A benzophenone compound, its preparation method and application

Abstract

The present invention belongs to the field of pharmaceutical technology, and specifically relates to a new compound and its application. The compound is a new benzophenone compound, and the application involved is the use of the above compound for preparing antioxidant drugs; the compound has an obvious antioxidant effect; moreover, the raw materials for preparing the compound are rich in resources, the extraction and separation technology is less difficult, the solvent can be recycled, and the production cost is low.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a novel ketone compound isolated from the fecal metabolites of rat after oral administration of Aspergillus terreus secondary metabolite butyrolactone I and its applications. Background Art

[0002] Butyrolactone-I is the main active secondary metabolite of the Aspergillus terreus fungus. It was first isolated from Aspergillus terreus var. africanus IFO 8835 in 1977. Because of the five-membered unsaturated lactone ring in its structure, it was named butyrolactone I.

[0003] There have been reports in the literature that butyrolactone I has definite selective cell cycle-dependent kinase (CDK) inhibitory activity, and its in vitro inhibitory mechanisms on cdc2 and CDK2 have also been elucidated. It is a potential anti-tumor candidate drug. Butyrolactone I and its derivatives have also been proven to be a class of multi-functional natural active substances with a wide range of pharmacological activities.

[0004] After orally administering butyrolactone I to rats, we collected their urine and feces, and used various chemical and chromatographic means to isolate and identify the metabolites of butyrolactone I in vivo, speculate on the transformation process of butyrolactone I in vivo, and simultaneously determine the pharmacological activities of these metabolites to understand the structural changes after drug metabolism and transformation and the relationship between these changes and efficacy and toxicity. Summary of the Invention

[0005] The purpose of the present invention is to provide a benzophenone compound, its preparation method and applications.

[0006] The present invention isolates a novel benzophenone compound and its applications from the fecal metabolites of rat after oral administration of Aspergillus terreus secondary metabolite butyrolactone I. The chemical structural formula of the benzophenone compound is (I):

[0007]

[0008] Among them, R1 is selected from hydrogen, (C1-C6) alkyl, (C1-C6) alkoxy, halogenated (C1-C4) alkyl or (C1-C4) alkoxy, (C1-C6) alkylthio, amino substituted by single or double (C1-C6 alkyl), (C1-C6) alkylamido, (C1-C6) alkylsulfinyl, (C1-C6) alkylacyl, halogen.

[0009] Furthermore, the difference in the number of carbon atoms between R1 and R2 is within 3.

[0010] Furthermore, the difference in the number of carbon atoms between R3 and R4 is within 3.

[0011] Furthermore, R1 is hydrogen and / or R2 is hydrogen.

[0012] Furthermore, R3 is methyl and / or R4 is methyl.

[0013] Furthermore, its chemical structural formula (Ⅱ)

[0014]

[0015] is named butyrolactone metabolite I.

[0016] A pharmaceutical composition, the compound and its pharmaceutically acceptable salts as active ingredients and pharmaceutically acceptable excipients.

[0017] Use of the pharmaceutical composition or the compound in the preparation of antioxidant health foods and / or antioxidant drugs.

[0018] A preparation method of the compound: (1) After orally administering butyrolactone I to an animal, collect its urine and / or feces, and extract with an organic solvent; (2) Use chromatography to elute and separate to obtain butyrolactone metabolite I.

[0019] The preparation method, the animal is a rodent; and / or the organic solvent is ethyl acetate.

[0020] The preparation method, the chromatography is (1) using silica gel column chromatography and then eluting, and / or (2) C18 reverse phase column chromatography and then eluting.

[0021] The raw materials for the preparation of the compound of the present invention are rich in resources, the extraction and separation technology is less difficult, the solvent can be recycled, and the production cost is low.

