CN117683002A - A kind of phloroglucinol derivative and its preparation method and application - Google Patents

Abstract

The invention relates to a phloroglucinol derivative, a preparation method thereof and application thereof in preparation of antibacterial drugs. The molecular structure of the phloroglucinol derivative is shown as formula I, and the configuration is 7S,1′R,4′S,5′R,7′R,10′R. The phloroglucinol derivative is formed by heterozygosis of acyl phloroglucinol and gordonane type sesquiterpene, and is a chemical entity with a novel framework structure. The compound has low concentration resistance to Staphylococcus aureus and methicillinGram-positive bacteria such as staphylococcus aureus and vancomycin-mediated drug-resistant staphylococcus aureus have remarkable inhibition effect, low toxicity to eukaryotic cells and good safety.

Description

A kind of phloroglucinol derivative and its preparation method and application

Technical field

The invention belongs to the technical field of natural medicines and chemical medicines, and specifically relates to a phloroglucinol derivative and its preparation method and application.

Background technique

Bacterial infection is a common disease and frequently-occurring disease. At present, clinical infections with drug-resistant bacteria are becoming increasingly serious, and bacterial resistance is showing a trend towards multi-drug resistance, including methicillin-resistant Staphylococcus aureus (MRSA), ampicillin-resistant Streptococcus pneumoniae (PRSP), and many more. Drug-resistant Mycobacterium tuberculosis (MDR-TB) is spreading rapidly. The increasing number of drug-resistant bacterial infections and the gradual increase in mortality indicate that traditional antibacterial drugs can no longer meet the needs of modern clinical treatment. Therefore, research and development of new antibacterial drugs has important clinical application value.

Contents of the invention

Based on this, the purpose of the present invention is to provide a new antibacterial drug.

In order to achieve the above-mentioned object, the present invention includes the following technical solutions.

On the one hand, the present invention provides a phloroglucinol derivative or a pharmaceutically acceptable salt thereof. The molecular structure of the phloroglucinol derivative is shown in Formula I, with a configuration of 7 S , 1′ R ,4′ S ,5′ R ,7′ R ,10′ R ,

.

In a second aspect, the present invention provides a method for preparing the phloroglucinol derivative, comprising the following steps:

Crush the above-ground part of Australian snow plum, and extract it by percolation with an ethanol aqueous solution with a volume concentration of 85-98%. The extract is concentrated and extracted with petroleum ether, and then the extract is separated and purified to obtain the phloroglucinol derivative. .

Wherein, the separation and purification includes: concentrating the extract and then sequentially passing through silica gel column chromatography, Sephadex LH-20 chromatography column, octadecylsilane bonded silica gel column chromatography, and preparative HPLC separation and purification.

In a third aspect, the present invention provides the use of the phloroglucinol derivative or a pharmaceutically acceptable salt thereof in the preparation of antibacterial drugs.

Specifically, the phloroglucinol derivative or its pharmaceutically acceptable salt is used in the preparation of a drug that inhibits bacterial growth.

Application of the phloroglucinol derivative or pharmaceutically acceptable salt thereof according to the present invention in the preparation of medicaments for treating and/or preventing bacterial infections.

In a fourth aspect, the present invention provides an antibacterial drug prepared from active ingredients and pharmaceutically acceptable excipients. The active ingredients include the phloroglucinol derivatives of the present invention or pharmaceutically acceptable auxiliary materials thereof. of salt.

The invention has the following beneficial effects:

(1) The present invention discovers, isolates and extracts a novel phloroglucinol derivative from Australian snow plum. This compound is formed by the hybridization of acyl phloroglucinol and gorgonane type sesquiterpene. Sesquiterpene Part of it has a 6/6/5 cage structure and is a chemical entity with a novel skeleton structure.

(2) The present invention found that the low concentration of the phloroglucinol derivative of the present invention has significant effects on Gram-positive bacteria such as Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and vancomycin-mediated resistance to Staphylococcus aureus. Antibacterial activity; in particular, it has significant inhibitory activity against multidrug-resistant bacteria and can be used as a new chemical entity to prepare new antibacterial drugs to treat diseases caused by drug-resistant bacterial infections.

(3) The phloroglucinol derivative of the present invention has low toxicity to eukaryotic cells, high safety, and good medicinal properties.

Description of the drawings

Figure 1 is the ¹ H NMR spectrum of phloroglucinol derivatives.

