Pleuromutilin-siderophore antibacterial coupling compound as well as preparation method and application thereof

Abstract

The invention discloses a pleuromutilin-siderophore antibacterial coupling compound as well as a preparation method and application thereof, and belongs to the field of medicinal chemistry. In the pleuromutilin-siderophore antibacterial coupling compound, a pleuromutilin antibiotic is used as an active component in the antibacterial coupling compound, and a siderophore or a siderophore analogue is used as a bacterial targeting carrier. The antibacterial activity of the compound on pathogenic microorganisms (Gram-positive bacteria or Gram-negative bacteria) is determined by using a trace broth dilution method, and experimental results show that the compound has good antibacterial activity on the pathogenic microorganisms (Gram-positive bacteria or Gram-negative bacteria), is wide in antibacterial spectrum, and can be developed and applied as a potential antibacterial drug.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemistry, and specifically relates to a pleuromutilin-siderophore antibacterial coupling compound and a preparation method and application thereof. Background Art

[0002] Siderophore is a small molecule iron ion chelate secreted by bacteria. After chelating with iron ions, it is recognized by specific outer membrane receptors and transported to the cytoplasm for bacterial use. Siderophore antimicrobial conjugate compounds are a new type of antibacterial preparation, which is composed of siderophores and antibiotics connected by specific chemical bonds. This compound can utilize the bacteria's specific recognition and transport mechanism of siderophores to effectively transport antibiotics into bacterial cells, thereby enhancing the antibacterial effect of antibiotics and overcoming bacterial resistance.

[0003] Antibiotics play an important role in the iron carrier antibacterial coupling compound, and will affect the antibacterial activity, targeted delivery, antibacterial spectrum and drug resistance of the iron carrier antibacterial coupling compound. At present, the antibiotics used in the iron carrier antibacterial coupling compound mainly include β-lactam antibiotics, macrolide antibiotics and quinolone antibiotics, etc., β-lactam antibiotics such as Cefiderocol, quinolone antibiotics such as norfloxacin. Although these existing antibiotics can ensure that the iron carrier antibacterial coupling compound has good stability and antibacterial activity, the antibacterial spectrum of the formed iron carrier antibacterial coupling compound is still narrow, resulting in its limited clinical application. Therefore, it is urgent to develop new iron carrier antibacterial coupling compounds that can take into account both antibacterial activity and antibacterial spectrum to meet the needs of fighting against multiple bacterial infections in clinical treatment. Summary of the invention

[0004] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a pleuromutilin-siderophore antibacterial coupling compound and its preparation method and application, so as to solve the technical problem that the existing siderophore antibacterial coupling compounds cannot take into account both antibacterial activity and antibacterial spectrum.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention discloses a pleuromutilin-siderophore antibacterial coupling compound, which is a compound of formula I, or a stereoisomer, tautomer, enantiomer, diastereomer, homologue, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof of the compound of formula I, or a mixture of any proportion of stereoisomers, tautomers, enantiomers, diastereomers, homologues, solvates, metabolites or pharmaceutically acceptable salts of the compound of formula I;

[0007]

[0008] Among them, R 1 Selected from aliphatic hydrocarbons substituted with -OH, 2 Substituted aliphatic hydrocarbons, containing -OH and -NH 2 Substituted aliphatic hydrocarbons, aromatic rings containing -OH, 2 Substituted aromatic rings and -OH and -NH 2 Any one of the substituted aromatic rings; Linker is selected from an aromatic heterocycle containing amide, or selected from an aromatic ring containing amide and halogen substitution; R 2 , R 3 , R 4 , R 5 and R 6 At least two adjacent substituents are selected from -OH or -OAc, and the substituents at the remaining positions are independently selected from -H or -Cl.

