Derivatives of dihydroxanthene-containing parent nucleus with antibacterial activity and preparation method and application thereof

Abstract

The application belongs to the field of medicine, and particularly discloses a derivative containing a dihydro stilbene parent nucleus for resisting helicobacter pylori and a preparation method thereof. The derivative is prepared from licorice stems and leaves as raw materials, through alcohol extraction, macroporous resin, silica gel column chromatography, and preparation liquid phase extraction separation and identification, and a new natural isopentenyl dihydro stilbene compound is obtained. The new compound has good activity on two kinds of drug-resistant and sensitive helicobacter pylori, and can be used for preparing a drug for resisting helicobacter pylori.

Description

Technical Field

[0001] This invention relates to a method for preparing dihydrozirconium compounds and their anti-Helicobacter pylori activity, belonging to the pharmaceutical field. Background Technology

[0002] Helicobacter pylori is a Gram-negative, spiral-shaped or curved microaerophilic bacterium. It colonizes the human gastric mucosa, causing chronic inflammation and is a significant pathogenic factor for chronic gastritis and peptic ulcers. It is also closely related to the development of gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma. In 1994, the World Health Organization classified Helicobacter pylori as a Group 1 carcinogen for gastric cancer, making it the first officially recognized carcinogenic bacterium in the world. The infection rate of Helicobacter pylori in the global general population exceeds 50%, and almost no other chronic infectious disease infects 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. It is noteworthy that my country is a high-incidence area for gastric cancer, with nearly half of the world's gastric cancer cases occurring in China.

[0003] Eradication of Helicobacter pylori can not only promote the healing of peptic ulcers, reduce the incidence of ulcer complications, prevent or delay gastric mucosal atrophy, and inhibit the occurrence and development of intestinal metaplasia, but also reduce the incidence of gastric cancer by one-third. Currently, bismuth-based quadruple therapy is recommended. However, due to the continuous emergence of drug-resistant strains of Helicobacter pylori, conventional treatment strategies are becoming increasingly difficult to eradicate, necessitating the discovery of novel anti-Helicobacter pylori drugs or lead compounds.

[0004] Licorice, also known as Glycyrrhiza uralensis, is a legume belonging to the genus Glycyrrhiza, including Glycyrrhiza inflata and Glycyrrhiza glabra. uralensis Fisch G. inflata Bat G. glabra L The dried roots and rhizomes of licorice have the effects of invigorating qi and replenishing the middle jiao, resolving phlegm and relieving cough, detoxifying, relieving spasms and pain, and moderating the properties of other herbs. They are used to treat spleen and stomach weakness, fatigue, palpitations, shortness of breath, cough with excessive phlegm, abdominal and limb spasms and pain, carbuncles and boils, and to alleviate the toxicity and harshness of other drugs. They have a long history of clinical application and are one of the commonly used medicinal materials. The stems and leaves of licorice are non-medicinal parts of licorice and are mostly discarded in the field, not being rationally utilized. Previous studies have shown that licorice stems and leaves are rich in isopentenyl compounds, and some studies have shown that isopentenylation can enhance the antibacterial effect of these compounds. Therefore, we conducted component separation to obtain novel and highly effective anti-Helicobacter pylori lead compounds. Summary of the Invention

[0005] The technical problem solved by this invention is: This invention provides a novel method for preparing dihydropyridines and their application in anti-Helicobacter pylori. The compounds 1, 3, 6-7, and 9 obtained have good activity against both drug-resistant and sensitive Helicobacter pylori, 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.

[0006] Derivatives containing a dihydrozirconium-like core that combat Helicobacter pylori include compounds with the following general structural formula:

[0007] R1, R2, R3, R4, R5, R6, and R7 can all be hydrogen, acyl, isopentenyl, hydroxyl, methoxy, or aldehyde.

