Derivative comprising dihydrostilbene parent nucleus with antibacterial activity, preparation method therefor, and use thereof

Abstract

The present invention relates to the field of pharmaceuticals. Particularly, disclosed are a derivative comprising a dihydrostilbene parent nucleus for resisting Helicobacter pylori and a preparation method therefor. After liquorice stems and leaves, as starting materials, are subjected to alcohol extraction, macroporous resin, silica gel column chromatography and preparative liquid chromatography separation, and identification are carried out to obtain a novel natural isopentenyl dihydrostilbene compound. Moreover, the new compound has good activity against two drug-resistant and sensitive Helicobacter pylori strains, and can be used for preparing a drug for resisting Helicobacter pylori.

Description

Derivatives containing dihydrostilbene nuclei with antibacterial activity, and preparation methods and applications thereof Technical Field

[0001] The invention relates to a preparation method of a dihydrostilbene compound and its anti-Helicobacter pylori activity, belonging to the field of medicine. Background Art

[0002] Helicobacter pylori is a Gram-negative, microaerophilic bacterium with a spiral or curved shape. It colonizes the gastric mucosa, causing chronic inflammation. It is a major causative factor for chronic gastritis and gastroduodenal ulcers, and is closely associated with the development of diseases such as gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma. In 1994, the World Health Organization designated Helicobacter pylori as a Class I carcinogen for gastric cancer, making it the first bacterium officially recognized as a carcinogen. Helicobacter pylori infects over 50% of the global population, while few chronic infectious diseases infect more than half of the world's population. According to relevant statistical analysis, the infection rate of Helicobacter pylori in my country is as high as 55.82%, affecting nearly 800 million people. Notably, my country has a high incidence of gastric cancer, accounting for nearly half of all gastric cancer cases worldwide. Among the new cancer cases caused by pathogen infection in 2018, the number of new cases of Helicobacter pylori infection was 810,000, with an incidence rate of 8.7 / 100,000, which has exceeded human papillomavirus (8 / 100,000), hepatitis B virus (4.1 / 100,000) and hepatitis C virus (1.7 / 100,000). In China, the incidence rate of cancer caused by Helicobacter pylori infection is 15.6 / 100,000, far exceeding the world average.

[0003] In 2016, the "Fifth National Consensus Report on the Management of Helicobacter pylori Infection" stated that eradication therapy is recommended for confirmed Helicobacter pylori infection, regardless of symptoms and complications. Eradication of Helicobacter pylori not only promotes the healing of peptic ulcers, reduces the incidence of ulcer complications, prevents or delays gastric mucosal atrophy, and inhibits the development and progression of intestinal metaplasia, but also reduces the incidence of gastric cancer by one-third. Currently, my country recommends a bismuth quadruple therapy. However, due to the continuous emergence of drug-resistant strains of Helicobacter pylori, conventional treatment strategies are increasingly difficult to eradicate Helicobacter pylori, and the discovery of new anti-Helicobacter pylori drugs or lead compounds is urgently needed.

[0004] Licorice is the dried root and rhizome of the legume plants Glycyrrhiza uralensis, Glycyrrhiza inflata Bat. and Glycyrrhiza uralensis Fisch., G. inflata Bat., G. glabra L., which have the effects of invigorating qi and tonifying the middle, removing phlegm and relieving cough, detoxifying, relieving pain, and alleviating the properties of medicines. It has a long history of being used to treat spleen and stomach weakness, fatigue, palpitations and shortness of breath, cough and sputum, abdominal and limb cramps and pain, carbuncles and sores, and to relieve drug toxicity and potency. It has a wide range of clinical applications and is one of the commonly used medicinal materials. Licorice stems and leaves are non-medicinal parts of licorice and are mostly discarded in the fields without being used reasonably. Previous studies have shown that licorice stems and leaves are rich in isopentenyl compounds, and studies have shown that isopentenylation can enhance the antibacterial effect of the compound. In summary, we separated its components in order to obtain new and highly effective anti-Helicobacter pylori lead compounds.

[0005] Summary of the Invention

[0006] The technical problem solved by the present invention is: the present invention provides a preparation method of a novel dihydrostilbene and its application in anti-Helicobacter pylori. The prepared compounds 1, 3, 6-7, and 9 have good activity against both resistant and sensitive Helicobacter pylori, with an MIC of 4-8 μg / mL. They can be used to prepare anti-Helicobacter pylori drugs or as lead compounds for the development of anti-Helicobacter pylori drugs.

[0007] Derivatives containing a dihydrostilbene core for resisting Helicobacter pylori, including compounds of the following general structural formula:

[0008] Among them, R1, R2, R3, R4, R5, R6, and R7 can all be hydrogen, acyl, isopentenyl, hydroxyl, methoxy, or aldehyde.

