A small molecule compound or a pharmaceutically acceptable salt thereof, its use and extraction method

Abstract

The present invention discloses a small molecule compound or a pharmaceutically acceptable salt thereof, its use, and an extraction method. The small molecule compound is represented by formula (I). The present invention also discloses the use of the small molecule compound or a pharmaceutically acceptable salt thereof in the preparation of a drug-resistant bacteria inhibitor. The small molecule compound has an MIC of 50 μg / mL against drug-resistant Staphylococcus aureus and can be used in the preparation of a drug-resistant bacteria inhibitor.

Description

Technical Field

[0001] The present invention relates to a small molecule compound or a pharmaceutically acceptable salt thereof, use thereof and an extraction method thereof, and belongs to the field of medicine. Background Art

[0002] Zanthoxylum avicennae (Lam.) DC. is a small deciduous tree in the Rutaceae family. It is a highly valuable wild resource with significant medicinal value. Zanthoxylum avicennae contains volatile oils, novel small molecules, coumarins, lignans, flavonoids, and terpenes, exhibiting anti-tumor, anti-inflammatory, antibacterial, analgesic, and antioxidant properties. These properties make it an ideal source for plant extracts or active ingredients.

[0003] Staphylococcus aureus (SA) is a Gram-positive commensal bacterium and opportunistic pathogen that primarily inhabits the skin and mucous membranes. Approximately 30% of healthy adults are carriers of S. aureus. S. aureus has become a common cause of hospital-acquired infections because it readily acquires antibiotic resistance. Despite the development of effective strategies to control drug-resistant pathogens, drug-resistant bacteria continue to emerge and spread globally at a rapid rate.

[0004] my country boasts a rich resource of traditional Chinese medicine, and many of its ingredients have been shown to have antibacterial or antimicrobial properties. Some of these herbs indirectly exert their antibacterial or antimicrobial effects by boosting the body's immune system, reducing the toxic side effects of antibiotics, and enhancing the effectiveness of antimicrobial drugs, potentially opening up new avenues for the development of new inhibitors for drug-resistant bacteria. Summary of the Invention

[0005] Objectives of the invention: The first objective of the present invention is to provide a small molecule compound or a pharmaceutically acceptable salt thereof. The second objective of the present invention is to provide the application and extraction method of the above-mentioned small molecule compound or a pharmaceutically acceptable salt thereof.

[0006] Technical solution: A small molecule compound or a pharmaceutically acceptable salt thereof according to the present invention, wherein the small molecule compound is represented by formula (I):

[0007]

[0008] The use of the small molecule compound or a pharmaceutically acceptable salt thereof in the preparation of a drug-resistant bacteria inhibitor is further described, wherein the drug-resistant bacteria is Staphylococcus aureus.

[0009] Furthermore, the effective concentration of the small molecule compound is above 50 μg / mL.

[0010] The pharmaceutical composition of the present invention comprises the small molecule compound or a pharmaceutically acceptable salt thereof.

[0011] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0012] The method for extracting the above-mentioned small molecule compound described in the present invention comprises the following steps:

[0013] (1) Extraction: Weigh the Zanthoxylum bungeanum raw material, crush it, extract it, combine the extracts, and filter it to obtain the total extract;

[0014] (2) Concentration: Concentrate the total extract to remove the organic solvent to obtain a concentrated paste;

[0015] (3) Separation: The concentrated paste is sequentially subjected to normal phase silica gel column chromatography, ODS column chromatography, and high performance liquid chromatography to obtain a small molecule compound of formula (I); the normal phase silica gel column chromatography separation first uses petroleum ether-ethyl acetate as the mobile phase, and gradient elution is performed in a volume ratio of 1:0, 50:1, 30:1, 15:1, 10:1, 8:1, 6:1, 3:1, 1:1, and 0:1, and each volume ratio is eluted 5 times. The fractions obtained with the gradient of 0:1 are combined and then eluted with dichloromethane. -Acetone was used as the mobile phase, and gradient elution was performed in a volume ratio of 1:0, 50:1, 30:1, 15:1, 10:1, 8:1, 6:1, 3:1, 1:1, and 0:1, with each volume ratio eluted 5 times, and the fractions obtained from the 36th to 47th elutions were subjected to ODS column chromatography separation; the ODS column chromatography separation was performed with a methanol aqueous solution with a concentration of 15% to 100% as the mobile phase for gradient elution; the high performance liquid chromatography separation was performed with a 60% methanol aqueous solution as the mobile phase for elution.

[0016] Furthermore, the raw materials described in step (1) include roots and stems of Zanthoxylum bungeanum.