[0009] The second aspect of the present invention discloses a method for preparing the above-mentioned pleuromutilin-siderophore antibacterial coupling compound, comprising the following steps:

[0010] 1) Using pleuromutilin and p-toluenesulfonyl chloride as raw materials, the intermediate I is obtained by reaction;

[0011] Among them, intermediate I is

[0012] 2) using 3-amino-5-halogenated benzoic acid and di-tert-butyl dicarbonate as raw materials, or using 5-aminonicotinic acid and di-tert-butyl dicarbonate as raw materials, to react to obtain intermediate II;

[0013] Among them, intermediate II is X is F, Cl or Br;

[0014] 3) using 5-amino-2-mercaptocyclohexane-1-ol and the intermediate I obtained in step 1) as raw materials, or using 3-aminothiophenol and the intermediate I obtained in step 1) as raw materials, to react to obtain intermediate III;

[0015] Among them, intermediate III is

[0016] 4) using the intermediate II obtained in step 2) and the intermediate III obtained in step 3) as raw materials, reacting to obtain intermediate IV;

[0017] Among them, intermediate IV is X is F, Cl or Br;

[0018] 5) Using the intermediate IV obtained in step 4) as a raw material, removing the tert-butyloxycarbonyl group to obtain intermediate V;

[0019] Among them, intermediate V is X is F, Cl or Br;

[0020] 6) When R 2 , R 3 , R 4 , R 5 and R 6 When at least two adjacent substituents are taken from -OAc, 2,3-diacetoxybenzoic acid and the intermediate V obtained in step 5) are used as raw materials to react to obtain a pleuromutilin-siderophore antibacterial coupling compound;

[0021] When R 2 , R 3 , R 4 , R 5 and R 6 When at least two adjacent substituents are taken from -OH, 2,3-dimethoxybenzoic acid and the intermediate V obtained in step 5) are used as raw materials for reaction, and then aluminum chloride is added to continue the reaction to obtain a truncated pleuromutilin-siderophore antibacterial coupling compound.

[0022] The third aspect of the present invention discloses the use of the above-mentioned pleuromutilin-siderophore antibacterial coupling compound in the preparation of drugs for treating infectious diseases.

[0023] Preferably, the infectious disease is a disease caused by infection with pathogenic microorganisms, and the pathogenic microorganisms are Gram-positive bacteria or Gram-negative bacteria.

[0024] Preferably, the preparation is used alone or in combination with other antibacterial drugs, or mixed with pharmaceutically acceptable excipients and diluents to form tablets, capsules, granules, syrups, premixes or pellets for oral administration, or prepared as ointments or injections for non-oral administration.

[0025] The fourth aspect of the present invention discloses a pharmaceutical composition, which uses the above-mentioned pleuromutilin-siderophore antibacterial coupling compound as an active ingredient.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention provides a pleuromutilin-siderophore antibacterial coupling compound, which is formed by coupling pleuromutilin and siderophore. In the compound, 1) the pleuromutilin antibiotics and the siderophore have good chemical compatibility, can be stably coupled together, and the antibacterial activity of the pleuromutilin antibiotics is not affected; 2) the siderophore can guide the pleuromutilin antibiotics to effectively locate and act on bacteria, thereby enhancing the antibacterial effect; 3) the siderophore and the pleuromutilin antibiotics have synergistic antibacterial effects, have excellent antibacterial properties against both Gram-positive bacteria and Gram-negative bacteria, and have a wider antibacterial spectrum; 4) due to the precise targeting effect of the siderophore, the effect of the pleuromutilin antibiotics is more concentrated on the bacteria, thereby avoiding the ineffective diffusion of the drug to non-target cells, reducing possible side effects, and having good safety; 5) by combining the design of the siderophore, the bacterial resistance mechanism can be bypassed, thereby improving the therapeutic effect of the pleuromutilin antibiotics against resistant strains. Experiments have shown that the truncated pleuromutilin-siderophore antibacterial conjugate compound has low toxicity and good antibacterial activity against pathogenic microorganisms (Gram-positive bacteria or Gram-negative bacteria). Compared with traditional siderophore antibacterial conjugate compounds, it has better antibacterial effect and a broader antibacterial spectrum, and can be developed and applied as a potential antibacterial drug.