[0009] As a preferred example, the above-mentioned anti-Helicobacter pylori derivative containing a dihydrostilbene core has the structural formula:

[0010] The method for preparing the anti-Helicobacter pylori derivative containing a dihydrostilbene core provided by the present invention comprises the following steps:

[0011] (1) Weighing the stem and leaf medicinal materials of Glycyrrhiza uralensis, crushing them, adding ethanol or methanol aqueous solution to soak the medicinal materials at room temperature, heating and refluxing to extract, filtering the extracts, collecting and combining them, and concentrating them under reduced pressure until there is no alcohol taste, thereby obtaining a concentrated liquor of Glycyrrhiza uralensis stem and leaf;

[0012] (2) The concentrated solution obtained in step (1) was applied to a macroporous resin column and eluted with ethanol and water in different volume ratios to obtain six sub-streams Fr.1-1–Fr.1-5;

[0013] (3) The eluate fraction Fr.1-4 obtained in step (2) was applied to a silica gel column and eluted with a mixed solvent of petroleum ether and ethyl acetate in different volume ratios to obtain 13 sub-fractions Fr.2-1–Fr.2-13;

[0014] (4) The eluate fraction Fr.2-4 obtained in step (3) was purified by C 18 - Medium-pressure preparative gradient elution was performed to obtain five sub-fractions Fr.3-1–Fr.3-5; fraction Fr.3-3 was separated by gel column and purified by preparative high-performance liquid chromatography to obtain compound 3;

[0015] (5) The eluate fractions Fr.2-5 obtained in step (3) were subjected to MCI-medium pressure preparative phase gradient elution to obtain 8 sub-fractions Fr.8-1–Fr.8-16; fraction Fr.8-4 was subjected to preparative high performance liquid chromatography to obtain compound 8; fraction Fr.8-8 was subjected to preparative high performance liquid chromatography to obtain compound 6; fraction Fr.8-11 was subjected to preparative high performance liquid chromatography to obtain compounds 1 and 9; fraction Fr.8-12 was subjected to preparative high performance liquid chromatography to obtain compounds 2 and 7; fraction Fr.8-13 was subjected to semi-preparative high performance liquid chromatography to obtain 4 and 5.

[0016] As a preferred embodiment, the method for preparing the above-mentioned anti-Helicobacter pylori derivative containing a dihydrostilbene core comprises the following steps:

[0017] (1) Weigh the stem and leaf medicinal materials of Glycyrrhiza uralensis, crush them, add ethanol or methanol aqueous solution to soak the medicinal materials at room temperature, and extract them under reflux at 105°C for 3 times, each time for 2 hours. The extracts are filtered, collected and combined, and concentrated under reduced pressure until there is no alcohol taste, thereby obtaining the Glycyrrhiza uralensis stem and leaf concentrate;

[0018] (2) The concentrated solution obtained in step (1) was subjected to macroporous resin column chromatography with a column volume of 20 L and gradient eluted with methanol-water with a volume concentration of 0-100% to obtain fractions Fr.1-1–Fr.1-5;

[0019] (3) The fraction Fr.1-4 obtained in step (2) was subjected to silica gel column chromatography with a column volume of 10 L and eluted with a petroleum ether / ethyl acetate system with a volume ratio of 6:1, 3:1, 1:1, 1:2, and 0:1 to obtain fractions Fr.2-1–Fr.2-13;

[0020] (4) The eluate fraction Fr.2-4 obtained in step (3) was purified by C 18- Medium-pressure preparative gradient elution, the mobile phase is pure water A-methanol B, using a 45-100% B gradient elution for 300 min; the flow rate is 25 mL / min, and 5 sub-fractions Fr.3-1–Fr.3-5 are obtained; the fraction Fr.3-3 is chromatographed on a gel column (AB-8, 1.5 cm × 210 cm) and eluted with methanol. After methanol elution, preparative high-performance liquid chromatography is performed with pure water A-methanol B as the mobile phase, and the volume ratio isocratic elution is 28:32 to obtain compound 3 in the time period of 76-80 min;