Figure 2 is the ¹³ C NMR spectrum of phloroglucinol derivatives.

Figure 3 shows the ¹ H- ¹ H COZY spectrum of phloroglucinol derivatives.

Figure 4 is the HSQC spectrum of phloroglucinol derivatives.

Figure 5 shows the HMBC spectrum of phloroglucinol derivatives.

Figure 6 shows the NOESY spectrum of phloroglucinol derivatives.

Figure 7 is a single crystal X-ray diffraction structure diagram of a phloroglucinol derivative.

Figure 8 shows the measured ECD and calculated ECD fitting diagrams of phloroglucinol derivatives.

Detailed ways

In order to facilitate an understanding of the invention, the invention will be described more fully below. The invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that a thorough understanding of the present disclosure will be provided.

Experimental methods without specifying specific conditions in the following examples usually follow conventional conditions or conditions recommended by the manufacturer. Various commonly used chemical reagents used in the examples are all commercially available products.

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings commonly understood by those skilled in the technical field belonging to the present invention. The terms used in the description of the present invention are only for the purpose of describing specific embodiments and are not used to limit the present invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Furthermore, as used herein, the term "or" is an inclusive "or" symbol and is equivalent to the term "and/or" unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for other factors not described to be based upon, unless the context clearly dictates otherwise. Furthermore, throughout this specification, the meanings of "a," "an," and "the" include plural referents. The meaning of "in" includes "in" and "on".

In order to develop more new antibacterial drugs to overcome the problem that existing clinical drugs cannot meet the demand for drug-resistant bacterial infections, in one embodiment of the present invention, a phloroglucinol derivative or a pharmaceutically acceptable derivative thereof is provided The molecular structure of the phloroglucinol derivative is shown in formula I, and the configuration is 7 S , 1′ R , 4′ S , 5′ R , 7′ R , 10′ R ,

.

In another embodiment of the present invention, a method for preparing the phloroglucinol derivative is provided, comprising the following steps:

Crush the above-ground part of Australian snow plum, and extract it by percolation with an ethanol aqueous solution with a volume concentration of 85-98%. The extract is concentrated and extracted with petroleum ether, and then the extract is separated and purified to obtain the phloroglucinol derivative. .

In some embodiments, the volume concentration of the ethanol aqueous solution is 90-95%.

In some embodiments, the number of percolation extractions is 4-6 times, and the ratio of ethanol aqueous solution and Australian snow plum each time is 0.8-1.2L:1kg.

In some embodiments, the separation and purification includes: concentrating the extract and then sequentially performing silica gel column chromatography, Sephadex LH-20 chromatography column separation and purification, octadecylsilane bonded silica gel column chromatography, and preparative HPLC separation. purification.

In another embodiment of the present invention, there is provided the use of the phloroglucinol derivative or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting bacterial growth.

In another embodiment of the present invention, there is provided the use of the phloroglucinol derivative or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and/or preventing bacterial infections.

The bacteria described in the present invention may be non-drug-resistant bacteria, or may be single-drug-resistant bacteria or multi-drug-resistant bacteria.

The phloroglucinol derivative of the present invention or its pharmaceutically acceptable salt has better antibacterial activity against Gram-positive bacteria, such as very excellent antibacterial activity against Staphylococcus aureus, Enterococcus aureus, etc.; wherein Staphylococcus aureus includes Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus, Staphylococcus epidermidis, etc.; enterococci include Enterococcus faecalis, Enterococcus faecium, etc.

In another embodiment of the present invention, an antibacterial drug is also provided, which is prepared from active ingredients and pharmaceutically acceptable excipients. The active ingredients include the phloroglucinol derivative of the present invention or its Pharmaceutically acceptable salt.

Among them, the dosage of the active ingredients contained is within the safe and effective dose range. The "safe and effective dose" means that the amount of active ingredients is sufficient to significantly improve the condition without causing serious side effects.

"Pharmaceutically acceptable excipients" refer to one or more compatible solid or liquid filler or gel substances that are suitable for human use and must be of sufficient purity and low enough toxicity.

"Compatibility" here refers to the ability of each component in the composition or preparation to be blended with the active ingredients of the present invention and with each other without significantly reducing the efficacy of the active ingredients.