[0028] The invention provides a method for preparing a pleuromutilin-siderophore antibacterial coupling compound, which has low cost, strong operability and scalability, and is convenient for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The graphs show the antibacterial effects of compounds 1 (rows E, F, and G) and 2 (rows B, C, and D) of the present invention on E. coli CMCC44103 in CAMHB culture medium;

[0030] Figure 2 The graph shows the antibacterial effect of compound 1 (rows E, F, and G) and 2 (rows B, C, and D) of the present invention on E. coli CMCC 44103 in ID-CAMHB medium;

[0031] Figure 3 is the MIC heat map of the present invention;

[0032] Figure 4 The graph shows the cytotoxicity test results of compounds 1, 12, retapamulin, and cefiderocol. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used in the text are the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definition in this specification shall prevail.

[0034] The present invention provides a pleuromutilin-siderophore antibacterial coupling compound, which is a compound shown in formula I, or a stereoisomer, tautomer, enantiomer, diastereomer, homologue, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof of the compound shown in formula I, or a mixture of any proportion of stereoisomers, tautomers, enantiomers, diastereomers, homologues, solvates, metabolites or pharmaceutically acceptable salts of the compound shown in formula I, including a racemic mixture;

[0035]

[0036] Among them, R 1 Selected from aliphatic hydrocarbons substituted with -OH, 2 Substituted aliphatic hydrocarbons, containing -OH and -NH 2 Substituted aliphatic hydrocarbons, aromatic rings containing -OH, 2 Substituted aromatic rings and -OH and -NH 2 Any of the substituted aromatic rings, preferably fragment 1 or fragment 2:

[0037]

[0038] Linker is selected from any one of an aromatic heterocycle containing amide and an aromatic ring containing amide or halogen substitution, preferably any one of Linker 1-4:

[0039]

[0040] R 2 , R 3 , R 4 , R 5 and R6 6 In 6, at least two adjacent substituents are selected from -OH or -OAc, and the substituents at the remaining positions are independently selected from -H or -Cl, preferably any one of fragments 3-5:

[0041]

[0042] The pharmaceutically acceptable salt is a salt formed by the pleuromutilin-siderophore antibacterial coupling compound and hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid or aspartic acid. The pharmaceutically acceptable salts of representative derivatives are shown below:

[0043]

[0044] The present invention also provides a method for preparing the above-mentioned pleuromutilin-siderophore antibacterial coupling compound, and the specific steps are as follows:

[0045] (1) Using pleuromutilin as a raw material, the C-14 hydroxyl group of pleuromutilin is activated with p-toluenesulfonyl chloride under the catalysis of composite catalyst I to obtain intermediate I;

[0046]

[0047] The equivalent ratio of pleuromutilin to p-toluenesulfonyl chloride is 1:2; the solvent used in the synthesis of intermediate I is acetonitrile; the composite catalyst I is composed of N,N-diisopropylethylamine (DIEA) and 4-dimethylaminopyridine (DMAP), the amount of DMAP is the catalytic equivalent, and the amount of DIEA is 1.5 equivalents; the reaction is stirred at room temperature;

[0048] (2) using 3-amino-5-halogenated benzoic acid (or 5-aminonicotinic acid) and di-tert-butyl dicarbonate as raw materials, reacting under the catalysis of composite catalyst II to obtain intermediate II;

[0049]

[0050] Wherein, X in the structural formula of 3-amino-5-halogenobenzoic acid is -F, -Cl or -Br, and the equivalent ratio of 3-amino-5-halogenobenzoic acid (or 5-aminonicotinic acid) to di-tert-butyl dicarbonate is 1:1.1; the composite catalyst II used is composed of triethanolamine (TEA) and DMAP, the amount of DMAP used is the catalytic equivalent, and the amount of DIEA used is 3.0 equivalents; the solvent used in the synthesis process of intermediate II is ethyl acetate (EA), and the reaction is stirred at 0°C;

