Bibenzyl succinate glycoside compound as well as preparation method and application thereof

Abstract

The invention discloses three bibenzyl succinate glycoside compounds as well as a preparation method and application thereof, and provides the bibenzyl succinate glycoside compounds as shown in formulas I, II and III or pharmaceutically acceptable salts thereof, which can effectively inhibit the content of one or more of TNF-alpha, IL-1beta or NO in RAW264.7 cells, have remarkable anti-inflammatory activity, and can be used for preparing anti-inflammatory drugs. The compound can effectively inhibit proliferation of one or more of human gastric cancer cells ASGC-7901, human breast cancer cells MCF-7 or human ovarian cancer cells A2780, has remarkable anti-tumor activity, and can be used for preparing drugs for preventing, treating or relieving inflammatory diseases and malignant tumor diseases. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a succinic acid bibenzyl ester compound and a preparation method and application thereof. Background Art

[0002] Inflammation refers to the defensive response of living tissues with vascular systems to damaging agents. Central to the inflammatory process is the vascular response, manifested primarily as redness, swelling, heat, pain, and dysfunction. Inflammation is implicated in the pathogenesis of numerous diseases, including cardiovascular and cerebrovascular diseases, diabetes, and rheumatoid arthritis.

[0003] Malignant tumors are a general term for a group of diseases that can affect any part of the body. A defining characteristic of malignant tumors is the rapid generation of abnormal cells that grow beyond their usual boundaries and invade adjacent parts of the body and spread to other organs, a process known as metastasis. Examples include breast cancer, stomach cancer, lymphoma, and ovarian cancer.

[0004] Pleione bulbocodioides (Franch.) Rolfe and Pleione yunnanensis (Rolfe) Rolfe are members of the Orchidaceae family. They have a sweet, slightly spicy flavor and cooling properties. They have the effects of clearing heat, detoxifying, and resolving phlegm and dispersing stagnation. Bibenzyl succinate is the main component of both Pleione bulbocodioides and Pleione yunnanensis (Rolfe) Rolfe. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a succinate bibenzyl ester compound with anti-inflammatory and anti-tumor activity, as well as its preparation method and application. The present invention provides three succinate bibenzyl ester compounds such as Formulas I, II, and III, or pharmaceutically acceptable salts thereof. These compounds can effectively inhibit the content of one or more of TNF-α, IL-1β, or NO in RAW264.7 cells, exhibiting significant anti-inflammatory activity, and can effectively inhibit the proliferation of one or more of human gastric cancer cells ASGC-7901, human breast cancer cells MCF-7, or human ovarian cancer cells A2780, exhibiting significant anti-tumor activity. They can be used to prepare drugs for preventing, treating, or alleviating one or more of inflammatory diseases and malignant tumors.

[0006] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0007] The present invention provides a succinic acid bibenzyl ester compound or a pharmaceutically acceptable salt thereof as shown in Formula I, II, or III.

[0008]

[0009] The present invention provides a method for preparing a succinic acid bibenzyl glycoside compound, which comprises the following steps: extracting and separating from a plant of the genus Pleione to obtain one or more of the succinic acid bibenzyl glycoside compounds represented by formulas I, II, and III; the plant of the genus Pleione is Pleione bulbocodioides (Franch.) Rolfe and / or Pleione yunnanensis (Rolfe) Rolfe.

[0010] In the present invention, preferably, the above-mentioned dibenzyl succinate compound is isolated from an extract of a plant of the genus Cyperus. Preferably, the extract of a plant of the genus Cyperus is extracted from the dried pseudobulbs of the plant of the genus Cyperus.

[0011] In the present invention, the process of separating the extract from the genus Cyperus may include the following steps:

[0012] The extract of the genus Allium is sequentially subjected to polyamide column chromatography, high pressure liquid chromatography and high performance liquid chromatography (HPLC) to obtain the succinic acid bibenzyl ester glycoside compounds shown in formulas I, II and III.

[0013] When the extract of the genus Glechoma is subjected to polyamide column chromatography, a mixed solvent of an alcohol solvent and water is used as an eluent for elution, and ultra-high performance liquid chromatography analysis is collected. R = 39min, 43min, 45min of component A;

[0014] The polyamide used in the polyamide column chromatography is 80-120 mesh polycaprolactam. 200g of sample is loaded with 2kg of polyamide filler. The column is 1m long and 0.1m in diameter. Each column holds 1kg of polyamide, and 100g of sample is loaded each time, in two batches.

[0015] The t R The value refers to the t measured under the following conditions R Value: Column is Waters Acquity HSS T3 (2.1*100mm,1.8μm); injection volume was 2μL; column temperature was 30℃; mobile phase was water (A)-methanol:acetonitrile (1:1) (B), and the gradient of gradient elution was: 0-10min, 80%-74%A; 10-20min, 74%A; 20-30min, 74%-58%A; 30-45min, 58%A; 45-55min, 58%-20%A; wavelength was 221nm; flow rate was 0.3mL / min.

[0016] In a certain embodiment, in the polyamide column chromatography, the alcohol solvent is methanol, and the elution is gradient elution. Preferably, the volume ratio of the eluent methanol-water in the gradient elution is 0, 20%, 40%, 60%, and 100%, respectively. The amount of mixed solvent used in each gradient is 3 times the column volume (v / v) (that is, the volume of the solvent used is 3 times the volume of the polyamide column), and a total of 13 components are obtained (except for methanol elution, each component is an eluent of one column volume, and methanol elution is to combine three column volumes into one component) and the 9th component is collected (the eluent collected when the amount of eluent is the 9th column volume, that is, the eluent collected when the amount of 40% methanol is 3 times the column volume).

