Application of myrtle ketone derivative in preparation of antibacterial drug and antibacterial drug

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of a myrtle ketone derivative in preparation of an antibacterial drug and the antibacterial drug. A novel myrtle ketone derivative is synthesized, the myrtle ketone derivative has inhibitory activity on gram-positive bacteria, and a compound 27 is the most effective DNA (deoxyribonucleic acid) gyrase and Topo IV inhibitor. Biological activity determination shows that the compound 27 not only has the advantage of rapid sterilization on methicillin-resistant staphylococcus aureus, but also has low toxicity, is not easy to induce bacterial drug resistance, and shows a synergistic effect with first-line antibacterial drugs such as ofloxacin, amikacin, cefpiramide and ceftazidime; this makes it a potential candidate for novel antibacterial agents.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to application of myrtle ketone derivatives in preparing 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 drugs with 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 separation. They are highly homologous, have similar structures and functions, show 40% DNA sequence similarity overall, and have significantly similar active sites. Their absence in mammals and their key role 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, Myrtus genus, is an evergreen shrub widely distributed in the Mediterranean region. It has long been used as a folk medicine for disinfection and is also planted in southern my country. However, little research has been done on the use of myrtus communis in antibacterial applications, so it is necessary to provide information on the use of myrtus communis in antibacterial applications. Summary of the invention

[0005] In order to solve the above problems, the present invention provides the use of myrtle ketone 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, (CH 2 )2 OH, isopropyl, (CH 2 ) 3 Cl, CH 2 O(CH 2 ) 2 OMe, (CH 2 ) 3 CH(OEt) 2 、isobutyryl、(CH 2 ) 2 CH 2 , (CH 2 ) 6 OH, benzyl, CH 2 C 6 F 5 4-NO 2 C 6 H 4 CH 2 , (CH 2 ) 3 C 6 H 5 , (CH 2 ) 2 NMe 2 , (CH 2 ) 2 Nt 2 , (CH 2 ) 3 Nt 2 , (CH 2 ) 3 NBu 2 , Any one of .

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

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

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

[0012] Preferably, the antimicrobial 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 also includes 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 on Gram-positive bacteria and can be used for preparing antibacterial drugs.

[0023] The present invention evaluates the inhibitory effects of myrtle derivatives on DNA gyrase and Topo IV, wherein compound 27 is the most effective inhibitor of DNA gyrase and Topo IV. Biological activity assays show that compound 27 not only has the advantage of rapid sterilization against methicillin-resistant Staphylococcus aureus (MRSA), but also has low toxicity, is not easy to induce bacterial resistance, and shows synergistic effects with first-line antibacterial drugs such as ofloxacin, amikacin, cefpiramide and ceftazidime, which may make it a potential candidate for a new antibacterial drug.

[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 1The inhibitory activity of compound 27 at different concentrations is shown, wherein A shows the inhibition of Escherichia coli gyrase supercoiling activity, 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, lanes 4-8 are various concentrations of relaxed pHOT1 DNA, enzyme and compounds, B shows the inhibition of DNA relaxation catalyzed by topo IV of Escherichia coli, D or the inhibition of DNA relaxation catalyzed by topo IV of 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, 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 (25μM) or ETP (25μM) as positive control, lanes 4-8 are different concentrations of pBR322 DNA, enzyme and compound 27, abbreviated as: CIP, ciprofloxacin, CPT, camptothecin, ETP, etoposide, Re, relaxed pBR322 DNA, Sc, supercoiled pBR322 DNA.

[0026] Figure 2 Schematic 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 dotted lines (3D map) and green lines (2D map), and the length (λ) is marked around the lines.