[0008] As a preferred embodiment, the above-described derivative containing a dihydrozirconium-like core for combating Helicobacter pylori has the following structural formula:

[0009]

[0010] The method for preparing the anti-Helicobacter pylori derivative containing a dihydrozirconium core provided by the present invention includes the following steps: (1) Weigh out the licorice stem and leaf medicinal material, crush it, soak the medicinal material in ethanol or methanol aqueous solution at room temperature, heat and reflux to extract, filter the extract and collect and combine it, concentrate it under reduced pressure until there is no alcohol taste, and obtain licorice stem and leaf concentrate. (2) The concentrate obtained in step (1) is loaded onto a macroporous resin column and eluted with ethanol and water at different volume ratios to obtain sub-streams Fr.1-1–Fr.1-5; (3) The eluent fraction Fr.1-4 obtained in step (2) was loaded onto 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; (4) The eluent obtained in step (3) is fractionated Fr.2-4 and subjected to C 18 - Medium-pressure gradient elution was used to obtain five fractions Fr.3-1–Fr.3-5; fraction Fr.3-3 was separated by gel column chromatography and purified by preparative high-performance liquid chromatography to obtain compound 3; (5) The eluent fractions Fr.2-5 obtained in step (3) were subjected to MCI-medium pressure preparative phase gradient elution to obtain 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; and fraction Fr.8-13 was subjected to semi-preparative high performance liquid chromatography to obtain compounds 4 and 5.

[0011] As a preferred embodiment, the preparation method of the above-described anti-Helicobacter pylori derivative containing a dihydrozirconia-like core includes the following steps: (1) Weigh out the licorice stem and leaf medicinal material, crush it, soak the medicinal material in ethanol or methanol aqueous solution at room temperature, and extract it by hot reflux at 105℃ 3 times, 2h each time. After filtering the extract, collect and combine it, and concentrate it under reduced pressure until there is no alcohol taste to obtain licorice stem and leaf concentrate. (2) The concentrate obtained in step (1) was subjected to macroporous resin column chromatography with a column volume of 20 L and eluted with a methanol-water gradient of 0-100% by volume to obtain fractions Fr. 1-1 – Fr. 1-5; (3) The fraction Fr.1-4 obtained in step (2) was subjected to silica gel column chromatography with a column volume of 10 L. The fractions were eluted sequentially with petroleum ether / ethyl acetate systems in 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 eluent obtained in step (3) is fractionated Fr.2-4 and subjected to C 18 - Medium-pressure gradient elution was performed using pure water A-methanol B as the mobile phase, with a gradient elution of 45-100% B for 300 min at a flow rate of 25 mL / min, yielding 5 fractions Fr.3-1–Fr.3-5. Fraction Fr.3-3 was subjected to gel column chromatography (AB-8, 1.5 cm × 210 cm) with methanol elution. After methanol elution, preparative high-performance liquid chromatography was performed with pure water A-methanol B as the mobile phase at a volume ratio of 28:32, eluting isocratically, yielding compound 3 in a time period of 76-80 min. (5) The eluent fractions Fr.2-5 obtained in step (3) were prepared by MCI-medium pressure elution with pure water A-methanol B as the mobile phase, using gradient elution: 45-100% B; time: 300 min; flow rate: 25 mL / min, to obtain 16 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, using a volume ratio of 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, using a volume ratio of 57:43 isocratic elution for 90 min, and compound 8 was obtained in the time period of 76-80 min. Compound 6 was obtained within a short time interval. Fractions Fr.8-11 were subjected to preparative high-performance liquid chromatography (HPLC) with pure water A-methanol B as the mobile phase and eluted isocratically at a volume ratio of 61:39 for 70 min, yielding compounds 1 and 9 at time intervals of 48-60 min and 60-64 min, respectively. Fraction Fr.8-12 was subjected to preparative HPLC with pure water A-methanol B as the mobile phase and eluted isocratically at a volume ratio of 61:39 for 80 min, yielding compounds 2 and 7 at time intervals of 58-62 min and 66-71 min, respectively. Fraction Fr.8-13 was subjected to semi-preparative HPLC with pure water A-methanol B as the mobile phase and eluted isocratically at a volume ratio of 68:32 for 70 min, yielding compounds 4 and 5 at time intervals of 54-58 min and 60-63 min, respectively.

[0012] The activity assays used the microbroth dilution method to evaluate the anti-Helicobacter pylori activity of each compound, with clinically used metronidazole, levofloxacin, clarithromycin, and vancomycin selected as positive control drugs. The results showed that compounds 1, 3, 6-7, and 9 exhibited good activity against both drug-resistant and susceptible Helicobacter pylori strains, with MICs of 4–8. μ g / mL, comparable to the positive control drug metronidazole, can be used to prepare anti-Helicobacter pylori drugs or as a lead compound for the development of anti-Helicobacter pylori drugs.