[0017] The component A is separated by high performance liquid chromatography, and the ultra-high performance liquid chromatography analysis is collected. R = 39min, 43min, 45min of component B;

[0018] The HPLC injection volume was 5 mL; the column temperature was 25° C.; the stationary phase was C18 bonded silica gel; the HPLC column was 50 cm long, 5 cm in diameter, and had a particle size of 10 μm; the HPLC liquid phase conditions were as follows: the mobile phase was methanol (A)-water (B), the gradient elution was: 40%-90% A over 0-30 min; 100% A over 30-45 min, the wavelength was 221 nm, the flow rate was 70 mL / min, and the fractions separated after 0-15 min were collected;

[0019] The t R The value refers to the t measured under the following conditions R Value: Column is Waters Acquity HSS T3 (2.1*100mm,1.8μm); injection volume was 2μL; column temperature was 30℃; mobile phase was water (A)-methanol:acetonitrile (1:1) (B), gradient elution was: 0-10min, 80%-74%A; 10-20min, 74%A; 20-30min, 74%-58%A; 30-45min, 58%A; 45-55min, 58%-20%A, wavelength 221nm, flow rate 0.3mL / min.

[0020] The component B was prepared by HPLC to obtain R =16.5min of the succinic acid bibenzyl ester compound shown in Formula I and t R = Component C for 21–28 min;

[0021] The t R The value refers to the t measured under the following conditions RValues: The chromatographic column is Agilent Eclipse XDB-C18 (9.4*250mm, 5μm); the injection volume is 200μL; the column temperature is 25℃; the stationary phase is C18 bonded silica gel; the mobile phase is 45% methanol aqueous solution; the detection wavelength is 221nm; the flow rate is 3mL / min; the preparative liquid phase is LC3050N high performance liquid chromatograph.

[0022] In one embodiment, the component B is prepared by HPLC using an Agilent Eclipse XDB-C18 column (9.4*250 mm, 5 μm), an injection volume of 200 μL, a column temperature of 25°C, a C18 bonded silica gel stationary phase, a methanol-water solution mobile phase, preferably a 45% methanol-water solution, a detection wavelength of 221 nm, and a flow rate of 3 mL / min. The percentages are percentages by volume.

[0023] The component C was prepared by HPLC to obtain R = 13.2min and 15.5min of the succinic acid bibenzyl ester compound shown in Formula II and III;

[0024] The t R The value refers to the t measured under the following conditions R Value: Column is Prep C18 (19*250mm, 5μm); injection volume was 200μL; column temperature was 25℃; stationary phase was C18 bonded silica gel; mobile phase was 23% acetonitrile aqueous solution; detection wavelength was 221nm; flow rate was 10mL / min; preparative liquid chromatography was performed on an LC3050N high performance liquid chromatograph.

[0025] In one embodiment, the component C is prepared by HPLC using a chromatographic column. Prep C18 (19*250mm, 5μm), injection volume: 200μL; column temperature: 25°C; stationary phase: C18 bonded silica gel; mobile phase: acetonitrile-water solution; preferably 23% acetonitrile-water solution; detection wavelength: 221nm; flow rate: 10mL / min; the percentages are by volume.

[0026] In the present invention, the above-mentioned Cynanchum plant extract is prepared by conventional methods in the art. Preferably, it is prepared through the following Cynanchum plant extract preparation steps.

[0027] In a certain embodiment, the preparation method of the above-mentioned dibenzyl succinate compound can also include the following steps (preparation step of the Cymbidium plant extract): extracting the Cymbidium plant in a solvent at the reflux temperature of the solvent; obtaining the Cymbidium plant extract, filtering and drying to obtain the Cymbidium plant dry extract, suspending the Cymbidium plant dry extract in water, and extracting to obtain the Cymbidium plant extract.

[0028] In one embodiment, the solvent used in the extraction of the genus Cyperus at reflux temperature can be any solvent conventional in the art, such as a mixed solvent of an alcohol and water. Preferably, it is a mixed solvent of ethanol and water, more preferably a 60% ethanol-water solution, where the percentages are by volume.

[0029] In one embodiment, during the extraction of the Cymbidium plant at the solvent reflux temperature, the volume-to-mass ratio of the solvent to the Cymbidium plant can be conventional in the art, preferably (8-15):1, for example 10:1.

[0030] In one embodiment, during the extraction of the Cymbidium plant at the solvent reflux temperature, the Cymbidium plant is extracted 1-3 times, for example, 2 times.

[0031] In one embodiment, during the extraction of the Cymbidium plant at the solvent reflux temperature, the extraction time of the Cymbidium plant is 0.5-2 hours, preferably 1 hour.

[0032] In one embodiment, during the extraction, petroleum ether, dichloromethane, ethyl acetate and n-butanol are used in sequence to collect the n-butanol extract, which is the extract of the genus Cyperus.

[0033] In one embodiment, the mass ratio of the dry extract of the genus Allium to water is 1:(1.2-3), preferably 1:2.

[0034] In one embodiment, the extraction is performed three times with petroleum ether, dichloromethane, ethyl acetate, and n-butanol, respectively. In one embodiment, the extraction is performed with a volume ratio of 1:1 between the volume of each extraction of petroleum ether, dichloromethane, ethyl acetate, and n-butanol and water.

[0035] The same experimental method as above can also be used to isolate the succinic acid bibenzyl ester glycoside compound as shown in Formulas I, II, and III described in the present application from Pleione yunnanensis (Rolfe) Rolfe. The present invention provides a pharmaceutical composition comprising:

[0036] (i) the succinate bibenzyl ester compound as shown in Formula I, II, or III, or a pharmaceutically acceptable salt thereof; and

[0037] (ii) at least one pharmaceutically acceptable excipient.

[0038] The present invention also provides a pharmaceutical composition comprising:

[0039] (i) the bibenzyl succinate compound of Formula I, II, or III or a pharmaceutically acceptable salt thereof;

[0040] (ii) nonsteroidal anti-inflammatory active ingredients or anti-tumor active ingredients; and

[0041] (iii) at least one pharmaceutically acceptable excipient.

[0042] The bibenzyl succinate compound as shown in Formula I, II, or III or a pharmaceutically acceptable salt thereof can be used in a therapeutically effective amount.

[0043] The non-steroidal anti-inflammatory active ingredient may be an anti-inflammatory ingredient that does not contain a steroidal structure, preferably one or more of indomethacin, aspirin and quercetin;

[0044] The anti-tumor active ingredient is preferably one or more of doxorubicin, paclitaxel and cisplatin.