[0027] Figure 3 The data are the drug resistance results, 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 drug resistance of Staphylococcus aureus ATCC25923. The data are the mean standard deviation (error bars), and each experiment was repeated at least 3 times independently. DETAILED DESCRIPTION

[0028] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope 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 creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are 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 BrukerAvance 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 MPA100Optimelt automatic melting point system without correction. The purity of all bioactive test compounds analyzed by HPLC exceeded 95%. The analysis conditions were: detection wavelength 220nm, flow rate 1.0mL / min, elution with 50%-15% PBS buffer (pH3) 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 assay was performed using relaxed pHOT1 DNA as substrate. Briefly, 1U DNA gyrase from E. coli or S. aureus and 0.5μg relaxed pHOT1 DNA were incubated in 30μL supercoiling assay buffer at 37°C for 60 min in the presence or absence of the compound. After incubation, 3μL 2mg / mL proteinase k was added to terminate the reaction. Then, 5μL of the reaction solution was mixed with 1μL 6× DNA loading buffer and analyzed by electrophoresis on a 1% agarose gel at 3V / cm in 1× TAE buffer. The gel was stained with 1× Gel Red, visualized with a UV transilluminator, and analyzed with AlphaEaseFC software. Among them, the supercoil 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 Topo IV was incubated with 0.5μg of supercoiled pBR322 DNA for relaxation assay in 30μL reaction buffer at 37°C for 30 min. The reaction was terminated by adding 30μL chloroform / isoamyl alcohol (24:1). 5μL of the reaction solution was mixed with 1μL 6× DNA loading buffer and analyzed by electrophoresis 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 with 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. The reaction mixture containing 0.5 μg of supercoiled pBR322 DNA in buffer was incubated with 1U of recombinant human DNA Topo I or Topo IIα in the absence or presence of the test compound at 37°C for 30 minutes. The reaction was terminated by adding Topo stop buffer. Then 4 μL of 6× loading buffer was added and analyzed on a 0.8% agarose gel in 4.6 v / cm TBE buffer for 1.5 h. The gel was stained with 1× Gel Red for 30 minutes and then observed with a UV transilluminator. The reaction mixture for supercoiled pBR322 DNA in Topo I relaxation experiment is: 10mM Tris-HCl, pH 7.5, 50mM KCl, 5mM MgCl2, 15μg / mL BSA, 40μg / mL DTT; the reaction mixture for supercoiled pBR322 DNA in Topo II relaxation experiment is: 10mMTris-HCl, pH 7.9, 50mM NaCl, 50mM KCl, 5mM MgCl2, 1mM EDTA, 1mMATP and 15mg / mL BSA. The termination buffer for Topo I experiment is 10% SDS solution containing 0.2% bromophenol blue and 0.2% xylene cyanol; the termination buffer for Topo II experiment is 7mM EDTA)

[0037] 4. Molecular docking

[0038] Molecular docking analysis of the protein targets of the selected ligands 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 sites where ligands bind to 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. Meanwhile, the crystal structure of the A subunit of Escherichia coli DNA gyrase 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 RCSB PDB (https: / / www.rcsb.org / ) for molecular docking studies and then processed by adding hydrogen atoms and deleting all water molecules. Binding affinity (kcal / mol) was calculated by using CB-Dock2, which projects the best docking model with the least energy. Molecular visualization is a key aspect of analysis and communication of modeling studies. The details of ligand-receptor interactions were revealed using PyMol2 software (free version) and LigPlot+ (version: 2.2.8) software.

[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 for 1 hour with stirring.

[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 thereto, and the reaction was refluxed at 65°C for 18 hours. After the reaction was completed, 50 mL of water was added to the reaction system, and hydrochloric acid was added to acidify, and ethyl acetate was extracted (20 mL×3), and the reaction solution was concentrated under reduced pressure after drying with anhydrous sodium sulfate, and the residue was purified by 200-300 mesh silica gel column chromatography (ethyl acetate: petroleum ether = 1:5) to obtain compound 1.

