Application of myrtle derivatives in preparation of antibacterial drugs and antibacterial drugs

Abstract

The present invention belongs to the field of pharmaceutical technology and specifically relates to the use of myrtle ketone derivatives in the preparation of antibacterial drugs and the antibacterial drugs themselves. A novel class of myrtle ketone derivatives synthesized in the present invention exhibits inhibitory activity against Gram-positive bacteria, among which compound 27 is the most potent DNA gyrase and Topo IV inhibitor. Biological activity assays demonstrate that compound 27 not only exhibits rapid bactericidal activity against methicillin-resistant Staphylococcus aureus, but also exhibits low toxicity, is less likely to induce bacterial resistance, and exhibits synergistic effects with first-line antibacterial drugs such as ofloxacin, amikacin, cefpiramide, and ceftazidime, making it a potential candidate for a new antibacterial drug.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and particularly relates to the application of myrone derivatives in the preparation of antibacterial drugs and the antibacterial drugs. Background Art

[0002] The use of antibiotics has made a significant contribution to the fight against human infectious diseases. However, the misuse and abuse of antimicrobial drugs has led to a sharp increase in bacterial resistance, posing a serious threat to public health. In recent years, various new ultra-resistant strains have been discovered, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). However, until now, these resistant strains have been difficult to treat with limited options. Vancomycin (Van) remains one of the first-line drugs for the treatment of MRSA infections, but in recent years, MRSA strains that are completely resistant to Van have emerged, and there is an urgent need to develop new antimicrobial drugs.

[0003] Blocking DNA replication is an effective and promising antibacterial mechanism. DNA gyrase and topoisomerase IV (Topo IV) are mature and proven targets for antibacterial drug discovery. It provides ideas for the research and development of new drugs and can avoid some existing drug resistance mechanisms. DNA gyrase and Topo IV are both IIA topoisomerases present in bacteria, playing an important role in bacterial DNA replication and chromosome segregation. They are highly homologous, have similar structures and functions, and show 40% DNA sequence similarity overall and have significantly similar active sites. The absence of both in mammals and their key roles in the bacterial DNA replication cycle make the two enzymes suitable targets for the development of new selective antibacterial drugs.

[0004] Myrtus communis L., Myrtaceae, genus Myrtus, is an evergreen shrub widely distributed in the Mediterranean region. It has long been used as a folk disinfectant and is also cultivated in southern my country. However, little research has been conducted on the antibacterial uses of myrtle ketone. Therefore, research is needed to understand the antibacterial uses of myrtle ketone. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides the use of myrtle derivatives in the preparation of antibacterial drugs and the antibacterial drugs.

[0006] The use of myrtle ketone derivatives in the preparation of antibacterial drugs, the structural formula of the myrtle ketone derivatives is as follows:

[0007]

[0008] Wherein, R is methyl, ethyl, (CH2)2OH, isopropyl, (CH2)3Cl, CH2O(CH2)2OMe, (CH2)3CH(OEt)2, isobutyryl, (CH2)2CHMe2, (CH2)6OH, benzyl, CH2C6F5, 4-NO2C6H4CH2, (CH2)3C6H5, (CH2)2NMe2, (CH2)2NEt2, (CH2)3NEt2, (CH2)3NBu2, Any one of .

[0009] Preferably, the structural formula of the myrtle derivative is as follows:

[0010] Formula (1): Formula (2): Formula (3):

[0011] Preferably, the antibacterial drug includes a compound having the structural formula of the formula (1), the formula (2) or the formula (3).

[0012] Preferably, the antibacterial drug inhibits Gram-positive bacteria.

[0013] Preferably, the Gram-positive bacteria are Staphylococcus aureus, MRSA, Streptococcus pneumoniae or hemolytic Streptococcus.

[0014] Preferably, the antibacterial drug inhibits DNA gyrase.

[0015] Preferably, the antibacterial drug inhibits Topo IV.

[0016] An antibacterial drug comprising any one of the compounds described above.

[0017] Preferably, the drug is the compound, or a salt thereof, or a stereoisomer thereof.

[0018] Preferably, the medicine further comprises pharmaceutically acceptable excipients.

[0019] Preferably, the pharmaceutically acceptable excipient includes a diluent.

[0020] Preferably, the diluent is sterile saline.

[0021] Compared with the prior art, the present invention is beneficial in that:

[0022] The invention synthesizes a new type of myrtle ketone derivatives, which have inhibitory activity against Gram-positive bacteria and can be used for preparing antibacterial drugs.

[0023] This study evaluated the inhibitory effects of myrtle derivatives on DNA gyrase and Topo IV, with compound 27 being the most potent inhibitor. Bioactivity assays demonstrated that compound 27 not only exhibited rapid bactericidal activity against methicillin-resistant Staphylococcus aureus (MRSA), but also exhibited low toxicity, was less likely to induce bacterial resistance, and exhibited synergistic effects with first-line antibacterial drugs such as ofloxacin, amikacin, cefpiramide, and ceftazidime, potentially making it a potential candidate for a novel antibacterial agent.

[0024] The present invention facilitates the development of new antibiotics based on myrtone-based DNA gyrase and Topo IV inhibitors to help overcome the growing trend of multidrug-resistant bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The inhibitory activity of compound 27 at different concentrations is shown, wherein A shows the inhibition of Escherichia coli gyrase supercoiling activity, and C shows the inhibition of Staphylococcus aureus gyrase supercoiling activity. In Figures A and C, lane 1 is relaxed pHOT1 DNA, lane 2 is relaxed pHOT1 DNA and enzyme, lane 3, relaxed pHOT1 DNA, enzyme and CIP (25 μM) is used as a positive control, and lanes 4-8 are relaxed pHOT1 DNA, enzyme and compounds at various concentrations. B shows the inhibition of DNA relaxation catalyzed by topo IV in Escherichia coli, and D shows the inhibition of DNA relaxation catalyzed by topo IV in Staphylococcus aureus. In Figures B and D, lane 1 is pBR322 DNA, lane 2 is pBR322 DNA and enzyme, lane 3, pBR322 DNA, enzyme and CIP (25 μM) is used as a positive control, and lanes 4-8 are pBR322 DNA, enzyme and different concentrations of compound 27, E shows the inhibitory effect on DNA relaxation catalyzed by human topo I, and F shows the inhibitory effect on DNA relaxation catalyzed by human topo Iα. In Figures E and F, lane 1 is pBR322 DNA, lane 2 is pBR322 DNA and enzyme, lane 3, pBR322 DNA, enzyme and CPT (25 μM) or ETP (25 μM) as positive control, lanes 4-8 are different concentrations of pBR322 DNA, enzyme and compound 27, abbreviations: CIP, ciprofloxacin, CPT, camptothecin, ETP, etoposide, Re, relaxed pBR322 DNA, Sc, supercoiled pBR322 DNA.

[0026] Figure 2Schematic diagram of the molecular docking model, where A is the 3D and 2D maps of the molecular docking of compound 27 with Escherichia coli DNA gyrase, and B is the 3D and 2D maps of the molecular docking of Escherichia coli DNA Topo IV. Hydrogen bonds are represented by red dashed lines (3D map) and green lines (2D map), and the length (λ) is marked around the lines.

[0027] Figure 3 Figure 2 is the drug resistance result, where A is the time-kill kinetic study of VAN and compound 27 against methicillin-resistant Staphylococcus aureus virus, and B is the study of norfloxacin (NFX) and compound 27 on the resistance of Staphylococcus aureus ATCC25923. The data are the mean standard deviation (error bars), and each experiment was repeated at least 3 times. DETAILED DESCRIPTION

[0028] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0029] All chemical reagents used for synthesis were purchased from local commercial suppliers and used without further purification unless otherwise stated. The chemical reaction process was monitored by silica gel GF254 thin layer chromatography. NMR spectra were measured using Bruker Avance III 400MHz and 500MHz instruments (Germany). HRMS analysis was performed on a SHIMADZU LCMS-IT-TOF mass spectrometer. The melting point was determined in an open capillary of an MPA100 Optimelt automatic melting point system without correction. The purity of all bioactive test compounds analyzed by HPLC was greater than 95%. The analysis conditions were: detection wavelength 220nm, flow rate 1.0mL / min, and elution with 50%-15% PBS buffer (pH 3) and 50%-85% MeOH within 30 minutes.

[0030] The experiments involved in the present invention were carried out as follows:

[0031] 1. DNA gyrase supercoiling experiment

[0032] DNA gyrase supercoiling assays were performed using relaxed pHOT1 DNA as a substrate. Briefly, 1 U of DNA gyrase from Escherichia coli or Staphylococcus aureus and 0.5 μg of relaxed pHOT1 DNA were incubated in 30 μL of supercoiling assay buffer at 37°C for 60 minutes in the presence or absence of the compound. Following incubation, the reaction was terminated by the addition of 3 μL of 2 mg / mL proteinase K. Then, 5 μL of the reaction solution was mixed with 1 μL of 6× DNA loading buffer and analyzed by electrophoresis on a 1% agarose gel at 3 V / cm in 1× TAE buffer. The gel was stained with 1× Gel Red, visualized with a UV transilluminator, and analyzed using AlphaEaseFC software. Among them, the supercoiling assay buffer consists of 35mM Tris-HCl, pH 7.5, 24mM KCl, 4mM MgCl2, 2mM DTT, 1.8mM spermidine, 1mM ATP, 6.5% glycerol and 0.1mg / mL BSA; the 6× DNA loading buffer consists of 30mM EDTA, 36% glycerol, 0.05% xylene cyanol FF, 0.05% bromophenol blue; and the 1× TAE buffer consists of 40mM Tris-acetate, 1mM EDTA, pH 8.5.

[0033] 2. Topoisomerase IV relaxation assay

[0034] 1U of Topo IV was incubated with 0.5μg of supercoiled pBR322 DNA for relaxation experiments in 30μL of reaction buffer at 37°C for 30 minutes. The reaction was terminated by adding 30μL of chloroform / isoamyl alcohol (24:1). 5μL of the reaction solution was mixed with 1μL of 6× DNA loading buffer and analyzed on a 1% agarose gel at 5V / cm. Relaxation experiments were performed in 1× TAE buffer. The gel was stained with 1× Gel Red, visualized with a UV transilluminator, and analyzed using AlphaEaseFC software. The reaction buffer consisted of 20mM HEPES-KOH, pH 7.6, 50mM potassium glutamate, 5mM magnesium acetate, 5mM dithiothreitol, 1mM ATP, and 25μg / mL BSA.