[0045] The selection of the pharmaceutical excipients varies depending on the route of administration and the characteristics of the action, and is usually a filler, diluent, binder, wetting agent, disintegrant, lubricant, emulsifier or suspending agent.

[0046] In a certain embodiment, the succinate-bis(benzyl) ester compound shown in Formula I, II, or III or a pharmaceutically acceptable salt thereof is the sole active ingredient.

[0047] In a certain embodiment, the concentration ratio of the dibenzyl succinate compound or its pharmaceutically acceptable salt as shown in Formula I, II, or III and the non-steroidal anti-inflammatory active ingredient (anti-tumor active ingredient) is preferably (10-30):1.

[0048] The present invention also provides a use of the bibenzyl succinate compound represented by Formula I, II, or III as described above, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as described above, in the preparation of a drug or inhibitor for preventing, treating, or alleviating a disease;

[0049] The drug is a drug for preventing, treating or alleviating diseases related to one or more of TNF-α, IL-1β or NO;

[0050] Alternatively, the drug is a drug for preventing, treating or alleviating inflammatory diseases or malignant tumor diseases;

[0051] The inhibitor is one or more inhibitors of TNF-α, IL-1β or NO.

[0052] In one embodiment, the disease associated with one or more of TNF-α, IL-1β or NO is an inflammatory disease or a malignant tumor disease.

[0053] In one embodiment, the inflammatory disease is rheumatoid arthritis, pharyngitis, otitis media, gastritis or periodontitis.

[0054] In a certain embodiment, the malignant tumor disease is breast cancer, ovarian cancer, lymphoma or gastric cancer; preferably breast cancer, ovarian cancer or gastric cancer.

[0055] In the application, one or more inhibitors of TNF-α, IL-1β or NO can be used in mammalian organisms; they can also be used in vitro, mainly for experimental purposes, for example: as a standard sample or control sample for comparison, or prepared into a kit according to conventional methods in the art to provide rapid detection of the inhibitory effect of TNF-α, IL-1β or NO.

[0056] The present invention also provides a method for preventing, treating or alleviating inflammatory diseases or malignant tumors, comprising administering (a therapeutically effective amount of) substance A to a subject in need thereof, wherein substance A is a succinic acid bibenzyl ester compound represented by formula I, II, or III as described above, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above.

[0057] The inflammatory disease or malignant tumor is as described in any of the above schemes.

[0058] The present invention also provides a method for inhibiting cell secretion of one or more of TNF-α, IL-1β or NO under in vitro, in vivo or ex vivo conditions, comprising the following steps: contacting the succinate bibenzyl ester compound shown in Formula I, II or III or a pharmaceutically acceptable salt thereof with cells.

[0059] In one embodiment, the cells can be conventional cells in the art, preferably RAW264.7 cells.

[0060] The present invention also provides a method for inhibiting tumor cell proliferation in vitro, in vivo or ex vivo, comprising the following steps: contacting the succinate bibenzyl ester compound as shown in Formula I, II, or III as claimed in claim 1 or a pharmaceutically acceptable salt thereof with cells.

[0061] In a certain embodiment, the cells can be conventional cells in the art, preferably human gastric cancer cell ASGC-7901, human breast cancer cell MCF-7, and human ovarian cancer cell A2780.

[0062] The pharmaceutical compositions of the present invention can be prepared according to the disclosed content using any method known to those skilled in the art, such as conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or lyophilizing processes.

[0063] The administration mode of the pharmaceutical composition of the present invention is not particularly limited. Representative administration modes include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration. Examples of solid oral preparations include, but are not limited to, granules, tablets, and capsules.

[0064] Unless otherwise specified, all technical and scientific terms used herein have the standard meanings in the art to which the claimed subject matter belongs. If there are multiple definitions for a term, the definition herein shall prevail.

[0065] In the present invention, "inflammatory disease" refers to the defensive response of living tissues containing blood vessels to damaging factors, typically manifested as redness, swelling, heat, pain, and functional impairment. Examples of such inflammatory diseases include rheumatoid arthritis, pharyngitis, otitis media, gastritis, or periodontitis. During the inflammatory process, the damaging factors directly or indirectly damage tissue cells. Furthermore, the inflammatory and exudative reactions dilute, kill, and surround the damaging factors. Simultaneously, the regeneration of parenchymal and interstitial cells allows the damaged tissue to repair and heal. Therefore, inflammation can be considered a unified process of both damage and resistance to damage.

[0066] "Malignant tumor" in this context is a general term for a group of diseases that can affect any part of the body. A defining characteristic of malignant tumors is the rapid generation of abnormal cells, which grow beyond their normal boundaries and invade adjacent parts of the body and spread to other organs, the latter of which is called metastasis. Examples include breast cancer, gastric cancer, lymphoma, and ovarian cancer.

[0067] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0068] The reagents and raw materials used in the present invention are commercially available.

[0069] The positive progress of the present invention is that the newly discovered succinic acid bibenzyl ester compound represented by Formula I, II, and III or a pharmaceutically acceptable salt thereof can effectively inhibit the content of one or more of TNF-α, IL-1β, or NO in RAW264.7 cells, and inhibit the proliferation of one or more of human gastric cancer cells ASGC-7901, human breast cancer cells MCF-7, or human ovarian cancer cells A2780, indicating that it has significant anti-inflammatory and anti-tumor activities. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1The succinic acid bibenzyl ester compound shown in formula I prepared in Example 1 is shown in FIG. 1 H- 1 H COSY spectrum (DMSO, 600 MHz).

[0071] Figure 2 The HSQC spectrum (DMSO, 600 MHz) of the dibenzyl succinate compound represented by Formula I prepared in Example 1 is shown.

[0072] Figure 3 The HMBC spectrum (DMSO, 600 MHz) of the dibenzyl succinate compound represented by Formula I prepared in Example 1 is shown.

[0073] Figure 4 The HPLC chart for confirming the absolute sugar configuration of the succinate bibenzyl ester compound represented by Formula I, II, and III prepared in Example 1 is shown.

[0074] Figure 5 The succinic acid bibenzyl ester compound shown in formula II prepared in Example 1 is shown in FIG. 1 H- 1 H COSY spectrum (DMSO, 600 MHz).

