Bibenzyl derivatives, their preparation methods and applications

Abstract

The present invention relates to the field of natural medicines, and particularly relates to bibenzyl derivatives, a preparation method thereof and applications. The present invention provides a pair of bibenzyl derivatives with anti-inflammatory activity, a preparation method thereof and applications. The present invention isolates two bibenzyl derivatives shown in Formula I and Formula II from the aerial parts of Dendrobium hercoglossum. The bibenzyl derivatives can effectively inhibit the production of NO by LPS-induced RAW264.7 cells, have significant anti-inflammatory activity, and have broad development and application prospects in the preparation of anti-inflammatory drugs.

Description

Technical Field

[0001] The present invention relates to the field of natural medicines, and particularly to bibenzyl derivatives and their preparation methods and applications. Background Art

[0002] Inflammation is a defensive response of the body to external stimuli or tissue damage, which can eliminate harmful stimuli, repair damaged tissues, and restore tissue structure and function. However, excessive or inappropriate inflammation will trigger various diseases, such as diabetes, sepsis, atherosclerosis, autoimmune diseases, and even tumors.

[0003] Currently, steroidal hormones and non-steroidal drugs (such as aspirin, diclofenac, and ibuprofen) are commonly used clinically to treat inflammation. Although these drugs have significant effects, they may cause serious side effects after administration, such as osteoporosis caused by glucocorticoids, induced and aggravated infections, gastrointestinal reactions, kidney damage, and liver damage caused by non-steroidal anti-inflammatory drugs. Therefore, it is of great significance to search for and develop anti-inflammatory drugs with low toxicity and significant anti-inflammatory effects. Summary of the Invention

[0004] In view of this, the present invention provides bibenzyl derivatives and their preparation methods and applications. The present invention provides bibenzyl derivatives with significant anti-inflammatory activity and their preparation methods and applications. The present invention discovers through experiments that, taking the model of LPS-induced RAW264.7 to produce NO for anti-inflammatory activity evaluation, the compounds shown in Formula I and / or Formula II can inhibit the production of NO, reflecting anti-inflammatory activity. It shows that the above compounds can be used to prepare medicines for treating and / or alleviating inflammation.

[0005] In order to achieve the above invention objectives, the present invention provides the following technical solutions:

[0006] The present invention provides bibenzyl derivatives, which include compounds having the structure shown in Formula I and / or compounds having the structure shown in Formula II;

[0007]

[0008] In some specific embodiments of the present invention, the bibenzyl compounds further include one or more of their derivatives, their analogs, their pharmaceutically acceptable salts, their prodrugs, or their metabolites.

[0009] The present invention also provides a preparation method of the bibenzyl derivatives, which includes the following steps:

[0010] Step 1: Take the aerial part of Dendrobium hercoglossum, crush it, extract it with an ethanol aqueous solution, filter, and concentrate to obtain an ethanol extract;

[0011] Step 2: Prepare a suspension by mixing the ethanol extract with water at a volume ratio of 1:1, and successively extract it with petroleum ether, ethyl acetate, and n-butanol. Concentrate each extract to obtain extracts at each level.

[0012] Step 3: Take the ethyl acetate extract, and trace and separate the compound with a characteristic ultraviolet absorption at 435 nm by liquid chromatography to obtain the bibenzyl derivative.

[0013] In some specific embodiments of the present invention, the ethanol aqueous solution includes an ethanol aqueous solution with a volume concentration of 95% or more.

[0014] In some specific embodiments of the present invention, the concentration temperature includes 50°C.

[0015] In some specific embodiments of the present invention, the preparation method of the bibenzyl derivative includes:

[0016] S1) After crushing the aerial parts (30.0 kg) of Dendrobium hercoglossum, extract it 3 times with an ethanol aqueous solution with a volume 2 times that of the raw material. After filtering the obtained extracts, combine and concentrate them into an ethanol extract.

[0017] S2) Prepare a suspension by mixing the ethanol extract with water at a volume ratio of 1:1, successively extract it with petroleum ether, ethyl acetate, and n-butanol, and then concentrate each extract to prepare an extract.

[0018] S3) Take the ethyl acetate extract and elute it through a normal-phase silica gel column under reduced pressure with petroleum ether - ethyl acetate (20:1 → 0:1, V / V) to obtain 10 fractions, denoted as Fr.1 - Fr.10.

[0019] S4) Through liquid chromatography analysis of the components of Fr.1 - Fr.10, it is found that two liquid chromatography peaks with a characteristic ultraviolet absorption at 435 nm appear in Fr.4.