[0035] 3. DNA topoisomerase I and IIα relaxation assay

[0036] All test compounds were dissolved in DMSO, and the DNA topoisomerase I and IIα inhibitory activity of each compound was determined. A reaction mixture containing 0.5 μg of supercoiled pBR322 DNA in buffer was incubated with 1 U of recombinant human DNA Topo I or Topo IIα at 37°C for 30 minutes in the absence or presence of the test compound. The reaction was terminated by adding Topo Stop Buffer. Then, 4 μL of 6× loading buffer was added, and the sample was analyzed on a 0.8% agarose gel in 4.6 v / cm TBE buffer for 1.5 hours. The gel was stained with 1× Gel Red for 30 minutes and visualized with a UV transilluminator. The reaction mixture for supercoiled pBR322 DNA in the Topo I relaxation experiment consisted of 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 5 mM MgCl2, 15 μg / mL BSA, and 40 μg / mL DTT. The reaction mixture for supercoiled pBR322 DNA in the Topo II relaxation experiment consisted of 10 mM Tris-HCl, pH 7.9, 50 mM NaCl, 50 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1 mM ATP, and 15 mg / mL BSA. The Topo I assay termination buffer consisted of 10% SDS solution containing 0.2% bromophenol blue and 0.2% xylene cyanol; the Topo II assay termination buffer consisted of 7 mM EDTA.

[0037] 4. Molecular docking

[0038] Molecular docking analysis of the protein target of the selected ligand was performed using the CB-Dock2 (https: / / cadd.labshare.cn / cb-dock2 / php / index.php) platform. CB-Dock2 is a molecular docking tool based on AutoDock Vina analysis that automatically identifies and analyzes the binding sites of ligands and receptors. It improves the accuracy of molecular docking while simplifying the docking process. The 2D chemical structure of 27 was constructed using ChemDraw software, which was then input into the CB-Dock2 platform and loaded with H atoms, and free energy minimization was performed separately. At the same time, the crystal structure of the Escherichia coli DNA gyrase A subunit in complex with SD8 (PDB ID: 4kcl)40 and the crystal structure of Escherichia coli DNA topoisomerase IV in complex with an inhibitor (PDB ID: 3FV5) were obtained from the RCSB PDB (https: / / www.rcsb.org / ) for molecular docking studies and then processed by adding hydrogen atoms and deleting all water molecules. Binding affinities (kcal / mol) were calculated using CB-Dock2, which projects the optimal docking model with the least energy. Molecular visualization is a key aspect of analysis and communication in modeling studies. Details of ligand-receptor interactions were revealed using PyMol2 (free version) and LigPlot+ (version 2.2.8).

[0039] Example 1

[0040] Synthesis of rac-4, rac-5a, and meso-5b

[0041] The synthesis diagram is as follows:

[0042]

[0043] Reagents and conditions: (a) Isobutyryl chloride, aluminum chloride, tetrahydrofuran, 65°C for 18 hours. (b) Methyl iodide, sodium methoxide, methanol, 65°C for 16 hours. (c) Diisobutylaluminum hydride, tetrahydrofuran, -80°C for 0.5 hours. (d) i) Sodium hydride, tetrahydrofuran, room temperature; ii) p-Toluenesulfonic acid, toluene, 100°C with stirring for 1 hour.

[0044] Specifically, 2 mL of isobutyryl chloride was dissolved in 5 mL of THF to obtain an isobutyryl chloride solution. 2.0 g of phloroglucinol, 8.4 g of aluminum chloride, and 100 mL of THF were added to a 250 mL single-necked flask, and the isobutyryl chloride solution was slowly added dropwise. The reaction was refluxed at 65°C for 18 hours. After the reaction was complete, 50 mL of water was added to the reaction system, and the mixture was acidified with hydrochloric acid. The mixture was extracted with ethyl acetate (20 mL x 3), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by 200-300 mesh silica gel column chromatography (ethyl acetate:petroleum ether = 1:5) to obtain compound 1.

[0045] To a 250 ml single-necked flask, add 1.0 g of compound 1, 1.4 g of sodium methoxide, and 50 mL of methanol. The mixture was refluxed at 65°C for 16 h. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the residue was purified by 200-300 mesh silica gel column chromatography (ethyl acetate:petroleum ether = 1:10) to obtain compound 2.

[0046] To a 100 ml single-necked flask, add 1.0 g of compound 2 and 30 mL of THF, place the flask at -78°C, add 2.5 mL of DIBAL-H, and react at -80°C for 0.5 h. After the reaction is complete, add 50 mL of water to the reaction system, acidify with hydrochloric acid, extract with dichloromethane (10 mL × 3), dry over anhydrous sodium sulfate, and concentrate the reaction solution under reduced pressure. The residue is purified by 200-300 mesh silica gel column chromatography (ethyl acetate: petroleum ether = 1:10) to obtain compound 3.

[0047] To a 150 mL single-necked flask, 2.0 g of compound 1, 7.0 g of compound 3, 0.5 g of NaHCO3, and 50 mL of THF were added and allowed to react for 1 h. 50 mL of water was added to the reaction system, and the mixture was acidified with hydrochloric acid. The mixture was extracted with ethyl acetate (10 mL x 3). After drying over anhydrous sodium sulfate, the reaction solution was concentrated under reduced pressure. 2.0 g of p-toluenesulfonic acid and 50 mL of toluene were added to the residue, and the mixture was refluxed for 1 h. The residue was concentrated under reduced pressure and purified by 200-300 mesh silica gel column chromatography (ethyl acetate:petroleum ether = 1:5) to obtain rac-4. This compound was identified as myrtucommulone B. rac-4 is a mixture of two stereoisomers, one pair of enantiomers, (+)-4 and one pair of enantiomers, (-)-4.

[0048] To a 150 mL single-necked flask were added 2.0 g of compound 1, 7.0 g of compound 3, 0.5 g of NaH, and 50 mL of THF. The reaction was carried out at 100°C for 1 h. 50 mL of water was added to the reaction system, and the mixture was acidified with hydrochloric acid. The mixture was extracted with ethyl acetate (10 mL × 3). After drying over anhydrous sodium sulfate, the reaction solution was concentrated under reduced pressure. 2.0 g of p-toluenesulfonic acid and 50 mL of toluene were added to the residue, and the mixture was refluxed for 1 h. The residue was concentrated under reduced pressure and purified by 200-300 mesh silica gel column chromatography (ethyl acetate:petroleum ether = 1:5) to obtain rac-5a and meso-5b. Rac-5a is a mixture of two stereoisomers, a pair of enantiomers, and a pair of enantiomers is (+)-5a and (-)-5a.

[0049] Compound 1: 2-methyl-1-(2,4,6-trihydroxyphenyl)propan-1-one, yellow viscous liquid, yield 94%; 1 HNMR (500MHz, DMSO-d6): δ12.28 (s, 2H), 10.36 (s, 1H), 5.86 (s, 2H), 3.92 (p, J=6.8Hz, 1H), 1.11 (d, J=6.8Hz, 6H); 13 CNMR (126MHz, DMSO-d6) δ209.4, 164.5, 164.3, 103.0, 94.9, 38.2, 19.2.

[0050] Compound 2: 5-hydroxy-4-isobutyryl-2,2,6,6-tetramethylcyclohex-4-ene-1,3-dione, yellow liquid, yield 95%; 1 HNMR (400MHz, Chloroform-d) δ3.80 (hept, J=6.8Hz, 1H), 1.45 (s, 6H), 1.37 (s, 6H), 1.19 (s, 3H), 1.17 (s, 3H); 13 CNMR (101MHz, Chloroform-d) δ210.0, 208.7, 199.4, 197.0, 108.3, 57.1, 52.4, 35.3, 24.4, 24.0, 19.2.

[0051] Compound 3: 2,2,4,4-tetramethyl-6-(2-methylpropylene)cyclohexane-1,3,5-trione, yellow liquid, yield 96%; 1 HNMR (400MHz, Chloroform-d) δ7.28 (s, 1H), 3.24 (t, J=7.0Hz, 1H), 1.34 (d, J=1.0Hz, 12H), 1.14 (s, 3H), 1.13 (s, 3H); 13C NMR (126MHz, Chloroform-d) δ208.9, 199.8, 196.6, 164.9, 130.6, 58.5, 58.2, 28.6, 22.4, 21.9, 21.8.

[0052] rac-4 (myrtucommunulone B), pale yellow solid, yield 78%, melting point 180.0-181.0℃. 1 HNMR (500MHz, CDCl3) δ13.39 (s, 1H), 6.32 (s, 1H), 4.36 (d, J=3.6Hz, 1H), 3.90 (hept, J=6.7Hz, 1H), 1.96-1.87 (m, 1H) , 1.62 (s, 3H), 1.45 (s, 3H), 1.42 (s, 3H), 1.39 (s, 3H), 1.28-1.23 (m, 6H), 0.82 (d, J = 7.0Hz, 3H), 0.78 (d, J = 7.0Hz, 3H); 13 C NMR (125MHz, CDCl3) δ212.0, 209.0, 198.8, 168.3, 164.9, 160.0, 153.5, 112.2, 103.9, 103.8, 100.7, 56.3, 47.5, 39.8, 34.9, 31.6, 25.3, 25.1, 24.3, 21.0, 19.0, 18.8, 17.8. HRMS(ESI)m / z:415.2116[M+H] + , calculate for C 24 H 31 O6415.2115.

[0053] The racemic compound was separated by chiral phase separation using HPLC (chiral column OD-H, iPrOH / n-hexane = 30:70, 0.5 mL / min, 15°C), and the absolute configuration of the enantiomer was determined by comparing the specific rotation with the literature. The product was a pale yellow solid with a yield of 39% and a melting point of 180.0-181.0°C. [α] 2 D 0 =+40.0 (c=1.0, CHCl3); (-)-4, pale yellow solid, yield 39%, melting point 180.0-181.0°C; [α] 2 D 0 =-38.9 (c=1.0, CHCl3).

[0054] rac-5a, white solid, yield 18%. 1H NMR (500MHz, CDCl3) δ4.51 (d, J=2.8Hz, 2H), 3.18 (q, J=5.6Hz, 1H), 2.00-1.94 (m, 2H), 1.52 (s, 6H), 1.43 (s, 6H), 1.38 (s, 6H), 1.35 (s, 6H) ), 1.26 (d, J = 2.0Hz, 6H), 1.28 (d, J = 2.4Hz, 3H), 0.87 (d, J = 2.0Hz, 3H), 0.86 (d, J = 2.0Hz, 3H) 0.79 (d, J = 2.1Hz, 3H), 0.77 (d, J = 2.1Hz, 3H); 13 C NMR (125MHz, CDCl3) δ212.0, 205.4, 198.5, 169.1, 152.3, 147.6, 111.3, 110.7, 108.6, 56.1, 47.7, 43.4, 35.24, 32.7, 25.1, 25.0, 24.9, 24.4, 19.3, 18.9, 17.9. HRMS(ESI)m / z:633.3444[M+H] + , calculate for C 38 H 49 O8633.6422.