[0013] This invention is the first to discover a novel dihydrozirconium from the stems and leaves of licorice, and provides extraction and separation techniques, structural identification methods, and applications of this new compound in combating Helicobacter pylori.

[0014] Beneficial effects

[0015] (1) The present invention provides a novel derivative containing a dihydrozirconium core with anti-Helicobacter pylori activity and its preparation method.

[0016] (2) This invention is the first to discover that dihydrozirconium derivatives have a good inhibitory effect on Helicobacter pylori. Compounds 1, 3, 6-7, and 9 have good activity against both drug-resistant and sensitive Helicobacter pylori, with MICs of 4–8. μ g / mL. It can be used to prepare anti-Helicobacter pylori drugs or as a lead compound for the development of anti-Helicobacter pylori drugs. Attached Figure Description

[0017] Figure 1 Compound 1 1 H NMR spectrum (500 MHz, Methanol- d 4) Figure 2 Compound 1 13 C10 NMR spectrum (125 MHz, Methanol-C10) d 4) Figure 3 Compound 2 1 H NMR spectrum (500 MHz, Methanol- d 4) Figure 4 Compound 2 13 C10 NMR spectrum (125 MHz, Methanol-C10) d 4) Figure 5 Compound 3 1 H NMR spectrum (500 MHz, Methanol- d 4) Figure 6 Compound 3 13 C10 NMR spectrum (125 MHz, Methanol-C10) d 4) Figure 7 Compound 4 1 H NMR spectrum (500 MHz, Methanol- d 4) Figure 8 Compound 4 13 C10 NMR spectrum (125 MHz, Methanol-C10) d 4) Figure 9 Compound 5 1 H NMR spectrum (500 MHz, Methanol- d 4) Figure 10 Compound 5 13 C10 NMR spectrum (125 MHz, Methanol-C10) d 4) Figure 11 Compound 6 1 H NMR spectrum (500 MHz, Methanol-d 4) Figure 12 Compound 6 13 C10 NMR spectrum (125 MHz, Methanol-C10) d 4) Figure 13 Compound 7 1 H NMR spectrum (500 MHz, Methanol- d 4) Figure 14 Compound 7 13 C10 NMR spectrum (125 MHz, Methanol-C10) d 4) Figure 15 Compound 8 1 H NMR spectrum (500 MHz, Methanol- d 4) Figure 16 Compound 8 13 C10 NMR spectrum (125 MHz, Methanol-C10) d 4) Figure 17 Compound 9 1 H NMR spectrum (500 MHz, Methanol- d 4) Figure 18 Compound 9 13 C10 NMR spectrum (125 MHz, Methanol-C10) d 4). Detailed Implementation

[0018] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the specific material ratios, process conditions, and results described in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0019] Example 1 1. Instruments and Materials 1.1 Instruments ACQUITY UPLC (Ultra-High Performance Liquid Chromatography) Waters Company, USA S-B50L Vertical Pressure Steam Sterilizer Jintan Medical Device Factory, Jiangsu Province <![CDATA[CHEETAH ® MP medium-pressure preparative chromatography]]> Tianjin Bona Aier Technology Co., Ltd. Adjustable pipette Eppendorf Company, USA Bruker AV-500 MRI scanner Bruker, Germany Microplate thermostatic oscillator Hangzhou Mio Instrument Co., Ltd. Shimadzu LC-20 AR Preparative High Performance Liquid Chromatography System Shimadzu Corporation of Japan 7500 Real-Time PCR System Applied Biosystems, Inc. R-300 Rotary Evaporator BUCHI Ltd., Switzerland IS-RDV1 type constant temperature oscillator American Precision Company PRX-150B Intelligent Artificial Climate Chamber Ningbo Saif Experimental Instruments Co., Ltd. PrimoStar Inverted Microscope German company ZEISS Quitix 125 D-1CN Electronic Balance Sartorius Scientific Instruments Co., Ltd. SIM-F140AY65-PC Ice Maker Panasonic Corporation, Japan Milli-Q Advantage System Ultrapure Water System Millipore, USA <![CDATA[Forma Series II water-jacketed CO₂ incubator]]> Thermo Technologies, Inc. 1300 series A2 clean bench Thermo Technologies, Inc. TDL-80-2B Centrifuge Shanghai Anting Co., Ltd. EnSpire Multifunctional Microplate Reader PerkinElmer, Inc. 1.2. Experimental Materials Hedera ODS preparative chromatographic column (10 nm, 5) μ m, 10×250 mm); Waters ACQUITY UPLC BEHC 18 (2.1 mm × 100 mm, 1.7) μm) chromatographic column; MCI GEL (CHP20, 75−150) μ m), the gel was Sephadex LH-20, the column chromatography silica gel (200-300 mesh); chromatographic grade acetonitrile, methanol and formic acid were purchased from Merck, Inc.; analytical grade petroleum ether, ethyl acetate, etc. were purchased from Nanjing Wanqing Chemical Reagent Co., Ltd.