[0017] Furthermore, the extraction method described in step (1) is organic solvent cold soaking extraction; the organic solvent is 90% to 95% ethanol aqueous solution; and the volume ratio of the raw material to the organic solvent is 1:2 to 15.

[0018] Furthermore, the concentration method described in step (2) is reduced pressure concentration, the concentration conditions are 45-60° C., 200 rpm, and the concentration instrument is a rotary evaporator.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following outstanding advantages: the small molecule compound of the present invention is extracted and separated for the first time, and its MIC against drug-resistant Staphylococcus aureus is 90 The concentration of 50 μg / mL can be used for the preparation of drug-resistant bacteria inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The TLC color pattern of the product prepared in Example 1 of the present invention (left) and the color pattern after spraying with 10% sulfuric acid-ethanol solution (right).

[0021] Figure 2 This is a structural diagram of key HMBC and key HH COSY related signals of the product prepared in Example 1 of the present invention.

[0022] Figure 3 This is a high-resolution mass spectrum of the product prepared in Example 1 of the present invention.

[0023] Figure 4 The product prepared in Example 1 of the present invention 1 H-NMR spectrum.

[0024] Figure 5 The product prepared in Example 1 of the present invention 13 C-NMR spectrum.

[0025] Figure 6 This is the HH COSY spectrum of the product prepared in Example 1 of the present invention.

[0026] Figure 7 This is the HSQC spectrum of the product prepared in Example 1 of the present invention.

[0027] Figure 8 This is the HMBC spectrum of the product prepared in Example 1 of the present invention.

[0028] Figure 9 This is the single crystal data of the product prepared in Example 1 of the present invention.

[0029] Figure 10 Circular dichroism spectrum of the product prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0030] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0031] Example 1 Extraction and separation of small molecule compounds

[0032] The Zanthoxylum avicennae was collected from Yunnan Province and was identified by Kunming Caizhi Biotechnology Co., Ltd. as the dried rhizome of Zanthoxylum avicennae (Lam.) DC., a plant of the Rutaceae family and the Zanthoxylum genus. The specimen is currently stored in the laboratory of the School of Pharmacy of Yangzhou University.

[0033] Instruments and parameters used in the experiment: Waters e2695 liquid chromatography, methanol as mobile phase A, water as mobile phase B; SunFire C 18(5 μm 4.6 x 250 mm) chromatographic column, column temperature 25°C, flow rate 2.0 ml / min, gradient elution (0-30 min, 15%-100%); Shanghai Airang Instrument Co., Ltd. CCA-1112A rotary evaporator.

[0034] (1) Weigh 30 kg of dried rhizomes of Zanthoxylum bungeanum, cut them into pieces, and crush them into 10-20 mesh sizes. Cold soak them in 90 L of 95% ethanol for 3 days, collect the extract, repeat the cold soaking for 3 times, and combine all the extracted filtrates to obtain the total extract.

[0035] (2) The extract was concentrated under reduced pressure using a vacuum film concentrator until there was no ethanol smell, thereby obtaining a concentrated paste of 95% ethanol extract.

[0036] (3) The concentrated paste was mixed with 200-300 mesh sample-mixing silica gel to obtain the stationary phase, with a mass ratio of 1:1.5; the separation silica gel was taken, and petroleum ether and the separation silica gel were mixed evenly and wet-packed into the column. The mass of the separation silica gel was about 5 times that of the sample-mixing silica gel; the stationary phase was then dry-loaded, and petroleum ether-ethyl acetate was used as the mobile phase to elute in sequence according to the concentration gradient. The volume ratio of petroleum ether-ethyl acetate used for gradient elution was 1:0, 50:1, 30:1, 15:1, 10:1, 8:1, 6:1, 3:1, 1:1, and 0:1. Three retention volumes were flushed for each gradient. The column volume was 5 L, and each 1 L was a fraction. Subsequently, the fractions of different polarities were recovered, and the fractions obtained with a gradient of 1:0-8:1 were combined into Fr.1, the fractions obtained with a gradient of 6:1-1:1 were combined into Fr.2, and the fractions obtained with a gradient of 0:1 were combined into Fr.3. Subsequently, the fractions were concentrated under reduced pressure on a rotary evaporator at 45°C and 200 rpm to finally obtain three refined components.

[0037] Mix Fr.3 with sample-mixed silica gel to form the stationary phase. The column loading method and sample mixing ratio were the same as above. After coherent loading of the stationary phase, a gradient elution using dichloromethane-acetone volume ratios of 1:0, 50:1, 30:1, 15:1, 10:1, 8:1, 6:1, 3:1, 1:1, and 0:1 was used, sequentially. Three retention volume flushes were performed for each gradient. Each column volume was 800 mL, and each 160 mL fraction was a fraction, for a total of 50 fractions. Fractions 1-5 were combined into Fr.3.1, fractions 6-12 into Fr.3.2, fractions 13-19 into Fr.3.3, fractions 20-25 into Fr.3.4, fractions 21-35 into Fr.3.5, fractions 36-47 into Fr.3.6, and fractions 48-50 into Fr.3.7. The mixture was then concentrated under reduced pressure on a rotary evaporator at 45°C and 200 rpm to obtain 7 subfractions.