[0075] Figure 6 The HSQC spectrum (DMSO, 600 MHz) of the dibenzyl succinate compound represented by Formula II prepared in Example 1 is shown.

[0076] Figure 7 The HMBC spectrum (DMSO, 600 MHz) of the dibenzyl succinate compound represented by Formula II prepared in Example 1 is shown.

[0077] Figure 8 The DEPT 135° spectrum (DMSO, 150 MHz) of the dibenzyl succinate compound represented by Formula II prepared in Example 1 is shown.

[0078] Figure 9 The succinic acid bibenzyl ester compound shown in formula III prepared in Example 1 is shown in FIG. 1 H- 1 H COSY spectrum (DMSO, 600 MHz).

[0079] Figure 10 The HSQC spectrum (DMSO, 600 MHz) of the dibenzyl succinate compound represented by Formula III prepared in Example 1 is shown.

[0080] Figure 11 The HMBC spectrum (DMSO, 600 MHz) of the dibenzyl succinate compound represented by Formula III prepared in Example 1 is shown.

[0081] Figure 12 The DEPT 135° spectrum (DMSO, 150 MHz) of the dibenzyl succinate compound represented by Formula III prepared in Example 1 is shown. DETAILED DESCRIPTION

[0082] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0083] Example 1 Preparation of the succinate bibenzyl ester compound shown in Formula I, II, and III

[0084] 1) Extraction: Dried pseudobulbs of Pleione bulbocodioides (Franch.) Rolfe were extracted by reflux with 60% ethanol (10 times the volume of the medicinal material, v / m). The extraction was repeated twice, each for one hour. The extracts were filtered, combined, and dried to obtain a Pleione bulbocodioides dry extract. The extract was suspended in twice the volume of water and extracted three times sequentially with petroleum ether, dichloromethane, ethyl acetate, and n-butanol, using the same volume of solvent as water each time. The n-butanol extract was obtained, which is the Pleione bulbocodioides extract.

[0085] 2) Column chromatography: The obtained Herba Lysimachiae extract was subjected to polyamide column chromatography (the polyamide was 80-120 mesh polycaprolactam, 200 g sample was loaded with 2 kg of polyamide filler, the column length was 1 m, and the diameter was 0.1 m. Each column accommodated 1 kg of polyamide, and 100 g was loaded each time, in two batches), and gradient elution was performed with water, 20% methanol, 40% methanol, 60% methanol, and methanol, in sequence. The amount of mixed solvent used in each gradient was 3 times the column volume (v / v) (i.e., the volume of solvent used was 3 times the volume of the polyamide column), and a total of 13 components were obtained (except for methanol elution, each component was an eluent of one column volume, and methanol elution was to combine three column volumes into one component), and component 9 was collected (i.e., the eluent collected when the amount of eluent was the 9th column volume, i.e., the eluent collected when the amount of 40% methanol was 3 column volumes), and analyzed by ultra performance liquid chromatography. R = 39min, 43min, 45min of component A;

[0086] The t R The value refers to the t measured under the following conditions R Value: Column is Waters Acquity HSS T3 (2.1*100 mm, 1.8 μm); injection volume 2 μL; column temperature 30°C; mobile phase water (A)-methanol:acetonitrile (1:1) (B); gradient elution: 0-10 min, 80%-74% A; 10-20 min, 74% A; 20-30 min, 74%-58% A; 30-45 min, 58% A; 45-55 min, 58%-20% A; wavelength 221 nm; flow rate 0.3 mL / min;

[0087] After concentration under reduced pressure, the product was separated by high performance liquid chromatography (column length 50 cm, diameter 5 cm, particle size 10 μm) (injection volume: 5 mL; column temperature: 25°C; stationary phase: C18 bonded silica gel; mobile phase: methanol (A)-water (B), gradient elution (0-30 min, 40%-90% A; 30-45 min, 100% A), detection wavelength 221 nm; flow rate 70 mL / min), and the fraction aged 0-15 min, i.e., fraction B, was collected and analyzed by ultra-performance liquid chromatography. R =39min,43min,45min;

[0088] The t R The value refers to the t measured under the following conditions R Value: Column is Waters Acquity HSS T3 (2.1*100mm,1.8μm); injection volume was 2μL; column temperature was 30℃; mobile phase was water (A)-methanol:acetonitrile (1:1) (B), gradient elution (0-10min, 80%-74%A; 10-20min, 74%A; 20-30min, 74%-58%A; 30-45min, 58%A; 45-55min, 58%-20%A, wavelength 221nm, flow rate 0.3mL / min).

[0089] 3) HPLC preparation: The above component B was prepared by HPLC (chromatographic column model: Agilent Eclipse XDB-C18 (9.4*250mm, 5μm)); injection volume: 200μL; column temperature: 25°C; stationary phase: C18 bonded silica gel; mobile phase: 45% methanol in water; detection wavelength: 221nm; flow rate: 3mL / min; preparative liquid chromatography: LC3050N high performance liquid chromatograph, to obtain component C (t R =21–28min component) and t R=16.5min The compound of bibenzyl succinate shown in Formula I: (2R)-1-{4-β-D-[(E)-3-cinnamoyl-glucopyranosyloxy]benzyloxy}-4-(4-β-D-glucopyranosyloxybenzyloxy)-2-O-β-D-glucopyranosyl-2-isosuccinate (t R =16.5min).

[0090] The above component C was prepared by HPLC (chromatographic column model: Prep C18 (19*250mm, 5μm); injection volume: 200μL; column temperature: 25℃; stationary phase: C18 bonded silica gel; mobile phase: 23% acetonitrile aqueous solution; detection wavelength: 221nm; flow rate: 10mL / min; preparative liquid chromatography: LC3050N high performance liquid chromatography, obtain t R =13.2min and 15.5min of the dibenzyl succinate compounds shown in Formula II and III: (2R)-1-{4-β-D-[(E)-6-cinnamoyl-glucopyranosyloxy]benzyloxy}-4-(4-β-D-glucopyranosyloxybenzyloxy)-2-O-β-D-glucopyranoside-2-isosuccinate, (2R)-1-{4-β-D-[(E)-2-cinnamoyl-glucopyranosyloxy]benzyloxy}-4-(4-β-D-glucopyranosyloxybenzyloxy)-2-O-β-D-glucopyranoside-2-isosuccinate.

