CN106588947B - Isoamylene radical chromocor class compound in jackfruit and its anti-osteoporosis new application - Google Patents

Abstract

The invention relates to a class of prenyl flavonoids and their application in the preparation of anti-osteoporosis drugs. The present invention separates seven prenyl flavonoids from Artocarpus heterophyllus , a plant of the genus Moraceae, which are 5'-hydroxycudraflavone A, artochamin B, artelastoxanthone, artoindonesianin Q, kuwanon T, artoheteroid A and artoheteroid B. Activity tests show that the compound of the present invention has strong inhibitory activity on cathepsin K, an important therapeutic target of osteoporosis, with an _IC50 of 0.88-93.9 mM, and can be further used for the preparation and treatment of osteoporosis medicine.

Description

Prenyl flavonoids in jackfruit and their new application in anti-osteoporosis

technical field

The invention belongs to the field of medicine and relates to new applications of medicines, in particular to the new application of prenyl flavonoids in jackfruit in the preparation of medicines for treating osteoporosis.

Background technique

Osteoporosis (Osteoporosis, OP) is a systemic bone disease characterized by degeneration of bone microstructure, increased fragility and susceptibility to fracture. With the extension of human life expectancy and the arrival of an aging society, the incidence of OP is increasing year by year. According to epidemiological statistics, the incidence of OP has accounted for 10% of the total number of middle-aged and elderly people in the world, and it has become a common and frequently-occurring disease that seriously threatens the health of middle-aged and elderly people. The number of elderly people in my country ranks first in the world. There are currently 90 million OP patients, accounting for 7.01% of the total population. Most of them are in our country. Therefore, how to effectively treat OP and prevent the occurrence of fragility fractures has become an urgent problem to be solved in the medical field.

Modern medicine believes that OP is an imbalance in the coupling of bone resorption and bone formation in the process of bone metabolism, which leads to an imbalance in calcium and phosphorus metabolism in the human body and a gradual decrease in bone density, resulting in clinical symptoms. Cathepsin K (CatK) is a cysteine protease abundantly expressed in osteoclasts, which has the strongest ability to degrade type I collagen, osteopontin and osteonectin, which account for 95% of the bone matrix. , plays a key role in the mechanism of osteoclast-mediated bone resorption. Both animal experiments and clinical studies have confirmed that CatK inhibitors can effectively inhibit bone resorption. Therefore, in recent years, CatK has been identified as an important molecular target for the treatment of OP, and the screening of new anti-OP drugs targeting this enzyme has become one of the hot spots in the field of new drug research.

Separating and identifying natural products with novel structures and significant activities from natural medicines has always been one of the main approaches for new drug discovery. Jackfruit has another name called "tree jackfruit", "wood jackfruit", "bull belly fruit", etc. It is a plant of the genus Jackfruit in the family Moraceae, and it is widely cultivated in various provinces in South my country and Taiwan. Jackfruit has multiple medicinal uses in the folk, such as treating anemia, asthma and skin diseases, relieving convulsions and ulcers, sedation, anti-syphilis, anthelmintic, etc., but there is no literature report on the application of jackfruit in the treatment of osteoporosis. In the preliminary activity screening of our research group, the 95% ethanol extract of jackfruit root showed strong inhibitory activity on human CatK. Therefore, the present invention intends to carry out in-depth research on the active extract, and previously discovered the CatK inhibitor, which provides a basis for the research and development of anti-osteoporosis drugs targeting the CatK target.

Contents of the invention

The purpose of the present invention is to provide a substance with CatK inhibitory activity, which provides a material basis for the research and development of anti-osteoporosis drugs targeting the CatK target. It specifically involves seven prenyl flavonoids extracted from jackfruit: 5′-hydroxycudraflavone A, artochamin B, artelastoxanthone, artoindonesianin Q, kuwanon T, artoheteroid A and artoheteroid B. Among them, artoheteroid A and artoheteroid B are new compounds that have not been reported in the literature.

A further object of the present invention is to provide a new application of the above-mentioned prenyl flavonoids in the preparation of drugs for treating osteoporosis.