[0021] (5) The eluate fraction Fr.2-5 obtained in step (3) was subjected to MCI-medium pressure preparation, with pure water A-methanol B as the mobile phase, gradient elution: 45-100% B; time: 300 min; flow rate 25 mL / min, to obtain 16 sub-fractions Fr.8-1–Fr.8-16; the fraction Fr.8-4 was subjected to preparative high performance liquid chromatography, with pure water A-methanol B as the mobile phase, volume ratio 45:55 isocratic elution for 65 min, and compound 8 was obtained in the time period of 52-56 min; Fr.8-8 was subjected to preparative high performance liquid chromatography, with pure water A-methanol B as the mobile phase, volume ratio 57:43 isocratic elution for 90 min, and compound 6 was obtained in the time period of 76-80 min; the fraction Fr.8-11 was subjected to preparative high performance liquid chromatography, with pure water A-methanol B as the mobile phase, volume ratio 61:39 isocratic elution for 70 m in, compounds 1 and 9 were obtained in the time periods of 48-60min and 60-64min, respectively; the fraction Fr.8-12 was subjected to preparative high performance liquid chromatography with pure water A-methanol B as the mobile phase, and isocratic elution was performed at a volume ratio of 61:39 for 80min, and compounds 2 and 7 were obtained in the time periods of 58-62min and 66-71min, respectively; the fraction Fr.8-13 was subjected to semi-preparative high performance liquid chromatography with pure water A-methanol B as the mobile phase, and isocratic elution was performed at a volume ratio of 68:32 for 70min, and 4 and 5 were obtained at 54-58min and 60-63min, respectively.

[0022] The anti-Helicobacter pylori activity of each compound was evaluated using the broth microdilution method, using clinically used metronidazole, levofloxacin, clarithromycin, and vancomycin as positive controls. The results showed that compounds 1, 3, 6-7, and 9 exhibited strong activity against both drug-resistant and drug-sensitive Helicobacter pylori strains, with MICs of 4–8 μg / mL, comparable to that of the positive control drug, metronidazole. These compounds could be used to prepare anti-Helicobacter pylori drugs or as lead compounds for their development.

[0023] The present invention discovers novel dihydrostilbenes from liquorice stems and leaves for the first time, and provides extraction and separation technology, structure identification method and application of the new compound in resisting Helicobacter pylori. Beneficial effects

[0024] (1) The present invention provides a novel dihydrostilbene-containing derivative oxalactamA with anti-Helicobacter pylori activity and a preparation method thereof.

[0025] (2) This invention first discovered that dihydrostilbenes have a strong inhibitory effect on Helicobacter pylori. Compounds 1, 3, 6-7, and 9 have good activity against both resistant and sensitive Helicobacter pylori strains, with MICs of 4–8 μg / mL. They can be used to prepare anti-Helicobacter pylori drugs or as lead compounds for the development of anti-Helicobacter pylori drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Compound 1 1 H NMR spectra (500 MHz, Methanol-d4)

[0027] Figure 2 Compound 1 13 C NMR spectra (125 MHz, Methanol-d4)

[0028] Figure 3 Compound 2 1 H NMR spectra (500 MHz, Methanol-d4)

[0029] Figure 4 Compound 2 13 C NMR spectra (125 MHz, Methanol-d4)

[0030] Figure 5 Compound 3 1 H NMR spectra (500 MHz, Methanol-d4)

[0031] Figure 6 Compound 3 13 C NMR spectra (125 MHz, Methanol-d4)

[0032] Figure 7 Compound 4 1 H NMR spectra (500 MHz, Methanol-d4)

[0033] Figure 8 Compound 4 13 C NMR spectra (125 MHz, Methanol-d4)

[0034] Figure 9 Compound 5 1 H NMR spectra (500 MHz, Methanol-d4)

[0035] Figure 10 Compound 5 13 C NMR spectra (125 MHz, Methanol-d4)

[0036] Figure 11 Compound 6 1H NMR spectra (500 MHz, Methanol-d4)

[0037] Figure 12 Compound 6 13 C NMR spectra (125 MHz, Methanol-d4)

[0038] Figure 13 Compound 7 1 H NMR spectra (500 MHz, Methanol-d4)

[0039] Figure 14 Compound 7 13 C NMR spectra (125 MHz, Methanol-d4)

[0040] Figure 15 Compound 8 1 H NMR spectra (500 MHz, Methanol-d4)

[0041] Figure 16 Compound 8 13 C NMR spectra (125 MHz, Methanol-d4)

[0042] Figure 17 Compound 9 1 H NMR spectra (500 MHz, Methanol-d4)

[0043] Figure 18 Compound 9 13 C NMR spectrum (125 MHz, Methanol-d4). DETAILED DESCRIPTION

[0044] The present invention can be better understood based on the following examples. However, it is readily understood by those skilled in the art that the specific material ratios, process conditions, and results described in the examples are merely illustrative of the present invention and should not and will not limit the present invention described in detail in the claims.

