TWI396533B - Prenylflavanone compounds and uses thereof - Google Patents

Description

Isoprene flavonoid compound and use thereof

This invention relates to novel isoprene flavonoid compounds and uses thereof.

Propolis is a mixture of resins that honeybees collect from tree buds, fruits, sap streams, or other plant sources. Propolis has been reported to contain a wide range of active ingredients and has a wide range of biological activities, including anti-tumor ^{( 3 )} , anti-oxidation ^{( 4 )} , anti-bacterial ^{( 5 )} , anti-viral ^{( 6 )} , anti-fungal ^{( 7 )} and anti-inflammatory activities ^{( 8 )} . Propolis from different regions can contain different active ingredients.

We have previously reported eight isoprene flavonoids isolated from Taiwan propolis, which are represented by the following structural formulas 1 to 8, respectively:

It has been reported that these propolis have a wide range of biological activities, including anti-cancer ^{( 10-l5 )} , antioxidant ^{( 10-16 )} and antimicrobial activity ^{( 16 )} . In addition, recent studies have confirmed that the active ingredients contained in Okinawan propolis are similar to those contained in Taiwan propolis ^{( 17-18 )} .

Tissue protein deacetylase (HDAC, Histone deacetylase) is a deacetylation of the ε-amine group which catalyzes the amino acid residue of the N-terminal of the tissue protein. Certain HDAC inhibitors have been identified and demonstrated in preclinical and clinical settings for patients with different types of cancer ^(19-21) . According to the current model of the anti-cancer mechanism of HDAC inhibitors, this inhibitor triggers the over-acetylation of core tissue proteins, thereby stimulating chromatin remodeling and activation of silent genes, such as tumor suppressor genes, resulting in Inhibition of tumor cell growth ^26-27 .

There is still a need to find novel ingredients from multifunctional propolis and to evaluate their useful physiological activities.

In one aspect, the invention provides a novel isoprenoid flavonoid compound represented by a structural formula selected from the group consisting of:

In another aspect, the invention provides a pharmaceutical composition comprising at least one of the above isoprene flavonoid compounds.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning meaning meaning

In one aspect, the invention provides an isoprene flavonoid compound isolated from Taiwan propolis.

A specific example of the isoprene flavonoid compound of the present invention is 5,7,3',4'-tetrahydroxy-6-farnesyl flavonoid (referred to as "propolis I", which is represented by the following formula:

₃₀ H ₃₆ O ₆ and a molecular weight compounds of formula I having the formula C is 492.25Da.

Another specific embodiment of the isoprenoid flavonoid compound is 5,7,4'-trihydroxy-6-geranyl flavonoid (referred to as "propolis J"), which is represented by the following formula:

The compound of formula II has the molecular formula C ₂₅ H ₂₈ O ₅ and has a molecular weight of 408.19 Da.

The isoprene flavonoid compound of the present invention has an inhibitory effect on HDAC enzyme activity and growth of cancer cells, and preferably has an inhibitory effect on growth of breast cancer cells.

Accordingly, in another aspect, the invention provides a pharmaceutical composition comprising an HDAC inhibitor comprising at least one compound of the invention and a pharmaceutically acceptable excipient or carrier.

In still another aspect, the invention provides a pharmaceutical composition for inhibiting the growth of cancer cells comprising at least one compound of the invention and a pharmaceutically acceptable excipient or carrier.

The invention also provides a pharmaceutical composition for treating a disease associated with deproteinization of tissue proteins comprising at least one compound of the invention and a pharmaceutically acceptable excipient or carrier.

In addition, many known HDAC inhibitors have been shown to have neuroprotective effects ^{( 30} , ^{31 )} , suggesting that HDAC inhibitors can be used to treat neurodegenerative diseases. Examples of such neurodegenerative diseases include, but are not limited to, multiple sclerosis (MS) ^{( 32 )} , Huntington's disease (HD, Huntington's disease) ^{( 33} , ³⁹ , ^{40 )} , spinal muscular muscles. atrophy ^{(SMA, spinal muscular atrophy) (} 34,35,36), spinal and bulbar muscular atrophy (SBMA, spinal and bullar muscular atrophy ) (37) and amyotrophic lateral sclerosis (ALS, amyotrophic lateral sclerosis) ^{( 38 )} .