Some examples of pharmaceutically acceptable excipients (or carriers) include cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants ( Such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween ®), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

The method of administration of the active ingredients or pharmaceutical compositions or pharmaceutical preparations of the present invention is not particularly limited. Representative methods of administration include but are not limited to: oral, transdermal, rectal, parenteral (intravenous, intramuscular or subcutaneous), etc. That is, the dosage forms of its pharmaceutical preparations include but are not limited to: capsules, granules, tablets, pills, powders, drops, ointments, patches, liniments, sprays, powders, suppositories, sustained-release agents, injections, etc. .

Solid dosage forms for oral administration include capsules, granules, tablets, pills, powders, etc. In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or excipient or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients:

(a) Fillers or compatibilizers such as starch, lactose, sucrose, glucose, mannitol and silicic acid;

(b) Binders such as hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic;

(c) humectants, for example, glycerin;

(d) Disintegrating agents, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate;

(e) Retarder, for example, paraffin;

(f) Absorption accelerators, such as quaternary ammonium compounds;

(g) Wetting agents, such as cetyl alcohol and glyceryl monostearate;

(h) adsorbents, such as kaolin; and

(i) Lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain buffering agents.

The solid dosage form can also be prepared using coating and shell materials, such as enteric casings and other materials known in the art. They may contain opacifying agents and the release of the active ingredient in such compositions may be in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxy substances.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredients, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances. Besides these inert diluents, the compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.

Suspensions may contain, in addition to the active ingredient, suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these substances and the like.

Compositions for parenteral injection may contain physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

The present invention will be described in further detail below with reference to specific examples.

Example 1 Separation and extraction of phloroglucinol derivatives

The separation and extraction of phloroglucinol derivatives includes the following steps:

(1) Weigh 50.4 kg of the dried Australian Thryptomene saxicola aerial part, crush it into coarse powder, extract it by percolation with 95% (v/v) ethanol aqueous solution 5 times, 50 L each time, combine the percolates and Concentrate under reduced pressure until there is no alcohol smell, and the total extract obtained is about 12 kg. Add water and suspend the extract according to the ratio of water:extract to 3L:1kg, and then extract it with petroleum ether 5 times. The amount of petroleum ether used each time is the same volume of water added. The extract is concentrated under reduced pressure to remove the petroleum ether to obtain petroleum ether. Ether extraction part 1.9kg.

(2) Perform silica gel column chromatography on the petroleum ether extraction part, using petroleum ether-ethyl acetate as the eluent, and the volume ratio of petroleum ether to ethyl acetate is 100:0, 100:1, 100:3, 100: 5. Elute with an elution gradient of 100:7, 100:10, 100:30, 100:50, 100:100 and 0:100. Analyze by thin layer chromatography (TLC) and combine similar fractions to obtain 10 Mainstream portion Fr. 1～Fr. 10. Fraction Fr. 7 (petroleum ether/ethyl acetate 100:7 elution fraction, 200 g) was concentrated and then subjected to silica gel column chromatography. The volume ratio of petroleum ether and ethyl acetate was 100:0, 100: 1. Elute with an elution gradient of 100:3, 100:5, 100:7, 100:10, 100:30, 100:50, and 100:100 to obtain 6 sub-fractions Fr. 7A ~ Fr. 7F. .

(3) Concentrate the sub-fraction Fr. 7C (31.1 g) and load it on the Sephadex LH-20 chromatographic column, using dichloromethane-methanol (volume ratio 1:1) as the mobile phase, and the flow rate is 0.5 mL/min. Carry out elution, collect the eluate, and obtain 10 fractions Fr. 7C-1 ~ Fr. 7C-10 through TLC analysis. Concentrate fraction Fr. 7C-5-6 (3.9 g) and perform reverse-phase ODS column chromatography, using methanol-water (MeOH-H ₂ O) as the eluent, and the volume ratio of methanol to water is 60:40. Elute with an elution gradient of 70:30, 80:20, 90:10, and 100:0, and collect the elution fraction with a methanol-water volume ratio of 90:10. The eluent was concentrated and dissolved in methanol, and then separated and purified by reversed-phase preparative HPLC. The chromatographic column was a Phenomenex C ₁₈ column (4.6 × 250 mm, 5 μm), with methanol-water at a volume ratio of 90:10. Eluent, the flow rate is 3 mL/min for elution, and the eluent with a retention time of 25.4 min is collected. After concentration, the phloroglucinol derivative (10.6 mg) is obtained. The vanillin-concentrated sulfuric acid reaction (TLC) appears purple. red.

