Benzoxane compound as well as preparation method and application thereof

Abstract

The invention discloses a series of benzoxane compounds as well as a preparation method and application thereof, novel benzoxane compounds 1a and 1b are extracted and separated from dry roots and rhizomes of a Gentiana plant Gentiana (Gentiana scabra Bge.), chiral resolution of the benzoxane compounds is realized for the first time, and the novel benzoxane compounds 1a and 1b have the advantages that the chiral resolution of the benzoxane compounds 1a and 1b is realized; and the anti-inflammatory activity evaluation is carried out on the obtained enantiomers 1a and 1b. The compounds 1a and 1b show significant nitric oxide (NO) release inhibitory activity, can significantly inhibit mRNA expression of cytokines IL-1beta, IL-6 and COX-2 in an inflammatory cell model, and show good development potential as lead compounds of anti-inflammatory drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to benzo[a]oxane compounds, their preparation methods, and applications. Background Technology

[0002] Benzo[a]oxoheterocyclic compounds are a class of organic compounds with a fused structure of a benzene ring and a seven-membered oxygen-containing heterocycle. These compounds are relatively rare in the natural product world, and have only been sporadically discovered in Gentiana species to date. Since Cheng Yiyu's team first reported the isolation of gentioxepine from Gentiana species in 2013, and reported (±)-3′-hydroxybenzo[a]oxoheterocyclic compounds in 2020, this invention is the first to achieve chiral resolution of benzo[a]oxoheterocyclic compounds, and the anti-inflammatory activity of the resulting enantiomers 1a and 1b was evaluated.

[0003] Gentian (hereinafter referred to as gentian) is a plant of the Gentianaceae family, namely Gentiana linearifolia. Gentiana manshurica Kitag.), Gentian ( Gentiana scabra Bge.), Gentiana triflora ( Gentiana triflora Pall.) or Gentiana scabra ( Gentiana rigescens Gentian (Fragrance) is the dried root and rhizome of the plant. The first three types are commonly known as gentian, mainly produced in Liaoning and other places; the last type is commonly known as hard gentian, mainly produced in Yunnan and other places. Gentian's medicinal use was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), listed as a medium-grade herb. It is bitter and cold in nature, and has the effects of clearing heat and drying dampness, and purging liver and gallbladder fire. It is mainly used to treat damp-heat in the lower burner. Clinically, it is widely used for herpes zoster, hypertension, sudden deafness, and various inflammatory diseases. Gentian Decoction for Draining Liver Fire, with gentian as the principal ingredient, is widely used clinically to treat damp-heat type liver diseases. Literature reports that gentian contains various chemical components such as iridoid glycosides, flavonoids, xanthones, triterpenoids, and phenolic acids. Iridoid glycosides, as a characteristic chemical component type of gentian, have been isolated and identified from gentian. Extensive pharmacological activity studies have focused on monomeric components such as gentiopicrin, swertiamarin, and swertiamarin, attracting considerable attention from scholars both domestically and internationally. Modern pharmacological studies have shown that gentian possesses various biological activities, including hepatoprotective, anti-inflammatory, and anti-tumor effects. Gentianoside can reduce the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of rats fed a high-fat, high-sugar diet, thus protecting liver function and increasing peroxisome proliferator-activated receptor (PPAR) levels. α It reduces the expression of α-coenzyme A oxidase 1 (ACOX1), promotes fatty acid oxidation in the liver, reduces the expression of key enzymes in gluconeogenesis, and decreases hepatic gluconeogenesis; swertiamarin can improve the pathological symptoms of non-alcoholic fatty liver disease (NAFLD) mice by regulating lipid metabolism and inflammatory response; swertiamarin can alleviate hepatic oxidative stress and reduce the level of hepatic inflammatory cytokines, thereby alleviating hepatic steatosis in NAFLD model mice.