The 7 prenyl flavonoids of the present invention have the following chemical structures:

Compounds of the present invention are prepared by the following methods:

The jackfruit root medical material (17Kg) was extracted by leakage with 95% ethanol, and the extract was concentrated under reduced pressure to obtain 1.5Kg of extract. The extract was suspended in water, extracted successively with petroleum ether, chloroform, ethyl acetate and n-butanol, and concentrated to dryness respectively. Take 532g of the extract from the chloroform extraction site, put it on a macroporous adsorption resin column (column size: 15*55cm, 5.3Kg), elute with ethanol-water gradient, and finally wash with acetone. The fractions eluted with ethanol-water (3:2) were subjected to reverse-phase silica gel column chromatography with ethanol-water (2:5, 9:10) to obtain 6 fractions: A-F. Fraction C was subjected to MCI column chromatography with ethanol-water (3:5, 9:10) to obtain fractions C1-C7. C4 and C7 obtained 7 prenylflavones by preparative separation respectively.

It is confirmed by activity screening experiments that the prenyl flavonoids in jackfruit of the present invention have obvious inhibitory activity on CatK. Among them, artelastoxanthone has a stronger activity, with a half inhibitory concentration (IC ₅₀ ) of 0.88 μM.

The prenyl flavonoids in jackfruit described in the present invention can be further prepared into a medicine for treating osteoporosis.

Description of drawings

Fig. 1 is the proton nuclear magnetic resonance spectrum ( ¹ H NMR) of the new compound artoheteroid A of the present invention.

Fig. 2 is the carbon nuclear magnetic resonance spectrum ( ¹³ H NMR) of the new compound artoheteroid A of the present invention.

Fig. 3 is the proton nuclear magnetic resonance spectrum ( ¹ H NMR) of the new compound artoheteroid B of the present invention.

Fig. 4 is the carbon nuclear magnetic resonance spectrum ( ¹³ H NMR) of the new compound artoheteroid B of the present invention.

Figure 5 is the main HMBC (H→C) correlation of the novel compounds artoheteroids A-B of the present invention.

Detailed ways

Through the specific examples given below, the present invention can be further clearly understood.

Example 1 Preparation of Prenyl Flavonoids in Jackfruit

Get jackfruit root medical material (17Kg), use 95% ethanol seepage extraction, extracting solution is concentrated under reduced pressure to obtain extractum 1.5Kg. The extract was suspended in 2L of water, extracted successively with petroleum ether, chloroform, ethyl acetate and n-butanol (volume ratio 1:1), and concentrated to dryness respectively. Get 532g of chloroform extraction part extractum, mix sample with HP-20 macroporous adsorption resin (weight ratio 1: 1), put on HP-20 macroporous adsorption resin column (column specification: 15*55cm, 5.3Kg), with ethanol-water Gradient elution (0-95%), and finally washed with acetone. Take 60% ethanol eluted fractions and perform ODS reverse-phase silica gel column chromatography with ethanol-water (2:5, 9:10) to obtain 6 fractions: A～F. Fraction C was subjected to MCI column chromatography with ethanol-water (3:5, 9:10) to obtain fractions C1-C7. The C4 and C7 fractions are further processed to obtain the 7 prenyl flavones of the present invention, the specific steps are as follows:

(1) Fraction C4 was applied to a Sephadex LH-20 gel column (eluted with methanol), and the resulting fraction was subjected to reverse-phase C18 preparative chromatography to obtain compounds 2 (8 mg), 3 (10 mg), and 4 (6 mg).

(2) Fraction C7 was subjected to reverse phase C18 preparative chromatography to obtain compounds 1 (2 mg), 5 (33 mg), 6 (10 mg) and 7 (11 mg).

Example 2 Structural Identification of Prenyl Flavonoids in Jackfruit

The isolated monomers were identified as seven prenylated flavonoids by high-resolution mass spectrometry (HR-ESI-MS) and nuclear magnetic resonance (1D NMR and 2D NMR). Among them, compounds 1-5 are known compounds, and compounds 6 and 7 are two new compounds that have not been reported in literature.