[0045] 1.0 g of compound 1, 1.4 g of sodium methoxide and 50 mL of methanol were added to a 250 ml single-necked flask and refluxed at 65°C for 16 h. After the reaction was completed, 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] 1.0 g of compound 2 and 30 mL of THF were added to a 100 ml single-necked flask, which was placed at -78 °C, and 2.5 mL of DIBAL-H was added to react at -80 °C for 0.5 h. After the reaction was completed, 50 mL of water was added to the reaction system, and hydrochloric acid was added to acidify, and dichloromethane was extracted (10 mL × 3), and the reaction solution was concentrated under reduced pressure after drying over anhydrous sodium sulfate. 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 3.

[0047] Add 2.0g of compound 1, 7.0g of compound 3, 0.5g of NaH and 50mL of THF to a 150mL single-necked flask and react for 1h. Add 50mL of water to the reaction system, add hydrochloric acid to acidify, extract with ethyl acetate (10mL×3), dry with anhydrous sodium sulfate, and concentrate the reaction solution under reduced pressure. Add 2.0g of p-toluenesulfonic acid and 50mL of toluene to the residue and reflux for 1h. The residue is concentrated under reduced pressure and purified by 200-300 mesh silica gel column chromatography (ethyl acetate: petroleum ether = 1:5) to obtain rac-4. The compound was determined to be myrtucommulone B. rac-4 is a mixture of two stereoisomers, a pair of enantiomers, and a pair of enantiomers is (+)-4, (-)-4.

[0048] To a 150 mL single-necked flask, 2.0 g of compound 1, 7.0 g of compound 3, 0.5 g of NaH and 50 mL of THF were added, and the reaction was carried out at 100 ° C for 1 h. 50 mL of water was added to the reaction system, and hydrochloric acid was added to acidify, and ethyl acetate was extracted (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 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 (500 MHz, DMSO-d 6 ): 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 (126 MHz, DMSO-d 6 )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-methylpropylidene)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), light yellow solid, yield 78%, melting point 180.0-181.0℃. 1 HNMR (500 MHz, CDCl 3 )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, 3 H), 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 (125 MHz, CDCl 3 )δ212.0, 209.0, 198.8, 168.3, 164.9, 160.0, 153.5, 112.2, 103.9, 103.8, 10 0.7, 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 O 6 415.2115.

[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 (+)-4, light yellow solid, yield 39%, melting point 180.0-181.0°C; [α] 2 D 0 = +40.0 (c = 1.0, CHCl 3 ); (-)-4, pale yellow solid, yield 39%, melting point 180.0-181.0℃; [α] 2 D 0 = -38.9 (c = 1.0, CHCl 3 ).

[0054] rac-5a, white solid, yield 18%. 1 H NMR (500 MHz, CDCl 3 )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 ; 13 C NMR (125 MHz, CDCl 3 )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 O 8 633.6422.

[0055] The racemic compound was separated by 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); the nuclear magnetic resonance (NMR) spectrum and the HRMS (ESI) spectrum were consistent with each other and had no difference. 1H NMR (400 MHz, CDCl 3 )δ4.53 (d, J=3.2Hz, 2H), 3.18 (p, J=6.0Hz, 1H), 2.00-1.91 (m, 2H), 1.45 (s, 6H), 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, CDCl 3 )δ211.7, 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 0.2g meso-5b, 0.4g tin tetrachloride and 20mL dichloroethane were added to a 50mL single-necked flask and refluxed at 85°C for 6h. 20mL of water was added to the reaction system, and hydrochloric acid was added for acidification. The reaction solution was extracted with ethyl acetate (5mL×3) and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The residue was purified by 200-300 mesh silica gel column chromatography (ethyl acetate: petroleum ether=1:10) to obtain the target product, wherein the target product (+)-6a was obtained when the raw material was (+)-5a, the target product (+)-6a was obtained when the raw material was (+)-5a, and the target product meso-6b was obtained when the raw material was meso-5b.