[0055] The racemic compound was resolved using chiral HPLC (ChiralCel OD-H, iPrOH / n-hexane = 30:70, 0.5 mL / min, 15°C), and the absolute configuration of the enantiomers was determined by comparing the specific rotation, ECD spectrum, and literature. (+)-5a, white solid, melting point 228.8-230.7°C; [α] 2 D 0 = +39.5 (c = 1.0, chcl3); (-)-5a, white solid, melting point 228.8-230.7 ° C; [α] 2 D 0 =-37.3 (c=1.0, CHCl3).

[0056] meso-5b, white powder, yield 37%, melting point 229.0-230.0℃, [α] 2 D 0 =0 (c=0.1, MeOH); nuclear magnetic resonance (NMR) spectrum and HRMS (ESI) spectrum were consistent with each other and showed no difference. 1H NMR (400MHz, CDCl3) δ4.53 (d, J=3.2Hz, 2H), 3.18 (p, J=6.0Hz, 1H), 2.00-1.91 (m, 2H), 1.45 (s, 6 H), 1.43 (s, 6H), 1.42 (s, 6H), 1.26 (d, J = 4.8Hz, 6H), 1.24 (d, J = 7.6Hz, 6H), 0.81-0.77 (m, 12H); 13 C NMR (125 MHz, CDCl3) δ 211.7, 204.2, 198.5, 168.6, 151.9, 147.4, 110.8, 110.1, 108.6, 55.8, 47.4, 42.6, 35.0, 32.2, 25.1, 24.7, 24.7, 24.3, 19.1, 18.5, 17.9. This compound is a meso compound.

[0057] Example 2

[0058] Synthesis of compound 6

[0059] The synthesis diagram is as follows:

[0060]

[0061] Wherein, a: tin tetrachloride, dichloromethane, reflux at 85℃ for 6 hours

[0062] Specifically, (+)-5a, (-)-5a or 0.2 g of meso-5b, 0.4 g of tin tetrachloride, and 20 mL of dichloroethane were added to a 50 mL single-necked flask and refluxed at 85°C for 6 h. 20 mL of water was added to the reaction system, and the mixture was acidified with hydrochloric acid. The mixture was extracted with ethyl acetate (5 mL x 3) and dried over anhydrous sodium sulfate. The reaction solution was then concentrated under reduced pressure, and the residue was purified by 200-300 mesh silica gel column chromatography (ethyl acetate:petroleum ether = 1:10) to obtain the target product. When the starting material is (+)-5a, the target product (+)-6a was obtained; when the starting material is (+)-5a, the target product (+)-6a was obtained; and when the starting material is meso-5b, the target product meso-6b was obtained.

[0063] rac-6a, yield 90%, melting point 208.9-210.0℃. 1 HNMR (400MHz, CDCl3) δ6.58 (s, 1H), 4.35 (d, J=3.5Hz, 2H), 1.98-1.93 (m, 2H), 1.49 (s, 6H), 1.47(s, 6H), 1.42(s, 6H), 1.28(s, 6H), 0.84(s, 3H), 0.82(s, 3H), 0.81(s, 3H), 0.79(s, 3H);13 C NMR (125MHz, CDCl3) δ212.5, 198.1, 171.3, 169.0, 151.3, 111.1, 108.7, 96.0, 56.1, 47.6, 35.1, 32.9, 25.5, 25.3, 24.6, 24.2, 19.3, 19.0.HRMS(ESI)m / z:561.2837[MH] - , calculate for C 34 H 41 O7561.2858.

[0064] (+)-6a, white solid, yield 89%, melting point 208.9-210.0℃, [α] 2 D 0 =+37.4 (c=1.0, chcl3); (-)-6a, white solid, yield 91%, melting point 208.9-210.0°C, [α] 2 D 0 =-35.1 (c=1.0, CHCl3).

[0065] meso-6b, white solid, yield 93%, melting point 209.8-211.0°C; 1 H NMR (400MHz, CDCl3) δ6.54 (s, 1H), 4.42 (d, J=3.9Hz, 2H), 1.97-1.91 (m, 2H), 1.45 (s, 6H), 1 .44(s, 6H), 1.36(s, 6H), 1.25(s, 6H), 0.87(s, 3H), 0.85(s, 3H), 0.78(s, 3H), 0.77(s, 3H); 13 C NMR (125MHz, CDCl3) δ212.4, 198.3, 169.2, 151.5, 151.3, 111.4, 108.5, 96.2, 56.1, 4 7.6, 35.3, 32.8, 25.3, 24.83, 24.78, 24.6, 19.4, 19.0.HRMS(ESI)m / z:561.2820[MH] - , calculate for C 34 H 41 O7561.2858.

[0066] Example 3

[0067] Synthesis of Compound 7-Compound 34

[0068] The synthesis diagram is as follows:

[0069]

[0070] Wherein, a: cesium carbonate, RX (X = Cl or Br), tetrahydrofuran, room temperature reaction

[0071] Specifically, 0.2 g of meso-5b, cesium carbonate, compound A, and 20 mL of tetrahydrofuran were added to a 50 mL single-necked flask and reacted at room temperature for 4 h. 20 mL of water was added to the reaction system, and the mixture was acidified with hydrochloric acid. The mixture was extracted with ethyl acetate (5 mL × 3) and dried over anhydrous sodium sulfate. The reaction solution was then concentrated under reduced pressure, and the residue was purified by 200-300 mesh silica gel column chromatography to obtain compounds 7 to 34, respectively. The corresponding relationships between the raw materials used and the obtained compounds are shown in Table 7:

[0072] Table 7 Correspondence between the raw materials used and the obtained compounds

[0073]

[0074]

[0075] The compound information is shown in Table 8 below:

[0076] Table 8 Compound information

[0077]

[0078]

[0079]

[0080] Compound 7: (12R,14S)-6-isobutyryl-12,14-diisopropyl-13-methoxy-2,2,4,4,8,8,10,10-octamethyl-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 93%, melting point 216.8-218.0°C; 1 H NMR (500MHz, CDCl3) δ4.43 (d, J=3.4Hz, 2H), 4.05 (s, 3H), 3.14 (p, J=6.9Hz, 1H), 1.93-1.86 (m, 2H), 1.53 (s, 6H ), 1.43 (s, 6H), 1.41 (s, 6H), 1.39 (s, 6H), 1.25 (d, J = 7.0Hz, 6H), 0.82 (d, J = 7.0Hz, 6H), 0.72 (d, J = 6.9Hz, 6H); 13C NMR (125MHz, CDCl3) δ212.2, 204.4, 197.8, 168.0, 156.2, 147.7, 116.2, 113.7, 110.6, 62.8, 56.1 ,47.6,42.8,36.3,32.9,25.5,25.4,24.9,24.2,20.3,18.2,17.8.HRMS(ESI)m / z:647.3598[M+H] + , calculate for C 39 H 51 O8647.3578.

[0081] Compound 8: (12R,14S)-13-Ethoxy-6-isobutyryl-12,14-diisopropyl-2,2,4,4,8,8,10,10-octamethyl-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 93%, melting point 219.0-220.3°C; 1 H NMR (500MHz, CDCl3) δ4.25 (d, J=3.7Hz, 2H), 3.88 (q, J=7.0Hz, 2H), 3.17 (p, J=6.9Hz, 1H), 1.98-1.91 (m, 2H), 1.52 (s, 6H), 1.4 8 (t, J=7.0Hz, 3H), 1.41 (s, 6H), 1.37 (s, 6H), 1.35 (s, 6H), 1.29 (d, J=6.9Hz, 6H), 0.84 (d, J=6.9Hz, 6H), 0.74 (d, J=6.9Hz, 6H); 13 C NMR (125MHz, CDCl3) δ212.0, 205.5, 197.8, 168.0, 156.0, 147.5, 115.4, 114.1, 111.3, 71.2, 56.2, 47.6, 43.3, 35.8, 33.5, 25.2, 24.83, 24.78, 24.5, 19.4, 19.0, 17.8, 16.2. HRMS(ESI)m / z:661.3949[M+H] + , calculate for C 40 H 53 O8661.3735.

[0082] Compound 9: (12R,14S)-13-(2-hydroxyethoxy)-6-isobutyryl-12,14-diisopropyl-2,2,4,4,8,8,10,10-octamethyl-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 47%, melting point 229.0-230.4°C; 1 H NMR (400MHz, CDCl3) δ13.46 (s, 1H), 4.31 (d, J=3.5Hz, 2H), 4.13-4.11 (m, 2H), 4.04-4.02 (m, 2H), 3.90 (q, J=6.8Hz, 1H), 1.90 -1.86 (m, 1H), 1.86-1.82 (m, 1H), 1.43 (s, 6H), 1.38 (s, 6H), 1.27-1.23 (m, 18H), 0.80 (d, J=6.9Hz, 6H), 0.76 (d, J=6.9Hz, 6H); 13 C NMR (125MHz, CDCl3) δ212.6, 206.8, 197.9, 168.6, 155.4, 149.5, 111.3, 111.2, 107.1, 7 1.1, 70.7, 56.1, 47.7, 42.5, 35.9, 35.1, 32.4, 25.0, 24.9, 24.8, 24.6, 19.5, 19.0, 18.3. HRMS(ESI)m / z:677.3724[M+H] + , calculate for C 40 H 53 O9677.3684.

[0083] Compound 10: (12R,14S)-6-isobutyryl-13-isopropoxy-12,14-diisopropyl-2,2,4,4,8,8,10,10-octamethyl-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11

[0084] (2H,10H)-tetraketone (10), white solid, yield 85%, melting point 219.0-221.1°C; 1H NMR (500MHz, CDCl3) δ4.86-4.77 (m, 1H), 4.40 (d, J=3.0Hz, 2H), 3.21-3.11 (m, 1H), 1.98-1.87 (m, 2H), 1.53 (s, 6 H), 1.42 (s, 9H), 1.40 (s, 9H), 1.37 (s, 6H), 1.25 (d, J = 3.5Hz, 6H), 0.81 (d, J = 7.1Hz, 6H), 0.67 (d, J = 7.0Hz, 6H); 13 C NMR (125MHz, CDCl3) δ212.2, 204.6, 197.8, 168.4, 152.8, 147.7, 117.0, 113.2, 110.5, 75.4 , 56.1, 47.6, 42.8, 35.7, 33.1, 25.5, 25.4, 24.9, 24.3, 22.8, 22.6, 20.6, 18.2, 18.0, 17.3. HRMS(ESI)m / z:675.3861[M+H] + , calculate for C 41 H 55 O8675.3891.