[0038] Fr.3.6 was mixed with ODS octadecylsilane to obtain the stationary phase in a mass ratio of 1:2; 100% methanol and ODS were mixed evenly and wet-packed on the column in a ratio of 5 times the amount of ODS used for sample mixing; the stationary phase was then dry-loaded and eluted using methanol-water solution as the mobile phase according to a concentration gradient, with three retention volumes for each gradient; the concentrations of methanol-water solution used for gradient elution were 15%, 20%, 30%, 40%, 60%, 70%, 80%, 90%, and 100%.

[0039] Subsequently, the fractions of various concentrations were recovered and concentrated under reduced pressure on a rotary evaporator at 45°C and 200 rpm. The fractions of all concentrations obtained above were subjected to semi-preparative HPLC using a semi-preparative C column. 18 Chromatographic column, Kromasil, 5μm4.6x250mm, flow rate 2mL / min, 60% methanol / water mixed solvent as mobile phase, finally obtained as the small molecule compound of formula I (t R =34.1min), TLC chart as shown Figure 1 , a single spot was observed in the figure, indicating that the purity was above 95%.

[0040] Example 2 Identification of small molecule compounds

[0041] The small molecule compound prepared in Example 1 was identified as light yellow crystals, which were dissolved in methanol. The molecular formula was determined by high-resolution mass spectrometry to be C 16 H 20 NaO7(m / z 347.1108[M+Na] + , calculated value 347.1101)( Figure 3 ).according to 1 H-NMR (CD3OD) spectrum, 20 hydrogens were observed, including methylene hydrogen signals connected to the chiral center δ4.09 (1H, d, J = 10 Hz, H-10), δ3.95 (1H, d, J = 10 Hz, H-10), two oxymethylene δ4.20 (1H, s, H-7), δ4.55 (1H, s, H-8), one aromatic hydrogen δ6.55 (1H, s, H-1), two methylene δ2.80 (2H, td, J = 8.0 Hz, H-11), δ2.57 (2H, td, J = 8.0 Hz, H-12), one methyl δ1.58 (3H, s, H-CH3), two methoxy δ3.66 (3H, s, H-OCH3), δ3.80 (3H, s, H-OCH3). According to 13C-NMR (CD3OD) spectrum, a total of 16 carbons, including six benzene ring carbons, a quaternary carbon 87.6 (C-9), two methoxy carbons 52.0 (OCH3-13), 61.0 (OCH3-4), a methyl carbon 16.8 (CH3-9) signal, two methylene 26.5 (C-11), 35.1 (C-12), two oxymethylene carbons 76.1 (C-7), 82.2 (C-8), a methylene carbon connected to the chiral center 71.8 (C-10), and a carbonyl carbon 175.6 (C-13). Combined 13 C and HSQC-NMR (CD3OD) spectra ( Figure 6 ) shows that there is 1 carbonyl carbon and 6 quaternary carbons. In the HMBC-NMR (CD3OD) spectrum ( Figure 7 ) observed that H-11 was associated with C-1, C-6, and C-12, and H-12 was associated with C-11 and C-13, which revealed that a methyl propionate group was bound to C-6. The present application also performed single crystal diffraction analysis on the small molecule ( Figure 8 ), in addition, the small molecule structure has a chiral center, which is calculated by circular dichroism ( Figure 9 ) determined its configuration to be (7S,8S,9S). In summary, the structure of this small molecule compound was determined.

[0042] The hydrocarbon spectrum data are as follows: 1 H-NMR (CD3OD): δ6.55(1H,s,H-1),4.20(1H,s,H-7),4.55(1H,s,H-8),4.09(1H,d,J=10Hz,H-10),3.95(1H,d,J=10Hz,H-10) ,2.80(2H,td,J=8.0Hz,H-11),2.57(2H,td,J=8.0Hz,H-12),3.66(3H,s,H-OCH3),1.58(3H,s,H-CH3),3.80(3H,s,H-OCH3). 13 C-NMR (CD3OD): δ123.7(C-1),145.9(C-2),118.3(C-3),136.1(C-4),150.3(C-5),120.7(C-6),76.1(C-7),82.2(C-8),87.6(C-9),71.8(C-10),26.5(C-11),35.1(C-12),175.6(C-13),52.0(OCH3-13),16.8(CH3-9),61.0(OCH3-4). The spectrum is shown in Figure 2. Figure 4-5 shown.