Example 2 Structural identification of phloroglucinol derivatives

The phloroglucinol derivatives separated and extracted in Example 1 were subjected to ultraviolet, infrared, mass spectrometry and nuclear magnetic resonance detection according to conventional methods. The results are as follows:

UV (CHCl ₃ ) λ _max (log ε ) 204 (4.04), 279 (4.03) nm.

IR (KBr) ν _max 3440, 2955, 2932, 2867, 1633, 1442, 1382, 1363, 1304,1180, 1143, 1064, 999, 966, 839 cm ^-1 .

HR-ESI-MS m/z 487.2691 [M + H] ⁺ (calculated for C ₂₈ H ₃₉ O ₇ : 487.2690).

The hydrogen spectrum ( ¹ H NMR) and carbon spectrum ( ¹³ C NMR) data are shown in Table 1.

^{The 1} H NMR, ¹³ C NMR, ¹ H- ¹ H COSY, HSQC, HMBC and NOESY nuclear magnetic resonance spectra are shown in Figures 1 to 6.

Single crystals of phloroglucinol derivatives were obtained in a mixed solution of methanol and methylene chloride using the solvent evaporation method. Select crystals with good crystal faces and suitable sizes for single crystal X-ray diffraction analysis. The instrument used is a Rigaku single crystal diffractometer, and the light source is the radiation of the Cu K α target at λ = 1.54184 Å. The collected diffraction data were analyzed using the SHELXTL 6.10 program package and Olex21.2 software. The X-ray diffraction structure is shown in Figure 7. The measured and calculated ECD fitting results are shown in Figure 8.

According to the above physical and chemical data, NMR data, single crystal X-ray diffraction data, and measured and calculated ECD fitting results, it can be seen that the structure of the phloroglucinol derivative prepared in Example 1 is as shown in Formula I, and the configuration is 7S , 1′ R ,4′ S ,5′ R ,7′ R ,10′ R .

;

Table 1. ^1H (400 MHz) and ^13C (100 MHz) NMR data of phloroglucinol derivatives

;

;

Note: The solvent is CDCl ₃ , the unit of δ is ppm, and the unit of J is Hz.

Example 3 Inhibitory effects of phloroglucinol derivatives on various bacteria

S. aureus ATCC29213, methicillin-resistant Staphylococcus aureus ATCC33591 (MRSA), vancomycin-mediated resistance to S. aureus Mu50 (VISA), S. epidermidis ATCC12228 , E. faecalis ATCC29212, E. faecium 13-01, E. coli ATCC25922, Ps. aeruginosa , the above strains were purchased from the Institute of Medical Biotechnology, Chinese Academy of Medical Sciences .

The broth microdouble dilution method was used to determine the minimum inhibitory concentration (MIC) of the compound's in vitro antibacterial effect. The specific operation method is as follows:

(1) Bacterial culture: Cultivate experimental bacteria in Mueller-Hinton (MH) broth medium and reserve when they grow for 8-12 hours to approximately 0.5 Mcfarland concentration (1×10 ⁸ CFU).

(2) Dissolve the sample to be tested in ethanol and dilute it to 1000 μg/mL with culture medium. Continue to use serial dilutions of the culture medium to make the sample concentration range from 160 μg/mL to 0.156 μg/mL. In addition, ampicillin and vancomycin at a certain concentration were prepared as positive controls.

(3) Add 100 μL of drug-containing culture medium and 100 μL of bacteria-containing culture medium of different concentrations to each well of the 96-well plate to make the final bacterial liquid concentration 5 × 10 ⁴ CFU. Use tryptone soy broth culture medium ( TSB) plus bacterial liquid as a negative control (100 μL each of TSB culture medium and bacterial liquid), and TSB broth medium without bacterial liquid as a blank control (200 μL TSB culture medium). The 96-well plate was sealed and placed Incubate in a constant temperature incubator at 37°C for 24 hours.

(4) Taking the obvious growth of bacteria in the negative control wells as the premise, through naked eye observation, the lowest concentration of the drug without obvious growth of bacteria in the wells after adding the drug is the MIC (μg/mL) of the drug. The experiment was repeated three times in parallel. The results See Table 2.