[0004] Inflammation is a protective defense response of the body to damage from various inflammatory factors, with vascular reactions at its core. However, when this process gets out of control, especially when it transforms into chronic inflammation, it changes from a protective mechanism into a pathogenic factor, potentially leading to a variety of serious diseases, including rheumatoid arthritis, diabetes, cardiovascular disease, and even cancer. The occurrence of these diseases is closely related to the release of a series of endogenous chemical factors—namely, inflammatory mediators. Among them, nitric oxide (NO), produced by inducible nitric oxide synthase (iNOS), plays a central role. It directly participates in the inflammatory process and synergistically amplifies the effects of other inflammatory mediators; its metabolic abnormalities are widely involved in diseases of multiple systems, including the nervous, respiratory, and circulatory systems. Therefore, targeting and inhibiting the release of key inflammatory mediators such as NO has become one of the most promising and important therapeutic strategies in modern anti-inflammatory drug development. Summary of the Invention

[0005] The technical problem solved by this invention is to provide a benzo[a]oxo heterojunction compound, its preparation method, and its application. The novel benzo[a]oxo heterojunction compound involved in this invention is derived from the genus *Gentiana* (*Gentiana* var. *spinosa*). Gentiana scabra The compound was extracted and isolated from the dried roots and rhizomes of *B. ge.*, and its anti-inflammatory activity was evaluated using a lipopolysaccharide (LPS)-induced Raw 264.7 inflammatory cell model. This invention relates to novel compounds and their pharmaceutically acceptable salts or carriers or excipients, which can be used to prepare anti-inflammatory drugs.

[0006] To achieve the aforementioned objectives, the technical solution adopted by this invention includes: The benzo[a]oxo compounds are characterized by being compounds 1a and 1b, specifically selected from compounds represented by formulas 1a and 1b below:

[0007] Among them, formulas 1a and 1b each contain a seven-membered oxygen-containing ring and a three-membered oxygen-containing ring; The compounds shown in formulas 1a and 1b are named as follows: Equation 1a: ; Formula 1b: .

[0008] The method for preparing the benzo[a]oxane compound is characterized by comprising the following steps: 1) The dried roots and rhizomes of gentian were pulverized and extracted by heating and reflux with ethanol solution. The extracts were combined and concentrated under reduced pressure to obtain the total extract. 2) Disperse the total extract obtained in step 1) in distilled water, separate it by AB-8 macroporous adsorption resin column chromatography, elute with a gradient of ethanol-water solution with a volume concentration of 10%~95%, collect the 95% ethanol-water solution eluent, and concentrate under reduced pressure to obtain extract 1. 3) The extract 1 obtained in step 2) was separated by ODS reversed-phase column chromatography and eluted with a methanol-water gradient of volume ratio of 40:60~100:0 to obtain 6 eluents, which were concentrated under reduced pressure and named (Fr.A~F). 4) The Fr.E obtained in step 3) was separated by silica gel column chromatography, and eluted with a dichloromethane-methanol gradient of 150:1 to 0:100 (v / v) to obtain 5 eluent fractions. The eluent fractions were concentrated under reduced pressure and named (Fr.E1 to E5). 5) Fr.E2 was prepared by preparative HPLC. The detection wavelength was 235 nm, the mobile phase was methanol-water with a volume ratio of 80:20, and the flow rate was 3 mL / min. Compound 1 was obtained by separation. 6) Compound 1 was chirally separated into a pair of enantiomers 1a and 1b by using a high-performance liquid chromatograph with a chiral stationary phase packed on a Chiralpak® IE column, a detection wavelength of 235 nm, a mobile phase of methanol-water with a volume ratio of 80:20, and a flow rate of 1 mL / min.

[0009] In step 1), the volume fraction of the ethanol solution is 75%, the amount is 3 times the weight of the gentian material, the extraction is carried out by heating and reflux 3 times, and the extraction time is 2 hours each time.

[0010] The distilled water in step 2) is 10 times the total mass of the extract; the volume concentration of the ethanol-water solution in step 2) is 10%, 30%, 50% and 95%, and the column volume is denoted as BV, with each gradient elution being 8 BV.

[0011] The methanol-water solution volume ratio in step 3) is 40:60, 50:50, 60:40, 70:30, 90:10, and 100:0. The column volume is denoted as BV, and each gradient elution is 5 BV.