[0091] Example 2 Identification of the succinate bibenzyl ester compounds shown in Formula I, II, and III

[0092] 1) Structural identification of the bibenzyl succinate compound shown in Formula I:

[0093] ESI-MS m / z 1017.2293[MH] - , the molecular weight of the compound was determined to be 1018.

[0094] 1 H-NMR spectrum shows a group of methylene signals δ H 3.10 (1H, d, J = 17.4 Hz, H-3a) and 2.9 9 (1H, d, J = 17.4 Hz, H-3b), 2 methyl signals δ H 0.77 (3H, d, J = 6.0 Hz, Me-7) and 0.84 (3H, d, J = 6.0 Hz, Me-8), and a methine signal δ H 1.71 (1H, m, H-6). HMBC shows that H-3 and two carbonyl carbons δ C 172.28(C-1) and δ C170.08 (C-4) and oxygen-linked carbon δ C 79.62 (C-2) correlation. HMBC showed δ H 1.73 (1H, m, H-5a), δ H 1.69 (1H, m, H-5b) and C-2, δ C 23.89(C-6), δ C 23.28 (C-7), δ C 24.32 (C-8), thus obtaining the fragment structure of 2-hydroxy-2-isobutylsuccinic acid.

[0095] 1 H-NMR showed two groups of AA′BB′ signals: δ H 7.31(2H,d,J=8.4Hz,H-2′,H-6′), δ H 7.27(2H,d,J=8.4Hz,H-2″″,H-6″″), δ H 7.06(2H,d,J=8.4Hz,H-3′,H-5′), δ H 7.02 (2H, d, J = 8.4 Hz, H-3″″, H-5″″); 2 groups of oxymethylene signals: δ H 5.09(1H,d,J=12.0Hz,H-7′a), δ H 4.98(1H,d,J=12.0Hz,H-7′b), δ H 4.98(1H,d,J=12.0Hz,H-7″″a), δ H 4.90 (1H, d, J = 12.0 Hz, H-7″″b); HMBC showed H2-7′ and δ C 129.14(C-1′), δ C 129.95 (C-2′ / C-6′) correlation, H2-7″″ and δ C 129.05(C-1″″), δ C 130.06 (C-2"" / C-6"") correlation confirmed the presence of two 4-oxobenzyloxy fragments. 1 H-NMR shows three terminal protons: δ H 5.09(1H,d,J=7.8Hz,H-1″), δ H 4.86(1H,d,J=7.8Hz,H-1″″′), δ H 4.67 (1H, d, J = 7.8 Hz, H-1″″″). At the same time, according to δ HThere are overlapping peaks of oxymethylene and oxymethylene between 2.90 and 5.07, indicating the presence of three β-glucose structures. HMBC shows that the terminal protons H-1″ and H-1″″′ are respectively C 157.21 (C-4′) and δ C 157.47 (C-4") is related, indicating the presence of two gastrodin fragments. H2-7' is related to C-1, and H2-7"" is related to C-4, confirming that these two fragments are ester-linked to the parent nucleus isobutyl succinic acid.

[0096] also, 1 H-NMR shows that there is a single substituted benzene ring: δ H 7.74(2H,m,H-2″′,H-6″′), δ H 7.44(2H,m,H-3″′,H-5″′), δ H 7.44 (1H, m, H-4″′); a group of trans-coupled double bond hydrogen signals δ H 7.68 (1H, d, J = 16.2 Hz, H-7″′) and δ H 6.68 (1H, d, J = 16.2 Hz, H-8″′). HMBC showed that H-7″′ and δ C 134.29 (C-1″′) is related to H-7″′, H-8″′ and carbonyl carbon δ C 165.94 (C-9″′), thus confirming the cinnamoyl structural fragment, further combined with HMBC, HSQC and 1 H- 1 The binding position was determined by HCOSY. 1 H- 1 HCOSY showed that H-2″ was correlated with H-3″, and H-2″ was correlated with H-1″; HMBC showed that δ H 5.07 (1H, m, H-3″) and δ C 100.05(C-1″) and δ C 165.94 (C-9″′), thus confirming that the cinnamoyl structure is connected to the 3″ position of glucose connected to the benzene ring, which is consistent with the mass spectrometry fragmentation pattern, thus confirming the planar structure of Formula I.

[0097] The absolute configuration of glucose was determined by derivatization after hydrochloric acid hydrolysis: 5 mg of the compound was added to 1N HCl (2.0 ml) and refluxed at 80°C for 6 h. The product was extracted with ethyl acetate (5 ml x 3 times). The aqueous layer was evaporated to dryness under reduced pressure. The sample was then derivatized with 5 mg of D-glucose, L-glucose, and D-galactose standards. The sample was dissolved in anhydrous pyridine (1.0 ml) and L-cysteine ​​methyl ester hydrochloride (3.0 mg) was added. The derivatization reaction was continued at 60°C for 1 h. Phenyl isothiocyanate (5.0 μl) was added to the reaction product and the reaction was continued at 60°C for 1 h. The product was evaporated to dryness under reduced pressure and dissolved in 1 ml of methanol. HPLC analysis was performed at a detection wavelength of 250 nm using a mobile phase of 78% MeCN-22% H2O at a flow rate of 1 ml / min. The sugars of all three new compounds were D-glucose. This compound is dibenzyl succinate. Based on the biogenic pathway analysis of this type of compound, the absolute configuration of C-2 was determined to be R. Therefore, the three-dimensional structure of the succinic acid bibenzyl ester compound shown in Formula I is as follows:

[0098]

[0099] 2) Structural identification of the bibenzyl succinate compound shown in Formula II:

[0100] ESI-MS m / z 1017.2293[MH] - , the molecular weight of the compound was determined to be 1018.