[0043] Example 3 MIC Detection

[0044] Broth microdilution method for MIC determination:

[0045] (1) The test strain was drug-resistant Staphylococcus aureus, which was provided by the Central Laboratory of the Affiliated Hospital of Yangzhou University.

[0046] After the strain was revived and rejuvenated, it was cultured in a 37°C incubator at 120 rpm until the OD 600 0.8, at which point the strain is in the logarithmic growth phase. Adjust the bacterial solution to a turbidity of 0.5 McFarland units (OD 625 0.08-0.13), and then diluted with pure water at a ratio of 2:3 to make the bacterial content 1×10 8 cfu / mL.

[0047] (2) Preparation of small molecule compound: 0.1 mg of the small molecule prepared in Example 1 was taken, 18 μL of DMSO was added and mixed, and the solution concentration was prepared to 5.5 mg / mL.

[0048] (3) Use a 96-well microdilution plate to conduct the experiment. Add 200 μL of the bacterial solution prepared in step (1) to well A, and add 100 μL of the bacterial solution to wells BG; add 2 μL of the drug solution prepared in step (2) to well A, mix well, and then draw 100 μL of the mixed solution from well A to well B. After mixing well, draw 100 μL of the mixed solution from well B to well C, and dilute the solution to well G in a multiple-fold manner. Discard the remaining 100 μL. Make the crude drug equivalent concentration (cdc) ck (k = 1, 2…7) of AG be 100, 50, 25, 12.5, 6.25, 3.125, 1.5625 μg / mL, respectively. Repeat the above steps three times.

[0049] (4) Results and Discussion: All three groups of results showed that when the concentration of the small molecule solution was 100 and 50 μg / mL, the growth of drug-resistant Staphylococcus aureus was significantly inhibited, and no viable bacteria grew. Therefore, the MIC of this small molecule compound against drug-resistant Staphylococcus aureus was 50 μg / mL. This indicates that this small molecule compound has an inhibitory effect on drug-resistant Staphylococcus aureus.

Claims

1. A small molecule compound or a pharmaceutically acceptable salt thereof, characterized in that: The small molecule compound is shown in formula (I):

2. Use of the small molecule compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug-resistant bacteria inhibitor, characterized in that: The drug-resistant bacteria is Staphylococcus aureus.

3. The use according to claim 2, characterized in that The effective concentration of the small molecule compound is above 50 μg / mL.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the small molecule compound according to claim 1 or a pharmaceutically acceptable salt thereof.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition further includes pharmaceutically acceptable excipients.

6. A method for extracting small molecule compounds according to claim 1, characterized in that: The following steps are involved: (1) Extraction: Weigh the Zanthoxylum bungeanum raw material, crush it, extract it, combine the extracts, and filter it to obtain the total extract; (2) Concentration: Concentrate the total extract to remove the organic solvent to obtain a concentrated paste; (3) Separation: The concentrated paste is sequentially subjected to normal phase silica gel column chromatography, ODS column chromatography, and high performance liquid chromatography to obtain a small molecule compound of formula (I); the normal phase silica gel column chromatography separation first uses petroleum ether-ethyl acetate as the mobile phase, and gradient elution is performed in a volume ratio of 1:0, 50:1, 30:1, 15:1, 10:1, 8:1, 6:1, 3:1, 1:1, and 0:1, and each volume ratio is eluted 5 times. The fractions obtained with the gradient of 0:1 are combined and then eluted with dichloromethane. -Acetone was used as the mobile phase, and gradient elution was performed in a volume ratio of 1:0, 50:1, 30:1, 15:1, 10:1, 8:1, 6:1, 3:1, 1:1, and 0:1, with each volume ratio eluted 5 times, and the fractions obtained from the 36th to 47th elutions were subjected to ODS column chromatography separation; the ODS column chromatography separation was performed with a methanol aqueous solution with a concentration of 15% to 100% as the mobile phase for gradient elution; the high performance liquid chromatography separation was performed with a 60% methanol aqueous solution as the mobile phase for elution.

7. The extraction method according to claim 6, characterized in that The raw materials described in step (1) include the roots and stems of Zanthoxylum bungeanum.

8. The extraction method according to claim 6, characterized in that The extraction method described in step (1) is organic solvent cold soaking extraction; the organic solvent is 90% to 95% ethanol aqueous solution; and the volume ratio of the raw material to the organic solvent is 1:2 to 15.

9. The extraction method according to claim 6, characterized in that The concentration method described in step (2) is reduced pressure concentration, the concentration conditions are 45-60° C., 200 rpm, and the concentration instrument is a rotary evaporator.

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