[0012] In step 4), the silica gel column has a mesh size of 200-300. The dichloromethane-methanol solution is used in sequence at volume ratios of 150:1, 100:1, 50:1, 10:1, and 0:100, with the column volume recorded as BV. Each ratio elutes 10 BV.

[0013] A pharmaceutical composition comprising the aforementioned benzoxoxane compound and its pharmaceutically acceptable salt or pharmaceutically acceptable carrier or excipient.

[0014] The pharmaceutical composition is used in the preparation of anti-inflammatory drugs.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: 1) This invention is the first to isolate and purify two new benzo[a]oxo-type compounds 1a and 1b from gentian, and the first to achieve chiral resolution of benzo[a]oxo-type compounds.

[0016] 2) This invention uses LPS-induced Raw264.7 cells to evaluate the anti-inflammatory effects of monomeric compounds. Compounds 1a and 1b exhibited significant nitric oxide (NO) release inhibitory activity and significantly inhibited the mRNA expression of cytokines IL-1β, IL-6, and COX-2 in an inflammatory cell model, demonstrating good development potential as lead compounds for anti-inflammatory drugs. Attached Figure Description

[0017] Figure 1 This is a high-resolution mass spectrum of compound 1 in Example 1 of the present invention; Figure 2 The above is the 1H NMR spectrum of compound 1 in Example 1 of this invention; Figure 3 The image shows the carbon NMR spectrum of compound 1 in Example 1 of this invention. Figure 4 This is a graph showing the effects of compounds 1a and 1b on the mRNA expression of five cellular inflammatory factors in Example 2 of this invention. Figure 4 A represents the mRNA expression level of IL-1β; Figure 4 B represents the mRNA expression level of TNF-α; Figure 4 C represents the mRNA expression level of COX-2; Figure 4 D represents the mRNA expression level of IL-6; Figure 4 E represents the mRNA expression level of iNOS. Detailed Implementation

[0018] The present invention will be further described below with reference to specific embodiments. These embodiments are merely exemplary and do not limit the scope of protection of the present invention.

[0019] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0020] Example 1: Method for separating and purifying benzo[a]oxo compounds from gentian. 1) 9.0 kg of dried roots and rhizomes of Gentiana scabra Bge. were crushed and extracted three times with 3 times the volume of 75% ethanol solution by heating and reflux for 2 hours each time. The extracts were combined, the solvent was recovered under reduced pressure at 45°C, and the extract was concentrated to obtain the total extract.

[0021] 2) Disperse the extract obtained in step 1) into 10 times its weight of distilled water, and separate it by AB-8 macroporous adsorption resin column chromatography. The column volume is recorded as BV. Elute with gradients of 10%, 30%, 50% and 95% ethanol-water solutions, with 8 BV eluted for each gradient. Collect the 95% ethanol-water solution eluent and concentrate it under reduced pressure to extract 1 (80.0 g).

[0022] 3) The extract 1 obtained in step 2) was separated by ODS reversed-phase column chromatography, with the column volume denoted as BV. Elution was performed using methanol-water gradients at volume ratios of 40:60, 50:50, 60:40, 70:30, 90:10, and 100:0, with 5 BV eluted per gradient. The eluted fractions were developed using silica gel thin-layer chromatography on a GF254 HPLC system with petroleum ether:ethyl acetate = 5:1 as the developing solvent. Similar components were combined to obtain six eluates, which were concentrated under reduced pressure and named (Fr.A~F).

[0023] 4) The Fr.E (10.3 g) obtained in step 3) was mixed with 100-200 mesh silica gel at a mass ratio of 1:1 and separated by chromatographic separation on a 200-300 mesh silica gel column. The column volume was recorded as BV. A gradient elution was performed sequentially using dichloromethane-methanol (150:1, 100:1, 50:1, 10:1, 0:100, V / V), with 10 BV eluted at each ratio. The eluent was developed using silica gel thin-layer chromatography on a GF254 chromatogram with petroleum ether:ethyl acetate = 5:1. Similar components were combined to obtain five eluent fractions, which were concentrated under reduced pressure and named (Fr.E1~E5).