[0085] Compound 11: (12R,14S)-13-(3-chlorooxo)-6-isobutyryl-12,14-diisopropyl-2,2,4,4,8,8,10,10-octamethyl-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 51%, melting point 244.9-245.6°C; 1 H NMR (500MHz, CDCl3) δ4.42 (d, J=1.5Hz, 2H), 4.11-4.08 (m, 1H), 3.87 (q, J=6.3Hz, 1H) , 3.72 (q, J=6.2Hz, 1H), 3.14 (p, J=7.0Hz, 1H), 2.43 (p, J=6.0Hz, 1H), 2.35 (p, J=6.0H z, 1H), 1.94-1.87 (m, 3H), 1.54 (s, 6H), 1.43 (s, 6H), 1.41 (s, 6H), 1.39 (s, 6H), 1.26 ( d, J=7.0Hz, 6H), 0.84 (d, J=2.5Hz, 3H), 0.82 (d, J=2.5Hz, 3H), 0.70 (d, J=6.8Hz, 6H); 13C NMR (125MHz, CDCl3) δ212.1, 204.3, 197.9, 168.2, 154.5, 147.8, 116.9, 114.1, 110.3, 7 1.1, 56.0, 47.7, 42.8, 41.3, 36.5, 32.9, 29.6, 25.6, 25.4, 24.9, 24.1, 18.1, 17.8, 17.5. HRMS(ESI)m / z:709.3711[M+H] + , calculate for C 41 H 54 O8Cl 709.3502.

[0086] Compound 12: (12R,14S)-6-isobutyryl-12,14-diisopropyl-2,2,4,4,4,8,8,10,10-octamethyl-13-(oxiran-2-ylmethoxy)-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone (12), white solid, yield 78%, melting point 253.9-255.9°C; 1 H NMR (500MHz, CDCl3) δ4.31 (d, J=17.0Hz, 2H), 4.24 (d, J=3.8Hz, 1H), 4.19 (d, J=13. 2Hz, 1H), 3.58-3.53(m, 1H), 3.16(p, J=6.8Hz, 1H), 2.93-2.89(m, 1H), 2.77-2.74(m , 1H), 1.97-1.94(m, 1H), 1.93-1.90(m, 1H), 1.51(s, 6H), 1.41(s, 6H), 1.37(s, 6H), 1.35 (s, 6H), 1.29 (d, J=6.6Hz, 6H), 0.82 (d, J=11.3Hz, 6H), 0.74 (d, J=13.3Hz, 6H); 13 C NMR (125MHz, CDCl3) δ211.9, 205.5, 197.7, 168.0, 155.2, 147.6, 115.4, 114.6, 111.2, 7 5.7, 56.2, 50.4, 47.6, 44.8, 43.3, 35.8, 33.4, 25.6, 24.9, 24.8, 24.4, 19.2, 19.1, 17.8. HRMS(ESI)m / z:689.3796[M+H] + , calculate for C 41 H 53 O9689.3684.

[0087] Compound 13: (12R,14S)-13-(cyclopropylmethoxy)-6-isobutyryl-12,14-diisopropyl-2,2,4,4,8,8,10,10-octamethyl-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 92%, melting point 229.0-230.5°C; 1 H NMR (500MHz, CDCl3) δ4.25 (s, 1H), 4.25 (s, 1H), 4.00 (d, J=7.1Hz, 2H), 3.20-3.09 (m, 1H), 2.58-2.55 (m, 1H), 2.50 (q, J=6.5Hz, 1H), 1.50 (s, 6H ), 1.40 (s, 6H), 1.36 (s, 6H), 1.34 (s, 6H), 1.28 (d, J = 7.0Hz, 6H), 1.24 (s, 1H), 1.16-1.14 (m, 4H), 0.80 (d, J = 6.9Hz, 6H), 0.72 (d, J = 6.9Hz, 6H); 13 C NMR (125MHz, CDCl3) δ212.0, 205.4, 197.7, 161.3, 155.8, 147.5, 115.4, 114.1, 111.2, 7 4.5, 56.2, 47.5, 43.3, 35.7, 33.4, 25.1, 25.0, 24.8, 24.3, 19.1, 19.0, 18.9, 18.3, 17.7. HRMS(ESI)m / z:687.3979[M+H] + , calculate for C 42 H 55 O8687.3891.

[0088] Compound 14: (12R,14S)-6-isobutyryl-12,14-diisopropyl-13-((2-methoxyethoxy)methoxy)-2,2,4,4,8,8,10,10-octamethyl-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 82%, melting point 229.4-230.7°C; 1H NMR (400MHz, CDCl3) δ5.10 (s, 2H), 4.31 (d, J=3.9Hz, 2H), 4.08-4.03 (m, 2H), 3.65-3.62 (m, 2H), 3.38 (s, 3H), 3.15 (q, J=6.9Hz, 1H) ; 13 C NMR (125MHz, CDCl3) δ212.0, 205.0, 197.6, 168.1, 154.5, 147.5, 115.5, 114.6, 111.3, 100.1 , 71.9, 69.9, 59.2, 56.2, 47.6, 43.3, 35.6, 33.6, 25.1, 24.8, 24.7, 24.6, 19.2, 19.0, 17.78. HRMS(ESI)m / z:721.4016[M+H] + , calculate for C 42 H 57 O 10 721.3946.

[0089] Compound 15: (12R,14S)-13-(2-(1,3-dioxolan-2-yl)ethoxy)-6-isobutyryl-12,14-diisopropyl-2,2,4,4,4,8,8,10,10-octamethyl-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 89%, melting point 236.7-237.1°C; 1 H NMR (400MHz, CDCl3) δ5.25 (t, J=4.9Hz, 1H), 4.46 (d, J=3.4Hz, 2H), 4.18-4.09 (m, 2H), 4.12-4.05 (m, 2H), 3.99-3.97 (m, 2H), 3.16 (q, J=7.2Hz, 1H), 2 .36-2.26(m, 2H), 1.98-1.91(m, 2H), 1.56(s, 6H), 1.45(s, 6H), 1.43(s, 6H ), 1.41 (s, 6H), 1.28 (s, 6H), 0.85 (d, J = 6.9Hz, 6H), 0.71 (d, J = 6.8Hz, 6H); 13C NMR (125MHz, CDCl3) δ212.2, 204.4, 197.8, 168.2, 154.7, 147.8, 117.0, 113.9, 110.4, 101.9, 7 0.8, 65.2, 56.0, 47.7, 42.8, 36.5, 35.0, 32.9, 25.5, 25.4, 24.9, 24.2, 20.6, 18.1, 17.9, 17.5. HRMS(ESI)m / z:733.3966[M+H] + , calculate for C 43 H 57 O 10 733.3946.

[0090] Compound 16: (12R,14S)-13-(4,4-diethoxybutoxy)-6-isobutyryl-12,14-diisopropyl-2,2,4,4,8,8,10,10-octamethyl-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 87%, melting point 239.5-240.4°C; 1 H NMR (400MHz, CDCl3) δ4.64 (t, J=5.4Hz, 1H), 4.44 (d, J=3.4Hz, 2H), 4.15 (q, J=7. 1Hz, 2H), 3.75-3.70 (m, 2H), 3.61-3.55 (m, 2H), 3.16 (q, J=6.5Hz, 1H), 2.06-1.99 (m, 4H), 1.94-1.90 (m, 2H), 1.56 (s, 6H), 1.45 (s, 6H), 1.43 (s, 6H), 1.41 (s, 6H), 1.27 (s, 6H), 0.91 (t, J=6.4Hz, 6H), 0.85 (d, J=6.9Hz, 6H), 0.71 (d, J=6.8Hz, 6H); 13 C NMR (125MHz, CDCl3) δ212.0, 202.4, 197.6, 168.0, 153.1, 147.6, 116.7, 110.2, 102.6, 100.0, 68.7, 61. 3, 61.2, 55.9, 47.5, 42.7, 36.3, 32.8, 30.2, 29.3, 25.4, 25.2, 24.8, 24.0, 20.5, 18.0, 17.7, 17.3, 15.4. HRMS(ESI)m / z:799.4375[M+Na] + , calcdfor C 46 H 64 O 10Na 799.4392.

[0091] Compound 17: (12R,14S)-6-isobutyryl-12,14-diisopropyl-2,2,4,4,4,8,8,10,10-octamethyl-1,3,9,11-tetraoxo-1,2,3,4,8,9,10,11,12,14-decahydropyrano[3,2-b]xanthene-13-isobutyrate, white solid, yield 63%, melting point 219.0-220.1°C; 1 H NMR (500MHz, CDCl3) δ4.11 (d, J=3.5Hz, 2H), 3.04 (p, J=7.0Hz, 1H), 2.72 (p, J=7.0Hz, 1H), 1.86-1.83 (m, 2H), 1.61 (s, 6H), 1.4 2 (s, 6H), 1.37 (s, 6H), 1.36 (s, 6H), 1.27 (d, J = 6.9Hz, 6H), 1.23 (d, J = 5.8Hz, 6H), 0.78 (d, J = 2.8Hz, 6H), 0.76 (d, J = 2.7Hz, 6H); 13 C NMR (125MHz, CDCl3) δ211.7, 204.2, 197.5, 174.4, 167.6, 153.2, 146.0, 114.1, 110.4, 10 9.2, 56.2, 47.4, 42.7, 35.3, 34.6, 33.1, 25.1, 24.9, 24.8, 24.6, 19.6, 18.9, 18.3, 17.9. HRMS(ESI)m / z:703.3894[M+H] + , calculate for C 42 H 55 O9703.3841.

[0092] Compound 18: (12R,14S)-6-isobutyryl-13-(isopentyloxy)-12,14-diisopropyl-2,2,4,4,8,8,10,10-octamethyl-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 54%, melting point 224.4-225.1°C; 1H NMR (500MHz, CDCl3) δ4.44-4.43 (m, 2H), 4.33-4.30 (m, 1H), 3.96-3.94 (m, 1H), 3.17-3.13 (m, 1H), 1.92-1.90 (m, 2H), 1.86-1.77 (m, 3H), 1.5 3 (s, 6H), 1.43 (s, 6H), 1.41 (s, 6H), 1.39 (s, 6H), 1.25 (d, J = 2.9Hz, 6H), 1.05 (d, J = 3.2Hz, 6H), 0.84 (d, J = 3.1Hz, 6H), 0.70 (d, J = 3.1Hz, 6H); 13 C NMR (125MHz, CDCl3) δ212.2, 204.4, 197.9, 168.2, 155.3, 147.7, 116.9, 113.6, 110.3, 73.7, 56 .0, 47.7, 42.8, 39.5, 36.5, 32.9, 25.6, 25.4, 24.97, 24.92, 24.1, 23.0, 20.7, 18.1, 17.9, 17.4. HRMS(ESI)m / z:703.4182[M+H] + , calculate for C 43 H 59 O8703.4204.