[0063] rac-6a, yield 90%, melting point 208.9-210.0℃. 1 HNMR (400 MHz, CDCl 3)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 (125 MHz, CDCl 3 )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 O 7 561.2858.

[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, CHCl 3 ).

[0065] meso-6b, white solid, yield 93%, melting point 209.8-211.0°C; 1 H NMR (400 MHz, CDCl 3 )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 (125 MHz, CDCl 3)δ212.4, 198.3, 169.2, 151.5, 151.3, 111.4, 108.5, 96.2, 56.1, 47.6, 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 O 7 561.2858.

[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 the mixture was reacted at room temperature for 4 h. 20 mL of water was added to the reaction system, and hydrochloric acid was added to acidify the mixture. The mixture was extracted with ethyl acetate (5 mL × 3), and dried over anhydrous sodium sulfate. The reaction solution was concentrated under reduced pressure, and the residue was purified by 200-300 mesh silica gel column chromatography to obtain compounds 7-compound 34, respectively. The corresponding relationship between the raw materials used and the obtained compounds is 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 (500 MHz, CDCl 3 )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); 13 C NMR (125 MHz, CDCl 3 )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 O 8 647.3578.

[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 (500 MHz, CDCl 3)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), 1.48 (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 (125 MHz, CDCl 3 )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 O 8 661.3735.

[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 (400 MHz, CDCl 3 )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 (125 MHz, CDCl 3)δ212.6, 206.8, 197.9, 168.6, 155.4, 149.5, 111.3, 111.2, 107.1, 71.1, 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 O 9 677.3684.

[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; 1 H NMR (500 MHz, CDCl 3 )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, 6H), 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 (125 MHz, CDCl 3 )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 O 8 675.3891.

[0085] Compound 11: (12R, 14S)-13-(3-chlorooxy)-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 (500 MHz, CDCl 3 )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=6.0Hz, 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); 13 C NMR (125 MHz, CDCl 3 )δ212.1, 204.3, 197.9, 168.2, 154.5, 147.8, 116.9, 114.1, 110.3, 71.1, 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 O 8 Cl 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 (500 MHz, CDCl 3)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), 1.9 7-1.94(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 (125 MHz, CDCl 3 )δ211.9, 205.5, 197.7, 168.0, 155.2, 147.6, 115.4, 114.6, 111.2, 75.7, 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 O 9 689.3684.

[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 (500 MHz, CDCl 3 )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 (125 MHz, CDCl3 )δ212.0, 205.4, 197.7, 161.3, 155.8, 147.5, 115.4, 114.1, 111.2, 74.5, 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 O 8 687.3891.

[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; 1 H NMR (400 MHz, CDCl 3 )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), 1.94-1. 91 (m, 2H), 1.51 (s, 6H), 1.40 (s, 6H), 1.37 (s, 6H), 1.34 (s, 6H), 1.25 (s, 6H), 0.82 (d, J = 7.1Hz, 6H), 0.74 (d, J = 6.9Hz, 6H); 13 C NMR (125 MHz, CDCl 3 )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 (400 MHz, CDCl 3 )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); 13 C NMR (125 MHz, CDCl 3 )δ212.2, 204.4, 197.8, 168.2, 154.7, 147.8, 117.0, 113.9, 110.4, 101.9, 70.8, 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 (400 MHz, CDCl 3)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 (125 MHz, CDCl 3 )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, 5 5.9, 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 10 Na 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 (500 MHz, CDCl 3 )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), 1.42 (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 (125 MHz, CDCl3 )δ211.7, 204.2, 197.5, 174.4, 167.6, 153.2, 146.0, 114.1, 110.4, 109.2, 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 O 9 703.3841.

[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; 1 H NMR (500 MHz, CDCl 3 )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), 1.53(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 (125 MHz, CDCl 3 )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 O 8 703.4204.