[0022] The preparation method of the above compound is as follows:

[0023] (1) Extraction: Forty Wistar rats, half male and half female, weighing 200±20 g, were adaptively fed for 1 week and then administered 40 mg·kg-1 by gavage. They were placed in metabolic cages for rats, one rat per cage, and feces were collected for 24 h. During the experiment, the rats were fed with starch buns and given 4‰ normal saline and 1‰ glucose solution as drinking water. The feces collected every morning for 24 h were stored in a -80 °C refrigerator. The gavage administration was cumulative for 4 weeks, once a day. A total of 8 g of extract was obtained from the ethyl acetate extract of the feces.

[0024] (2) Isolation: The above extract was subjected to silica gel column chromatography and eluted with a gradient of dichloromethane-methanol system with a volume ratio of 100:0 - 100:50. Thin layer chromatography was used for detection, and the fractions containing the new compound were collected. Then, it was eluted with a gradient of petroleum ether-ethyl acetate system with a volume ratio of 100:0 - 0:100, and finally prepared by preparative liquid chromatography on a C18 reverse phase column and eluted with methanol-water with a volume ratio of 68:32 to obtain the new compound butyrolactone metabolite I.

[0025] The new compound butyrolactone metabolite I of the present invention is a colorless oily substance (methanol), UV (MeOH) λ max at 209 nm. HR-ESI-MS gave a [M+Na] + peak at m / z 333.1447, and the speculated molecular formula was C 20 H 22 O3. 1 1H-NMR and 13 13C-NMR data are shown in Table 1.

[0026] Experimental studies have shown that the compound of the present invention has obvious antioxidant effects. Therefore, it can be used to prepare antioxidant drugs.

[0027] Table 1 Butyrolactone metabolite I 1 1H and 13 13C nuclear magnetic resonance data

[0028]

[0029] Structural analysis of the new compound butyrolactone metabolite I.

[0030] As Figure 1 shown, the 1 1H NMR, 13 13C NMR, 2D-NMR (HSQC, HMBC) spectra, and HR-ESI-MS spectra of the new compound of the present invention were used to determine the compound structure. Specifically:

[0031] A colorless oily substance (methanol), UV (MeOH) λ max : 209 nm. The infrared spectrum (IR) indicated the presence of a hydroxyl group (3431 cm-1 ) and functional groups such as double bond (1638 cm -1 ). HR-ESI-MS gave a peak at m / z 333.1447 for [M+Na] + (calcd. 333.1467), and its molecular formula was determined to be C 20 H 22 O3.

[0032] 1 In the 1H-NMR (400 MHz, DMSO-d6) spectrum, a set of proton signals of an AA′BB′ coupling system was given in the aromatic region at δ H 6.91 (2H, d, J = 8.4 Hz, H-2′,6′), 6.68 (2H, d, J = 8.4 Hz, H-3′,5′), suggesting the presence of a para-substituted benzene structure; δ H 6.76 (1H, dd, J = 8.0, 2.0 Hz, H-6″), 6.75 (1H, d, J = 2.0 Hz, H-2″), 6.70 (1H, d, J = 8.0 Hz, H-5″), indicating the presence of a 1,3,4-trisubstituted benzene ring structure. From the two methyl signals δ H 1.65 (3H, s, H-10″) and 1.67 (3H, s, H-11″), a double bond hydrogen signal δ H 5.23 (1H, t, J = 7.2 Hz, H-8″) and the coupled hydrogen signal δ H 3.16 (2H, d, J = 7.2 Hz, H-7″), it was speculated that there was an isopentenyl fragment.

[0033] 13 The 13C-NMR (100 MHz, DMSO-d6) spectrum gave 14 aromatic or double bond carbon signals, which were in line with the number of carbons in the two benzene ring fragments and the isopentenyl fragment speculated above. Compared with butyrolactone I, the compound butyrolactone metabolite I had reduced carbon signals of -COOCH3 and the lactone structure fragment, and a ketone carbonyl carbon signal δ C 206.9 (C-2) and two methylene carbon signals δC 47.8 (C-1), 48.0 (C-3) appeared. Based on the above information, it was speculated that the lactone ring was opened and decarboxylation rearrangement occurred.