[0020] S5) Gradient elute the fraction Fr.4 through a C18 reversed-phase silica gel column with methanol - water (1:9 → 8:2, V / V) to obtain 5 fractions, denoted as Fr.4-1 - Fr.4-5.

[0021] S6) Through liquid chromatography analysis of the components of Fr.4-1 - Fr.4-5, it is found that one liquid chromatography peak with a characteristic ultraviolet absorption at 435 nm appears in each of the components Fr.4-2 and Fr.4-4.

[0022] S7) Elute the fraction Fr.4-2 through a normal-phase silica gel column with chloroform - methanol (70:1, V / V) to obtain the compound with the formula I structure having a characteristic ultraviolet absorption at 435 nm.

[0023] S8) The fraction Fr.4-4 was eluted through a normal-phase silica gel column with chloroform-methanol (90:1, V / V) to obtain a compound of formula II with a characteristic ultraviolet absorption at 435 nm.

[0024] The present invention also provides the use of any of the following in the preparation of a medicament for treating and / or alleviating inflammation or an inflammatory response;

[0025] (I), the bibenzyl derivative; and / or

[0026] (II), the bibenzyl derivative prepared by the preparation method.

[0027] In some specific embodiments of the present invention, the treating and / or alleviating inflammation or an inflammatory response includes inhibiting the production of pro-inflammatory factors.

[0028] In some specific embodiments of the present invention, the pro-inflammatory factors include one or more of tumor necrosis factor, interleukin, prostaglandin, nitric oxide or reactive oxygen species.

[0029] In some specific embodiments of the present invention, the pro-inflammatory factor includes nitric oxide.

[0030] In some specific embodiments of the present invention, the inflammatory response includes one or more of body redness, swelling, fever or pain.

[0031] The present invention also provides an anti-inflammatory medicament, which includes any of the following and a pharmaceutically acceptable excipient:

[0032] (I), the bibenzyl derivative; and / or

[0033] (II), the bibenzyl derivative prepared by the preparation method.

[0034] In some specific embodiments of the present invention, the dosage form of the anti-inflammatory medicament includes tablets, capsules, powder injections or suspensions.

[0035] The present invention also provides a method for treating and / or alleviating inflammation or an inflammatory response, by administering to a subject any of the following:

[0036] (I), the bibenzyl derivative; and / or

[0037] (II), the bibenzyl derivative prepared by the preparation method.

[0038] The present invention includes but is not limited to achieving the following beneficial effects:

[0039] The present invention provides a pair of compounds for treating and / or alleviating inflammation, having the structures shown in Formula I and / or Formula II, which are novel bibenzyl derivatives. Using the model of LPS-induced RAW264.7 to produce NO for anti-inflammatory activity evaluation, the compounds shown in Formula I and / or Formula II can inhibit the production of NO, reflecting anti-inflammatory activity. Therefore, it is indicated that the above compounds can be used to prepare drugs for treating and / or alleviating inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0041] Figure 1 1H NMR spectrum of the compound with the structure of Formula I prepared in Example 1; 1 1H NMR spectrum;

[0042] Figure 2 13C NMR spectrum of the compound with the structure of Formula I prepared in Example 1; 13 13C NMR spectrum;

[0043] Figure 3 HSQC spectrum of the compound with the structure of Formula I prepared in Example 1;

[0044] Figure 4 1H- 1 H COSY spectrum of the compound with the structure of Formula I prepared in Example 1; 1 1H-1H COSY spectrum;

[0045] Figure 5 HMBC spectrum of the compound with the structure of Formula I prepared in Example 1;

[0046] Figure 6 HRESIMS spectrum of the compound with the structure of Formula I prepared in Example 1;

[0047] Figure 7 Single crystal structure diagram of the compound with the structure of Formula I prepared in Example 1;

[0048] Figure 8 1H NMR spectrum of the compound with the structure of Formula II prepared in Example 1; 1 1H NMR spectrum;

[0049] Figure 9 13C NMR spectrum of the compound with the structure of Formula II prepared in Example 1; 13 13C NMR spectrum;

[0050] Figure 10 HSQC spectrum of the compound with the structure of Formula II prepared in Example 1;

[0051] Figure 11Showing the compound of formula II prepared in Example 1 1 H- 1 H COSY spectrum;

[0052] Figure 12 Showing the HMBC spectrum of the compound of formula II prepared in Example 1;

[0053] Figure 13 Showing the HRESIMS spectrum of the compound of formula II prepared in Example 1. Detailed implementation manners

[0054] The present invention discloses bibenzyl derivatives, their preparation methods and applications. Those skilled in the art can draw on the content of this article and appropriately improve process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0055] As one of the traditional precious medicinal materials, Dendrobium can be used as a drug for treating diseases such as gastrointestinal tract and cardiovascular diseases. A variety of chemical components have been isolated from Dendrobium, such as biphenyls, phenanthrenes, fluorenones, sesquiterpenes and alkaloids, etc. These components have been found to have functions such as anti-tumor, anti-inflammatory, anti-thrombotic, hypoglycemic and immunomodulatory effects.