[0020] 2. The preparation method of the present invention includes the following steps: (1) Weigh 5 kg of licorice stem and leaf medicinal material, crush it, add 10 L of 75% ethanol aqueous solution to soak the medicinal material at room temperature for 1 h, and extract it by hot reflux at 105℃ 3 times, 2 h each time. After filtering the extract, collect and combine it, and concentrate it under reduced pressure until there is no alcohol taste to obtain licorice stem and leaf concentrate. (2) The concentrate obtained in step (1) was subjected to macroporous resin column chromatography with a column volume of 20 L and eluted with a methanol / water (0-100%) gradient system for 400 L to obtain fractions Fr. 1-1 – Fr. 1-5.

[0021] (3) The fraction Fr.1-4 obtained in step (2) was subjected to silica gel column chromatography with a column volume of 10 L. The fractions were eluted sequentially with petroleum ether / ethyl acetate systems in 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 eluent obtained in step (3) is fractionated Fr.2-4 and subjected to C 18 - Medium-pressure gradient elution was performed using pure water A-methanol B as the mobile phase, with a gradient elution of 45-100% B for 300 min at a flow rate of 25 mL / min, yielding 5 fractions Fr.3-1–Fr.3-5. Fraction Fr.3-3 was subjected to gel column chromatography (AB-8, 1.5 cm × 210 cm) with methanol elution. After methanol elution, preparative high-performance liquid chromatography was performed with pure water A-methanol B as the mobile phase at a volume ratio of 28:32, eluting isocratically, yielding compound 3 in a time period of 76-80 min. (5) The eluent fractions Fr.2-5 obtained in step (3) were prepared by MCI-medium pressure elution with pure water A-methanol B as the mobile phase, using gradient elution: 45-100% B; time: 300 min; flow rate: 25 mL / min, to obtain 16 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, using a volume ratio of 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, using a volume ratio of 57:43 isocratic elution for 90 min, and compound 8 was obtained in the time period of 76-80 min. Compound 6 was obtained within a short time interval. Fractions Fr.8-11 were subjected to preparative high-performance liquid chromatography (HPLC) with pure water A-methanol B as the mobile phase and eluted isocratically at a volume ratio of 61:39 for 70 min, yielding compounds 1 and 9 at time intervals of 48-60 min and 60-64 min, respectively. Fraction Fr.8-12 was subjected to preparative HPLC with pure water A-methanol B as the mobile phase and eluted isocratically at a volume ratio of 61:39 for 80 min, yielding compounds 2 and 7 at time intervals of 58-62 min and 66-71 min, respectively. Fraction Fr.8-13 was subjected to semi-preparative HPLC with pure water A-methanol B as the mobile phase and eluted isocratically at a volume ratio of 68:32 for 70 min, yielding compounds 4 and 5 at time intervals of 54-58 min and 60-63 min, respectively.