[0045] Example 1

[0046] 1. Instruments and Materials

[0047] 1.1 Instrument

[0048] Experimental Materials

[0049] Hedera ODS preparative chromatography column (10 nm, 5 μm, 10 × 250 mm); Waters ACQUITY UPLC BEH C 18(2.1 mm × 100 mm, 1.7 μm) chromatographic column; MCI GEL (CHP20, 75-150 μm), Sephadex LH-20 gel, column chromatography silica gel (200-300 mesh); chromatographic grade acetonitrile, methanol, and formic acid were purchased from Merck, USA; analytical grade petroleum ether, ethyl acetate, etc. were purchased from Nanjing Wanqing Chemical Reagent Co., Ltd.

[0050] 2. The preparation method of the present invention comprises the following steps:

[0051] (1) Weigh 5 kg of licorice stem and leaf medicinal materials, crush them, add 10 L of 75% ethanol aqueous solution to soak the medicinal materials at room temperature for 1 hour, and extract them under reflux at 105°C for 2 hours each time. The extracts are filtered, collected and combined, and concentrated under reduced pressure until there is no alcohol taste to obtain licorice stem and leaf concentrate;

[0052] (2) The concentrated solution obtained in step (1) was subjected to macroporous resin column chromatography with a column volume of 20 L and gradient elution of 400 L with a methanol / water (0-100%) system to obtain fractions Fr.1-1–Fr.1-5.

[0053] (3) The fraction Fr.1-4 obtained in step (2) was subjected to silica gel column chromatography with a column volume of 10 L and eluted with a petroleum ether / ethyl acetate system with a volume ratio of 6:1, 3:1, 1:1, 1:2, and 0:1 to obtain fractions Fr.2-1–Fr.2-13;

[0054] (4) The eluate fraction Fr.2-4 obtained in step (3) was purified by C 18 - Medium-pressure preparative gradient elution, the mobile phase is pure water A-methanol B, using a 45-100% B gradient elution for 300 min; the flow rate is 25 mL / min, and 5 sub-fractions Fr.3-1–Fr.3-5 are obtained; the fraction Fr.3-3 is chromatographed on a gel column (AB-8, 1.5 cm × 210 cm) and eluted with methanol. After methanol elution, preparative high-performance liquid chromatography is performed with pure water A-methanol B as the mobile phase, and the volume ratio isocratic elution is 28:32 to obtain compound 3 in the time period of 76-80 min;

[0055] (5) The eluate fraction Fr.2-5 obtained in step (3) was subjected to MCI-medium pressure preparation, using pure water A-methanol B as the mobile phase, gradient elution: 45-100% B; time: 300 min; flow rate 25 mL / min, to obtain 16 sub-fractions Fr.8-1-Fr.8-16; the fraction Fr.8-4 was subjected to preparative high performance liquid chromatography, using pure water A-methanol B as the mobile phase, volume ratio 45:55 isocratic elution for 65 min, and compound 8 was obtained in the time period of 52-56 min; Fr.8-8 was subjected to preparative high performance liquid chromatography, using pure water A-methanol B as the mobile phase, volume ratio 57:43 isocratic elution for 90 min, and compound 6 was obtained in the time period of 76-80 min; the fraction Fr.8-4 was subjected to preparative high performance liquid chromatography, using pure water A-methanol B as the mobile phase, volume ratio 57:43 isocratic elution for 90 min, and compound 6 was obtained in the time period of 76-80 min; Fraction Fr.8-11 was subjected to preparative high performance liquid chromatography with pure water A-methanol B as the mobile phase and isocratic elution at a volume ratio of 61:39 for 70 minutes, and compounds 1 and 9 were obtained at 48-60 minutes and 60-64 minutes, respectively; fraction Fr.8-12 was subjected to preparative high performance liquid chromatography with pure water A-methanol B as the mobile phase and isocratic elution at a volume ratio of 61:39 for 80 minutes, and compounds 2 and 7 were obtained at 58-62 minutes and 66-71 minutes, respectively; fraction Fr.8-13 was subjected to semi-preparative high performance liquid chromatography with pure water A-methanol B as the mobile phase and isocratic elution at a volume ratio of 68:32 for 70 minutes, and 4 and 5 were obtained at 54-58 minutes and 60-63 minutes, respectively.