[0034] The HSQC spectrum gave information on all directly connected hydrogen-carbon in the structure, as shown in Table 1.

[0035] In the HMBC spectrum, the proton signal δ H 3.61 (2H, s, H-1) was correlated with the carbon signals δ C 206.9 (C-2), 125.2 (C-1'), 130.9 (C-2'), δH 3.59 (2H, s, H-3) is related to the carbon signal δ C 206.9 (C-2), 128.2 (C-6″) and C-2′ (δc 130.9), and H-7″ (δ H 3.16) is related to the carbon signals C-4″ (δc 154.0), C-3″ (δc 127.8) and C-8″ (δc 123.3). H-10″ (δ H 1.67) is related to C-9″ (δc 131.5) and C-8″ (δc 123.3). H-11″ (δ H 1.65) is related to C-9″ (δc 131.5) and C-8″ (δc 123.3). The signal of the active hydrogen 4′-OH (δ H 9.21) is related to C-4′ (δc 156.5) and C-5′ (δc 115.6). 4″-OH (δ H 9.19) is related to C-4″ (δc 154.0) and C-5″ (δc 115.1). The structure of the compound is determined to be 1-(4-hydroxy-3-(3-methylbut-2-enyl)phenyl)-3-(4-hydroxyphenyl)propan-2-one.

[0036] This compound is a new dibenzoylmethane compound not reported in the literature. The assignment of the relevant NMR data is shown in Table 1. Description of the Drawings

[0037] Figure 1 The 1 1H NMR spectrum of the new compound;

[0038] Figure 2 The 13 13C NMR spectrum of the new compound;

[0039] Figure 3 The HSQC spectrum of the new compound;

[0040] Figure 4 The HMBC spectrum of the new compound;

[0041] Figure 5 The HR-ESI-MS spectrum of the new compound. Detailed Embodiments

[0042] The present invention will be further described below by way of examples to enable those skilled in the art to understand the present invention more comprehensively, but the present invention is not limited in any way.

[0043] Example 1: 40 Wistar rats, half male and half female, weighing 200±20 g. After 1 week of adaptive feeding, they were gavaged with 40 mg·kg -1 , and were respectively placed in rat metabolic cages, 1 rat per cage, and urine and feces were collected for 24 h. During the experiment, the rats were fed with starch buns and given 4% normal saline and 1% glucose solution as drinking water. The feces collected for 24 h every morning were stored in a -80 °C refrigerator. Cumulative gavage administration was carried out for 4 weeks, once a day, and a total of about 14 g of the drug was administered. A total of 8 g of extract was obtained from the ethyl acetate extract of the feces. The extract was subjected to silica gel column chromatography and eluted with a gradient of dichloromethane-methanol system with a volume ratio of 100:0 - 100:50. Thin layer chromatography was used for detection, and the fractions containing the new compound were collected. Then, it was eluted with a gradient of petroleum ether-ethyl acetate system with a volume ratio of 100:0 - 0:100. Finally, it was prepared by preparative liquid chromatography on a C18 reverse phase column and eluted with methanol-water with a volume ratio of 68:32 at a flow rate of 3 ml / min and a detection wavelength of 210 nm to obtain 80 mg of the new compound butyrolactone metabolite ketone I.

[0044] Example 2: Antioxidant activity experiment of the new compound butyrolactone metabolite ketone I

[0045] 1. Experimental materials and instruments

[0046] Instruments: Microplate reader: Thermo scientific, USA; 96-well plate: Nest Biotech, USA; Precision electronic balance: Shimadzu, Japan; Micropipette: 100 - 1000 μL, Thermo, USA;

[0047] Reagents: DPPH (Sigma), ABTS (J&K Chemical Ltd.), PBS (Solarbio), L-ascorbic acid (Aldrich Chemical Co., Inc.).