[0093] Compound 19: (12R,14S)-13-((6-hydroxyhexyl)oxy)-6-isobutyryl-12,14-diisopropyl-2,2,4,4,8,8,10,10-octamethyl-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 75%, melting point 195.5-196.7°C; 1 H NMR (500MHz, CDCl3) δ4.26 (d, J=3.9Hz, 2H), 4.00-3.93 (m, 1H), 3.80 (t, J=6.1Hz, 2H), 3.44-3.39 (m, 1H), 3.17 (p, J=7.0Hz, 1H), 1.96-1.92 (m, 2H), 1.89-1.85 (m, 3 H), 1.73-1.68(m, 2H), 1.52(s, 6H), 1.41(s, 6H), 1.37(s, 6H), 1.35(s, 6H), 1.29( d, J=7.0Hz, 6H), 1.26-1.24 (m, 3H), 0.82 (d, J=6.8Hz, 6H), 0.75 (d, J=7.0Hz, 6H); 13C NMR (125MHz, CDCl3) δ211.9, 205.4, 198.0, 168.3, 155.9, 147.6, 115.4, 114.1, 111.2, 75.0, 63.1 , 56.2, 47.6, 43.3, 35.8, 33.4, 30.3, 29.8, 25.8, 25.7, 25.2, 25.0, 24.8, 24.4, 19.2, 19.1, 17.8. HRMS(ESI)m / z:733.4349[M+H] + , calculate for C 44 H 61 O9733.4310.

[0094] Compound 20: (12R,14S)-13-(benzyloxy)-6-isobutyryl-12,14-diisopropyl-2,2,4,4,8,8,10,10-octamethyl-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 49%, melting point 236.6-237.1°C; 1 H NMR (500MHz, CDCl3) δ7.55-7.34 (m, 5H), 4.80 (s, 2H), 4.33 (d, J=3.5Hz, 2H), 3.17 (p, J=6.9Hz, 1H), 1.96-1.93 (m, 2H), 1 .52 (s, 6H), 1.40 (s, 6H), 1.38 (s, 6H), 1.35 (s, 6H), 1.29 (d, J = 7.0Hz, 6H), 0.79 (d, J = 7.0Hz, 6H), 0.71 (d, J = 6.5Hz, 6H); 13 C NMR (125MHz, CDCl3) δ211.8, 205.3, 197.5, 167.8, 155.1, 147.5, 135.9, 128.7, 128.62, 128.27, 1 15.5, 114.2, 111.0, 77.2, 56.0, 47.4, 43.2, 35.7, 33.4, 25.0, 24.8, 24.7, 24.2, 19.1, 19.0, 17.6. HRMS(ESI)m / z:723.4185[M+H] + , calculate for C 45 H 55 O8723.3891.

[0095] Compound 21: (12R,14S)-6-isobutyryl-12,14-diisopropyl-2,2,4,4,8,8,10,10-octamethyl-13-((perfluorophenyl)methoxy)-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 59%, melting point 229.6-231.1°C; 1 H NMR (500MHz, CDCl3) δ5.21 (s, 2H), 4.18 (d, J=3.4Hz, 1H), 4.12 (d, J=7.2Hz, 1H), 3.16 (p, J=6.9Hz, 1H), 1.85-1.80 (m, 2H), 1.47 (s, 5H), 1.36 (s, 12H), 1.33 (s, 6H), 1.13 (d, J = 7.1Hz, 3H), 1.11 (d, J = 6.9Hz, 3H), 0.72 (d, J = 7.2Hz, 6H), 0.69 (d, J = 7.2Hz, 6H); 13 C NMR (125MHz, CDCl3) δ212.3, 205.9, 197.8, 168.4, 154.2, 149.8, 147.7 (m, J=59.5Hz), 146.9 (m, J=38.5Hz), 146.8 (m, J=35.5Hz), 144.9 (m, J=15.5Hz), 144.8 (m, J=15.0Hz), 111.3, 111.1, 109.3, 108.5, 56.2, 47.6, 42.6, 35.0, 32.3, 29.8, 25.0, 24.8, 24.7, 24.6, 19.1, 18.1, 17.5; 19 F NMR (CDCl3): δ-142.15 (2F, F o ), -151.40(1F, F p ), -160.84(2F, F m ). HRMS(ESI)m / z:813.3408[M+H] + , calculate for C 45 H 49 O8F5813.3420.

[0096] Compound 22: (12R,14S)-6-isobutyryl-12,14-diisopropyl-2,2,4,4,8,8,10,10-octamethyl-13-((4-nitrobenzyl)oxy)-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 76%, melting point 186.8-187.7°C; 1H NMR (500MHz, CDCl3) δ7.59 (d, J=8.5Hz, 2H), 7.58 (d, J=8.5Hz, 2H), 6.32 (s, 2H), 4.38 (d, J=3.4Hz, 2H), 3.12 (p, J=7.0Hz, 1H), 1.99-1. 94 (m, 2H), 1.51 (s, 6H), 1.41 (s, 6H), 1.40 (s, 6H), 1.36 (s, 6H), 1.20 (d, J = 7.0Hz, 6H), 1.18 ((d, J = 7.0Hz, 6H)), 0.78 (d, J = 4.0Hz, 6H); 13 CNMR (125MHz, CDCl3) δ211.7, 205.2, 197.7, 168.1, 154.6, 148.1, 147.7, 143.2, 128.7, 124.2, 11 5.5, 114.9, 111.1, 75.7, 56.2, 47.6, 43.4, 35.9, 33.6, 25.1, 25.0, 24.8, 24.2, 19.3, 19.0, 17.7. HRMS(ESI)m / z:768.3786[M+H] + , calculate for C 45 H 54 O 10 N 768.3742.

[0097] Compound 23: (12R,14S)-6-isobutyryl-12,14-diisopropyl-2,2,4,4,8,8,10,10-octamethyl-13-(3-phenylpropoxy)-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 86%, melting point 195.2-196.5°C; 1 HNMR (500MHz, CDCl3) δ7.34-7.30 (m, 5H), 4.44 (d, J=3.4Hz, 2H), 4.31-4.2 7(m, 1H), 3.99-3.90(m, 1H), 3.18-3.10(m, 1H), 2.99-2.87(m, 2H), 2.31-2. 21(m, 2H), 1.92-1.82(m, 2H), 1.54(s, 6H), 1.43(s, 6H), 1.41(s, 6H), 1.39( s, 6H), 1.25 (d, J=6.9Hz, 6H), 0.81 (d, J=7.0Hz, 6H), 0.67 (d, J=6.9Hz, 6H); 13CNMR (125MHz, CDCl3) δ212.2, 204.4, 197.8, 168.2, 155.1, 147.7, 141.3, 128.7, 128.6, 126.2, 116.9, 113. 7, 110.3, 74.5, 56.0, 47.7, 42.8, 36.4, 32.9, 32.33, 32.27, 25.6, 25.4, 24.9, 24.1, 20.6, 18.1, 17.8, 17.4. HRMS(ESI)m / z:751.4218[M+H] + , calculate for C 47 H 59 O8751.4204.

[0098] Compound 24 (12R,14S)-13-(2-(dimethylamino)ethoxy)-6-isobutyryl-12,14-diisopropyl-2,2,4,4,8,8,10,10-octamethyl-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 68%, melting point 229.7-230.8°C; 1 H NMR (400MHz, CDCl3) δ4.34 (d, J=3.7Hz, 2H), 4.01-4.06 (m, 2H), 3.94-3.91 (m, 2H), 3.16 (p, J=7.0Hz, 1H), 2.38 (s, 6H), 2.00-1.97 (m, 2H), 1.51 (s, 6H), 1.40 (s, 6H), 1.37 (s, 6H), 1.34 (s, 6H), 1.29 (d, J = 7.0Hz, 6H), 0.80 (d, J = 6.9Hz, 6H), 0.75 (d, J = 6.9Hz, 6H); 13 C NMR (125MHz, CDCl3) δ212.0, 205.4, 197.7, 168.0, 155.8, 147.5, 115.5, 114.2, 111.1, 7 3.2, 59.3, 56.2, 47.6, 46.0, 43.3, 35.8, 33.4, 25.2, 24.9, 24.8, 24.4, 19.2, 19.0, 17.7. HRMS(ESI)m / z:704.4216[M+H] + , calculate for C 42 H 58 O8N704.4157.

[0099] Compound 25: (12R,14S)-13-(2-(diethylamino)ethoxy)-6-isobutyryl-12,14-diisopropyl-2,2,4,4,8,8,10,10-octamethyl-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 81%, melting point 236.2-237.1°C; 1 H NMR (500MHz, CDCl3) δ4.42 (d, J=3.4Hz, 2H), 4.13-4.09 (m, 2H), 4.07-4.04 (m, 1H ), 3.44-3.40(m, 1H), 3.16-3.11(m, 1H), 3.06-3.03(m, 1H), 1.95-1.91(m, 2H), 1 .62-1.53(m, 2H), 1.53(s, 6H), 1.42(s, 6H), 1.40(s, 6H), 1.37(s, 6H), 1.25(d, J =4.5Hz, 6H), 1.13 (t, J = 7.1Hz, 6H), 0.83 (d, J = 6.9Hz, 6H), 0.68 (d, J = 6.8Hz, 6H); 13 C NMR (125MHz, CDCl3) δ212.2, 204.4, 197.8, 168.2, 154.9, 147.7, 116.9, 113.8, 110.2, 73.6, 56.0, 52.8, 48.0 , 47.6, 42.8, 36.4, 32.9, 25.5, 25.4, 24.9, 24.1, 20.7, 18.1, 17.8, 17.4, 14.3.HRMS(ESI)m / z:732.4533[M+H] + , calculate for C 44 H 62 O8N732.4470.