[0056] 3. Structural analysis of the compound:

[0057] Compound 1 is an orange-yellow oil with an optical rotation value of +5.0(c 0.08,MeOH); HR-ESI-MS m / z 369.2066[M+H] + , C 23 H 29 O4 + The calculated value is 369.2066), indicating that the molecular formula is C 23 H 28 O4, with 10 unsaturated groups. Compound 1 is highly similar to 2-(3-methyl-2-butenyl)-3,5,4'-trihydroxy-bibenzyl. Analysis of its carbon and hydrogen spectra revealed a group of hydrogen signals of isobutyryl, δ H 2.80dd, 1.28d, 1.27d and a group of carbon signals δ C 177.4, 35.4, 19.3, 19.3. In the HMBC spectrum, δ H 3.11(H-7) and δ C144.0 (C-1), 123.9 (C-2), 151.2 (C-3), which can be determined to be 2-isopentenyl substitution. C The upfield shift at 151.2 (C-3) confirmed the presence of a 3-O-isobutyryl substitution. The structure of the compound was determined to be 2-isopentenyl-3-isobutyryl-5,4'-dihydroxystilbene. Compound 1 is a new natural product and was named uralensisol F.

[0058] Compound 2 is an orange-yellow oil with an optical rotation value of +2.5(c 0.08,MeOH); HR-ESI-MS m / z 383.2215[M+H] + , C 24 H 31 O4 + The calculated value is 383.2222), indicating that the molecular formula is C 24 H 30 O4, with 10 degrees of unsaturation. Compound 2 is highly similar to compound 1. Analysis of its carbon and hydrogen spectra revealed a group of characteristic signals of 2-methylbutyryl group δ H 2.80dd, 1.28d, 1.27d and δ C 177.4, 35.4, 19.3, 19.3. In the HMBC spectrum, δ H 3.11(H-7) and δ C 144.1 (C-1), 123.9 (C-2), 151.2 (C-3), which can be determined to be 2-isopentenyl substitution. C The upfield shift at 151.2 (C-3) confirmed the presence of a 3-O-2-methylbutyryl group. The structure of the compound was determined to be 2-isopentenyl-3-acetoxy-5,4'-dihydroxystilbene. Compound 2 is a new natural product and was named uralensisol G.

[0059] Compound 3 is an orange-yellow oil with an optical rotation value of +40.0(c 0.08,MeOH); HR-ESI-MS m / z 313.1818[M+H] + , C 20 H 25 O3 + The calculated value is 313.1804), indicating that the molecular formula is C 20 H 24O3, has 9 unsaturated degrees. Compound 3 is highly similar to compound 2-(3-methyl-2-butenyl)-3,5,4'-trihydroxy-bibenzyl. The main difference is that there is a group of methoxy hydrogen signals δ H 3.67 (3H, s). In the HSQC spectrum, δ H 3.67(3H,s) and δ C 55.5 is directly related. In the HMBC spectrum, δ H 3.67(3H,s) and δ C A long-range correlation of 159.6 confirmed that the methoxy group was substituted at position 5. Compound 3 is a new natural product and was named uralensisol I.

[0060] Compound 4 is an orange-yellow oil with an optical rotation value of [α] 2 D 0 , +2.5(c 0.08,MeOH); HR-ESI-MS m / z 409.2385[M+H] + , C 26 H 33 O4 + The calculated value is 409.2379), indicating that the molecular formula is C 26 H 32 O4, has 11 unsaturated groups. The A ring of compound 4 is similar to that of compound 9. In addition, 1 The H NMR (500 MHz, Methanol-d4) spectrum showed a group of mutually coupled hydrogen signals, δ H 6.76 (dd, J = 2.2, 8.7 Hz, H-6'), 6.64 (d, J = 8.7 Hz, H-5'), 6.77 (d, J = 2.2 Hz, H-2') and a group of isopentenyl hydrogen signals, δ H 3.24 (d, J = 7.3 Hz, H-7'), 5.05 (t, J = 7.3 Hz, H-5'), 1.65 (s, H-11'), 1.70 (s, H-10'). In the HMBC spectrum, δ H 3.24 (d, J = 7.3 Hz, H-7') and δ C 154.2, 129.0, 130.5 correlation, confirming that the B ring is 3-isopentenyl-4'-hydroxyl substituted. C 151.2(C-3) shifts to the upfield and is related to δ C The residues 26.0 (C-7') were related to the residues 3-O-acetyl. Compound 4 is a new natural product and was named uralensisol J.

[0061] Compound 5 is an orange-yellow oil with an optical rotation value of -2.5(c 0.08,MeOH); HR-ESI-MS m / z 439.2488[M+H] + , C 27 H 35 O5 + The calculated value is 439.2484), indicating that the molecular formula is C 27 H 34 O5 has 11 unsaturated groups. Compound 5 is highly similar to compound 4. 13 C NMR (125MHz, Methanol-d4) spectrum of δ C The signals at 145.2 (C-4') and 150.8 (C-5') indicate that the hydroxyl group of the hydroxyl group is substituted with o-diphenol. 1 The H NMR (500 MHz, Methanol-d4) spectrum showed a group of methoxyl hydrogen signals δ H 3.73 (3H, s). In the HMBC spectrum, δ H 3.73 (3H, s) and δ C The long-range correlation of 145.2 was confirmed to be 4'-methoxy substituted. Compound 5 is a new natural product and was named uralensisol K.

