steroids, and triterpene compounds. ing. (Chen, CH & Yang, SWJNat. Prod. 1995, 58, 1655-1661; Cherng, IH et al., Phytochemistry 1996, 41, 263-267, and Shen, CC et al., J. Chin. Med. 2003, 14, 247 -258).

First, the present invention relates to three new labdan (half-day alkylated) diterpene compounds (labdane) isolated from the fruit body of Antrodia camphorata.
diterpenoids1-3). That is, 19-hydroxylabda-8 (17), 13-diene-16,15-olide (19-hydroxylabda-8 (17), 13-dien-16, 15-olide.) (1), 3β, 19 -Dihydroxylabda-8 (17), 11E-diene-16,15-oride (3β, 19-dihydroxylabda-8 (17), 11E-dien-16,15-olide.) (2), and 13-epi -3β, 19-dihydroxylabda-8 (17), 11E-diene-16,15-olide (13-epi-3β, 19-dihydroxylabda-8 (17), 11E-dien-16, 15-olide.) (3) and four more known compounds: 19-hydroxylabda-8 (17), 13-diene-16, 15-olide (19-hydroxylabda-8 (17), 13-dien -16, 15-olide.) (4), 14-deoxy-11,12-didehydroandrographolide. (5), 14-deoxyandrographoloid, And abietic acid. In the in vitro analysis system evaluation, the effects of these seven kinds of separated compounds on nerve cells were evaluated, thereby completing the present invention.

The purpose and features of the present invention will be described in part below, and will be described in a clear and easy-to-understand manner.

An object of the present invention is to provide a novel diterpene compound and a biologically active derivative isolated from the fruit body of Antrodia camphorata.

For purposes of the present invention, the novel compounds characteristic of the present invention are represented by the following structural formula:
[Chemical Formula 3] or [Chemical Formula 4], and pharmaceutically acceptable bases, solvates, biologically active derivatives and the like are contained.

The second object of the present invention relates to a pharmaceutical composition of one or more compounds of the present invention, or pharmaceutically acceptable bases, or solvates, hydrates and derivatives. In a first aspect according to the present invention, there is provided a pharmaceutical composition used for treating, preventing and ameliorating nerve cell damage such as Alzheimer's disease (AD, also known as senile dementia). is there.

In a second aspect according to the present invention, the composition of the present invention comprises one or more compounds of the present invention, or pharmaceutically acceptable bases or solvates, hydrates, derivatives other than derivatives. And therapeutic drugs.

In a third aspect according to the invention, the composition of the invention comprises a compound of the invention, or a pharmaceutically acceptable base or solvate, hydrate, biologically active equivalent, derivative and pharmaceutically Contains acceptable carriers, diluents, excipients.

In a preferred embodiment of the invention, the composition of the invention takes the form of a pharmaceutical composition or a single drug dose. The pharmaceutical composition or single drug dose in the present invention contains one or more kinds of components, which are adjusted in relative amounts and in the pharmaceutical composition or drug dose format, for example, proliferative diseases such as cancer. Used for the treatment and prevention. Suitable pharmaceutical compositions and dosage forms include compounds of formula 1, 2, 3, 4 or 5, 14-deoxyandrographoloid and abietic acid, or pharmaceutically acceptable bases or solvates thereof , Hydrates, derivatives, and combinations with one or more types of certain active agents.

Other features of the present invention will be described in detail in the specific examples shown below.

Hereinafter, specific examples of the present invention will be described. These specific examples are provided by interpretation of the invention and do not limit the present invention. Other changes and modifications including these may be made within the spirit and scope of the present invention.

In the following examples, specific rotations are recorded with a DIP-1000 polarimeter manufactured by JASCO (JASCO Corporation). IR spectra are recorded on a Perkin-Elmer 983G spectrometer. ¹ H and NMR spectra and ¹³ C NMR spectra are recorded on a Varian Unity Plus-400 nuclear magnetic resonance apparatus. EIMS (electron ionization mass spectrometry) and HREIMS (high resolution electron ionization mass spectrometry) are measured by Finnigan TSQ-46C manufactured by JEOL and SX-102A mass spectrometer manufactured by JEOL. Place the extract in silica gel (Merck70-230mesh, 230-400mesh), perform chromatographic analysis, semi-prepared normal phase HPLC column [250x10mm,
Purify with LCD Refracto Monitor III on Licrosorb (Si 60 (7 μm)). The obvious peaks are, in order, δ (ppm), chemical shift, multiplicity (s: single peak, d: double peak, t: triple peak, q: quadruple peak, m: multiple peak, brs: broad single peak), The unit is Hertz (Hz) coupling constant (J) and proton number.
[Example 1] Separation of novel diterpene compounds and their structural analysis and identification