[0048] 2. Test methods

[0049] DPPH method:

[0050] Accurately weigh 3.94 mg of DPPH, dissolve it with absolute ethanol, and make up the volume to 10 ml in a brown volumetric flask. Then dilute it 5 times to prepare a solution with a concentration of 0.2 mM. Measure its absorbance at 517 nm to make the absorbance value around 0.7. L-ascorbic acid and the compound to be tested are respectively prepared with absolute ethanol into solutions with concentrations of 100, 50, 25, 10, 5, and 1 μg / ml. Use a pipette to respectively pipette 100 μL and place them in a 96-well plate. Subsequently, add 100 μL of the sample solution to be tested with different concentrations dissolved in ethanol (100, 50, 25, 10, 5, 1 μg / ml) with a volume of 100 μL respectively. After thorough mixing of each, let it stand in the dark at room temperature for 30 min, and measure the absorbance value of each well with a microplate reader at 517 nm; the activity of each compound in scavenging DPPH free radicals is calculated using the following formula:

[0051] DPPH scavenging activity (%) = [1 - (S 样品 - SB 空 ) / (C 对照 - CB 空白 )] × 100%.

[0052] Among them, sample S: 100 μL of sample solution + 100 μL of DPPH;

[0053] Negative control C: 100 μL of absolute ethanol + 100 μL of DPPH;

[0054] Blank control CB: 200 μL of absolute ethanol.

[0055] Positive control: L-ascorbic acid

[0056] ABTS method:

[0057] Preparation of 0.01 mol / L PBS: 0.395 g of NaCl, 0.1 g of KCl, 0.12 g of KH2PO4, 0.9 g of K2HPO4, dissolve in 400 ml of distilled water, adjust the pH to 7.4 with HCl, and finally make up the volume to 500 ml, and place it in the refrigerator for refrigerated storage for later use.

[0058] Preparation of potassium persulfate (2.45 mmol / L): Weigh 0.0066 g of potassium persulfate and make up the volume to 10 ml with distilled water.

[0059] Preparation of ABTS solution: Weigh 0.0384 g of ABTS and make up the volume to 10 ml with the prepared PBS.

[0060] Generation of ABTS+· radical: Mix the prepared potassium persulfate solution and ABTS solution evenly at a volume ratio of 1:1, and place them in the dark at room temperature for 12 - 16 h to generate ABTS+·. Dilute this solution with PBS (0.01 M) with a pH of 7.4. When its absorbance at 734 nm reaches 0.70 (±0.02), measure its absorbance after equilibration at 30 °C for 30 min.

[0061] L-ascorbic acid and the test compound are respectively prepared into concentrations of 100, 50, 25, 10, 5, 2, 1 μg / ml with absolute ethanol.

[0062] Place the ABTS+· (150 μL) solution in a 96-well plate, add 100 μL of the test sample ethanol solution at different concentrations (100, 50, 25, 10, 5, 2, 1 μg / ml), mix well and let it stand in the dark for 20 min, then measure the absorbance value of each well at 734 nm with an enzyme-labeled instrument; the ABTS·+ radical scavenging activity of the compound is calculated by the following formula:

[0063] ABTS+· scavenging activity (%) = [1 - (S 样品 - SB 空 ) / (C 对照 - CB 空白 )] × 100%.

[0064] Among them, sample S: 100 μL of sample solution + ABTS· + 150 μL;

[0065] Negative control C: 100 μL of absolute ethanol + 150 μL of PBS;

[0066] Blank control CB: 100 μL of absolute ethanol + ABTS· + 150 μL.

[0067] Positive control: L-ascorbic acid

[0068] 3. Experimental results

[0069] The antioxidant ability of compound butyrolactone metabolite ketone I was evaluated by DPPH and ABTS radical scavenging methods. The results showed (Table 2) that it showed significant radical scavenging ability.

[0070] Table 2 Antioxidant activity of butyrolactone metabolite ketone I (IC 50 , μM)

[0071]

[0072] Vc a(ascorbic acid) was used as positive control in test of antioxidant activities.

[0073] b ND: Not detect。

Claims

1. A benzophenone compound, characterized in that, The structural formula (I) of the compound is as follows: 。

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