[0101] Ⅱ and I are isomers. The NMR data of Ⅱ are similar to those of I. The main difference is that the chemical shifts of C-6″ and H-6″ are shifted to the downfield, suggesting that the cinnamoyl structure may be connected to the H-6″ position. HMBC shows δ H 4.44(1H,d,J=11.4Hz,H-6″a), δ H 4.23 (1H, d, J = 11.4 Hz, H-6″b) and carbonyl carbon δ C 166.13 (C-9″′), confirming that the phenylpropionic acid structure is connected to the 6-position of glucose. The method for determining the absolute configuration of glucose is the same as in I, and it is D-glucose. This compound is succinic acid bibenzyl ester glycoside. Based on the analysis of the biogenic pathway of this type of compound, it can be determined that the absolute configuration of C-2 is R. Therefore, the stereostructure of the succinic acid bibenzyl ester glycoside compound shown in Formula II is as follows:

[0102]

[0103] 3) Structural identification of the bibenzyl succinate compound shown in Formula III:

[0104] ESI-MS m / z 1017.2685[MH] -, the molecular weight of the compound was determined to be 1018.

[0105] Ⅲ is an isomer with Ⅱ and I. The NMR data of Ⅲ are also similar to those of I, the main difference is that H-2″ is significantly shifted to the downfield, suggesting that the cinnamoyl structure may be connected to the H-2″ position. HMBC shows δ H 4.94 (1H, m, H-2″) and carbonyl carbon δ C 165.39 (C-9″′) and terminal carbon δ C 98.19 (C-1″′), indicating that the cinnamoyl structure is attached to the 2-position of glucose. The determination of the absolute configuration of glucose is the same as in I, which is D-glucose. This compound is succinic acid bibenzyl ester glycoside. Based on the biosynthetic pathway of this type of compound, the absolute configuration of C-2 can be determined to be R. Therefore, the stereostructure of the succinic acid bibenzyl ester glycoside compound shown in Formula III is as follows:

[0106]

[0107] 4) Spectral data

[0108] Table 1 Compounds I, II, and III 1 H and 13 C NMR data (chemical shifts (δ) in ppm, coupling constants (J) in Hz)

[0109]

[0110]

[0111]

[0112]

[0113] a 1 H-NMR (600MHz DMSO-d6) and 13 C-NMR (150 MHz DMSO-d6); c Overlapping signals were identified by HSQC.

[0114] Example 3 Anti-inflammatory activity of the succinate-bisbenzyl ester compounds represented by formula I, II, and III

[0115] 1. Instrument

[0116] Microplate reader (BioTek, EPOCH); CO2 incubator (Thermo 3111); adjustable pipette (Eppendorff); microscope (Olympus CX23); biological safety cabinet (Heal Force, HFsafe-1200LC).

[0117] 2. Materials

[0118] Cell culture dish: Corning, 430167; 96-well cell culture plate: Corning, 3599; 75 cm 2 Cell culture flask: Corning, 43063; DMEM medium (Gibco, C11995500CP), RPMI 1640 culture medium (Gibco, C11875500BT); fetal bovine serum (FBS, Gibco, 2110875CP); antibacterial-antifungal agent (Antibiotic-Antimycotic, Lifetechnologies, 15240-112); phosphate buffered saline (PBS), pH 7.4 (Gibco, 10010-500BT); trypsin-EDTA (0.25%) (Gibco, 25200-056); bovine serum albumin (Lifetechnologies, 15561012); mouse TNFα ELISA kit, MultiSciences, cat. no. EK282 / 3; mouse IL-1β ELISA kit, MultiSciences, cat. no. EK201B / 3; lipopolysaccharide (LPS, Beyotime, S1732); total nitric oxide detection kit (Nanjing Jiancheng, A013-2).

[0119] 3. Methods

[0120] 1) Cells were routinely cultured in DMEM medium supplemented with 10% FBS. 20,000 cells were seeded in 96-well cell culture plates at a concentration of 10% serum.

[0121] 2) On the second day, add the drugs and negative controls prepared in the culture medium according to the experimental settings and allow the cells to react for 2 hours. LPS was diluted to 10 μg / mL in DMEM with 10% FBS and added to a 96-well cell culture plate at 10 μL / well. The cells were cultured for another 8 hours.

[0122] 3) According to the requirements of the kit, use a multifunctional microplate reader to measure the absorbance value, perform the concentration test of each indicator, calculate the concentration of each indicator, and calculate the inhibition rate of each indicator produced by different concentrations of drugs on LPS-stimulated cells, so as to obtain the IC of different compounds. 50 .

[0123] 4) Accurately weigh the sample to be tested and indomethacin, and dilute them to the working concentration using assay buffer before the experiment.

[0124] 4. Calculation

[0125]

[0126] 5. Experimental Results

[0127] 5.1 Initial screening

[0128] Table 2. Inhibition rate of NO release in RAW264.7 cells by dibenzyl succinate compounds represented by formula I, II, and III at 30 μM

[0129]

[0130] 5.2IC 50 Value determination

[0131] Table 3. IC values ​​of the inhibitory effects of dibenzyl succinate compounds represented by formula I, II, and III and indomethacin on TNF-α, IL-1β, and NO in RAW264.7 cells 50 surface

[0132]

[0133] The results are shown in Table 2 and Table 3. The results show that the succinic acid bibenzyl ester compounds shown in Formula II and III of the present invention have strong inhibitory activity against TNF-α, IL-1β, and NO, and their IC 50 The values ​​were all less than 15 μM, indicating that the bibenzyl succinate compounds represented by Formulas II and III have a significant inhibitory effect on inflammation. Therefore, the bibenzyl succinate compounds represented by Formulas II and III of the present invention can provide a new approach for preventing, treating, or alleviating inflammatory diseases and can also be developed as anti-inflammatory drugs.

[0134] Example 4 Antitumor Activity of Bis(2-Benzyl)-5-Succinate Compounds Represented by Formulas I, II, and III

[0135] 1. Instrument

[0136] Microplate reader (BioTek, EPOCH); CO2 incubator (Thermo 3111); adjustable pipette (Eppendorff); microscope (Olympus CX23); biological safety cabinet (Heal Force, HFsafe-1200LC).