[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 (500 MHz, CDCl 3 )δ4.26 (d, J=3.9Hz, 2H), 4.00-3.93 (m, 1H), 3.80 (t, J=6.1Hz, 2H), 3.44-3.3 9(m, 1H), 3.17(p, J=7.0Hz, 1H), 1.96-1.92(m, 2H), 1.89-1.85(m, 3H), 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); 13 C NMR (125 MHz, CDCl 3 )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 O 9 733.4310.

[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 (500 MHz, CDCl 3)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 (125 MHz, CDCl 3 )δ211.8, 205.3, 197.5, 167.8, 155.1, 147.5, 135.9, 128.7, 128.62, 128.27, 115.5, 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 O 8 723.3891.

[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 (500 MHz, CDCl 3 )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 (125 MHz, CDCl 3)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 O 8 F 5 813.3420.

[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; 1 H NMR (500 MHz, CDCl 3 )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, CDCl 3)δ211.7, 205.2, 197.7, 168.1, 154.6, 148.1, 147.7, 143.2, 128.7, 124.2, 115.5, 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 (500 MHz, CDCl 3 )δ7.34-7.30(m, 5H), 4.44(d, J=3.4Hz, 2H), 4.31-4.27(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); 13 CNMR (125MHz, CDCl 3 )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, 7 4.5, 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 O 8 751.4204.

[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 (400 MHz, CDCl 3 )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 (125 MHz, CDCl 3 )δ212.0, 205.4, 197.7, 168.0, 155.8, 147.5, 115.5, 114.2, 111.1, 73.2, 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 O 8 N704.4157.

[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 (500 MHz, CDCl 3)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=4.5 Hz, 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 (125 MHz, CDCl 3 )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 O 8 N732.4470.

[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; 1 H NMR (400 MHz, CDCl 3)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 (125 MHz, CDCl 3 )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 O 8 N 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 (500 MHz, CDCl 3 )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);13 C NMR (125 MHz, CDCl 3 )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 O 8 N 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 (400 MHz, CDCl 3 )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 (125 MHz, CDCl 3 )δ212.2, 204.4, 197.8, 168.2, 154.9, 147.7, 116.9, 113.8, 110.2, 71.1, 59.0, 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 O8 N 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 (400 MHz, CDCl 3 )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, CDCl 3 )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 O 8 N 758.4626.

[0104] Compound 30: (12R, 14S)-13-(2-(azepine-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; 1 H NMR (400 MHz, CDCl 3)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 (125 MHz, CDCl 3 )δ212.0, 205.3, 197.6, 167.9, 155.8, 147.5, 115.2, 114.1, 111.2, 70.3, 57.7, 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 O 8 N 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 (400 MHz, CDCl 3 )δ4.41(d, J=3.4Hz, 2H), 4.10-4.09(m, 2H), 3.58-3.54(m, 3H), 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), 1.5 3(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 (125 MHz, CDCl 3 )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 O 8 N802.5252.

[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 (500 MHz, CDCl 3 )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, CDCl 3 )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 for C 45 H 62 O8 N 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 (400 MHz, CDCl 3 )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), 1.28(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 (125 MHz, CDCl 3 )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 O 8 N 2 773.4735.

[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 (500 MHz, CDCl 3 )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 (125 MHz, CDCl 3 )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] The inhibitory activity was determined on 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 the unbound oligonucleotides, the enzyme assay was performed in a reaction volume of 30 μL of buffer; for DNA gyrase activity assay, the buffer was: 35 mM Tris-HCl, pH 7.5, 4 mM MgCl2, 24 mM KCl, 2 mM DTT, 1.8 mM spermidine, 1 mM ATP, 6.5% glycerol, 0.1 mg / mL albumin; for Topo IV activity assay, the buffer was: 40 mM hEPES-KOH, pH 7.6, 100 mM potassium glutamate, 10 mM magnesium acetate, 10 mM DTT, 1 mM ATP, 0.05 mg / mL albumin. The buffer contained 1 U of DNA gyrase (from E. coli or Staphylococcus aureus) or Topo IV (from E. coli or Staphylococcus aureus), 0.5 μg of relaxed pNO1 plasmid (for DNA gyrase assay) or supercoiled pNO1 plasmid (for Topo IV assay). 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 plasmids were 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 tested by streptavidin-coated 96-well microtiter plates with ciprofloxacin as a positive control. The compounds were tested at 6 concentrations of 10 μM, 2 μM, 0.4 μM, 0.08 μM, 0.016 μM, and 0.0032 μM. The half-inhibitory concentration values, defined as the concentration of the compound that inhibits 50% of the DNA gyrase or Topo IV activity, were 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 mean ± SEM of at least three independent experiments. Abbreviations: CIP, ciprofloxacin. “ / ” indicates “not applicable”.