[0056] The present invention provides a pair of bibenzyl derivatives with significant anti-inflammatory activity and novel structures, including: the compound of formula I and / or the compound of formula II:

[0057]

[0058] The present invention provides a preparation method of the bibenzyl derivative with anti-inflammatory activity described in the above technical solution, including:

[0059] S1) After crushing the aerial parts of Dendrobium hercoglossum (30.0 Kg), extract it 3 times with an ethanol aqueous solution with a volume 2 times that of the raw material, and after filtering the obtained extraction liquid, combine and concentrate it into an ethanol extract;

[0060] S2) Make a suspension with a volume ratio of ethanol extract to water of 1:1, and extract it successively with petroleum ether, ethyl acetate and n-butanol, and then concentrate each extraction liquid to make an extract;

[0061] S3) Take the ethyl acetate extract and elute it through a normal-phase silica gel column under reduced pressure with petroleum ether - ethyl acetate (20:1 → 0:1, V / V) to obtain 10 fractions, denoted as Fr.1 - Fr.10;

[0062] S4) By performing liquid chromatography analysis on fractions Fr.1 to Fr.10, it was found that two liquid peaks with a characteristic ultraviolet absorption at 435 nm appeared in Fr.4.

[0063] S5) The fraction Fr.4 was eluted through a C18 reverse-phase silica gel column with a gradient of methanol-water (1:9 → 8:2, V / V) to obtain 5 fractions, denoted as Fr.4-1 to Fr.4-5;

[0064] S6) By performing liquid chromatography analysis on fractions Fr.4-1 to Fr.4-5, it was found that a liquid peak with a characteristic ultraviolet absorption at 435 nm appeared in fractions Fr.4-2 and Fr.4-4, respectively.

[0065] S7) The fraction Fr.4-2 was eluted through a normal-phase silica gel column with chloroform-methanol (70:1, V / V) to obtain a compound of formula I with a characteristic ultraviolet absorption at 435 nm.

[0066] S8) The fraction Fr.4-4 was eluted through a normal-phase silica gel column with chloroform-methanol (90:1, V / V) to obtain a compound of formula II with a characteristic ultraviolet absorption at 435 nm.

[0067] In the present invention, the anti-inflammatory activity was evaluated using the model of LPS-induced RAW264.7 to produce NO. The results showed that the compounds shown in formula I and formula II could effectively inhibit the production of NO, demonstrating anti-inflammatory activity.

[0068] In the present invention, the RAW264.7 is a mouse monocyte macrophage leukemia cell.

[0069] The LPS is lipopolysaccharide, which is the main component of the cell wall of Gram-negative bacteria.

[0070] Based on this, the present invention provides the use of the above compounds or the compounds prepared by the above preparation method for inhibiting the production of pro-inflammatory factors.

[0071] Preferably, in the present invention, the pro-inflammatory factors include one or more of tumor necrosis factor (TNF-α), various interleukins, prostaglandin (PG), nitric oxide (NO), reactive oxygen species (ROS), etc.

[0072] The present invention provides the use of the above compounds or the compounds prepared by the above preparation method as NO production inhibitors.

[0073] The present invention provides the use of the above compounds or the compounds prepared by the above preparation method in the preparation of drugs for treating and / or alleviating inflammation or inflammatory reactions.

[0074] Preferably, the inflammatory response of the present invention includes one or more of body redness, swelling, fever, and pain.

[0075] The present invention provides a drug for treating and / or alleviating inflammatory response, comprising a compound of formula I structure and / or a compound of formula II structure, and a pharmaceutically acceptable adjuvant;

[0076] Compared with the prior art, the present invention provides a pair of compounds for treating and / or alleviating inflammation, having the structures shown in formula I and / or formula II, which are novel dibenzyl derivatives. Using LPS-induced RAW264.7 to produce NO as a model for anti-inflammatory activity evaluation, the compounds shown in formula I and / or formula II can inhibit the production of NO, reflecting anti-inflammatory activity. Therefore, it is indicated that the above compounds can be used to prepare drugs for treating and / or alleviating inflammation.

[0077] Unless otherwise specified, the raw materials and reagents used in the dibenzyl derivatives, their preparation methods and applications provided by the present invention can be purchased from the market.