[0051] (3) using the intermediate I obtained in step (1) and 5-amino-2-mercaptocyclohexane-1-ol (or 3-aminothiophenol) as raw materials, reacting under the catalysis of the composite catalyst III to obtain the intermediate III;

[0052]

[0053] The equivalent ratio of intermediate I to 5-amino-2-ylcyclohexane-1-ol (or 3-aminothiophenol) is 1:1.5; the solvent used in the synthesis of intermediate III is N,N-dimethylformamide (DMF), and the composite catalyst III is composed of K 2 CO 3 and KI, the amount of KI is catalytic equivalent, K 2 CO 3 The dosage is 2.0 equivalents; the reaction is stirred at 60°C;

[0054] (4) using the intermediate II obtained in step (2) and the intermediate III obtained in step (3) as raw materials, reacting to obtain intermediate IV, wherein X = -F, -Cl or -Br;

[0055]

[0056] The equivalent ratio of intermediate III to intermediate II is 1:1.05; the solvent used in the synthesis of intermediate IV is a mixed solvent of tetrahydrofuran (THF) and dichloromethane (DCM); the condensation agent used is (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) (EDCI), the amount of EDCI used is 1.05 equivalents; the catalyst used is DMAP, and the amount of DMAP used is catalytic equivalents; the reaction is stirred at room temperature;

[0057] (5) Using the intermediate IV obtained in step (4) as a raw material, trifluoroacetic acid (TFA) is added to obtain intermediate V;

[0058]

[0059] The equivalent ratio of intermediate IV to TFA is 1:2; the reaction is stirred at 0°C;

[0060] (6) using the intermediate V obtained in step (5) and 2,3-dimethoxybenzoic acid (or 2-chloro-3,4-dimethoxybenzoic acid) as raw materials, reacting to obtain intermediate VI;

[0061]

[0062] The intermediate V prepared in step (5) and 2,3-diacetoxybenzoic acid are used as raw materials to react to obtain compound 3, compound 6, compound 9, compound 12, compound 15, compound 18, compound 21 (X = -Br) and compound 24:

[0063]

[0064] The equivalent ratio of intermediate V to 2,3-dimethoxybenzoic acid (or 2-chloro-3,4-dimethoxybenzoic acid or 2,3-diacetoxybenzoic acid) is 1:1.05; the solvent used in the synthesis of intermediate VI and compounds 3, 6, 9, 12, 15, 18, 21 and 24 is DMF, the condensation agent used is EDCI, the amount of EDCI used is 1.05 equivalents, the catalyst used is DMAP, and the amount of DMAP used is catalytic equivalents; the reaction is stirred at room temperature;

[0065] (7) using the intermediate VI obtained in step (6) as a raw material, reacting with aluminum chloride to obtain the remaining compounds, namely compounds 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22 and 23;

[0066]

[0067] Wherein, the equivalent ratio of intermediate VI to aluminum chloride is 1:3; and the reaction is stirred under reflux conditions.

[0068] The invention also discloses the use of the pleuromutilin-siderophore antibacterial coupling compound in preparing medicines for treating infectious diseases caused by pathogenic microorganisms.

[0069] The infectious disease is an infectious disease caused by Gram-positive bacteria or Gram-negative bacteria in humans or animals.

[0070] The Gram-positive bacteria are: Methicillin-resistant S.aureus ATCC 33591, Methicillin-resistant S.aureus ATCC 43300, S.aureus ATCC 29213 or Methicillin-resistant S.epidermidis ATCC 51625; the Gram-negative bacteria are A.baumannii ATCC 19606, S.enterica ATCC 14028, E.coli ATCC 25922 or E.coli CMCC 44103.

[0071] The pleuromutilin-siderophore antibacterial coupling compound is used alone or mixed with a pharmaceutically acceptable excipient or diluent to form tablets, capsules, granules, syrups, premixes or pellets for oral administration, or is made into ointments or injections for non-oral administration.