[0100] Compound 26: (12R,14S)-13-(3-(diethylamino)propoxy)-6-isobutyryl-12,14-diisopropyl-2,2,4,4,8,8,10,10-octamethyl-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 83%, melting point 233.7-234.5°C; 1H NMR (400MHz, CDCl3) δ4.42 (d, J=3.5Hz, 2H), 4.31-4.24 (m, 2H), 4.19-4.15 (m, 1H), 2. 80-2.76(m, 3H), 2.58-2.54(m, 1H), 2.12-2.09(m, 3H), 1.96-1.93(m, 2H), 2.58-2.54 (m, 1H), 1.69-1.66 (m, 1H), 1.53 (s, 6H), 1.43 (s, 6H), 1.41 (s, 6H), 1.38 (s, 6H), 1.25 (d, J=6.9Hz, 6H), 1.10 (t, J=7.1Hz, 6H), 0.83 (d, J=7.0Hz, 6H), 0.69 (d, J=6.8Hz, 6H); 13 C NMR (125MHz, CDCl3) δ212.2, 204.4, 197.8, 168.2, 155.0, 147.7, 116.8, 111.2, 110.3, 73.8, 56.0 , 49.5, 47.7, 46.9, 42.8, 36.4, 32.9, 29.8, 25.6, 25.4, 24.9, 24.1, 20.6, 18.1, 17.8, 17.4, 11.5. HRMS(ESI)m / z:746.4679[M+H] + , calculate for C 45 H 64 O8N 746.4626.

[0101] Compound 27 (12R,14S)-6-isobutyryl-12,14-diisopropyl-2,2,4,4,8,8,10,10-octamethyl-13-(2-(pyrrolidin-1-yl)ethoxy)-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 88%, melting point 239.3-240.2°C; 1 H NMR (500MHz, CDCl3) δ4.31 (d, J=3.4Hz, 2H), 3.48-3.38 (m, 2H), 3.16 (p, J=6.8Hz, 1H), 3.00-2.95 (m, 1H), 2.72-2.61 (m, 3H), 1.99-1.94 (m, 2H) ), 1.70-1.57 (m, 6H), 1.51 (s, 6H), 1.40 (s, 6H), 1.37 (s, 6H), 1.34 (s, 6H), 1.28 (d, J = 6.9Hz, 6H), 0.80 (d, J = 6.9Hz, 6H), 0.74 (d, J = 6.9Hz, 6H); 13C NMR (125MHz, CDCl3) δ212.0, 205.4, 197.6, 168.0, 155.8, 147.5, 115.5, 114.1, 111.1, 74.1 , 56.2, 56.0, 54.8, 47.6, 43.3, 35.8, 33.4, 25.2, 24.9, 24.8, 24.4, 23.7, 19.2, 19.0, 17.7. HRMS(ESI)m / z:730.4367[M+H] + , calculate for C 44 H 60 O8N 730.4313.

[0102] Compound 28: (12R,14S)-6-isobutyryl-12,14-diisopropyl-2,2,4,4,8,8,10,10-octamethyl-13-(2-(piperidin-1-yl)ethoxy)-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 71%, melting point 245.5-246.4°C; 1 H NMR (400MHz, CDCl3) δ4.42 (d, J=3.5Hz, 2H), 4.10-4.08 (m, 2H), 3.14 (p, J=7 .0Hz, 1H), 2.96-2.90 (m, 2H), 2.62-2.59 (m, 2H), 1.95-1.91 (m, 2H), 1.64-1 .60(m,5H),1.53(s,6H),1.43(s,6H),1.40(s,6H),1.38(s,6H),1.30-1.27 (m, 3H), 1.24 (d, J = 7.0Hz, 6H), 0.84 (d, J = 7.0Hz, 6H), 0.68 (d, J = 6.8Hz, 6H); 13 C NMR (125MHz, CDCl3) δ212.2, 204.4, 197.8, 168.2, 154.9, 147.7, 116.9, 113.8, 110.2, 71.1, 5 9.0, 56.1, 55.3, 47.7, 42.8, 36.4, 32.9, 25.6, 25.5, 25.4, 24.9, 24.2, 21.2, 20.7, 17.4, 14.3. HRMS(ESI)m / z:744.4526[M+H] + , calculate for C 45 H 62 O8N 744.4470.

[0103] Compound 29: (12R,14S)-6-isobutyryl-12,14-diisopropyl-2,2,4,4,8,8,10,10-octamethyl-13-(3-(piperidin-1-yl)propoxy)-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 93%, melting point 246.6-247.5°C; 1 H NMR (400MHz, CDCl3) δ4.24 (d, J=3.9Hz, 2H), 3.86-3.83 (m, 3H), 3.16 (p, J=7.1Hz, 1H), 2.67-2.63 (m, 1H), 2.50-2.44 (m, 5H), 1.95-1.91 (m, 2H) ), 1.51 (s, 6H), 1.40 (s, 6H), 1.37 (s, 6H), 1.35 (s, 6H), 1.29 (d, J = 7.0Hz, 6H), 1.26-1.24 (m, 8H), 0.81 (d, J = 6.9Hz, 6H), 0.74 (d, J = 6.8Hz, 6H); 13 CNMR (125MHz, CDCl3) δ211.9, 205.2, 197.5, 167.9, 155.6, 147.4, 115.2, 114.0, 111.0, 73.8, 56.0 , 54.52, 54.51, 47.4, 43.2, 35.7, 33.3, 27.7, 25.7, 25.0, 24.8, 24.7, 24.3, 24.0, 19.1, 19.0, 17.6. HRMS(ESI)m / z:758.4667[M+H] + , calculate for C 46 H 64 O8N 758.4626.

[0104] Compound 30: (12R,14S)-13-(2-(azepan-1-yl)ethoxy)-6-isobutyryl-12,14-diisopropyl-2,2,4,4,8,8,10,10-octamethyl-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 48%, melting point 246.8-247.4°C; 1H NMR (400MHz, CDCl3) δ4.29 (d, J=3.8Hz, 2H), 4.13-4.05 (m, 2H), 3.65-3.62 ( m, 1H), 3.16 (p, J=7.0Hz, 1H), 3.09-3.00 (m, 2H), 2.89-2.83 (m, 3H), 1.97-1 .93(m, 2H), 1.70-1.65(m, 8H), 1.51(s, 6H), 1.40(s, 6H), 1.37(s, 6H), 1.35 (s, 6H), 1.29 (d, J = 7.0Hz, 6H), 0.81 (d, J = 6.9Hz, 6H), 0.74 (d, J = 6.9Hz, 6H); 13 C NMR (125MHz, CDCl3) δ212.0, 205.3, 197.6, 167.9, 155.8, 147.5, 115.2, 114.1, 111.2, 70.3, 5 7.7, 56.2, 55.9, 47.5, 43.3, 35.8, 33.5, 26.4, 25.6, 25.2, 24.9, 24.8, 24.4, 19.2, 19.1, 17.8. HRMS(ESI)m / z:758.4681[M+H] + , calculate for C 46 H 64 O8N 758.4626.

[0105] Compound 31: (12R,14S)-13-(3-(dibutylamino)propoxy)-6-isobutyryl-12,14-diisopropyl-2,2,4,4,8,8,10,10-octamethyl-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 80%, melting point 249.3-250.6°C; 1 H NMR (400MHz, CDCl3) δ4.41 (d, J=3.4Hz, 2H), 4.10-4.09 (m, 2H), 3.58-3.54 (m, 3 H), 3.14 (p, J=7.0Hz, 1H), 2.53-2.50 (m, 1H), 1.98-1.96 (m, 2H), 1.92-1.83 (m, 10H), 1.53(s, 6H), 1.42(s, 6H), 1.40(s, 6H), 1.38(s, 6H), 1.30-1.28(m, 2H), 1 .24 (d, 6H), 0.93 (t, J = 7.3Hz, 6H), 0.82 (d, J = 7.0Hz, 6H), 0.68 (d, J = 6.8Hz, 6H); 13C NMR (125MHz, CDCl3) δ212.2, 204.5, 197.8, 168.2, 155.1, 147.7, 116.8, 113.7, 110.3, 74.0, 56.0, 53 .8, 50.8, 47.6, 42.8, 36.4, 32.9, 30.4, 27.0, 25.5, 25.4, 24.9, 24.1, 20.6, 18.1, 17.8, 17.4, 14.21. HRMS(ESI)m / z:802.5278[M+H] + , calculate for C 49 H 72 O8N802.5252.

[0106] Compound 32: (12R,14S)-6-isobutyryl-12,14-diisopropyl-2,2,4,4,4,8,8,10,10-octamethyl-13-(3-morpholinopropoxy)-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 82%, melting point 248.8-250.1°C; 1 H NMR (500MHz, CDCl3) δ4.23 (d, J=3.9Hz, 2H), 3.86-3.83 (m, 2H), 3.65-3.55 ( m, 4H), 3.15 (q, J=7.0Hz, 1H), 2.66-2.63 (m, 2H), 2.49-2.43 (m, 3H), 1.96-1 .92(m, 2H), 1.68-1.65(m, 3H), 1.51(s, 6H), 1.40(s, 6H), 1.36(s, 6H), 1.34 (s, 6H), 1.28 (d, J=7.0Hz, 6H), 0.81 (d, J=6.9Hz, 6H), 0.73 (d, J=6.9Hz, 6H); 13 CNMR (125MHz, CDCl3) δ211.8, 205.2, 197.5, 167.9, 155.6, 147.4, 115.2, 114.1, 111.0, 73.4, 67. 0, 56.0, 55.5, 53.7, 53.7, 47.4, 43.2, 35.7, 33.3, 27.0, 25.0, 24.8, 24.6, 24.2, 19.1, 18.9, 17.6. HRMS(ESI)m / z:760.4465[M+H] + ,calcd fo rC 45 H 62 O8N 760.4419.

[0107] Compound 33: (12R,14S)-6-isobutyryl-12,14-diisopropyl-2,2,4,4,4,8,8,10,10-octamethyl-13-(3-(4-methylpiperazin-1-yl)propoxy)-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 75%, melting point 256.3-257.7°C; 1 H NMR (400MHz, CDCl3) δ4.24 (d, J=3.8Hz, 2H), 4.13-4.08 (m, 2H), 3.16 (p, J=7.0Hz, 1H ), 2.68-2.64(m, 2H), 2.58-2.54(m, 4H), 2.31-2.29(m, 1H), 2.04(s, 3H), 1.96-1.91 (m, 2H), 1.71-1.66 (m, 4H), 1.51 (s, 6H), 1.40 (s, 6H), 1.37 (s, 6H), 1.34 (s, 6H), 1.2 8(d, J=6.8Hz, 6H), 1.18-1.15 (m, 1H), 0.82 (d, J=6.8Hz, 6H), 0.74 (d, J=6.9Hz, 6H); 13 C NMR (125MHz, CDCl3) δ212.0, 205.4, 197.7, 168.0, 155.7, 147.6, 115.3, 114.2, 111.2, 71.1, 56.2 , 55.1, 55.1, 53.0, 47.6, 45.9, 43.3, 35.8, 33.4, 26.8, 25.2, 24.9, 24.8, 24.4, 19.3, 19.0, 17.7. HRMS(ESI)m / z:773.4790[M+H] + , calculate for C 46 H 65 O8N2773.4735.