Among them, compound 1 was identified as 5′-hydroxycudraflavone A. C ₂₅ H ₂₂ O ₇ , yellow amorphous powder. ¹ HNMR (CD ₃ OD, 600MHz) δppm 7.15 (1H, s, H-6'), 6.65 (1H, d, J=10.0Hz, H-16), 6.39 (1H, s, HH-3'), 6.35(1H, s, H-8), 6.10(1H, d, J=9.0Hz, H-11), 5.69(1H, d, J=10.0Hz, H-17), 5.44(1H, d, J =9.0Hz, H-12), 1.97 (3H, s, H-15), 1.70 (3H, s, H-14), 1.47 (6H, s, H-19, 20); ¹³ CNMR (CD ₃ OD , 150MHz) δppm: 157.7(C-2), 110.5(C-3), 179.6(C-4), 157.8(C-5), 106.5(C-6), 160.3(C-7), 96.0(C -8), 157.3(C-9), 106.3(C-10), 70.2(C-11), 122.3(C-12), 139.7(C-13), 26.0(C-14), 18.7(C- 15), 116.1 (C-16), 129.5 (C-17), 79.1 (C-18), 28.5 (C-19, 20), 107.6 (C-1′), 153.5 (C-2′), 105.4 (C-3'), 152.6 (C-4'), 142.0 (C-5'), 109.9 (C-6').

Among them, compound 2 was identified as atochamin B. C ₂₅ H ₂₄ O ₇ , yellow amorphous powder. ¹ H NMR (CD ₃ OD, 600MHz) δppm 7.15 (1H, s, H-6'), 6.43 (1H, s, H-6), 6.36 (1H, s, H-3'), 6.11 (1H, d, J=9.0Hz, H-11), 5.44(1H, d, J=9.0Hz, H-12), 5.24(1H, t, J=7.2Hz, H-2"), 3.30(2H,d , J=7.2Hz, H-1″), 1.95 (3H, s, H-15), 1.79 (3H, s, H-5″), 1.71 (3H, s, H-14), 1.68 (3H, s, H-4″); ¹³ C NMR (CD ₃ OD, 150MHz) δppm 157.2 (C-2), 109.9 (C-3), 179.6 (C-4), 160.1 (C-5), 99.9 (C -6), 163.0(C-7), 107.4(C-8), 156.4(C-9), 105.5(C-10), 70.2(C-11), 122.5(C-12), 139.5(C- 13), 26.0(C-14), 18.7(C-15), 22.2(C-1″), 123.5(C-2″), 132.0(C-3″), 26.0(C-4″), 17.9 (C-5″), 108.0 (C-1′), 152.4 (C-2′), 105.4 (C-3′), 153.0 (C-4′), 141.9 (C-5′), 110.5 (C -6').

Among them, compound 3 was identified as artelastoxanthone. C ₂₅ H ₂₂ O ₇ , yellow amorphous powder. ¹ H NMR (CD ₃ OD, 600MHz) δppm 6.77 (1H, d, J = 10.0Hz, H-16), 6.45 (1H, d, J = 2.0Hz, H-8), 6.21 (1H, d, J =2.0Hz, H-6), 5.70(1H, d, J=10.0Hz, H-17), 4.68(1H, s, H-14α), 4.27(1H, s, H-14β), 3.96(1H , d, J=6.3Hz, H-12), 3.36 (1H, dd, J=6.7, 15.8Hz, H-11α), 2.44 (1H, dd, J=6.7, 15.8Hz, H-11β), 1.80 (3H, s, H-15), 1.50 (3H, s, H-20), 1.49 (3H, s, H-19); ¹³ C NMR (CD ₃ OD, 150MHz) δppm 162.1 (C-2), 112.2(C-3), 181.6(C-4), 163.0(C-5), 100.0(C-6), 165.4(C-7), 95.0(C-8), 158.1(C-9), 105.0 (C-10), 22.6(C-11), 38.4(C-12), 145.6(C-13), 112.0(C-14), 21.9(C-15), 117.7(C-16), 129.9( C-17), 78.5(C-18), 28.1(C-19), 28.1(C-20), 108.2(C-1′), 146.3(C-2′), 111.5(C-3′), 145.8 (C-4'), 137.6 (C-5'), 129.7 (C-6').