Accordingly, the present invention provides a neuroprotective pharmaceutical composition comprising at least one compound of the invention and a pharmaceutically acceptable excipient or carrier.

The invention also provides a pharmaceutical composition for treating a neurodegenerative disease comprising at least one compound of the invention and a pharmaceutically acceptable excipient or carrier. Neurodegenerative diseases are preferably multiple sclerosis (MS), Huntington's disease (HD), spinal muscular atrophy (SMA), spinal medullary muscular dystrophy (SBMA) or amyotrophic lateral sclerosis (ALS).

In making the compositions of the present invention, the compounds of the present invention as active ingredients are usually diluted or enclosed in a vehicle in the form of a capsule, sachet, paper or other container. Excipients are generally excipients suitable for administration to humans or other mammals. Examples of suitable excipients include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium citrate, microcrystals. Cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methylcellulose. Furthermore, the compositions of the present invention may be in the form of a troche, a pill, a powder, a suspension, an emulsion, a solution, a syrup, an aerosol (in solid form or in a liquid base), an ointment, a capsule or a sterile packaged powder.

The composition can be administered to a patient via an oral route, for example, including a human, a monkey, a dog, etc., or administered as an injectable preparation via the parenteral route. Typically, the compositions of the present invention contain from about 125 mg to about 250 mg of a compound of the invention (propolis I or J, or both). The adult dose is preferably between about 500 mg and 1000 mg per day, which may be administered in the form of a single dose or divided doses.

The features of the present invention are more specifically described with reference to the specific embodiments of the invention which

Example 1: Preliminary extraction

200 g of TW-I grade Taiwan propolis (collected from the honeycomb of Tainan, Taiwan) ⁽²⁹⁾ was homogenized by stirring at a low temperature. The homogenized sample was then rinsed 3 times with 1.0 L of deionized water and the residue was extracted 3 times with 95% ethanol. The filtered ethanol extract was evaporated to dryness under reduced pressure to give a brown powder (135.6 g) which was stored at -20 ° C for further purification.

Example 2: Isolation and purification of compounds

The brown powder obtained from the ethanol extract was dissolved in methanol and applied to a Sephadex LH-20 column (Amersham Pharmacia Biotech AB, Uppsala, Sweden) using 95% ethanol as a solvent for elution. The effect of all of the eluate (including the distinction obtained by subsequent chromatography) on the proliferation of human breast cancer was examined, and the active fraction was again isolated by chromatography on a Sephadex LH-20 column and using 95% ethanol as the eluent. Next, the active fraction was subjected to cartridge chromatography (Kiesel Gel 60, E. Merck, Darmstadt 1, Germany) using a n-hexane/EtOAc solvent system.

The most active fraction was purified by reverse preparative HPLC (n-hexane / EtOAc, 30:70). The experimental conditions were as follows: the column was Luna Phenomenex (C18, 250 x 4.6 mm); the solvent system was methanol/water (8:2); the flow rate was 1 mL/min; and the detection was carried out at UV 280 nm. The fractions having a residence time of 19.0 minutes (propolis I) and 10.5 minutes (propolis J) were collected, thereby separating propolis I (light yellow powder) and propolis J (light yellow liquid), respectively. Figure 3 shows the peaks of reverse preparative HPLC.

Example 3: Identification of propolis I and propolis J

The structures of propolis I and propolis J were analyzed using the following instruments: Perkin Elmer 1760-X IR-FT spectrophotometer; Hitachi 150-20 UV; Jasco J-710 spectropolarimeter; Finnigan MAT-95XH mass spectrometer (EI) ; and Bruker AV-500 spectrophotometer (using solvent spikes (MeOH- d 3) as a reference standard).

Propolis I

The physicochemical data of propolis I are as follows: [] ²⁰ _D +3.8 ( c 0.26, CH ₃ OH); IR v max (film) 3747, 3390, 2923, 1636 cm ^-1 ; UV (EtOH) λ max nm (log ε) 207.0 (1.65), 291 (0.73); CD (MeOH) 347 nm (Δε+3.19), 294 nm (Δε-3.98); HREIMS m/z 492.2512 (calculated for C ₃₀ H ₃₆ O ₆ : 492.2512); ¹ H and ¹³ C NMR.