[0072] The invention also discloses a pharmaceutical composition, which contains an effective amount of the pleuromutilin-siderophore antibacterial coupling compound, and the remainder is pharmaceutical excipients or other compatible drugs.

[0073] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0074] The following examples use conventional instruments and equipment in the art. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various raw materials used in the following examples, unless otherwise specified, are conventional commercial products, and their specifications are conventional specifications in the art.

[0075] 1. Specific Examples of Synthesizing Pleuromutilin-Siderophore Antibacterial Coupling Compounds 1 to 24

[0076] The structural formula of the representative compound of the present invention is shown below:

[0077]

[0078] Examples of the synthesis of the above compounds are given below.

[0079] Example 1

[0080] Compound 1: Preparation of (3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propylcyclopenta[8]annul-5-yl 2-((4-(3-(2,3-dihydroxybenzamide)-5-fluorobenzamide)-2-hydroxycyclohexyl)thio)acetate

[0081] (1) Preparation of intermediate Ⅰ

[0082]

[0083] Pleuromutilin (100.00 g, 264.19 mmol), p-toluenesulfonyl chloride (100.73 g, 528.38 mmol) and DMAP (3.23 g, 26.42 mmol) were dissolved in 500 mL of acetonitrile and stirred until the system was dissolved. DIEA (59.82 mL, 343.45 mmol) was added to the system and stirred at room temperature for 8 h. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, and the resulting solid was concentrated with saturated NaHCO 3 The mixture was washed with aqueous solution, filtered, and the filter cake was collected and dried under air at 55°C for 6 h to obtain 135.07 g of intermediate I with a yield of 95.98%.

[0084] 1H NMR (600MHz, DMSO-d6) δ7.77–7.73(m,2H),7.36(dq,J=8.4,0.9Hz,2H),5.72(tdq,J=11.4,1.9,0.9Hz,1H),5.19(dd,J=10.2,2.4Hz,1H),5.06(tq, J=6.0,1.5Hz,1H),4.99(dd,J=11.4,2.4Hz,1H),4.78–4.60(m,2H),3.51( dddt,J=8.1,6.2,3.1,1.5Hz,1H),2.93(d,J=7.2Hz,1H),2.42(d,J=0.9Hz ,3H),2.38(d,J=3.7Hz,1H),2.33(t,J=4.7Hz,2H),2.15(dd,J=12.6,5.7 Hz,1H),1.77(dt,J=12.5,4.7Hz,1H),1.73–1.66(m,2H),1.64–1.50(m,3H ),1.48(dt,J=12.5,4.7Hz,1H),1.45–1.29(m,2H),1.12(t,J=1.3Hz,3H), 1.05(d,J=1.7Hz,3H), 0.94(dd,J=6.6,1.5Hz,3H), 0.90(d,J=7.8Hz,3H).

[0085] 13 C NMR(151MHz,DMSO-d6)δ218.19,169.95,146.87,143.02,133.35,130.32,127.22,112.19,80.62,76.94,66.14,5 8.99,46.96,46.13,44.04,43.84,37.92,37.79,34.34,33.65,33.48,26.85,21.72,20.49,17.66,17.53,12.01.

[0086] (2) Preparation of intermediate II-1

[0087]

[0088] Dissolve 3-amino-5-bromobenzoic acid (20.00 g, 128.92 mmol), triethylamine (23.30 mL, 167.60 mmol) and DMAP (17.32 g, 141.81 mmol) in 60 mL of ethyl acetate and stir until the system is clear. Add di-tert-butyl dicarbonate (30.95 g, 141.81 mmol) to the system in batches, stir and react at 0°C for 2 h, and monitor the reaction progress by TLC. After the reaction is completed, the reaction solution is concentrated under reduced pressure to remove the solvent, and the resulting solid concentrated state is slurried with methyl tert-butyl ether at 15°C. Filter, collect the filter cake, and dry it at 40°C with air for 8 h to obtain 25.77 g of intermediate II-1, with a yield of 78.31%.