[0024] 5) Fr.E2 (980.5 mg) was prepared by preparative HPLC at a detection wavelength of 235 nm. Compound 1 (12.5 mg) was obtained by separation with methanol-water (80:20, V / V, 3 mL / min).

[0025] 6) Compound 1 was then chirally separated by high performance liquid chromatography (HPLC) at a detection wavelength of 235 nm using a Chiralpak® IE column (10 × 250 mm, 5 μm; Daicel Corporation, Tokyo, Japan) packed with a chiral stationary phase, with methanol-water (80:20, V / V, 1 mL / min) to obtain a pair of enantiomers 1a (5.5 mg) and 1b (6.5 mg).

[0026] The structures of compounds 1a and 1b were determined using physicochemical constants and modern spectroscopic techniques. Both are novel compounds not previously reported in the literature. Their structural formulas are shown below:

[0027] High-resolution mass spectra of compound 1, such as Figure 1 As shown; the 1H NMR spectrum of compound 1 is as follows. Figure 2 As shown; the carbon NMR spectrum of compound 1 is as follows. Figure 3 As shown.

[0028] Compounds 1a and 1b are enantiomers: 1a is named as follows: .

[0029] White powder (methanol); = +32.0 (c 0.05, MeOH); UV (MeOH) λ max (log ε)235 (3.82), 254 (3.49), 313 (2.74) nm; IR (KBr) ν max :2943, 1700, 1453, 1375 cm -1 ;HRESIMS m / z 249.0771 [M + H] + (calcd for C 13 H 13 O5, 249.0763), determined that the molecular formula of compound 1a is C 13 H 12 O5; 1 H (600 MHz, CDCl3) and 13 C-NMR (150 MHz, CDCl3) data are shown in Table 1; the structural formula is shown in Equation 1a above, which contains a seven-membered oxygen-containing ring and a three-membered oxygen-containing ring.

[0030] 1b is named as follows: .

[0031] White powder (methanol); = -28.0 (c 0.05, MeOH); other physicochemical data are the same as those of compound 1a. The structural formula is shown in formula 1b above.

[0032] Table 1. 1H and 1C NMR spectra of compound 1 (δ in ppm, 1 in Hz, CDCl3)

[0033] Example 2: In vitro anti-inflammatory activity of compounds 1a / 1b in macrophage Raw 264.7 cells. (1) Experiment on the inhibitory activity of compounds 1a / 1b on the release of nitric oxide (NO) from lipopolysaccharide-induced mouse monocytes / macrophages Raw 264.7 Mouse monocyte-macrophage Raw 264.7 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% heat-inactivated (56°C, 30 min) fetal bovine serum (FBS), 100 U / mL penicillin sodium (Gibco), and 100 μg / mL streptomycin (Gibco) and incubated at 37°C in a 5% CO2 incubator. Because NO is extremely unstable, it is rapidly metabolized into nitrite (NO3) in the cell culture supernatant. 2- Therefore, the Griess method was used to determine NO in the sample. 2- The concentration of NO was used as an indicator of NO levels. Griess Reagent A: 0.1% N-naphthylethylenediamine dihydrochloride dissolved in water; Griess Reagent B: 1% sulfanilamide dissolved in 5% H3PO4. Mix equal volumes of reagents A and B before use. Dilute Raw264.7 cells to 5 × 10⁻⁶ using RPMI 1640 medium. 5 Cells were seeded at a concentration of [cells / mL] into 96-well cell culture plates, with 200 μL of cell suspension added to each well. After culturing in a CO2 incubator for 1 hour, 0.4 μL of different concentrations of test samples (0.0 μM, 3.12 μM, 6.25 μM, 12.5 μM, 25.0 μM, 50.0 μM, 100.0 μM) dissolved in dimethyl sulfoxide (DMSO) were added to each well. An LPS group (containing LPS but no test sample, with a 0% inhibition rate of NO release) and a blank control group (containing only 0.4 μL of DMSO, with a 100% inhibition rate of NO release) were also included. Each sample was divided into four parallel wells. The cells were cultured at 37℃ in a 5% CO2 incubator for 24 h. 100 μL of the culture supernatant was transferred to an ELISA plate, centrifuged (1000×g, 4℃, 3 min), and 100 μL of Griess reagent was added. The reaction was carried out at room temperature in the dark for 10 min, and the absorbance at 540 nm was measured using an ELISA reader. Standard curves were plotted using NaNO2 at concentrations of 1, 5, 10, and 50 μmol / L. The NO content in the cell culture supernatant was calculated based on the NaNO2 standard curves. 2- The concentration was then used to calculate the inhibition rate of NO release by the test sample.