[0062] Compound 6 is an orange-yellow oil with an optical rotation value of +42.5(c 0.08,MeOH); HR-ESI-MS m / z 367.2278[M+H] + , C 24 H 31 O3 + The calculated value is 367.2273), indicating that the molecular formula is C 24 H 30 O3, with 10 degrees of unsaturation. Compound 6 is highly similar to gancaonin R, the main difference being that the B ring of compound 6 is not substituted with an ortho-diphenolic hydroxyl group. In compound 6, 1 The H NMR (500 MHz, Methanol-d4) spectrum showed a group of AABB-type hydrogen signals, δ H 6.70 (2H, d, J = 8.4 Hz) and δ H 7.00 (2H, d, J = 8.4 Hz). 13 C NMR (125MHz, Methanol-d4) spectrum of δ CThe signals at 130.1 (C-4') and 116.1 (C-5') suggest 1,4 substitution. Compound 6 is a new natural product and was named uralensisol L.

[0063] Compound 7 is an orange-yellow oil with an optical rotation value of +7.5(c 0.08,MeOH); HR-ESI-MS m / z 409.2379[M+H] + , C 26 H 33 O4 + The calculated value is 409.2379), indicating that the molecular formula is C 26 H 32 O4, with 10 degrees of unsaturation. Compound 7 is highly similar to 6, with the main difference being the presence of an acetoxy group in compound 7. 13 The C NMR (125 MHz, Methanol-d4) spectrum showed the characteristic signal of acetyl group δ C 171.4 and 18.2. In addition, the 3-carbon signal of compound 7 can be found. C 149.0(C-3) compared to 6δ C The upfield shift of 154.6 (C-3) confirmed that the acetoxy group was substituted at position 3. Compound 7 is a new natural product and was named uralensisol M.

[0064] Compound 8 is an orange-yellow oil with an optical rotation value of +52.5(c 0.08,MeOH); HR-ESI-MS m / z 259.0970[M+H] + , C 15 H 15 O4 + The calculated value is 259.0970), indicating that the molecular formula is C 15 H 14 O4, with 9 degrees of unsaturation. Compound 8 is highly similar to 3,5,4′-trihydroxy-bibenzyl, the main difference being the presence of an aldehyde group in compound 8. The characteristic signal of the aldehyde group is shown in its hydrogen and carbon spectra. H 9.89 (1H, s), δ C 193.9. In addition, the 3-carbon signal of compound 8 can be found. C 149.0(C-3) compared to 12δ C The upfield shift of 154.6 (C-3) confirmed that the acetoxy group was substituted at position 3. Compound 8 is a new natural product and was named uralensisol N.

[0065] Compound 9 is an orange-yellow oil with an optical rotation value of +52.5(c 0.08,MeOH); HR-ESI-MS m / z 367.2275[M+H] + , C 24 H 31 O3 + The calculated value is 367.2275), indicating that the molecular formula is C 24 H 30 O3, with 10 unsaturated degrees. Compound 9 is basically the same as compound 4. There is no acetyl characteristic signal in compound 9, and its δ C 157.0 (C-3) compared to compound 10δ C 151.2(C-3) shifts to the downfield. Then through 1 H- 1 The structure of the compound was further confirmed by H COSY, HSQC, and HMBC. Compound 9 is a new natural product and was named uralensisol O.

[0066] Example 2

[0067] The antibacterial activity test research of the present invention is carried out according to the following steps:

[0068] 1. Experimental Materials and Reagents

[0069] Helicobacter pylori strains HpG27, Hp159, Hp129, and Hp26695 were provided by Professor Bi Hongkai of Nanjing Medical University. Culture media and major reagents included Columbia medium, selective antibiotics (metronidazole, levofloxacin, clarithromycin), and serum.

[0070] 2. Culture Medium Preparation

[0071] Columbia liquid medium: Accurately weigh 29.0 g of Columbia liquid medium, dissolve it in 1000 mL of double-distilled water by heating. After the medium is completely dissolved, autoclave at 121°C for 15 min and set aside.

[0072] Columbia blood agar solid medium: Accurately weigh 39.0 g of Columbia blood agar solid medium and dissolve it in 1000 mL of double-distilled water. After the medium is completely dissolved, autoclave it at 121°C for 15 min. After autoclaving, cool it naturally to about 50°C, then quickly add 5% sterile defibrinated sheep blood, mix well, and pour it into a sterile plate while it is still hot.