[0089] 1 H NMR (600MHz, DMSO-d6) δ11.26(s,1H),8.36(s,1H),8.14(t,J=2.2Hz,1H),7.61(dt,J=8.1,2.2Hz,1H),7.53(dt,J=8.1,2.2Hz,1H),1.45(s,9H).

[0090] 13 C NMR(151MHz,DMSO-d6)δ168.71,162.24(d,J=305.0Hz),155.59,140.46(d,J=9.1Hz),131.21( d, J=10.6Hz), 116.65 (d, J=4.5Hz), 111.50 (d, J=24.2Hz), 109.13 (d, J=24.2Hz), 80.72, 28.42.

[0091] (3) Preparation of intermediate III-1

[0092]

[0093] Dissolve intermediate Ⅰ (50.00g, 93.86mmol) and 5-amino-2-ylcyclohexane-1-ol (20.73g, 140.79mmol) in 150mL DMF and stir until the system is clear. Add potassium carbonate (25.94g, 187.72mmol) and potassium iodide (23.37g, 140.79mmol) to the system, stir and react at 60℃ for 6h, and monitor the reaction progress by TLC. After the reaction is completed, add saturated ammonium chloride aqueous solution and ethyl acetate to the system, extract, collect and concentrate the organic phase, separate by rapid column chromatography (eluent is petroleum ether: ethyl acetate = 1:1), and obtain 40.59g intermediate Ⅲ-1 with a yield of 85.17%.

[0094] 1H NMR (600MHz, DMSO-d6) δ5.72 (tdq, J=10.8, 1.9, 0.9Hz, 1H), 5.16 (dd, J=9.6, 2.4Hz,1H),5.04(tq,J=5.7,1.5Hz,1H),4.97(dd,J=11.0,2.4Hz,1H),3.92– 3.83(m,1H),3.48(dtq,J=7.3,3.5,1.6Hz,1H),3.42(s,2H),3.34(dd,J=7.6 ,4.2Hz,1H),3.25(dd,J=7.1,4.0Hz,1H),3.16–3.10(m,2H),3.03(d,J=4.7Hz ,1H),2.86(d,J=7.2Hz,1H),2.37(d,J=3.1Hz,1H),2.30(t,J=4.3Hz,2H),2. 15(dd,J=12.6,5.7Hz,1H),1.86–1.75(m,3H),1.74–1.68(m,3H),1.65–1.50 (m,6H),1.49(dt,J=12.5,4.7Hz,1H),1.41–1.26(m,2H),1.10(t,J=1.3Hz,3 H), 1.06 (d, J = 1.8Hz, 3H), 0.96 (dd, J = 6.0, 1.5Hz, 3H), 0.92 (d, J = 6.3Hz, 3H).

[0095] 13 C NMR(151MHz,DMSO-d6)δ218.14,171.58,146.84,112.22,80.46,76.27,72.68,58.15,48.47,46.89,46.17,46.02, 44.61,43.93,37.83,37.77,37.29,34.95,33.34,33.25,32.93,32.19,26.35,25.42,20.48,17.58,17.39,12.10.

[0096] (4) Preparation of Intermediate IV-1

[0097]

[0098] Dissolve intermediate II-1 (5.28 g, 20.69 mmol) in a mixed solution of 30 mL of THF and DCM, and stir until the system is clear. Add EDCI (4.53 g, 23.64 mmol) and DMAP (2.89 g, 23.64 mmol), stir and react for 2 h, and monitor the carboxyl activation process by TLC. Then add intermediate III-1 (10.00 g, 19.70 mmol), continue to stir and react for 4 h, and monitor the reaction process by TLC. After the reaction is completed, add saturated sodium chloride aqueous solution to the system, extract, collect and concentrate the organic phase, separate by rapid column chromatography (eluent is dichloromethane: methanol = 20: 1), and obtain 9.20 g of intermediate IV-1 with a yield of 62.69%.