[0022] 3. Structural analysis of compounds:

[0023] Compound 1 is an orange-yellow oily substance with an optical rotation value of [ ]. α ]20 D, +5.0 ( c 0.08, MeOH); HR-ESI-MSm / z 369.2066 [M + H] + C 23 H 29 O4 + The calculated value is 369.2066), indicating the molecular formula is C. 23 H 28 O4 has 10 degrees of unsaturation. 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 set of hydrogen signals from isobutyryl groups. δ H 2.80 dd, 1.28 d, 1.27 d and a set of carbon signals δ CThe presence of 177.4, 35.4, 19.3, and 19.3 in the HMBC spectrum. δ H 3.11 (H-7) respectively with δ C The values ​​144.0 (C-1), 123.9 (C-2), and 151.2 (C-3) are related, which confirms that it is a 2-isopentenyl substitution. δ C 151.2 (C-3) Displacement towards the upper field, determined to be 3- O - Isobutyryl substitution. Based on the above, the structure of the compound is determined to be 2-isopentenyl-3-isobutyryl-5,4'-dihydroxyuridine. Compound 1 is a new natural product, named uralensisol F.

[0024]

[0025] Compound 2 is an orange-yellow oily substance with an optical rotation value of [ ]. α ]20 D, +2.5 ( c 0.08, MeOH); HR-ESI-MSm / z 383.2215 [M + H] + C 24 H 31 O4 + The calculated value is 383.2222), indicating the molecular formula is C. 24 H 30 O4 has 10 degrees of unsaturation. Compound 2 is highly similar to compound 1. Analysis of its carbon and proton spectra revealed a characteristic signal of a 2-methylbutyryl group. δ H 2.80 dd, 1.28 d, 1.27 d and δ C The presence of 177.4, 35.4, 19.3, and 19.3 in the HMBC spectrum. δ H 3.11 (H-7) respectively with δ C The values ​​144.1 (C-1), 123.9 (C-2), and 151.2 (C-3) are related, which confirms that it is a 2-isopentenyl substitution. δ C 151.2 (C-3) displacement towards the upper field, determined to be 3- O -2-Methylbutyryl substitution. Based on the above, the compound structure is determined to be 2-isopentenyl-3- O-2-Methylbutyryl-5,4'-dihydroxydihydrourethane. Compound 2 is a new natural product, named uralensisol G.

[0026]

[0027] Compound 3 is an orange-yellow oily substance with an optical rotation value of [ ]. α ]20 D, +40.0 ( c 0.08, MeOH); HR-ESI-MSm / 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 24 O3 has 9 degrees of unsaturation. Compound 3 is highly similar to compound 2-(3-methyl-2-butenyl)-3,5,4'-trihydroxy-bibenzyl. The main difference is that compound 3 contains a set 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 correlated. In the HMBC spectrum, δ H 3.67 (3H, s) and δ C 159.6 Long-range correlation determined that the methoxy group was substituted at the 5-position. Compound 3 is a novel natural product, named uralensisol I.

[0028]

[0029] Compound 4 is an orange-yellow oily substance with an optical rotation value of [ ]. α ]20 D, +2.5 ( c 0.08, MeOH); HR-ESI-MSm / z 409.2385 [M + H] + C 26 H 33 O4 + The calculated value is 409.2379), indicating the molecular formula is C. 26 H 32 O4 has 11 degrees of unsaturation. The A ring of compound 4 is similar to that of compound 9. Besides, 1 H NMR (500 MHz, Methanol-d 4) The spectrum shows a set of mutually coupled hydrogen signals. δ H 6.76 (dd, J = 2.2, 8.7 Hz, H-6'), 6.64 (d, J =8.7Hz, H-5'), 6.77 (d, J =2.2Hz, H-2') and a set 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 The correlations of 154.2, 129.0, and 130.5 confirm that ring B is substituted with 3-isopentenyl-4'-hydroxy. δ C 151.2 (C-3) shifts to a higher field, and is related to... δ C 26.0 (C-7') is relevant and determined to be 3- O - Acetyl substitution. Compound 4 is a new natural product, named uralensisol J.