[0073] 3. Recovery and cultivation of test strains

[0074] Remove the plate from the -80°C freezer and allow it to stand at room temperature. Accurately pipette 200 μL of the standard bacterial strain onto the solid culture medium and spread the strain evenly using an L-shaped glass rod. Place the culture dish containing the bacterial strain into a culture bag, then insert a microaerobic gas-producing bag into the culture bag. Quickly seal the bag and place it in a 37°C constant-temperature incubator for 72 hours. After the incubation period, identify each strain. Scrape the bacterial moss from the solid culture medium and transfer it to 50 mL of liquid culture medium to serve as the stock culture solution.

[0075] 4. MIC determination

[0076] (1) The concentration of the sample solution of each compound 1-9 obtained by the above separation and purification is 1 mg / mL.

[0077] (2) Preparation of MIC plate: First, add 176 μL of culture medium to the first well, then add 3.2 μL of antimicrobial drug, and dilute it to the seventh well in a doubling ratio; do not add drug to the eighth well, and retain 90 μL of culture medium as a control with bacteria but no drug.

[0078] (3) Preparation of bacterial suspension: Take the Helicobacter pylori growing in the logarithmic phase on the solid plate and make a bacterial suspension using BHI medium. Adjust the concentration OD 600 0.3(1×10 8 CFU / mL), diluted 10 times, 1×10 7 CFU / mL, reserve for future use.

[0079] (4) Take 10 μL of the inoculated bacterial solution and add it to wells 1-8 (the concentration of the bacterial solution in each well is about 1.0×10 6 CFU / mL). The results were determined after 72 h of incubation. The drug concentrations in wells 1 to 7 were 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL, respectively.

[0080] (5) The results are judged by the lowest drug concentration that completely inhibits bacterial growth in the small wells as MIC. The test is meaningful only when bacteria in the 8th well of the positive control well (i.e., without antibiotics) show obvious growth. When a single jump well appears in the microdilution method, the highest drug concentration that inhibits bacterial growth should be recorded. If multiple jump wells appear, the results should not be reported and the test should be repeated. The test should be repeated 3 times for each drug.

[0081] (6) Specific experimental results are shown in Table 1

[0082] Table 1 Minimum inhibitory concentration of compounds against Helicobacter pylori (MIC: μg / mL) MTZ, metronidazole; CLR, clarithromycin; LVX, levofloxacin.

[0083] Through activity screening, the present invention found that the prepared compounds 1, 3, 6-7, and 9 have good activity against both resistant and sensitive Helicobacter pylori, with an MIC of 4-8 μg / mL, which is equivalent to that of positive drugs. They can be used to prepare anti-Helicobacter pylori drugs or as lead compounds for the development of anti-Helicobacter pylori drugs.

Claims

1. A derivative containing a stilbene - type mother nucleus against Helicobacter pylori, characterized in that, It includes compounds with the following structural general formula: Among them, R1, R2, R3, R4, R5, R6, and R7 can all be hydrogen, acyl, isopentenyl, hydroxyl, methoxy, or aldehyde group.

2. The derivative containing a dihydrostilbene-based nucleus for anti-Helicobacter pylori according to claim 1, wherein, Its structural formula is as follows:

3. The preparation method of the derivative containing a dihydrostilbene-based parent nucleus for anti-Helicobacter pylori as claimed in claim 2, characterized in that, It includes the following steps: (1) Weigh the licorice stem and leaf medicinal materials, crush them, soak the medicinal materials in ethanol or methanol aqueous solution at room temperature, heat under reflux for extraction, filter the extraction solution, collect and combine it, and concentrate it under reduced pressure until the alcohol smell disappears to obtain the concentrated licorice stem and leaf solution; (2) Load the concentrated solution obtained in step (1) onto a macroporous resin column, and elute it with ethanol-water mixtures with different volume ratios to obtain 6 sub-streams, Fr.1-1–Fr.1-5; (3) Load the eluate fraction Fr.1-4 obtained in step (2) onto a silica gel column, and elute it with a mixed solvent of petroleum ether and ethyl acetate with different volume ratios to obtain 13 sub-streams, Fr.2-1–Fr.2-13; (4) The eluate fraction Fr.2-4 obtained in step (3) is subjected to medium-pressure preparative gradient elution on column C 18 to obtain five sub-fractions Fr.3-1–Fr.3-5; fraction Fr.3-3 is separated by gel column and purified by preparative high performance liquid chromatography to obtain compound 3; (5) Subject the eluate fraction Fr.2-5 obtained in step (3) to MCI-medium pressure preparative phase gradient elution to obtain 8 sub-fractions, Fr.8-1–Fr.8-16; Compound 8 is obtained from fraction Fr.8-4 by preparative high performance liquid chromatography; Compound 6 is obtained from fraction Fr.8-8 by preparative high performance liquid chromatography; Compounds 1 and 9 are obtained from fraction Fr.8-11 by preparative high performance liquid chromatography; Compounds 2 and 7 are obtained from fraction Fr.8-12 by preparative high performance liquid chromatography; Compounds 4 and 5 are obtained from fraction Fr.8-13 by semi-preparative high performance liquid chromatography.