[0108] Compound 34: (12R,14S)-13-(2-(1,3-dioxoindolin-2-yl)ethoxy)-6-isobutyryl-12,14-diisopropyl-2,2,4,4,8,8,10,10-octamethyl-4,8,12,14-tetrahydrochromeno[3,2-b]xanthene-1,3,9,11(2H,10H)-tetraone, white solid, yield 84%, melting point 316.4-317.8°C; 1H NMR (500MHz, CDCl3) δ7.86 (dd, J=5.4, 3.1Hz, 2H), 7.72 (dd, J=5.5, 3.1Hz, 2H), 4.23 (d, J=3.9Hz, 2H), 4.05-4.01 (m, 2H), 3.91-3.88 (m, 2H), 3.17 (p, J= 7.0Hz, 1H), 2.00-1.93 (m, 2H), 1.51 (s, 6H), 1.38 (s, 6H), 1.37 (s, 6H), 1.31 (s, 6H), 1.29 (d, J = 6.9Hz, 6H), 0.85 (d, J = 6.8Hz, 6H), 0.75 (d, J = 6.8Hz, 6H); 13 C NMR (125MHz, CDCl3) δ211.9, 205.2, 197.4, 168.2, 167.8, 155.4, 147.5, 133.9, 132.2, 123.3, 115.2, 114.2, 111.1, 72.7, 56.0, 47.4, 43.2, 35.7, 33.3, 29.7, 25.0, 24.7, 24.6, 24.2, 19.1, 18.9, 17.6.HRMS(ESI)m / z:806.4361[M+H] + , calculate for C 48 H 56 O 10 N806.4361.

[0109] Example 4

[0110] Inhibitory activity assay against DNA gyrase and Topo IV

[0111] Inhibitory activity was determined in 96-well microtiter plates coated with streptavidin. First, the plates were rehydrated with buffer (20 mM Tris-HCl, pH 7.6, 0.01% BSA, 0.05% Tween 20, 137 mM NaCl) and then the biotinylated oligonucleotides were immobilized. After washing away unbound oligonucleotides, enzyme assays were performed in a 30 μL reaction volume containing 1 U of DNA gyrase (from E. coli or S. aureus) or Topo IV (from E. coli or S. aureus) and 0.5 μg of either relaxed pNO1 plasmid (for DNA gyrase assays) or supercoiled pNO1 plasmid (for Topo IV assays). The reaction solution was incubated at 37°C for 30 minutes. After incubation, TF buffer (50mM NaOHAC, pH 5.0, 50mM NaCl and 50mM MgCl2) was added to terminate the enzymatic reaction. After an additional 30 min incubation at room temperature, during which time a triplex (biotin-oligonucleotide-plasmid) was formed, unbound plasmid was washed away using TF buffer and SybrGOLD in T10 buffer (10mMTris-HCl, pH 8.0 and 1mM EDTA). The plate was immediately read five times using a dynamic readout on a Flex Station 3 (Ex485 / Em535nm). IC50 values ​​were calculated using nonlinear regression analysis (GraphPad Prism5). Each experiment was performed independently at least 3 times.

[0112] result

[0113] The DNA gyrase and Topo IV inhibitory abilities of the synthesized compounds were assessed in streptavidin-coated 96-well microtiter plates, using ciprofloxacin as a positive control. The compounds were tested at six concentrations: 10 μM, 2 μM, 0.4 μM, 0.08 μM, 0.016 μM, and 0.0032 μM. The 50% inhibitory concentration (IC50), defined as the concentration of compound that inhibits DNA gyrase or Topo IV activity by 50%, was calculated and summarized in Tables 1 and 2.

[0114] Table 1 Structures, DNA gyrase and Topo IV inhibitory activities of compounds 5 and 6

[0115]

[0116]

[0117] Note: IC 50 Values ​​are expressed as the mean ± SEM of at least three independent experiments. Abbreviation: CIP, ciprofloxacin. “ / ” indicates “not applicable”.

[0118] Table 2 Structures, DNA gyrase and Topo IV inhibitory activities of compounds 7-34

[0119]

[0120]

[0121] Note: IC 50 Values ​​are expressed as mean ± SEM of at least three independent experiments. Abbreviation: CIP, ciprofloxacin.

[0122] “ / ” means “Not applicable”.

[0123] As shown in Table 1, meso-5b has higher DNA gyrase and Topo IV inhibition ability than other isomers (+)-5a and (-)-5a. The desisobutyl analogs have lower potency, and meso-5b has the strongest inhibitory effect. As shown in Table 2, the aminoalkyl substituent at position 13 seems to be more favored. Compound 27 is the most effective DNA gyrase and Topo IV inhibitor, with E. coli enzyme half-inhibitory concentration = 0.011±0.0018μM, E. coli IV half-inhibitory concentration = 0.083±0.0079μM, Staphylococcus aureus enzyme half-inhibitory concentration = 0.017±0.0032μM, Staphylococcus aureus IV half-inhibitory concentration = 0.021±0.0046μM. 13-alkoxy substitution cannot significantly enhance DNA gyrase and Topo inhibition. For example, compounds 7 to 23 showed DNA gyrase and Topo inhibition ability, IC 50 The values ​​ranged from 0.020 to 4.2 μM.

[0124] To evaluate the mechanism of antibacterial activity, DNA cyclospheric coiling assay was performed using DNA of compound 27 as substrate. CIP was used as positive control, e.g. Figure 1 A and Figure 1 As shown in Figure C, compound 27 has a strong inhibitory effect on the supercoiling activity of Escherichia coli DNA gyrase and Staphylococcus aureus DNA gyrase, which is significantly better than CIP (25μM). Using supercoiled pBR322DIA as a substrate, compound 27 was used to perform DNA topoisomerase IV (Topo IV) relaxation analysis. CIP was used as a positive control. Figure 1B and Figure 1 As shown in Figure D, compound 27 exhibited stronger inhibitory activity than CIP (25 μM). In Topo IV of E. coli and S. aureus, compound 27 retained inhibitory activity at 0.04 μM and 1 μM, and completely inhibited the relaxant activity at 5 μM.

[0125] To better investigate the selectivity of myrtosides, the inhibitory activity of compound 27 against human topoisomerase I (hTopoI) or human topoisomerase IIα (hTopo IIα) was tested. DNA TopoI and IIα relaxation assays were performed using supercoiled pBR322 as substrate. Camptothecin (CPT) and etoposide (ETP) were used as positive controls, respectively. Figure 1 E and Figure 1 As shown in Figure 5(F), at 25 μM, CPT and ETP almost completely inhibited the senescence activity of human Topo I and IIα, respectively. The inhibitory activity of compound 27 was much lower than that of CPT and ETP. At a concentration of 125 μM, compound 27 did not affect the catalytic activity of human DNA Topo I and IIα.

[0126] To observe the molecular interactions of the novel inhibitor with DNA gyrase (PDB ID: 4CKL) and Topo IV (PDB ID: 3FV5) proteins, molecular docking was performed, e.g. Figure 2 As shown in Figure 1, molecular docking revealed that compound 27 binds to the hydrophobic DNA binding groove of the DNA gyrase protein. The pentacyclic scaffold of compound 27 aligns along the hydrophobic cliff formed by Ile174; the pi-alkyl group includes residues His45 and Tyr266. The side chain of 13 extends into the narrow DNA binding channel of the DNA gyrase, effectively blocking the binding of the DNA substrate to Topo IV. Two hydrogen bonds exist between the carbon group at position 3 and the Arg91 residue (2.8 Å) and the carbon group at position 26 and the Lys42 residue (2.9 Å), which may account for the binding affinity between the enzyme and the inhibitor. There is also a van der Waals force associated with Gly173, Ala175, Ala33, Asp87, Thr88, Asn165, Ala84, Ala84, Gly170, and Gln267. Meanwhile, compound 27 can be fully embedded in the docking site and interacts with DNA Topo IV through hydrophobic bonds with Met74, Pro75, Ala86, and Leu89 residues; it forms a hydrogen bond with Arg93 (3.2A), which may contribute to the binding affinity of the enzyme and the inhibitor. There is also a van der W variable associated with Asn42, Asp45, Arg72, Gly73, Leu94, Ile116, and Thr163.

[0127] Example 5

[0128] In vitro antibacterial activity evaluation using the MIC method

[0129] MIC values ​​were determined using the broth dilution method. The initial concentration of the test compound was 32 μg / mL. A solution of the compound in DMSO (15 μL) was added to 285 μL of bacterial culture (5.0×10^5 cells / mL) in the first well of a 96-well plate. The solution was then diluted by doubling. Bacterial culture fluid containing the appropriate compound (150 μL) was discarded in the last well to ensure that the volume of bacterial culture in each well was 150 μL. Before measuring the absorbance value, the plate was cultured overnight at 37°C in an electric thermostatic incubator. The absorbance of the solution at 600 nm was recorded. Each experiment was performed independently at least 3 times.

[0130] result

[0131] Antimicrobial activity against Gram-negative strains was evaluated using ciprofloxacin (CIP), penicillin (PEN), and vancomycin (VAN) as positive controls. Minimum inhibitory concentrations (MICs) are shown in Table 3. Myrtle derivatives also exhibited low activity against Gram-negative and fungal strains, particularly those with 13-aminoalkyl substitutions, such as compounds 26, 27, and 28. Antimicrobial screening revealed that compound 27 exhibited strong activity against Gram-positive strains, with MIC values ​​of 0.25, 1, 0.125, and 0.5 μg / mL against Staphylococcus aureus (ATCC25923), MRSA (ATCC43300), Streptococcus pneumoniae (ATCC6305), and hemolytic Streptococcus (ATCC19615), respectively, representing 8, 1, 64, and 16 times that of Vancomycin.

[0132] Table 3 In vitro antibacterial activity of compounds 5-34

[0133]

[0134] Note: a: EC 50 Values ​​are expressed as the mean ± SEM of at least three independent experiments. Abbreviations: CIP, ciprofloxacin. PEN, penicillin. VAN, vancomycin. “ / ” indicates “not applicable.”