[0034] Experimental results showed that compounds 1a and 1b exhibited significant nitric oxide (NO) release inhibitory activity, with IC50 values ​​of 100%. 50 The values ​​were 1.72 ± 0.45 μM and 3.03 ± 0.25 μM, respectively, showing good development potential as a lead compound for anti-inflammatory drugs.

[0035] (2) Real-Time PCR was used to determine the effects of compounds 1a and 1b on the mRNA expression of inflammatory factors in LPS-Raw 264.7 cells. ① Cell processing and collection: Raw 264.7 cells were processed and collected according to the method shown in Example 2 (1).

[0036] ② Extraction and quantification of total RNA from cells: Add 300 μL of RNA lysis buffer to the cell pellet, pipette to disperse the pellet and mix well, then transfer to a 1.5 mL nuclease-free Eppendorf tube. Add 300 μL of RNA diluent and pipette to mix well. Incubate at room temperature for 5 min, then centrifuge at 12000 rpm for 5 min. Carefully aspirate the supernatant. Add 0.5 times the volume of anhydrous ethanol to the supernatant and mix by pipetting 3-4 times. Transfer the mixture to a centrifuge column, centrifuge at 12000 rpm for 1 min, discard the filtrate, add 600 μL of RNA washing buffer, centrifuge at 12000 rpm for 1 min, and discard the filtrate. Carefully add 50 μL of the prepared DNase I incubation solution to the center of the centrifuge column and incubate at room temperature for 15 min. Add 600 μL of RNA washing buffer, centrifuge at 12000 rpm for 1 min, and discard the filtrate. Repeat this operation twice. After the second centrifugation and discarding of the filtrate, place the centrifuge column back into the collection tube and centrifuge at 12000 rpm for 2 min. After completing all the above steps, place the centrifuge column in the collection tube and carefully add 30 μL of nuclease-free water to the center of the adsorption membrane. Let it stand at room temperature for 2 min, then centrifuge at 12000 rpm and 4℃ for 1 min. Add the eluent from the collection tube back into the centrifuge column, let it stand for 2 min, and centrifuge at 12000 rpm and 4℃ for 1 min. The collection tube now contains total RNA. Then, carefully pipette 1 μL of the RNA sample to be quantified and place it in the sample slot of the micro-volume biodetector to determine the total RNA concentration and the OD values ​​of the sample at 260 nm and 280 nm for total RNA quantification.

[0037] ③ Reverse transcription reaction: Add the reagents required for reverse transcription according to the dosage in Table 2 (the total amount of RNA added to each tube is 500 ng); the temperature parameters for the reverse transcription reaction include maintaining at 95℃ for 10 min, followed by maintaining at 95℃ for 15 seconds and then at 60℃ for 1 minute as one cycle, and perform 40 temperature cycles.

[0038] Table 2 Reagents for Reverse Transcription Reaction

[0039] ④Real-Time PCR reaction was performed based on the target gene nucleic acid sequence provided by the National Gene Bank (NCBI). The primer sequences are shown in Table 3.

[0040] Table 3. qPCR primers for IL-1β, IL-6, TNF-α, COX-2, and iNOS

[0041] ⑤ Results Analysis: Based on the Ct values ​​of the target gene and internal reference gene given by the Real-Time PCR instrument, the results were analyzed using... The relative expression level of the target gene is calculated using this method.