4. The preparation method of the derivative containing a dihydrostilbene parent nucleus for anti-Helicobacter pylori according to claim 3, characterized in that, It includes the following steps: (1) Weigh the licorice stem and leaf medicinal materials, crush them, soak the medicinal materials in ethanol or methanol aqueous solution at room temperature, heat under reflux at 105°C for 3 times, each time for 2 h, filter the extraction solution, collect and combine it, and concentrate it under reduced pressure until the alcohol smell disappears to obtain the concentrated licorice stem and leaf solution; (2) Perform macroporous resin column chromatography on the concentrated solution obtained in step (1), with a column volume of 20 L, and elute it with a methanol-water gradient with a volume concentration of 0-100% to obtain fractions Fr.1-1–Fr.1-5; (3) Perform silica gel column chromatography on the fraction Fr.1-4 obtained in step (2), with a column volume of 10 L, and elute it successively with petroleum ether / ethyl acetate systems with volume ratios of 6:1, 3:1, 1:1, 1:2, and 0:1 to obtain fractions Fr.2-1–Fr.2-13; (4) The eluate fraction Fr.2-4 obtained in step (3) is subjected to medium-pressure preparative gradient elution with mobile phase of pure water A - methanol B. A gradient elution of 45 - 100% B is carried out for 300 min at a flow rate of 25 mL / min to obtain 5 sub-fractions Fr.3-1 - Fr.3-5. The fraction Fr.3-3 is subjected to gel column chromatography on AB-8 type gel, eluted with methanol, and then subjected to preparative high-performance liquid chromatography with pure water A - methanol B as the mobile phase and isocratic elution at a volume ratio of 28:32 to obtain compound 3 in the time period of 76 - 80 min. 18 ​ (5) The eluate fraction Fr.2 - 5 obtained in step (3) was subjected to MCI medium - pressure preparation, with pure water A - methanol B as the mobile phase, gradient elution: 45 - 100% B; time: 300 min; flow rate 25 mL / min, to obtain 16 sub - fractions Fr.8 - 1–Fr.8 - 16; fraction Fr.8 - 4 was subjected to preparative high - performance liquid chromatography, with pure water A - methanol B as the mobile phase, isocratic elution at a volume ratio of 45:55 for 65 min, and compound 8 was obtained in the time period of 52 - 56 min; fraction Fr.8 - 8 was subjected to preparative high - performance liquid chromatography, with pure water A - methanol B as the mobile phase, isocratic elution at a volume ratio of 57:43 for 90 min, and compound 6 was obtained in the time period of 76 - 80 min; fraction Fr.8 - 11 was subjected to preparative high - performance liquid chromatography, with pure water A - methanol B as the mobile phase, isocratic elution at a volume ratio of 61:39 for 70 min, and compounds 1 and 9 were obtained in the time periods of 48 - 60 min and 60 - 64 min respectively; fraction Fr.8 - 12 was subjected to preparative high - performance liquid chromatography, with pure water A - methanol B as the mobile phase, isocratic elution at a volume ratio of 61:39 for 80 min, and compounds 2 and 7 were obtained in the time periods of 58 - 62 min and 66 - 71 min respectively; fraction Fr.8 - 13 was subjected to semi - preparative high - performance liquid chromatography, with pure water A - methanol B as the mobile phase, isocratic elution at a volume ratio of 68:32 for 70 min, and compounds 4 and 5 were obtained in the time periods of 54 - 58 min and 60 - 63 min respectively.

5. Use of the derivative containing a dihydrostilbene - type parent nucleus as claimed in claim 1 or 2 in the preparation of a medicament for treating Helicobacter pylori.

6. Use of the derivative containing a dihydrostilbene - type parent nucleus as claimed in claim 1 or 2 in the preparation of a medicament for treating drug - resistant and sensitive Helicobacter pylori.

7. The application according to claim 5 or 6, characterized in that, The derivative containing a dihydrostilbene - type parent nucleus is prepared into an oral preparation or a spray with a pharmaceutically acceptable carrier.

8. The application according to claim 7, wherein The oral preparations include tablets, pills, capsules, ointments, powders, mixtures, extracts, granules.

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