Among them, compound 4 was identified as artoindonesianin Q. C ₂₂ H ₂₂ O ₇ , yellow amorphous powder. ¹ H NMR (CD ₃ OD, 600MHz) δppm 6.75 (1H, s, H-6'), 6.62 (1H, s, H-3'), 6.46 (1H, d, J=2.2Hz, H-8) , 6.32(1H, d, J=2.2Hz, H-6), 5.08(1H, t, J=7.0Hz, H-12), 3.89(3H, s, MeO-7), 3.74(3H, s, MeO-4'), 3.04 (2H, d, J=6.7Hz, H-11), 1.61 (3H, s, H-15), 1.40 (3H, s, H-14); ¹³ C NMR (CD ₃ OD, 150MHz) δppm 163.0(C-2), 122.2(C-3), 183.6(C-4), 163.3(C-5), 98.8(C-6), 167.0(C-7), 92.8(C -8), 159.6(C-9), 106.1(C-10), 24.9(C-11), 122.6(C-12), 132.8(C-13), 25.9(C-14), 17.6(C- 15), 113.5(C-1'), 149.8(C-2'), 101.3(C-3'), 152.5(C-4'), 139.9(C-5'), 117.8(C-6') , 56.6 (7-OCH3), 56.3 (4'-OCH3).

Among them, compound 5 was identified as kuwanon T. C ₂₅ H ₂₆ O ₆ , yellow amorphous powder. ¹ H NMR (CD ₃ OD, 600MHz) δppm 6.91 (1H, d, J=8.3Hz, H-6'), 6.46 (1H, d, J=2.1Hz, H-8), 6.45 (1H, d, J=8.3Hz, H-5'), 6.19(1H, d, J=2.1Hz, H-6), 5.26(1H, m, H-2"), 5.10(1H, m, H-12), 3.40 (2H, d, J=7.0Hz, H-1″), 3.09 (2H, d, J=7.0Hz, H-11), 1.80 (3H, s, H-5″), 1.69 (3H, s , H-4"), 1.59 (3H, s, H-15), 1.35 (3H, s, H-14); ¹³ C-NMR (CD ₃ OD, 150MHz) δppm 163.2 (C-2), 122.1 ( C-3), 183.7(C-4), 163.6(C-5), 99.5(C-6), 165.5(C-7), 94.6(C-8), 159.3(C-9), 105.5(C -10), 113.8(C-1′), 154.7(C-2′), 117.7(C-3′), 159.0(C-4′), 108.1(C-5′), 128.8(C-6′ ), 23.3(C-1″), 124.0(C2″), 131.9(C-3″), 18.0(C-4″), 26.0(C-5″), 24.8(C-11), 122.6(C -12), 132.8 (C-13), 17.6 (C-14), 25.9 (C-15).

Among them, compound 6 is a red amorphous powder, HR-ESI-MS gives a quasi-molecular ion peak m/z 379.0821 ([MH] ^- , calculated value: 379.0823), and its molecular formula is determined to be C ₂₁ H ₁₆ O ₇ . Infrared spectrum (v _max 3355, 1654, 1633, 1614, 1512, 1450cm ^-1 ) and ultraviolet spectrum (λ _max 256, 308nm) showed that compound 6 had typical absorption characteristics of prenylflavonoids. The hydrogen spectrum of compound 6 shows the following proton signals: two hydrogen protons δ _H 6.53 (1H, d, J = 1.8Hz, H-8) and 6.32 (1H, d, J = 1.8Hz) coupled at the meta position on the benzene ring , H-6); a quinoid proton δ _H 6.02 (1H, s); a methoxyl δ _H 3.89 (MeO-4'); in the form of -CH ₂ -CH-C(CH ₃ )=CH ₂ fragments A partially cyclized isopentenyl δ _H occurred 4.79, 4.59 (each 1H, brs, H ₂ -14), 3.84 (1H, d, J=8.4Hz, H-12), 3.42 (1H, d, J = 16.8 Hz, H _a -11), 2.63 (1H, dd, J = 16.8, 8.4 Hz, H _b -11) and 1.82 (3H, s, H -15). ^{The 1} H-NMR spectrum of compound 6 is very similar to the known compound artomunoxanthone, the difference is that the proton of the 2,2-dimethylpyran ring spliced by artomunoxanthone at the C-7/C-8 position does not appear in the hydrogen spectrum of 6 signal, but one more aromatic proton signal δ _H 6.53 (1H, s, H-8). This indicates that there is no isopentenyl substitution at the C-8 position of 6 as in artomunoxanthone. This conclusion can be further explained in the ¹³ C-NMR spectrum of 6: The ¹³ C NMR and DEPT spectra of 6 show 21 carbon signals, namely 3 carbonyl carbons, 10 sp ² hybridized quaternary carbons (5 are oxidized ), 3 sp ² hybridized methine carbons, 1 sp ² hybridized methylene carbon, 1 sp ³ hybridized methine carbon, 1 sp ³ hybridized methylene carbon, 1 sp ³ hybridized methyl carbon and 1 methoxy group. Except for individual carbons (such as C-3, 5, 8, 1' and 6'), the chemical shift values of the ¹³ C-NMR spectrum of 6 are very similar to those of artomunoxanthone. Through HSQC and HMBC, we assigned the signals of all protons and carbon atoms of 6, and determined the connection positions of each substituent, and finally obtained the chemical structure of 6. Compound 6 is a new isopentenyl substituted flavone, which we named artoheteroid A.