From the ¹ H NMR spectrum, it is known that there are four olefin methyl groups (δ _H 1.52, 1.56, 1.63, 1.74), eight methylene protons (δ _H 1.87, 1.95, 2.05), and two benzylmethylene protons. (δ _H 3.20) and two vinyl protons (δ _H 5.04, 5.18) indicate the presence of farnesyl. In addition, ¹³ C NMR spectra showed 4 methyl groups (δ _c 16.1, 16.2, 17.8, 25.9), 5 methylene carbons (δ _c 21.8, 27.3, 27.8, 40.8, 40.9), and 3 third olefin carbons. (δ _c 124.1, 125.3, 125.6) and three quaternary olefin carbons (δ _c 132.0, 134.9, 135.8), which further support the presence of farnesyl. Furthermore, the ABX systems of δ _H 5.21 (1H, dd, J = 3.0, 13.0 Hz), 2.65 (1H, dd, J = 3.0, 17.0 Hz) and 3.02 (1H, dd, J = 13.0, 17.0 Hz) respectively A characteristic pattern showing the flavonoid skeleton at the H-2 and H-3 positions.

In addition, ¹ H and ¹³ C NMR assignments and correlations were determined based on the derivation of ¹ H- ¹ H COSY, HSQC and HMBC data. The HMBC spectrum of propolis I shows that the methylene signal of δ _H 3.20(H-1") is related to C-5 (δ _c 162.5) and C-7 (δ _c 165.9), respectively, suggesting that farnesyl is linked to C. -6 (Fig. 1) In addition, the CD spectrum shows that the configuration of C-2 is S-type.

Thus, propolis I was identified as 5,7,3',4'-tetrahydroxy-6-farnesylflavone, represented by the above formula I. This compound was isolated for the first time and was not disclosed in the published literature.

Propolis J

The physicochemical data of propolis J is as follows: [] ²⁰ _D +2.4 ( c 0.41, CH ₃ OH); IR v max (film) 3747, 3393, 2925, 2361, 1637 cm ^-1 ; UV (EtOH) λ max nm (log ε ) 209.0 (1.82), 293 ( 0.92); CD (MeOH) 328.9nm (Aε + 2.37), 289nm (Aε-3.98); HREIMS m / z 408.1944 (C 25 H 28 O ₅ the calculated value: 408.1937); ¹ H and ¹³ C NMR.

From the ¹ H NMR spectrum, it is known that there are three olefin methyl groups (δ _H 1.55, 1.61, 1.74), four methylene protons (δ _H 1.93, 2.04), and two benzylmethylene protons (δ _H 3.18). And 2 vinyl protons (δ _H 5.05, 5.18), which shows the presence of Geranyl. In addition, ¹³ C NMR spectra showed 3 methyl groups (δ _c 16.2, 17.7, 25.9), 3 methylene carbons (δ _c 21.8, 27.7, 40.9), and 2 tertiary olefin carbons (δ _c 123.9, 125.5 And two quaternary olefin carbons (δ _c 132.0, 135.3), which further support the presence of geranyl groups. Furthermore, δ _H 5.29 (1H, dd, J = 2.7, 13.0 Hz), 2.67 (1H, dd, J = 2.7, 17.0 Hz) and 3.08 (1H, dd, J = 13.0, 17.0 Hz) ABX systems respectively A characteristic pattern showing the flavonoid skeleton at the H-2 and H-3 positions.

In addition, ¹ H and ¹³ C NMR assignments and correlations were determined based on the derivation of ¹ H- ¹ H COSY, HSQC and HMBC data. The HMBC spectrum of propolis J shows that the methylene signal of δ _H 3.18(H-1") is related to C-5 (δ _c 162.5) and C-7 (δ _c 166.0), respectively, suggesting that the geranyl group is linked to C. -6 (Fig. 2) In addition, the CD spectrum shows that the configuration of C-2 is S-type.

Therefore, propolis J was identified as 5,7,4'-trihydroxy-6-geranylflavonoid, represented by the above formula H. This compound was isolated for the first time and was not disclosed in the published literature.