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

[0119]

[0120]

[0121] Note: IC 50 Values ​​are expressed as mean SEM of at least three independent experiments. Abbreviations: 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, S. aureus enzyme half-inhibitory concentration = 0.017±0.0032μM, S. 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 curling assay was performed using DNA of compound 27 as substrate. CIP was used as positive control. Figure 1 A and Figure 1 As shown in 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 D, compared with CIP (25 μM), compound 27 exhibited stronger inhibitory activity. In Topo IV of Escherichia coli and Staphylococcus aureus, compound 27 still retained inhibitory activity at 0.04 μM and 1 μM, and completely inhibited the relaxation activity at 5 μM.

[0125] In order to better study 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 analysis was 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 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] In order 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, as Figure 2 As shown in Figure A, molecular docking revealed that compound 27 binds to the hydrophobic DNA binding groove of the DNA gyrase protein. The five-ring scaffold of compound 27 is aligned along the hydrophobic cliff formed by Ile174; Pi-alkyl: His45, Tyr266 residues. The 13 side chain extends into the narrow DNA binding channel of DNA gyrase, effectively blocking the binding of the DNA substrate to Topo IV. There are two hydrogen bonds between the carbon group at position 3 and the Arg91 residue (2.8A), the carbon group at position 26 and the Lys42 residue (2.9A), which may be responsible for the binding affinity of the enzyme to 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 interact 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 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. The bacterial culture solution containing the appropriate compound (150 μL) was discarded in the last well to ensure that the volume of the 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] The antibacterial activity against Gram-negative strains was evaluated with ciprofloxacin (CIP), penicillin (PEN) and vancomycin (VAN) as positive controls. The minimum inhibitory concentration (MIC) is shown in Table 3. The myrtle derivatives also showed low activity against Gram-negative strains and fungal strains, especially the derivatives with 13-aminoalkyl substitution, such as compound 26, compound 27, compound 28, etc. Antibacterial screening showed that compound 27 had strong activity against Gram-positive strains, especially against Staphylococcus aureus (ATCC25923), MRSA (ATCC43300), Streptococcus pneumoniae (ATCC6305) and hemolytic Streptococcus (ATCC19615), with MIC values ​​of 0.25, 1, 0.125, and 0.5 μg / mL, respectively, which were 8 times, 1 times, 64 times, and 16 times that of Van, respectively.

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

[0133]

[0134] Note: a:EC 50 Values ​​are expressed as 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 3 times independently.