[0030]

[0031] Compound 5 is an orange-yellow oily substance with an optical rotation value of [ ]. α 20 D, -2.5 ( c 0.08, MeOH); HR-ESI-MSm / z 439.2488 [M + H] + C 27 H 35 O5 + The calculated value is 439.2484), indicating the molecular formula is C. 27 H 34 O5 has 11 degrees of unsaturation. Compound 5 is highly similar to compound 4. 13 C NMR (125 MHz, Methanol- d 4) In the score δ CSignals at 145.2 (C-4') and 150.8 (C-5') suggest ortho- and tho-diphenol hydroxyl substitution. Furthermore, 1 H NMR (500 MHz, Methanol- d 4) The spectrum shows a set of methoxy hydrogen signals. δ H 3.73 (3H, s). In the HMBC spectrum, δ H 3.73 (3H, s) and δ C 145.2 Long-range correlation confirmed a 4'-methoxy substitution. Compound 5 is a novel natural product, named uralensisol K.

[0032]

[0033] Compound 6 is an orange-yellow oily substance with an optical rotation value of [ ]. α ]20 D, +42.5 ( c 0.08, MeOH); HR-ESI-MSm / z 367.2278 [M + H] + C 24 H 31 O3 + The calculated value is 367.2273, indicating the molecular formula is C. 24 H 30 O3 has 10 degrees of unsaturation. Compound 6 is highly similar to gancaonin R, the main difference being the non-ortho-dihydroxyl substitution in the B ring of compound 6. In compound 6, 1 H NMR (500 MHz, Methanol- d 4) The spectrum shows a set 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 (125 MHz, Methanol- d 4) In the score δ C Signals at 130.1 (C-4') and 116.1 (C-5') suggest 1,4 substitution. Compound 6 is a novel natural product, named uralensisol L.

[0034]

[0035] Compound 7 is an orange-yellow oily substance with an optical rotation value of [ ]. α ]20 D, +7.5 ( c 0.08, MeOH); HR-ESI-MSm / z 409.2379 [M + H] + C 26 H 33 O4 + The calculated value is 409.2379), indicating the molecular formula is C. 26 H 32 O4 has 10 degrees of unsaturation. Compound 7 is highly similar to 6, the main difference being that compound 7 has an acetoxy group as a substitute. 13 C NMR (125 MHz, Methanol-) d 4) The spectrum shows the characteristic signal of the acetyl group. δ C 171.4 and 18.2. Additionally, a carbon signal at position 3 of compound 7 can be detected. δ C 149.0 (C-3) compared to 6 δ C The 154.6 (C-3) axis shifts to a higher field, thus determining that the acetoxy group is substituted at the 3-position. Compound 7 is a novel natural product, named uralensisol M.

[0036]

[0037] Compound 8 is an orange-yellow oily substance with an optical rotation value of [ ]. α ]20 D, +52.5 ( c 0.08, MeOH); HR-ESI-MSm / z 259.0970 [M + H] + C 15 H 15 O4 + The calculated value is 259.0970), indicating the molecular formula is C. 15 H 14 O4 has 9 degrees of unsaturation. Compound 8 is highly similar to 3,5,4′-trihydroxy-bibenzyl, the main difference being that compound 8 has an aldehyde substitution. Both compounds show characteristic aldehyde signals in their proton and carbon spectra. δ H 9.89 (1H, s). δ C 193.9. Furthermore, in the HMBC spectrum, δ H 9.89 (1H, s) and δ C113.3 (C-2) long-range correlation identified as 2-aldehyde substitution. Compound 8 is a novel natural product, named uralensisol N.

[0038]

[0039] Compound 9 is an orange-yellow oily substance with an optical rotation value of [ ]. α ]20 D, +52.5 ( c 0.08, MeOH); HR-ESI-MSm / z 367.2275 [M + H] + C 24 H 31 O3 + The calculated value is 367.2275, indicating the molecular formula is C. 24 H 30 O3 has 10 degrees of unsaturation. Compound 9 is essentially identical to compound 4. Compound 9 lacks the characteristic signal of an acetyl group, and its... δ C 157.0 (C-3) compared to compound 4 δ C 151.2 (C-3) shifts to a lower field. Then through... 1 H- 1 H COSY, HSQC, and HMBC further determined the structure of the compound. Compound 9 is a new natural product, named uralensisol O.

[0040] Example 2 The antibacterial activity test of the present invention was conducted according to the following steps: 1. Experimental Materials and Reagents Helicobacter pylori strains HpG27, Hp159, Hp129, and Hp26695 were provided by Professor Bi Hongkai of Nanjing Medical University. Culture media and main reagents: Columbia broth, selective antibiotics (metronidazole, levofloxacin, clarithromycin), serum, etc.