Example 4: Cell culture and cytotoxicity assay

Human breast cancer MCF-7 and MDA-MB-231 cells were purchased from the Food Industry Research Institute (Hsinchu, Taiwan). The cells were cultured in the following medium: Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS, Fetal boving serum), 1% dilution of penicillin-streptomycin, and 2 mM glutamic acid. The cells were maintained in humidified air at 37 ° C, 95% air and 5% CO ₂ . Among them, MDA-MB-231 cells were cultured in L-15 medium (Gibco) containing 10% fetal bovine serum (FBS), 1% dilution of penicillin-streptomycin, and 2 mM glutamic acid. The cells were maintained at 37 ° C, 100% air and 0% CO ₂ humidified air.

Propolis F, I and J were dissolved in Dimethyl sulfoxide and prepared at a fixed concentration of 10 mg/mL. Cells (1.5 x 10 ⁶ /pan) were grown in 100-mm plates for 14 hours, then treated with DMSO for 48 hours or treated with different concentrations of propolis (2.5, 5.0, 10 and 20.0 μg/mL) for 48 hours. Propolis is a small molecule compound having a molecular weight of 408 to 492 Da. These values are converted to individual molar concentrations.

The number of cells was counted and its viability was determined by the Trypan blue exclusion assay. The inhibitory activity of the compound is expressed as the IC ₅₀ value (the concentration required to induce 50% cytotoxicity), and the average value is obtained from three replicates.

Table 2 shows the ₅₀ pixel values I and J of propolis the IC ₅₀ values in the present invention, the cytotoxicity assay the obtained ([mu] M) and control group of propolis prime F IC.

Therefore, the compounds of the present invention (propolis I and J) were confirmed to have cytotoxic activity against human breast cancer cells.

Example 5: Inhibition of propolis I and J on the growth of cancer cells Cell staining

Two types of breast cancer cell lines MCF-7 (ER receptor positive) and MDA-MB-231 cells (ER receptor negative) were cultured under the conditions described in Example 5. These cells were treated with various concentrations of different propolis for 24 or 72 hours. Treated cells were stained with trypan blue and their viability was assessed.

Figure 4A shows the results of staining of MCF-7 cells treated with various concentrations of propolis for 24 hours. Figure 4B shows the results of staining of MDA-MB-231 cells treated with various concentrations of propolis for 24 hours. Figure 4C shows the staining results of MCF-7 cells treated with various concentrations of propolis for 72 hours. Figure 4D shows the staining results of MDA-MB-231 cells treated with various concentrations of propolis for 72 hours. Figure 4E shows the number of cells of MCF-7 cells treated with various concentrations of propolis for 72 hours.

Flow cytometry

Human breast cancer MCF-7 and MDA-MB-231 cells (1.5 x 10 ⁶ ) in 100-mm plates were treated with various concentrations of propolis F, I and J for 24 or 72 hours. Cells were trypsinized and harvested with ice-cold PBS. The cells were resuspended in 200 μL of PBS and fixed in 800 μL of ice-cold 100% ethanol, and then incubated overnight at -20 °C. The cells were collected by centrifugation, resuspended in 1 mL of low-tension buffer (0.5% Triton X-100 in PBS and 1 μg/mL RNase A), and incubated at 37 ° C for 30 minutes. Then 1 mL of PI solution (50 μg/mL) was added, and the mixture was allowed to stand at 4 ° C for 30 minutes. The cellular DNA content was then analyzed by a FACScan cytometer (Becton Dickinson).

Figure 5A shows the results of MCF-7 cells treated with various concentrations of propolis for 72 hours. Figure 5B shows the results of MDA-MB-231 cells treated with various concentrations of propolis for 24 hours. Figure 5C shows the results of MDA-MB-231 cells treated with various concentrations of propolis for 72 hours.

As shown in Figures 4 and 5, the compounds of the present invention were shown to have an inhibitory effect on the growth of cancer cells.