[0042] Experimental results showed that different concentrations of compounds 1a (1, 2, and 4 μM) and 1b (2.5, 5, and 10 μM) significantly inhibited the mRNA expression of cytokines IL-1β, IL-6, and COX-2 in an inflammatory cell model, but had no effect on the mRNA levels of TNF-α and iNOS. (See attached figures.) Figure 4 .

[0043] Compositions containing the compounds described in this invention, as well as their pharmaceutically acceptable salts or pharmaceutically acceptable carriers or excipients, can be used to prepare formulations suitable for oral or injectable applications, such as tablets, capsules, injections, powders, etc. Each dosage form can be prepared using conventional methods.

[0044] The above embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope thereof, but all such modifications and substitutions fall within the protection scope of the present invention.

Claims

1. A benzoxanoxane compound, characterized in that, Compounds 1a and 1b are specifically selected from the compounds shown in formulas 1a and 1b below: Among them, formulas 1a and 1b each contain a seven-membered oxygen-containing ring and a three-membered oxygen-containing ring; The compounds shown in formulas 1a and 1b are named as follows: Equation 1a: ; Formula 1b: .

2. The method for preparing the benzo[a]oxane compound as described in claim 1, characterized in that, The method includes the following steps: 1) The dried roots and rhizomes of gentian were pulverized and extracted by heating and reflux with ethanol solution. The extracts were combined and concentrated under reduced pressure to obtain the total extract. 2) Disperse the total extract obtained in step 1) in distilled water, separate it by AB-8 macroporous adsorption resin column chromatography, elute with a gradient of ethanol-water solution with a volume concentration of 10%~95%, collect the 95% ethanol-water solution eluent, and concentrate under reduced pressure to obtain extract 1. 3) The extract 1 obtained in step 2) was separated by ODS reversed-phase column chromatography and eluted with a methanol-water gradient of volume ratio of 40:60~100:0 to obtain 6 eluents, which were concentrated under reduced pressure and named (Fr.A~F). 4) The Fr.E obtained in step 3) was separated by silica gel column chromatography, and eluted with a dichloromethane-methanol gradient of 150:1 to 0:100 (v / v) to obtain 5 eluent fractions. The eluent fractions were concentrated under reduced pressure and named (Fr.E1 to E5). 5) Fr.E2 was prepared by preparative HPLC. The detection wavelength was 235 nm, the mobile phase was methanol-water with a volume ratio of 80:20, and the flow rate was 3 mL / min. Compound 1 was obtained by separation. 6) Compound 1 was chirally separated into a pair of enantiomers 1a and 1b by using a high-performance liquid chromatograph with a chiral stationary phase packed on a Chiralpak® IE column, a detection wavelength of 235 nm, a mobile phase of methanol-water with a volume ratio of 80:20, and a flow rate of 1 mL / min.

3. The method for preparing the benzo[a]oxane compound according to claim 2, characterized in that, In step 1), the volume fraction of the ethanol solution is 75%, the amount is 3 times the weight of the gentian material, the extraction is carried out by heating and reflux 3 times, and the extraction time is 2 hours each time.

4. The method for preparing the benzo[a]oxane compound according to claim 2, characterized in that, The distilled water in step 2) is 10 times the total mass of the extract; the volume concentration of the ethanol-water solution in step 2) is 10%, 30%, 50% and 95%, and the column volume is denoted as BV, with each gradient elution being 8 BV.

5. The method for preparing the benzo[a]oxane compound according to claim 2, characterized in that, The methanol-water solution volume ratio in step 3) is 40:60, 50:50, 60:40, 70:30, 90:10, and 100:

0. The column volume is denoted as BV, and each gradient elution is 5 BV.

6. The method for preparing the benzo[a]oxane compound according to claim 2, characterized in that, In step 4), the silica gel column has a mesh size of 200-300. The dichloromethane-methanol solution is used in sequence at volume ratios of 150:1, 100:1, 50:1, 10:1, and 0:100, with the column volume recorded as BV. Each ratio elutes 10 BV.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the benzoxoxane compound of claim 1 and its pharmaceutically acceptable salt or pharmaceutically acceptable carrier or excipient.

8. Use of the pharmaceutical composition of claim 7 in the preparation of an anti-inflammatory drug.

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