Antrodia
The body of camphorate No.2 is provided by Kang Jian Biotech Corp. Ltd., Nantau, Taiwan, Republic of China. The fungus was determined by Dr. Toshio Yori of Kang Jian Biotech Corp. Ltd. The proof of specimen (No.2) is stored at Kang Jian Biotech Corp. Ltd.
[Example 1-2]

Place the fruit body (2 kilograms) of turfgrass at room temperature and extract with methyl alcohol (40 liters) twice for 5 days. After evaporation of the solvent, the residue extract is mixed with water to a total volume of 1 liter. This phase was extracted three times with 1 liter of EtOAc, the combined organic phases were evaporated, the resulting black gel (150 grams) was loaded onto silica gel and chromatographed with a solution of alkylated ethyl acetate (EtOAc). I do. Elution was performed with a solution of the components alkylated with 30-40% EtOAc and purified using a silica gel column prepared using HPLC and an EtOAc / alkylated (3: 7) mixture as eluent to remove EtOAc-soluble components. obtain. This was subjected to repeated chromatographic analysis to find that compound 1 (8.2 mg, tR: 5'25 "), compound 2 (19.4 mg, tR: 8'40"), compound 3 (3.0 mg, tR: 8'45 " ) Three new purified compounds, and 19-hydroxylabda-8 (17), 13-diene-16,15-oride (compound 4, 32.4 mg, tR: 5'50 "), 14-deoxy -11,12-didehydroandrographoloid (compound 5, 5.5 mg, tR: 9'55 "), 14-deoxyandrographoloid (6.2 mg, tR: 9'10"), abietic acid (3.1 mg , TR: 11'25 "), four known half-day alkylated diterpene compounds. The structure of the known compounds is determined by comparison with the spectral data in the references.
[Example 1-3]

Compound 1 exhibits an amorphous powder upon separation and its ¹³ CNMR (shown in Table 1) and HREIMS data confirm that the molecular formula is C ₂₀ H ₃₂ O ₃ . The IR spectrum of Compound 1 confirms that the molecule contains a γ-lactone group (1772 cm ⁻¹ ) and a hydroxy group (3470 cm ⁻¹ ). In the ¹ HNMR spectrum (shown in Table 1), two primary methyl groups [δ _H 0.62 and 0.95 (each 3H, s)] and a γ-lactone group group [δ _H 4.16 (td, J = 8.8, 6.8 Hz) and 4.30 (td, J = 8.8, 2.8 Hz)], a pair of alkene protons [δ _H
4.50 and 4.80 (each 1H, brs)] two primary methyl alcohol protons [δ _H 3.36 and 3.72 (each 1H, d, J
= 11.2Hz)]. Compound 1 ¹ HNMR spectrum and the known compound 4 (Han, BH addition, J.Med. Chem. 1998,41,2626-2630 reference) is substantially similar to the, difference between the two H ₂ -15 and compound The only alkene proton in 4 is one. ¹³ CNMR data and DEPT (Distionless
enhancement by polarization
The transfer) spectral analysis, two CH _3, the CH ₂ 11, 3 a CH, 3 pieces of C, electromagnetic waves 20 containing one carbon atom of the carbonyl group of the lactone is apparent. HMBC (Heteronuclear multiple
According to the spectrum of bond correlaction spectroscopy, the carbon atom of the carbonyl group of this lactone is C-16. The protons of the methylene group of the two linked γ-lactone groups are related to C-13 of δ _C 39.5, C-14 of δ _C 29.6, and C-16 of δ _C 179.1. In the same experiment, the protons of the H ₂ -19 methylene group at δ 3.36 and 3.72, and C-3 at δ 35.4, C-4 at δ 39.6, C-5 at δ 57.1 and δ 27.1 There is an interaction between C-18. The NOESY (Nuclear Overhanser enhancement exchangespect roscopy) spectrum (shown in FIG. 1) confirms that the C-20 methyl group, H ₂ -11, and H ₂ -19 are located on the same side of the molecule. The above data proves that the chemical name of Compound 1 is 19-hydroxylabda-8 (17) -alkene-16,15-oride.
[Example 1-4]