Western ink stain analysis

Human breast cancer MCF-7 cells (1.5 x 10 ⁶ ) on 100-mm plates were treated with various concentrations of propolis F, I and J for 24 hours. After the treatment, the cells were collected and resuspended in 100 μL of the lysis buffer. An equal amount of protein (30 μg) was mixed with 2x sample buffer and analyzed by β-actin, Bid, p21, Ac-histone 3, CTPS and gelsolin with 12.5% SDS-PAGE. The protein was electroporated to an immobilon membrane (PVDF; Millipore Corp.) and the membrane was stained with a reversible dye, guanamine black (Sigma Chemical Co.) to confirm that the protein loading was the same. Blocking was then carried out overnight with a solution of 20 mM Tris-HCl (pH 7.4), 125 mM NaCl, 0.2% Tween 20 and 3% BSA. Specific antibodies used were anti-human Bid (1:500 rabbit polyclonal antibody; Cell Signaling Technology, Inc.), anti-Ac-tissue protein (1:1000 rabbit polyclonal antibody; Cell Signaling Technology, Inc.) , anti-p21 (1:1000 rat monoclonal antibody; BDPharmingen Technology, Inc.), anti-CTPS (1:1000 rat monoclonal antibody; Taiwan ABNOVA), anti-gelsolin (1:1000 rat) Monoclonal antibody; Sigma Chemical Co.) and anti-beta-actin (1:5000 rat monoclonal antibody; Cell Signaling Technology, Inc.). Proteins were detected by chemiluminescence (ECL, Amersham).

Example 6: Determination of HDAC enzyme activity Immunocytochemistry

MCF-7 cells were cultured on 6-well culture slides and treated with propolis F, I and J for 6 hours. SAHA, known as an HDAC inhibitor, was used as a positive control. The slides were fixed in an 80% methanol solution for 30 minutes at room temperature and then washed 3 times with PBS. The cells were permeabilized with 0.3% Triton X-100 for 30 minutes at room temperature, then blocked with 10% fetal bovine serum (FBS) in PBS for 1 hour at room temperature, followed by 1 at 4 °C and 1: The 500-dilute anti-acetylation tissue protein H3 (Cell Signaling) was incubated overnight. After washing 3 times with PBS, the slides were stained with a secondary antibody that binds anti-rabbit IgG for 1.0 hour, then washed 3 times with PBS, and mounted with a mounting medium (Sigma). In a parallel control experiment, no staining was observed with no antibody added. Slides were analyzed under a fluorescent microscope with an Olympus PM30 camera (Melville, NY). Figure 6A shows the results of an immunocytochemical study.

Western ink stain determination

Western blotting measurements were performed on Ac-tissue protein 3, p21, gelsolin, CTPS and β-actin according to the method described in Example 6. Figure 6B shows the results of Western ink stain determination.

As shown in Figures 6A and 6B, the compounds of the present invention demonstrated inhibition of HDAC enzymatic activity.

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The contents and embodiments of the present invention will be more readily understood by reference to the following drawings. The purpose of the present invention is illustrated by the accompanying drawings, which illustrate the preferred embodiments. However, it is to be understood that the invention is not limited to the preferred embodiments shown.

In each figure:

Figure 1 represents the important HMBC relationship between H and C in propolis I.

Figure 2 represents the important HMBC relationship between H and C in propolis J.

Figure 3 shows the peak values of reverse preparative HPLC in Example 2.

Figure 4 (A) shows the results of staining of MCF-7 cells treated with various concentrations of propolis for 24 hours, wherein (a) refers to the control group treatment; (b), (c) and (d) refers to the concentration 5, 10, and 15 μg/mL of propolis F treatment; (e), (f), and (g) are treated with propolis I at concentrations of 2.5, 5.0, and 7.5 μg/mL, respectively; and (h) (i) and (j) are treated with propolis J at concentrations of 5, 10 and 15 μg/mL, respectively.

Figure 4 (B) shows the results of staining of MDA-MB-231 cells treated with various concentrations of propolis for 24 hours, wherein (a) refers to the control treatment; (b), (c) and (d) refers to Treatment with propolis F at concentrations of 5, 10, and 15 μg/mL; (e), (f), and (g) are treated with propolis I at concentrations of 2.5, 5.0, and 7.5 μg/mL, respectively; ), (i) and (j) are treated with propolis J at concentrations of 5, 10 and 15 μg/mL, respectively.