[0137] 2. Materials

[0138] Cell culture dish: Corning, 430167; 96-well cell culture plate: Corning, 3599; 75 cm 2Cell culture flasks: Corning, 43063; Dulbecco's Modified Eagle's Medium (Gibco, C11995500CP), RPMI 1640 Medium (Gibco, C11875500BT); Fetal bovine serum (FBS, Gibco, 2110875CP); Antibiotic-Antimycotic (Life Technologies, 15240-112); Phosphate buffered saline (PBS), pH 7.4 (Gibco, 10010-500BT); Trypsin-EDTA (0.25%) (Gibco, 25200-056); Bovine serum albumin (Life Technologies, 15561012); Cell Titer Glo reagent (Promega, USA); human gastric cancer cell ASGC-7901, human breast cancer cell MCF-7, and human ovarian cancer cell A2780 were provided by Shanghai Biolei Biotechnology Co., Ltd.

[0139] 3. Methods

[0140] 1) Cells were routinely cultured in DMEM medium supplemented with 10% FBS. 20,000 cells were seeded in 96-well cell culture plates at a concentration of 10% serum.

[0141] 2) On the second day, add drugs and negative controls prepared in the culture medium according to the experimental settings.

[0142] 3) According to the kit requirements, use a multifunctional microplate reader to measure the absorbance value and calculate the IC value of each compound for tumor cell growth inhibition. 50 value.

[0143] 4) Accurately weigh the test sample and doxorubicin, and dilute them to the working concentration using assay buffer before the experiment.

[0144] 4. Calculation

[0145]

[0146] 5. Experimental Results

[0147] Table 4. IC values ​​of the inhibitory rates of the succinate-bibenzyl ester compounds represented by formula I, II, and III on tumor cell growth 50 value

[0148]

[0149] The results are shown in Table 4. The results indicate that the bibenzyl succinate compounds represented by Formulas I and II of the present invention have strong or moderate inhibitory activity against human gastric cancer cells ASGC-7901, human breast cancer cells MCF-7, and human ovarian cancer cells A2780, indicating that the bibenzyl succinate compounds represented by Formulas I and II have a certain inhibitory effect on tumors. Therefore, the bibenzyl succinate compounds represented by Formulas I and II of the present invention can provide a new approach for the prevention, treatment, or alleviation of malignant tumors and can also be developed as anti-tumor drugs.

Claims

1. A succinic acid bibenzyl ester compound as shown in formula I, II, or III, or a pharmaceutically acceptable salt thereof, 2. A method for preparing a succinic acid bibenzyl ester compound, characterized in that: The method comprises the following steps: extracting and separating from Pleione bulbocodioides (Franch.) Rolfe plants to obtain one or more of the succinic acid bibenzyl ester compounds as shown in formula I, II and III as claimed in claim 1; the Pleione bulbocodioides plants are Pleione bulbocodioides (Franch.) Rolfe and / or Pleione yunnanensis (Rolfe) Rolfe.

3. The method for preparing the bibenzyl succinate compound according to claim 2, wherein: The succinic acid bibenzyl ester glycoside compound is isolated from an extract of a plant of the genus Cyperus; preferably, the extract of a plant of the genus Cyperus is extracted from the dried pseudobulbs of the plant of the genus Cyperus; The separation process from the extract of the genus Allium plant comprises the following steps: subjecting the extract of the genus Allium plant to polyamide column chromatography, high pressure liquid chromatography and high performance liquid chromatography in sequence to obtain the succinic acid bibenzyl ester glycoside compound.

4. The method for preparing the bibenzyl succinate compound according to claim 3, wherein: It includes one or more of the following steps: ① When the extract of the genus Allium plant is subjected to polyamide column chromatography, a mixed solvent of an alcohol solvent and water is used as an eluent for elution, and the ultra-high performance liquid chromatography analysis is collected. R = 39min, 43min, 45min of component A; The polyamide used in the polyamide column chromatography is 80-120 mesh polycaprolactam, with a column length of 1 m and a diameter of 0.1 m; The R The value refers to the t measured under the following conditions R Value: Column is Waters Acquity HSS T3 (2.1*100mm, 1.8μm); injection volume 2μL; column temperature 30℃; mobile phase water (A)-methanol:acetonitrile (1:1) (B); gradient elution gradient: 0~10min, 80%~74%A; 10~20min, 74%A; 20~30min, 74%~58%A; 30~45min, 58%A; 45~55min, 58%~20%A; wavelength 221nm; flow rate 0.3mL / min; ② The component A is separated by high pressure liquid chromatography, and the ultra-high performance liquid chromatography analysis is collected. R = 39min, 43min, 45min of component B; The injection volume of the high pressure liquid chromatography: 5 mL; Column temperature: 25°C; stationary phase is C18 bonded silica gel; the chromatographic column of the high pressure liquid chromatography is 50 cm long, 5 cm in diameter, and has a particle size of 10 μm; the liquid phase conditions of the high pressure liquid chromatography are: the mobile phase is methanol (A)-water (B), and the gradient of the gradient elution is: 0-30min, 40%-90% A; 30-45min, 100% A; 45-50min: 40% A; wavelength 221nm; flow rate 70mL / min; collect 0-15min components; The R The conditions for determining the value are the same as those described in step ① R Value determination conditions; ③ The component B was prepared by high performance liquid chromatography to obtain t R =16.5min of the succinic acid bibenzyl ester compound as shown in Formula I and t R = 21–28min component C; The R The value refers to the t measured under the following conditions R Values: chromatographic column is Agilent Eclipse XDB-C18 (9.4*250mm, 5μm); injection volume is 200μL; column temperature is 25℃; stationary phase is C18 bonded silica gel; mobile phase is 45% methanol aqueous solution; detection wavelength is 221nm; flow rate is 3mL / min; ④ The component C was prepared by high performance liquid chromatography to obtain R = 13.2min and 15.5min of the succinic acid bibenzyl ester compounds shown in formula II and III; The R The value refers to the t measured under the following conditions R Value: Column is Prep C18 (19*250mm, 5μm); injection volume is 200μL; column temperature is 25℃; stationary phase is C18 bonded silica gel; mobile phase is 23% acetonitrile aqueous solution; detection wavelength is 221nm; flow rate is 10mL / min.