[0078] The following further elaborates the present invention in conjunction with examples:

[0079] Example 1 Preparation and Structure Identification of Compounds

[0080] 1.1 Instruments and Reagents

[0081] Bruker AV-500 superconducting nuclear magnetic resonance spectrometer (Bruker Corporation, Switzerland); Autospec300 mass spectrometer (VG Company, UK); analytical high performance liquid chromatograph (Agilent Company, USA); semi-preparative high performance liquid chromatograph (Dionex Company, USA); N-1000 (2L) vertical rotary evaporator and CA-1111 cooling water circulation device (Shanghai Ailang Instrument Co., Ltd.); SHZ-D(Ⅲ) circulating vacuum pump (Shanghai Longtuo Instrument and Equipment Co., Ltd.); AS220.R2 ten-thousandth electronic balance (RADWAG Wagi Elektroniczne); Sephadex LH-20 gel (Merck Co., Ltd.); C18 reversed-phase silica gel (20 - 45μm, Fuji Silysia Chemical Ltd, Japan); silica gel for column chromatography and thin layer chromatography silica gel plate (Qingdao Marine Chemical Factory); deuterated reagents and chromatographic methanol (Merck Company, Germany); common organic reagents such as 95% ethanol, redistilled methanol, ethyl acetate, chloroform, petroleum ether, acetone, etc. (Tianjin Kemiou, Tianjin Fuchen, Guangzhou Guanghua, etc. companies).

[0082] 1.2 Preparation and Structure Identification of Compounds

[0083] The Dendrobium hercoglossum samples were collected from the Dendrobium cultivation base in Baoshan City, Yunnan Province in March 2021. After being identified by Associate Researcher Huang Shengzhuo of the Institute of Biosciences, Chinese Academy of Tropical Agricultural Sciences, they were confirmed to be Dendrobium hercoglossum Rchb.f. of the genus Dendrobium Sw. in the family Orchidaceae. The specimens are stored in the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences.

[0084] After crushing the above-ground parts of Dendrobium hercoglossum, they were extracted three times with an ethanol aqueous solution with a volume concentration of 95% (at 25°C ± 5°C, for 3 days each time, and the ratio of medicinal materials to solvent used for extraction was 1:2). The obtained extraction solutions were filtered and combined, and then concentrated into an extract at 50°C. After that, water was added and stirred to make a 1:1 suspension, and then successively extracted with equal volumes of petroleum ether, ethyl acetate, and n-butanol until the extraction solutions were colorless; each extraction layer was evaporated and concentrated at 50°C to make an extract for use.

[0085] Take the ethyl acetate layer extract and pass it through a reduced-pressure normal-phase column (silica gel H), and use petroleum ether:ethyl acetate (20:1 → 0:1, V / V) for systematic gradient elution to obtain 10 fractions (Fr.1 - Fr.10). Through liquid chromatography analysis of the Fr.1 - Fr.10 components, it was found that two liquid-phase peaks with a characteristic ultraviolet absorption at 435 nm appeared in Fr.4.

[0086] The 4th fraction Fr.4 (3.3 g, petroleum ether:ethyl acetate = 8:1) was chromatographed on an ODS reversed-phase silica gel column, and gradient elution with methanol-water was carried out with a concentration change from 10% to 80% to obtain 5 fractions (Fr.4-1 - Fr.4-5). Through liquid chromatography analysis of the Fr.4-1 - Fr.4-5 components, it was found that a liquid-phase peak with a characteristic ultraviolet absorption at 435 nm appeared in the Fr.4-2 and Fr.4-4 components respectively.

[0087] The 2nd fraction Fr.4-2 (173.8 mg, 30% methanol) was gradient eluted with chloroform-methanol (70:1) on a normal-phase silica gel column (H silica gel) to obtain the compound of formula I with a characteristic ultraviolet absorption at 435 nm (17.9 mg).

[0088] The 4th fraction Fr.4-4 (295.9 mg, 40% methanol) was gradient eluted with chloroform-methanol (90:1) on a normal-phase silica gel column (H silica gel) to obtain the compound of formula II with a characteristic ultraviolet absorption at 435 nm (1.5 mg).