Figure 4 (C) shows the staining results of MCF-7 cells treated with various concentrations of propolis for 72 hours, wherein (a) refers to the control treatment; (b), (c) and (d) refer to the concentration respectively. Treatment with 5, 10 and 15 μg/mL of propolis F; (e), (f) and (g) means treatment with propolis I at concentrations of 2.5, 5.0 and 7.5 μg/mL; and (h), (i) and (j) are treated with propolis J at concentrations of 5, 10 and 15 μg/mL, respectively.

Figure 4 (D) shows the staining results of MDA-MB-231 cells treated with various concentrations of propolis for 72 hours, wherein (a) refers to the control treatment; (b), (c) refers to the concentration of 10 And 15 μg/mL of propolis F treatment; (d) and (e) are treated with propolis I at concentrations of 5.0 and 7.5 μg/mL, respectively; and (f) and (g) are at concentrations of 10 And 15 μg / mL of propolis J treatment.

Figure 4 (E) shows the number of MCF-7 cells treated with various concentrations of propolis for 72 hours.

Figure 5 (A) shows the results of flow cytometry of MCF-7 cells treated with various concentrations of propolis for 72 hours, wherein (a) refers to the control treatment; (b), (c) and (d) refers to Treatment with propolis F at concentrations of 5, 10, and 15 μg/mL; (e), (f), and (g) are treated with propolis I at concentrations of 2.5, 5.0, and 7.5 μg/mL, respectively; h), (i) and (j) are treated with propolis J at concentrations of 5, 10 and 15 μg/mL, respectively.

Figure 5 (B) shows the results of flow cytometry of MDA-MB-231 cells treated with various concentrations of propolis for 72 hours, wherein (a) refers to control treatment; (b), (c) and (d) Treatment with propolis F at concentrations of 5, 10, and 15 μg/mL; (e), (f), and (g) are treated with propolis I at concentrations of 2.5, 5.0, and 7.5 μg/mL, respectively; And (h), (i) and (j) are treated with propolis J at concentrations of 5, 10 and 15 μg/mL, respectively.

Figure 5 (C) shows the results of flow cytometry of MDA-MB-231 cells treated with various concentrations of propolis for 72 hours, wherein (a) refers to control treatment; (b) and (c) refers to concentration 10 and 15 μg/mL of propolis F treatment; and (d) and (e) are treated with propolis I at concentrations of 5.0 and 7.5 μg/mL, respectively; and (f) and (g) refer to concentration Treatment with 10 and 15 μg/mL of propolis J, respectively.

Figure 6 (A) shows the results of immunocytochemical studies in Example 7.

Fig. 6(B) shows the results of the Western blotting measurement in Example 7.

Claims (8)

Use of a compound of Formula I or Formula II for the manufacture of a medicament for the treatment of a disease associated with deacetylation of tissue proteins, wherein the structural formulas of Formulas I and II are as follows: A pharmaceutical composition for tissue protein deacetylase (HDAC) inhibitor comprising at least one compound as described in claim 1 and a pharmaceutically acceptable excipient or carrier. A pharmaceutical composition for inhibiting the growth of cancer cells as an HDAC inhibitor, comprising at least one compound as described in claim 1 and a pharmaceutically acceptable excipient or carrier. The pharmaceutical composition of claim 3, which is effective for inhibiting the growth of breast cancer cells. A pharmaceutical composition for treating a disease associated with deacetylation of a tissue protein, comprising at least one compound as described in claim 1 and a pharmaceutically acceptable excipient or carrier. A neuroprotective pharmaceutical composition comprising at least one compound as described in claim 1 and a pharmaceutically acceptable excipient or carrier. A pharmaceutical composition for treating a neurodegenerative disease, comprising at least one compound as described in claim 1 and a pharmaceutically acceptable excipient Or carrier. The pharmaceutical composition according to claim 7, wherein the neurodegenerative disease is selected from the group consisting of multiple sclerosis (MS), Huntington's disease (HD), spinal muscular atrophy (SMA), spinal cord medullary muscle. A group of atrophic (SBMA) and amyotrophic lateral sclerosis (ALS).