5. The method for preparing the bibenzyl succinate compound according to claim 4, characterized in that: The preparation of the extract of the genus Cymbidium plant comprises the following steps: extracting the genus Cymbidium plant in a solvent at the reflux temperature of the solvent; obtaining an extract of the genus Cymbidium plant, filtering and drying the extract of the genus Cymbidium plant to obtain a dry extract of the genus Cymbidium plant, suspending the dry extract of the genus Cymbidium plant in water, and extracting to obtain the extract of the genus Cymbidium plant.

6. The method for preparing the bibenzyl succinate compound according to claim 5, characterized in that: The preparation method meets one or more of the following conditions: (1) In the step ①, the alcohol solvent is methanol, and the elution is gradient elution. Preferably, the volume ratio of the eluent methanol-water in the gradient elution is 0, 20%, 40%, 60%, and 100%, respectively. The amount of the mixed solvent used in each gradient is 3 column volumes (v / v), and one column volume is taken as one component, and the 9th component is collected; (2) In step ③, the HPLC column is Agilent Eclipse XDB-C18 (9.4*250 mm, 5 μm), and the mobile phase is methanol-water solution; (3) In step ④, the chromatographic column of the high performance liquid chromatography is Prep C18 (19*250mm, 5μm); mobile phase is acetonitrile-water solution; (4) During the extraction of the genus Glechoma at the solvent reflux temperature, the solvent is a mixed solvent of an alcohol solvent and water; the mixed solvent of the alcohol solvent and water is preferably a mixed solvent of ethanol and water; (5) During the extraction of the Cymbidium plant at the solvent reflux temperature, the volume mass ratio of the solvent to the Cymbidium plant is (8-15):1; (6) During the extraction process of the plant of the genus Cymbidium at the solvent reflux temperature, the number of extractions of the plant of the genus Cymbidium is 1-3 times; (7) During the extraction of the plant from the genus Cymbidium at the solvent reflux temperature, the extraction time of the plant from the genus Cymbidium is 0.5-2h; (8) The mass ratio of the extract of the dried plant of the genus Dictyophora to water is 1:(1.2-3); In the extraction described in (9), extraction is carried out with petroleum ether, dichloromethane, ethyl acetate and n-butanol in sequence, and the n-butanol extract is collected to obtain the extract of the genus Allium.

7. The method for preparing the bibenzyl succinate compound according to claim 6, characterized in that: The preparation method meets one or more of the following conditions: (1) In step ③, the HPLC column is Agilent Eclipse XDB-C18 (9.4*250 mm, 5 μm), and the mobile phase is 45% methanol in water; (2) In step ④, the chromatographic column of the high performance liquid chromatography is Prep C18 (19*250mm, 5μm); mobile phase is 23% acetonitrile aqueous solution; (3) During the extraction of the plant of the genus Glechoma at a solvent reflux temperature, the solvent is a 60% ethanol aqueous solution; (4) During the extraction of the genus Cymbidium plant at the solvent reflux temperature, the volume mass ratio of the solvent to the genus Cymbidium plant is 10:1; (5) During the extraction of the plant from the genus Cymbidium at the solvent reflux temperature, the extraction times of the plant from the genus Cymbidium is 2 times; (6) During the extraction process of the plant of the genus Cymbidium at the solvent reflux temperature, the extraction time of the plant of the genus Cymbidium is 1 hour; (7) The mass ratio of the dry extract of the genus Cyperus to water is 1:2; (8) In the extraction, petroleum ether, dichloromethane, ethyl acetate and n-butanol are used for extraction three times each; In the extraction described in (9), the volume ratio of each extraction volume of petroleum ether, dichloromethane, ethyl acetate and n-butanol to water is 1:

1.

8. A pharmaceutical composition, characterized in that It satisfies one or both of the following scenarios: Option 1 includes: (i) a succinic acid bibenzyl ester compound as shown in Formula I, II, or III, or a pharmaceutically acceptable salt thereof; and (ii) at least one pharmaceutical excipient; and Option 2, which includes: (i) a succinic acid bibenzyl ester compound as shown in Formula I, II, or III, or a pharmaceutically acceptable salt thereof; (ii) non-steroidal anti-inflammatory active ingredients or anti-tumor active ingredients; and (iii) at least one pharmaceutical excipient; The non-steroidal anti-inflammatory active ingredient may be an anti-inflammatory ingredient without a steroidal structure, preferably one or more of indomethacin, aspirin and quercetin; the anti-tumor active ingredient is preferably one or more of doxorubicin, paclitaxel and cisplatin; The pharmaceutical excipients may be one or more of fillers, diluents, binders, wetting agents, disintegrants, lubricants, emulsifiers and suspending agents; Preferably, the succinic acid bibenzyl ester compound or its pharmaceutically acceptable salt as shown in Formula I, II, III is the only active ingredient; The concentration ratio of the succinic acid bibenzyl ester compound or its pharmaceutically acceptable salt as shown in Formula I, II, III and the non-steroidal anti-inflammatory active ingredient (anti-tumor active ingredient) is preferably (10-30):

1.

9. Use of a substance A in the preparation of a drug or inhibitor for preventing, treating or alleviating a disease; characterized in that: The substance A is the succinic acid bibenzyl ester compound as shown in formula I, II, III as claimed in claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as claimed in claim 8; The inhibitor is one or more inhibitors of TNF-α, IL-1β or NO; The drug is a drug used to prevent, treat or alleviate inflammatory diseases or malignant tumor diseases; Alternatively, the drug is a drug for preventing, treating or alleviating diseases associated with one or more of TNF-α, IL-1β or NO; Preferably, the succinic acid bibenzyl ester compound or a pharmaceutically acceptable salt thereof as shown in Formula I, II, or III is the sole active ingredient.

10. The use according to claim 9, characterized in that The disease related to one or more of TNF-α, IL-1β or NO is an inflammatory disease or a malignant tumor disease; The inflammatory disease is rheumatoid arthritis, pharyngitis, otitis media, gastritis or periodontitis; the malignant tumor disease is breast cancer, ovarian cancer, lymphoma or gastric cancer; preferably breast cancer, ovarian cancer or gastric cancer.

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