[0041] 2. Preparation of culture medium Columbia liquid medium: Accurately weigh 29.0 g of Columbia liquid medium, heat and dissolve it in 1000 mL of double-distilled water. After the medium is completely dissolved, autoclave at 121°C for 15 min and set aside.

[0042] Columbia blood agar solid medium: Accurately weigh 39.0 g of Columbia blood agar solid medium, heat and dissolve it in 1000 mL of double-distilled water. After the medium is completely dissolved, autoclave at 121°C for 15 min. After autoclaving, let it cool naturally to about 50°C, then quickly add 5% sterile defibrinated sheep blood, mix well, and pour it into sterile Petri dishes while hot.

[0043] 3. Recovery and culture of the tested bacterial strains Remove from -80°C freezer, place at room temperature, and accurately aspirate 200 μL of standard bacterial culture. μ Transfer the bacterial culture to a solid culture medium and spread it evenly using an L-shaped glass rod. Place the culture dish containing the culture into a culture bag, then add a microaerophilic gas-generating bag to the culture bag. Quickly seal the culture bag and place it in a 37°C incubator for 72 hours. After incubation, identify each strain, then scrape off the bacterial growth on the solid culture medium and transfer the bacterial growth to 50 mL of liquid culture medium as the original bacterial culture.

[0044] 4. Determination of MIC (1) Prepare sample solutions of compounds 1-9 obtained by separation and purification above with a concentration of 1 mg / mL.

[0045] (2) Preparation of MIC plate: Add culture medium 176 to the first well. μ L, plus 3.2 μ L-type antibiotic, serially diluted to well 7; well 8 without antibiotic, retain 90°C. μ L-culture medium was used as a control with added bacteria but no added drugs.

[0046] (3) Preparation of bacterial suspension: Helicobacter pylori in the logarithmic growth phase on solid plates was used to prepare a bacterial suspension in BHI medium and the concentration OD was adjusted. 600 It is 0.3 (1×10 8 CFU / mL), diluted 10 times, yielding 1×10⁻⁶ CFU / mL. 7 CFU / mL, for later use.

[0047] (4) Take 10g of the inoculation solution μ L was added to wells 1-8 (the bacterial concentration in each well was approximately 1.0 × 10⁻⁶). 6 (CFU / mL). Results were assessed after 72 hours of incubation. Drug concentrations in wells 1 through 7 were 16, 8, 4, 2, 1, 0.5, and 0.25, respectively. μ g / mL.

[0048] (5) Result Interpretation: The lowest drug concentration that completely inhibits bacterial growth within the well is defined as the MIC. The test is only meaningful if bacteria show significant growth in the 8th well (i.e., without antibiotic) of the positive control well. When a single skipped well occurs in the microdilution method, the highest drug concentration that inhibits bacterial growth should be recorded. If multiple skipped wells occur, the results should not be reported, and the test should be repeated. Each drug should be tested three times.

[0049] (6) The specific experimental results are shown in Table 1. Table 1. Minimum inhibitory concentration (MIC) of compounds against Helicobacter pylori: μ g / mL) 1 8 8 8 8 2 >16 >16 >16 >16 3 4 4 8 8 4 >16 >16 >16 >16 5 >16 >16 >16 >16 6 4 8 8 8 7 8 8 8 8 8 >16 >16 >16 >16 9 8 8 8 8 MTZ 2 1 32 ( R ) 16 ( R ) CLR 0.008 0.008 1 ( R ) 2 ( R ) LVX 0.25 0.25 8 ( R ) 8 ( R ) MTZ, metronidazole; CLR, clarithromycin; LVX, levofloxacin. Through activity screening, this invention found that compounds 1, 3, 6-7, and 9 exhibited good activity against both drug-resistant and sensitive Helicobacter pylori, with MICs of 4–8 μg / mL, comparable to positive control drugs. These compounds can be used to prepare anti-Helicobacter pylori drugs or as lead compounds for the development of anti-Helicobacter pylori drugs.