Among them, compound 7 is an orange amorphous powder. HR-ESI-MS gave a quasi-molecular ion peak m/z 395.0771 ([MH] ^- , calculated value: 395.0772), and its molecular formula was determined to be C ₂₁ H ₁₆ O ₈ . The UV, IR, and ¹ HNMR spectra of compound 7 and 6 are very similar, indicating that 7 also belongs to prenyl flavonoids. ^{The 13} C NMR spectrum of 7 shows three carbonyl carbon signals δc179.4, 184.9 and 183.7, further confirming that 7 and 6 have the same quinoid structural unit. Comparing the ¹³ C NMR data of 6 and 7, it can be seen that the carbon spectrum chemical shift value of 7 is basically consistent with that of 6. The only differences are C-1' and C-6'. The C-1′ and C-6′ signals of 7 appear at δc 56.2 and 67.9, respectively, and both have an upfield shift of more than 75ppm compared with 6. This shift comes from the shielding effect of the ternary oxygen ring at the C-1′/C-6′ position formed by the further oxidation of the quinone ring. Through HSQC and HMBC spectra, we assigned the signals of all protons and carbon atoms of 7, and determined the connection positions of each substituent, and finally obtained the chemical structure of 7. Compound 7 is also a new prenyl-substituted flavone, which we named artoheteroid B.

Table 1 Hydrogen spectrum and carbon spectrum data of neoprenylflavone artoheteroids A-B

Example 3 Inhibitory activity experiment of prenyl flavones in jackfruit to CatK

40 μL of CatK (diluted with 0.1% Brii35) was added to the 96-well plate. Then add 35μL buffer solution containing the compound to be tested (400mM NaH ₂ PO ₄ -Na ₂ HPO ₄ buffer, 88mM DTT and 4mM EDTA, pH 6.8), and incubate at 25° for 10 minutes to fully combine the compound with the enzyme . Then 25 μL of the substrate Z-GPR-AMC (diluted in 1% DMSO) was added to the mixture to start the reaction. The final concentrations of substrate and CatK were 5 μM and 5.6 μM, respectively, and the total volume of each well was 100 μL. After the reaction system was incubated at 37° C. for 2 h, 50 μL of reaction termination solution (containing 100 mM CH ₃ COOH-CH ₃ COONa, 100 mM Cl ₃ CCOONa, pH 4.3) was added to each well. An Omega microwell spectrophotometer (BMG LABTECH, Germany) was used to detect the fluorescence signal intensity of AMC in the reaction system under the excitation light of 355nm and the emission light of 460nm. The inhibition rate IP of each compound was calculated according to the fluorescence value. The inhibitory rate of multiple concentrations was determined, and the IC ₅₀ value was calculated using the new drug data statistical processing software (NDST-21st Century Edition).

The calculation formula of IP is:

In the formula, F1 is the background value after adding enzyme and sample; F2 is the fluorescence value of the sample well, including sample, enzyme, substrate and buffer; F3 is the blank value of only buffer and substrate; F4 is the fluorescence value of the enzyme Normal activity values with enzyme, substrate and buffer.

Table 2 is the experimental results of the CatK inhibitory activity (IC ₅₀ inμM) of seven prenyl flavonoids according to the present invention.

Table 2 Inhibitory activity of prenylflavones in jackfruit on CatK

Claims (2)

1. the prenyl flavones in jackfruit, its structural formula is as follows: . 2. The application of the prenyl flavonoids in Jackfruit or its pharmaceutically acceptable salts in the preparation of medicines for the treatment of osteoporosis, characterized in that: the prenyl flavonoids have the following structural formula chemical structure: .