Compound 2 exhibits an amorphous powder upon separation, and its molecular formula is determined to be C ₂₀ H ₃₀ O _{4 from} its HREIMS and ¹³ CNMR data (shown in Table 1). Positioning at the IR absorption peaks at 3381 cm ^-1 and 1777 cm ^-1 indicates the presence of hydroxy groups and γ-lactone functional groups. In its ¹ HNMR spectrum (shown in Table 1), two primary methyl groups [δ _H 0.72 and 1.22 (each 3H, s)], and protons of a γ-lactone group to which two methylene group protons are bonded. [δ _H 4.23 (td, J = 8.4, 6.8Hz), 4.34 (td, J =
8.4, 3.6 Hz)], a pair of terminal methylene protons [δ _H 4.50 and 4.74 (1H, d, J = 1.6 Hz each)], a pair of trans-coupled alkene protons [δ _H 5.50 ( dd, J = 15.6, 5.6 Hz), 5.64 (dd, J = 15.6, 9.6 Hz)], one proton of methylene group with alcohol [δ _H 3.45 (dd, J = 11.2, 4.4 Hz)], 2 Methylene group protons [δ _H 3.30 and 4.17 (each 1H, d, J
= 11.2Hz)]. ^{In the 1} HNMR spectrum, the electromagnetic waves of other methylene group protons and compound 5 (see Reddy, MK et al., Phytochemistry 2003, 62, 1271-1275) are similar. Since Compound 5 has one double bond, ^one more alkene electromagnetic wave appears in ¹ HNMR spectrum than Compound 2. Compared to compound 5, the ¹ H chemical shift of H-11 and H-12 in compound 2 moves to a higher region, and compound 2 does not show an obvious absorption peak in its UV spectrum. In the HMBC spectrum, the electromagnetic wave of H-3 (δ _H 3.45) is associated with C-18 and C-19, indicating that this hydroxy group is bonded onto C-3. The coupling constant (J = 11.2, 4.4 Hz) indicated by this observation, the H-3 proton is in an axial arrangement. In the NOESY spectrum, the electromagnetic wave of H-20 proton is related to H-11 and H-19, and H-11, H-19 and H-20 are all on the β-side. The above data proves that the chemical name of Compound 2 is 3β, 19-dihydroxylabda-8 (17), 11E-diene-16, 15-olide.
[Example 1-5]

Compound 3 also exhibits an amorphous powder upon separation, and its HREIMS and ¹³ CNMR data (shown in Table 1) confirm that its molecular formula is C ₂₀ H ₃₀ O ₄ . Located in the IR absorption peaks at 3391 cm ^-1 and 1775 cm ^-1 indicates the presence of hydroxy groups and γ-lactone functional groups. As a result of HMBC and HMQC spectra, the total structure of Compound 3 is the same as that of Compound 2. NOESY spectral analysis reveals the relative configuration of this molecule and the side chain located at C-9 and appears to be the same as compound 2. Since the data obtained from the analysis show that compound 3 is 13-epimerism of compound 2, this chemical name is 13-epi-3β, 19-dihydroxylabda-8 (17), 11E-diene-16 It is inferred to become 15-Oride.
[Example 2] Protective action of isolated compound against nerve cell damage

In this example, a primary cultured cell of a new rat cerebral cortex neuron obtained from the cerebral cortex of a young rat of Harlan Sprague-Dawley rat on the first day of life is used as a target cell. That is, after each young rat is anesthetized, the cerebral cortex is cut out and placed in 0.5 mg / mL kalin proteinase at 37 ° C. and decomposed for 15 minutes. Pipette the tissue from the Hibernate A medium (including B27 supplement) and crush to separate the cells. Spread cells to a culture plate coated with poly-D-lysine to a density of 5 × 10 ⁴ cells / cm ² , B27 supplement, 10 units / mL penicillin, 10 mg / mL streptomycin, 0.5 mg / mL Neurobasal culture medium (5% CO ₂ /9% O ₂ ) containing 5% glutathione is cultured for 3 days. The cells are then exposed to cytosine-β-D-arabinofuranoside (5 μM) for 1 day to inhibit non-neuronal cell growth. Experiments are performed using cells on day 5.
[Example 2-1]

MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium
Bromide) method is used to measure the killing effect of pure compounds obtained by isolation in Example 1 on Aβ _{25-35 -treated} cells and evaluate the protective activity of the neurons. The reductive splitting of the tetrazole salt MTT (bromine 3- (4,5-dimethylthiazol-2-yl) -2,5-difernithiazole) yields a purple dye located at an absorbance value of approximately 570 nm. Analyze dehydrogenase activity. The results are shown as a percentage compared to the absorbance value of the control group. The cortical neurons obtained by the above preparation and the solvent (0.1% DMSO, dimethyl sulfoxide, dimethyl sulfoxide (DMSO)) or various different concentrations of compounds are cultured together for 2 hours, and then exposed to 5 μMAβ for 40 hours. Cell viability is measured by MTT reduction analysis. Cells are cultured for 1 hour in basal medium containing 0.5 mg / mL MTT. After removing the culture medium, the formazan particles are dissolved in DMSO (Dimethyl sulfoxide). The absorbance value at 600 nm is measured and analyzed by ELISA (Enzyme-linked immunosorbent assay). The results of the experiments performed three times alone are expressed as mean ± SD, and data analysis is performed by posthoc multiple comparison of Bonferroni test by analysis of variance ANOVA.
[Example 2-2]

As the results shown in Table 2, all of compounds 1-5 can reduce the neuronal toxicity caused by Aβ- due to concentration-dependence. Of these compounds, these compounds were respectively 25.3% (compound 1), 29.5% (compound 2), 36.7% (compound 3), 28.9% (compound 4) at concentrations of 5, 10, 10, 10 and 20 μM, respectively. ), 29.5% (compound 5) cell death is reduced, so it is clear that protection against nerve cells is not damaged by Aβ.

The main NOESY relevance of Compound 1 is shown.

Claims (15)

A compound having the following structural formula, [Chemical Formula 1], and pharmaceutically acceptable bases, solvents are compounds, hydrates or biologically active derivatives.
A compound having the following structural formula: [Chemical Formula 2], wherein R is H, and pharmaceutically acceptable bases and solvents are derivatives of active substances or organisms.
Antrodia
3. A compound according to claim 1 or 2 isolated from the fruiting body of camphorata). The compound according to claim 1, which is 19-hydroxylabda-8 (17), 13-diene-16,15-oride or has a derivative equivalent to biological activity. The compound according to claim 2, which is 3β, 19-hydroxylabda-8 (17), 11E-diene-16,15-olide or has a derivative equivalent to biological activity. The compound according to claim 2, which is 13-epi-3β, 19-hydroxylabda-8 (17), 11E-diene-16,15-olide, or has a derivative equivalent to biological activity.  The compound according to claim 4, which is 19-hydroxylabda-8 (17), β-diene-16, 15-olide, or has a derivative equivalent to biological activity.  The compound according to claim 4, which is 14-deoxy-11,12-didehydroandrographoloid or has a derivative equivalent to biological activity. A pharmaceutical composition having a neuroprotective action, comprising at least the compound according to claim 1 and 2 and a pharmaceutically acceptable carrier, excipient, or diluent. 10. The pharmaceutical composition according to claim 9, wherein the compound is 19-hydroxylabda-8 (17), 11E-diene-16,15-olide, or has a derivative equivalent to biological activity. The pharmaceutical composition according to claim 9, wherein the compound is 3β, 19-hydroxylabda-8 (17), 11E-diene-16,15-olide, or has a derivative equivalent to biological activity. 10. The compound according to claim 9, wherein the compound is 13-epi-3β, 19-hydroxylabda-8 (17), 11E-diene-16,15-oride or has a derivative equivalent to biological activity. Pharmaceutical composition. 10. The pharmaceutical composition according to claim 9, characterized in that it is used for protecting nerve cells. 10. The pharmaceutical composition according to claim 9, wherein it is used for treatment or prevention of diseases involving nerve cells. The pharmaceutical composition according to claim 9, characterized in that it is used for the treatment or prevention of Alzheimer's disease.