Table 2. Inhibitory effects of phloroglucinol derivatives on various bacteria (MIC, μg/mL)

The results show that the phloroglucinol derivative of the present invention has significant antibacterial activity against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and vancomycin-resistant Staphylococcus aureus, with an MIC value of 0.156 μg/ mL, are significantly better than the positive drugs ampicillin, vancomycin and other commonly used clinical drugs, which is of great significance for the development of new drugs against drug-resistant bacteria; the phloroglucinol derivative of the present invention is effective against Staphylococcus epidermidis and Enterococcus faecalis , Enterococcus faecium has strong antibacterial activity, and its MIC value is 4 to 8 μg/mL.

The phloroglucinol derivative of the present invention is a new chemical entity whose molecular structure is completely different from existing antibacterial drugs. It has significant inhibitory activity against multidrug-resistant bacteria and can be used as a new chemical entity to prepare new antibacterial drugs.

Example 4 Effects of Phloroglucinol Derivatives on Normal Cells

Test method: Human embryonic kidney cells HEK 293 (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were cultured in DMEM medium containing 10% (v/v) fetal calf serum and double antibodies (100 U/mL each of penicillin and streptomycin) until In the logarithmic phase, wash with PBS and digest with 0.25% (w/v) trypsin. Suspend the cells in fresh DMEM culture medium, adjust the cell density to 1×10 ⁶ cells/mL, spread it on a 96-well plate, with 200 μL per well. After the cells adhere to the wall, add samples of different concentrations and incubate at 37°C and 5% CO ₂ conditions for 24 h. After the culture, 20 μL of 5mg/mL MTT solution was added to each well and the culture was continued for 4 h. Use a pipette to suck out the liquid in the well, add 100 μL DMSO to each well, shake gently for 10 min at room temperature, and use a microplate reader to detect the absorbance OD value of each well at a wavelength of 570 nm. Calculate the cell growth inhibition rate according to the following formula, and repeat the experiment at least 3 times. The formula for calculating the cell growth inhibition rate: inhibition rate (%) = (1-OD value of the drug-added group)/OD value of the control group × 100%. The results are shown in Table 3.

Table 3. Cytotoxicity of phloroglucinol derivatives against HEK 293

Experimental results show that at a high concentration of 200 μg/mL, the phloroglucinol derivative of the present invention has a cell growth inhibition rate of 9.61% on human normal embryonic kidney cells HEK 293, showing very low cytotoxicity.

The above-mentioned embodiments only express several implementation modes of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the scope of protection of the patent of the present invention should be determined by the appended claims.

Claims (10)

1. A phloroglucinol derivative or a pharmaceutically acceptable salt thereof, characterized in that the molecular structure of the phloroglucinol derivative is as shown in formula I, and the configuration is 7 S , 1′ R , 4′ S ,5′ R ,7′ R ,10′ R , . 2. A preparation method of phloroglucinol derivative according to claim 1, characterized in that it includes the following steps: Crush the above-ground part of Australian snow plum, and extract it by percolation with an ethanol aqueous solution with a volume concentration of 85-98%. The extract is concentrated and extracted with petroleum ether, and then the extract is separated and purified to obtain the phloroglucinol derivative. . 3. The preparation method of phloroglucinol derivatives according to claim 2, characterized in that the volume concentration of the ethanol aqueous solution is 90-95%; and/or, The number of percolation extractions is 4-6 times, and the ratio of ethanol aqueous solution and Australian snow plum each time is 0.8-1.2L:1kg; and/or, The separation and purification includes: concentrating the extract and then sequentially performing silica gel column chromatography, Sephadex LH-20 chromatography column separation and purification, octadecylsilane bonded silica gel column chromatography, and preparative HPLC separation and purification. 4. Use of the phloroglucinol derivative of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting bacterial growth. 5. Use of the phloroglucinol derivative of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and/or preventing bacterial infections. 6. The application according to claim 4 or 5, characterized in that the bacteria are drug-resistant bacteria. 7. The application according to claim 4 or 5, characterized in that the bacteria are Gram-positive bacteria. 8. The application according to claim 7, characterized in that the bacteria are Staphylococcus aureus and Enterococcus aureus. 9. The application according to claim 8, characterized in that the bacteria are Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, vancomycin-intermediate resistant Staphylococcus aureus, Staphylococcus epidermidis, fecal enteritis. cocci, Enterococcus faecium. 10. An antibacterial drug, characterized in that it is prepared from active ingredients and pharmaceutically acceptable excipients, and the active ingredients include the phloroglucinol derivative of claim 1 or a pharmaceutically acceptable salt thereof .