Claims

1. Application of derivatives containing dihydrozirconium cores in the preparation of drugs against Helicobacter pylori; The derivatives containing dihydrozirconia-like cores are compounds 1, 3, 6, 7, and 9 with the following structural formulas: 。 2. The application according to claim 1, characterized in that, Application of derivatives containing dihydrozirconium cores in the preparation of drugs against drug-resistant and susceptible Helicobacter pylori.

3. The application according to claim 1 or 2, characterized in that, The aforementioned derivative containing a dihydrozirconium-like core was prepared by the following method: (1) Weigh out the licorice stem and leaf medicinal material, crush it, soak the medicinal material in ethanol or methanol aqueous solution at room temperature, heat and reflux to extract, filter the extract and collect and combine it, concentrate it under reduced pressure until there is no alcohol taste, and obtain licorice stem and leaf concentrate. (2) The concentrate obtained in step (1) is loaded onto a macroporous resin column and eluted with methanol and water at different volume ratios to obtain sub-streams Fr.1-1–Fr.1-5; (3) The eluent obtained in step (2) was fractionated Fr.1-4, loaded onto 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; (4) The eluent obtained in step (3) is fractionated Fr.2-4 and subjected to C 18 - Medium-pressure gradient elution was used to obtain five fractions Fr.3-1 to Fr.3-5; fraction Fr.3-3 was separated by gel column chromatography and purified by preparative high-performance liquid chromatography to obtain compound 3; (5) The eluent fraction Fr.2-5 obtained in step (3) was subjected to MCI-medium pressure preparative phase gradient elution to obtain sub-fractions Fr.8-1–Fr.8-16; 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 compound 7.

4. The application according to claim 3, characterized in that, The aforementioned derivative containing a dihydrozirconium-like core was prepared by the following method: (1) Weigh out the licorice stem and leaf medicinal material, crush it, soak the medicinal material in ethanol or methanol aqueous solution at room temperature, and extract it by hot reflux at 105℃ 3 times, 2h each time. After filtering the extract, collect and combine it, and concentrate it under reduced pressure until there is no alcohol taste to obtain licorice stem and leaf concentrate. (2) The concentrate obtained in step (1) was subjected to macroporous resin column chromatography with a column volume of 20 L and eluted with a methanol-water gradient of 0-100% by volume to obtain fractions Fr. 1-1 – Fr. 1-5; (3) The fractions Fr.1-4 obtained in step (2) were subjected to silica gel column chromatography with a column volume of 10 L. The fractions were eluted sequentially with petroleum ether / ethyl acetate systems in volume ratios of 6:1, 3:1, 1:1, 1:2, and 0:1 to obtain fractions Fr.2-1 to Fr.2-13. (4) The eluent obtained in step (3) is fractionated Fr.2-4 and subjected to C 18 - Medium-pressure gradient elution was performed using pure water A-methanol B as the mobile phase, with a gradient elution of 45-100% B for 300 min at a flow rate of 25 mL / min, yielding 5 fractions Fr.3-1–Fr.3-5. Fraction Fr.3-3 was subjected to AB-8 gel column chromatography, eluted with methanol. After methanol elution, preparative high-performance liquid chromatography was performed using pure water A-methanol B as the mobile phase, with isocratic elution at a volume ratio of 28:32, yielding compound 3 in a time period of 76-80 min. (5) The eluent fractions Fr.2-5 obtained in step (3) were prepared by MCI-medium pressure elution with pure water A-methanol B as the mobile phase, using gradient elution: 45-100% B; time: 300 min; flow rate: 25 mL / min, to obtain 16 fractions Fr.8-1–Fr.8-16; Fr.8-8 was subjected to preparative high performance liquid chromatography with pure water A-methanol B as the mobile phase, using a volume ratio of 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, using a volume ratio of 61:39 isocratic elution for 70 min, and compound 6 was obtained in the time period of 48-60 min and 60-64 min, respectively. Compounds 1 and 9 were obtained in a time interval of min; 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 min, and compound 7 was obtained in a time interval of 66-71 min.

5. The application according to claim 1 or 2, characterized in that, Derivatives containing dihydrozirconia-like cores are prepared into oral formulations or sprays using pharmaceutically acceptable carriers.

6. The application according to claim 5, characterized in that, The oral preparations include tablets, pills, capsules, ointments, powders, mixtures, extracts, or granules.

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