[0089] The compounds of formula I and formula II prepared in Example 1 were subjected to structure identification, and the test results are as Figures 1 to 13 , and the high-resolution mass spectrum of the compound of formula I is m / z 422.1580 [M+H]+ , with the molecular formula C 24 H 23 NO 6 ; The high-resolution mass spectrometry m / z of the compound with the structure of Formula II: 408.1417 [M+H] + , with the molecular formula C 23 H 21 NO 6 ; The 1 H NMR (500 MHz) and 13 C NMR (125 MHz) data of the compound with the structure of Formula I and the compound with the structure of Formula II are shown in Table 1:

[0090] Table 1 NMR data of the compound with the structure of Formula I and the compound with the structure of Formula II

[0091]

[0092] a The solvent is deuterated methanol, b The solvent is deuterated chloroform

[0093] Example 2 Determination of the anti-inflammatory activities of the compound with the structure of Formula I and the compound with the structure of Formula II

[0094] RAW264.7 (mouse monocyte macrophage leukemia cells) was selected and 100 μL of cells with a concentration of 5×10 4 cells / mL were inoculated on a 96-well flat-bottom cell culture plate and cultured at 37 °C, 5% CO 2 , and under the condition of more than 90% humidity. After 24 h, 50 μL of the prepared test compound solution was added, and the cells were continuously cultured under this condition. After 1 h, 50 μL of the prepared LPS (final concentration 1 μg / mL) solution was added. After 24 h, 100 μL of the supernatant was taken from each well and transferred to a new 96-well plate. Then, 100 μL of Griess reagent was added to each well and mixed by the cross-cross method. The absorbance of each well was measured and recorded at a wavelength of 540 nm using an enzyme-linked immunosorbent assay reader. The NO inhibition rate was calculated according to the following formula. The control group was (quercetin) indomethacin, and the negative control group was DMSO. The test compound was serially diluted 5 concentration gradients. The concentration of the test compound was plotted on the abscissa and the inhibition rate was plotted on the ordinate to obtain the IC 50 value of the test compound.

[0095] Inhibition rate (%) = (C 2 - C 1 ) / (C 2 - C 0 ) × 100%;

[0096] Where: C 0 , C 1 , C 2Absorbance values of the blank control group (without LPS), experimental group, and model group (with LPS) measured at 540 nm, respectively. The inhibition rate at each concentration was calculated and a compound concentration-inhibition rate curve was plotted, and the half-inhibitory concentration (IC 50 value) of the compound against the production of NO by LPS-induced RAW264.7 was calculated. The detection results are shown in Table 2.

[0097] Table 2 Anti-inflammatory activities of the compounds of Formula I and Formula II

[0098] compound <![CDATA[IC 50 ±SD μM <!-- 6 -->]]> compound of formula I structure 7.50±1.49* compound of formula II structure 9.30±0.57* <![CDATA[Indomethacin a > 52.30±5.11

[0099] a Positive control; * P < 0.05 vs positive control group.

[0100] As can be seen from the above examples, the present invention has discovered a new class of bibenzyl derivatives with significant anti-inflammatory activity; this compound is derived from Dendrobium hercoglossum and has good application prospects in the development of anti-inflammatory drugs.

[0101] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Bibenzyl derivatives, characterized in that, it has a compound with the structure shown in Formula I and / or a compound with the structure shown in Formula II; 2. A method for preparing the bibenzyl derivative according to claim 1, characterized in that, it comprises the following steps: Step 1: Take the aerial part of Dendrobium hercoglossum, crush it, extract it with an ethanol aqueous solution, filter, and concentrate to obtain an ethanol extract; Step 2: Prepare a suspension by mixing the ethanol extract and water at a volume ratio of 1:1, and sequentially extract with petroleum ether, ethyl acetate, and n-butanol, and concentrate each extract to obtain extracts at each level; Step 3: Take the ethyl acetate extract, and use liquid chromatography to trace and separate the compound with a characteristic ultraviolet absorption at 435 nm to obtain the bibenzyl derivative according to claim 1.

3. Use of any of the following in the preparation of a drug for treating and / or alleviating inflammation or an inflammatory reaction; (I), the bibenzyl derivative according to claim 1; and / or (II), the bibenzyl derivative prepared by the preparation method according to claim 2.

4. The use according to claim 3, characterized in that, the treatment and / or alleviation of inflammation or an inflammatory reaction includes inhibiting the production of pro-inflammatory factors; the pro-inflammatory factor is nitric oxide.

5. The use according to claim 4, characterized in that, the inflammatory reaction includes one or more of body flushing, swelling, fever or pain.

6. An anti-inflammatory drug, characterized in that, it comprises any of the following and a pharmaceutically acceptable excipient: (I), the bibenzyl derivative according to claim 1; and / or (II), the bibenzyl derivative prepared by the preparation method according to claim 2.

7. The anti-inflammatory drug according to claim 6, characterized in that, the dosage form of the anti-inflammatory drug includes tablets, capsules, powder injections or suspensions.

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