[0135] Table 4 Cytotoxicity of compound 5-34 in normal human cells

[0136]

[0137] Note: GI 50 The value was defined as the compound concentration that resulted in 50% cell growth inhibition. Etoposide (ETP) was used as a positive control. Each experiment was repeated at least three times independently.

[0138] Example 6

[0139] Time-kill kinetic analysis

[0140] As described above, the killing rate and degree of antibacterial effect of myrtle ketone derivatives against MRSA were determined by time-kill kinetic analysis. Briefly, 1×10^6 CFU / mL of bacteria were incubated at different concentrations. During the incubation process, 50 μL aliquots were taken from each test tube every 0, 2, 8, 12, 16, 20 and 24 hours and serially diluted using 450 μL sterile saline solution (1:10). After each dilution, 10 μL was inoculated onto Mueller Hinton agar (MHA, Oxoid, Singapore) plates and incubated at 37°C for 24 hours. Then, the number of viable colonies was counted only from plates containing 30 to 300 colonies to calculate CFU / mL.

[0141] result

[0142] Encouraged by the promising in vitro antibacterial activity of compound 27, we conducted a time-kill kinetic study to evaluate the bactericidal properties of the most potent compound 27 against MRSA. Different concentrations (0.5×, 1×, 2×, and 4× the MIC) were added to a MRSA suspension. Subsequently, the dendritic bacterial suspension was harvested at different time points (2–24 h) for colony counts. Figure 3 Figure a shows the growth and killing patterns of MRSA by compound 27 and VAN, respectively. Overall, the time-kill curves for MRSA revealed a significant reduction in bacterial population size upon exposure to the antibiotic. After a short lag period, a slow decline was observed at almost all concentrations, with the lowest colony-forming unit (CFU) counts reached only after 24 hours of incubation. Only after 8 hours did concentrations above the MIC begin to effectively reduce bacterial density, with the greatest decrease occurring at 4 hours at the highest concentration. However, regrowth was observed at 0.5× the MIC, and a major decrease in CFUs was observed at concentrations of 2× the MIC or higher. At 4× the MIC (4.0 μg / mL), MRSA was killed within 20 hours. At 2× the MIC (2.0 μg / mL), MRSA was killed within 24 hours. There was no significant difference in the bactericidal properties between compound 27 and VAN. Both exhibited a pronounced, long-lasting bactericidal effect, with a clear concentration-dependent activity. At the same concentration, even at 1× the MIC, compound 27 exhibited a higher bactericidal rate than VAN. Furthermore, the bactericidal properties of compound 27 were dose-dependent. Therefore, compound 27 effectively killed MRSA, shortened the treatment time of bacterial infections, and avoided the development of bacterial resistance.

[0143] Example 7

[0144] Bacterial resistance research

[0145] Drug resistance was defined as a 4-fold increase over the initial MIC. Norfloxacin was used as a control drug. Staphylococcus aureus was cultured for 4 hours, inoculated onto fresh MHA plates containing the compound 27 at sub-inhibitory concentration (1 / 2 MIC), and cultured again at 37 °C for 17 hours. The MIC was obtained by the above method. The experiment was repeated 20 times.

[0146] Results

[0147] Antibiotic resistance has become an increasingly serious problem, and the number of deaths caused by diseases resulting from drug-resistant bacterial infections will increase year by year. The development of drug resistance is an important criterion for the design and evaluation of new antibacterial agents. To study the possibility of compound 27 inducing bacterial drug resistance, a 20-day bacterial drug resistance study was conducted on Staphylococcus aureus at sub-lethal concentrations of compound 27 ( Figure 3 B). After incubation of Staphylococcus aureus for 20 generations, the MIC value of compound 27 did not increase, indicating that compound 27 is not sensitive to the drug resistance of Staphylococcus aureus. In contrast, the fluoroquinolone antibiotic norfloxacin rapidly induced bacterial drug resistance, with the MIC value increasing by more than 64-fold on the 20th day ( Figure 3 B).

[0148] Example 8

[0149] Drug combination study

[0150] A drug combination study of myrtenal derivatives 26, 27, and 28 with different groups of related antibiotics (levofloxacin, ciprofloxacin, ofloxacin, amikacin, cefepime, ceftazidime, and gentamicin) against MRSA was performed using 96-well plates by the two-dimensional broth microdilution checkerboard method. The experiment was repeated, and the plates were incubated at 37 °C for 24 hours, and then evaluated using the fractional inhibitory concentration (FIC) index, which was calculated using the following formula: FIC index = (MIC of compound A in combination / MIC of compound A alone) + (MIC of compound B in combination / MIC of compound B alone). An FIC index ≤ 1 represents synergy, 1 < FIC index ≤ 2 represents additive effect, and FIC index > 2 represents antagonism.

[0151] Results

[0152] Combined therapy is currently a promising strategy for treating infections. Studies have shown that combinations of different types of clinical antibacterial drugs can enhance bactericidal efficacy, reduce or eliminate side effects, and even overcome drug resistance through different mechanisms of action. Therefore, in this study, we used combinations of myrtenal derivatives 26, compounds 27, 28, and several antibiotics at high concentrations to combat MRSA. All the tested compounds showed additive effects (1 < FIC ≤ 2) when combined with levofloxacin, indicating that these compounds may have the same targets as levofloxacin, such as acting on DNA gyrase and Topo IV. In addition,

[0153] 27 showed additive effects (1 < FIC ≤ 2) with ciprofloxacin and gentamicin. Derivatives 26 and 28 showed additive or antagonistic effects (2 < FIC) with ciprofloxacin and gentamicin. Therefore, these combinations could not effectively inhibit the growth of MRSA. Compound 27 showed synergistic effects when combined with ofloxacin (OFX), amikacin (AK), cefepime (FEP), and ceftazidime (CAZ). These combinations showed high activity with lower doses and expanded antibacterial activity. This result indicates that these compounds and these antibiotics can bind to different targets of the pathogen, thus enhancing antibacterial activity. The results are shown in Table 3. Finally, the combined use of traditional antibiotics and myrtenal derivatives can reduce their MIC values, thereby alleviating the impact of the multi-drug resistance problem.

[0154] Table 5 Combined effects of the most active myrtenal derivative 26, compound 27, 28 and different antibiotics against MRSA

[0155]

[0156] Note: Fractional inhibitory concentration index (FIC) = (MIC of compound A in combination / MIC of compound A alone) + (MIC of compound B in combination / MIC of compound B alone). FIC index ≤ 1, synergistic effect; 1 < FIC index ≤ 2, additive effect; FIC index > 2, antagonistic effect. Abbreviations: AK, amikacin; CAZ, ceftazidime; CIP, ciprofloxacin; FEP, cefepime; GN, gentamicin; LEV, levofloxacin; OFX, ofloxacin.

[0157] Example 9

[0158] Cytotoxicity produced in human normal cells

[0159] Normal human cell lines (including hepatocyte cell lines MIHA and HL7702), bronchial smooth muscle cell line HBSMC, and lung epithelial cell line BEAS-2B were routinely cultured in RPMI 1640 medium containing 10% fetal bovine serum and cultured in a humidified atmosphere of 5% CO2 at 37°C. For cytotoxicity assays, cells were seeded in 96-well plates (3.0 × 10 3 / well), 100 μL of culture medium was added and diluted in the range of 100M to 3.12M for 72h in the presence or absence of the specified drug (pre-dissolved in DMSO). Then, PBS buffer (20 μL, 2.5 mg / mL) was added to each well, and the resulting solution was further incubated for 4h. After siphoning out the culture medium, DMSO (200 μL) was used to dissolve the methyl crystals formed in each well. The absorbance of the solution at 570nm was recorded. Nonlinear regression analysis (GraphPad Prism 5) was used to calculate the GI50 value. Each experiment was performed independently at least 3 times.

[0160] result

[0161] In the research and development of new antibiotics, their toxic side effects on normal cells are also important criteria for the design and evaluation of new antibiotics. The cytotoxicity of the derivatives was initially evaluated using the MTT assay against normal human cells, hepatocytes (MIHA and HL7702), and lung cells (HBSMC and BEAS-2B), using ETP as a positive control. The GI50 values, defined as the concentration of compound that causes 50% cell growth inhibition, were calculated and summarized in Table 6. The results showed that all myristicin analogs exhibited moderate cytotoxicity at the micromolar level against MIHA, HL7702, HBSMC, and BEAS-2B cell lines. Compound 27, the most potent DNA gyrase and Topo IV inhibitor, exhibited lower cytotoxicity (MIHA: 47 ± 0.44 μM, HL7702: 33 ± 0.11 μM, HBSMC: 17 ± 0.87 μM, and BEAS-2B: 10 ± 0.81 μM).

[0162] Table 6 Cytotoxicity of Compound 5-34 in Normal Human Cells

[0163]

[0164]

[0165] Note: GI 50 The value was defined as the compound concentration that resulted in 50% cell growth inhibition. Etoposide (ETP) was used as a positive control. Each experiment was repeated at least three times independently.

[0166] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0167] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0168] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. The use of myrtle ketone derivatives in the preparation of antibacterial drugs, characterized in that: The structural formula of the myrone derivative is as follows: ， Among them, R is methyl, ethyl, (CH2)2OH, isopropyl, (CH2)3Cl, 、 、CH2O(CH2)2OMe、 , (CH2)3CH(OEt)2, isobutyryl, (CH2)2CHMe2, (CH2)6OH, benzyl, CH2C6F5, 4-NO2C6H4CH2, (CH2)3C6H5, (CH2)2NMe2, (CH2)2NEt2, (CH2)3NEt2, 、 、 、 、(CH2)3NBu2、 、 、 Any one of .

2. The use according to claim 1, characterized in that The structural formula of the myrtle derivative is as follows: Formula (1): , formula (2): , Formula (3): .

3. The use according to claim 2, characterized in that The antibacterial drug includes a compound having the structural formula of the formula (1), the formula (2) or the formula (3).

4. The use according to claim 1, characterized in that The antibacterial drug inhibits Gram-positive bacteria.

5. The use according to claim 4, characterized in that The Gram-positive bacteria are Staphylococcus aureus, MRSA, Streptococcus pneumoniae or hemolytic Streptococcus.

6. The use according to claim 1, characterized in that The antibacterial drug inhibits DNA gyrase.

7. The use according to claim 1, characterized in that The antimicrobial drug inhibits Topo IV.

8. An antibacterial drug, characterized in that The invention comprises the compound described in any one of claim 3.

9. The antibacterial drug according to claim 8, characterized in that The drug is the compound or a salt thereof.

10. The antibacterial drug according to claim 9, characterized in that The medicine also includes pharmaceutically acceptable excipients.

Images