[0138] Example 6

[0139] Time-kill kinetic analysis

[0140] As described above, the killing rate and degree of antibacterial effect of myrtle derivatives on MRSA were determined by time-kill kinetic analysis. In brief, 1×10^6CFU / mL of bacteria were incubated at different concentrations. During the culture process, 50μL aliquots were taken from each test tube every 0, 2, 8, 12, 16, 20 and 24 hours and serially diluted using 450μL sterile saline solution (1:10). After each dilution, 10μL was inoculated onto Mueller Hinton agar (MHA, Oxoid, Singapore) plates and cultured at 37°C for 24h. 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 good in vitro antibacterial activity of compound 27, we performed a time-kill kinetic test to evaluate the bactericidal performance of the strongest compound 27 against MRSA. Different concentrations (0.5×, 1×, 2×, and 4× MIC) were added to the MRSA suspension. Subsequently, the dendritic bacterial suspension was collected at different time points (2-24 h) for colony counting. Figure 3 a shows the growth and killing patterns of MRSA by compound 27 and VAN, respectively. Overall, the time-kill curves for MRSA show a significant reduction in the size of the bacterial population when exposed 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 only reached after 24 h of incubation. Only after 8 h did concentrations greater than the MIC begin to effectively reduce bacterial density, with the largest decline at 4 h at the highest concentration. However, regeneration was observed at 0.5×MIC, and a major decline in CFU was observed at concentrations of 2×MIC or higher. At 4 times the MIC (4.0 μg / mL), MRSA was killed within 20 h. At 2 times the MIC (2.0 μg / mL), MRSA was killed within 24 h. There was no significant difference in the bactericidal properties between compound 27 and VAN. Both had a significant and persistent bactericidal effect, and both had a significant concentration-dependent activity. At the same concentration, even at 1×MIC, compound 27 had a higher bactericidal rate than VAN. In addition, the bactericidal property of compound 27 was dose-dependent. Therefore, compound 27 effectively killed MRSA, shortened the treatment time of bacterial infection, 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 the control drug. Staphylococcus aureus was cultured for 4 hours, inoculated onto fresh MHA plates containing 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. Development of drug resistance is an important criterion for designing and evaluating 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 myrtenone derivatives 26, 27, and 28 with different groups of relevant 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 synergism, 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 classes of clinical antibacterial agents can enhance bactericidal efficacy, reduce or eliminate side effects, and even overcome drug resistance through different modes of action. Therefore, in this study, we used combinations of myrtenone derivatives 26, compounds 27, 28, and several antibiotics at high concentrations to combat MRSA. All the tested compounds in combination with levofloxacin showed additive effects (1 < FIC ≤ 2), 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 were not effective in inhibiting the growth of MRSA. Compound 27 showed synergistic effects in combination with ofloxacin (OFX), amikacin (AK), cefepime (FEP), and ceftazidime (CAZ). These combinations showed high activity with lower doses and an expanded antibacterial spectrum. This result indicates that these compounds and these antibiotics can bind to different targets of the pathogen, thereby enhancing antibacterial activity. The results are shown in Table 3. Finally, the combined use of traditional antibiotics and myrtenone derivatives can reduce their MIC values, thereby alleviating the impact of the multidrug resistance problem.

[0154] Table 5 Combinatorial effects of the most active myrtenone derivative 26, compound 27, 28 with 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 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 incubated at 37 °C in 5% CO 2For 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 at 6-fold doses 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 thyroxine crystals formed in each well. The absorbance of the solution at 570nm was recorded. GI50 values ​​were calculated using nonlinear regression analysis (GraphPad Prism 5). 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 preliminarily evaluated using ETP as a positive control, and the cytotoxicity of four synthetic compounds on normal human cells, hepatocytes MIHA, HL7702, lung cells HBSMC and BEAS-2B was tested by MTT method. The GI50 value, defined as the concentration of the compound that causes 50% cell growth inhibition, was calculated and summarized in Table 6. The results showed that all myrtle analogs showed moderate cytotoxicity on MIHA, HL7702, HBSMC and BEAS-2B cell lines at the micromolar level. The strongest DNA gyrase and Topo IV inhibitor compound 27 had lower cytotoxicity (MIHA: 47 ± 0.44 μM, HL7702: 33 ± 0.11 μM, HBSMC: 17 ± 0.87 μM, 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 a preferred embodiment.

[0167] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other 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 can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

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: 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 .

2. The use according to claim 1, characterized in that: The structural formula of the myrone derivative is as follows:

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 antimicrobial 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 and.

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, or a stereoisomer thereof.

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

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