[0099] 1 H NMR(600MHz,DMSO-d6)δ8.23(s,1H),8.04(t,J=2.2Hz,1H),7.68(dt,J=8.5,2.2Hz,1H),7 .59(dt,J=8.1,2.1Hz,1H),7.44(d,J=7.5Hz,1H),5.82–5.67(m,1H),5.15(dd,J=16.3,2. 4Hz,1H),5.04(tq,J=5.3,1.5Hz,1H),4.96–4.86(m,1H),3.93(dddd,J=9.3,6.0,4.6,2.9 Hz,2H),3.49(tdd,J=6.6,3.2,1.6Hz,1H),3.42(s,2H),3.13(d,J=4.1Hz,1H),2.94(dt,J= 5.8,4.8Hz,1H),2.89(d,J=7.2Hz,1H),2.37(d,J=3.6Hz,1H),2.30(t,J=4.1Hz,2H),2.14 (dd,J=11.2,5.3Hz,1H),2.03(dt,J=12.6,5.8Hz,1H),1.96–1.88(m,1H),1.85–1.65(m,7H ),1.61–1.53(m,3H),1.52(dt,J=12.5,4.7Hz,1H),1.45(s,9H),1.43–1.34(m,2H),1.10( t,J=1.1Hz,3H),1.07(d,J=1.4Hz,3H),0.93(dd,J=5.8,1.5Hz,3H),0.88(d,J=6.6Hz,3H).

[0100] 13C NMR(151MHz,DMSO-d6)δ218.31,171.40,167.47,163.09(d,J=303.5Hz),155.95,146.45,139.84( d,J=10.6Hz),135.12(d,J=9.1Hz),115.16(d,J=3.0Hz),112.71,111.12(d,J=24.2Hz),110.43(d, J=24.2Hz),80.63,80.10,76.28,72.50,58.83,46.93,46.67,46.31,46.07,44.65,43.61,37.28,3 7.14,36.56,34.12,33.89,33.14,32.13,28.73,28.42,26.53,26.19,20.37,17.75,17.20,12.34.

[0101] (5) Preparation of intermediate V-1

[0102]

[0103] Dissolve the intermediate IV-1 (9.00 g, 12.08 mmol) in 10 mL of DCM, stir until the system is clear, and cool the system to 0°C. Slowly add TFA (1.85 mL, 24.16 mmol) dropwise, stir and react for 4 hours, and monitor the reaction progress by TLC. After the reaction is completed, the reaction solution is concentrated under reduced pressure to remove the solvent, and the resulting solid concentrated state is slurried with ethyl acetate at 0°C. Filter, collect the filter cake, and air dry at 45°C for 4 hours to obtain 7.46 g of intermediate V-1, with a yield of 95.77%.

[0104] 1H NMR(600MHz,DMSO-d6)δ7.82(d,J=7.2Hz,1H),7.37(dt,J=8.1,2.1Hz,1H),7.22(t,J=2.6Hz,1H),6.79(dt,J=7.6,2.2Hz,1H),5.67(tdq,J=11.2,1.9,0.9Hz,1H),5.14(dd,J=9.6,2.4Hz,1H),5.02(ddt,J=5.3,4.1,1.5Hz,1H),4.94(dd,J=10.8,2.4Hz,1H),4.76(d,J=5.1Hz,1H),4.63(d,J=4.9Hz,1H),4.04–3.92(m,2H),3.48(dddt,J=8.6,6.6,3.1,1.6Hz,1H),3.45(s,2H),3.16(d,J=4.4Hz,1H),2.98(dt,J=6.0,4.8Hz,1H),2.90(d,J=6.6Hz,1H),2.36(d,J=3.1Hz,1H),2.29(t,J=4.6Hz,2H),2.16(dd,J=12.6,5.7Hz,1H),2.04(dt,J=11.8,6.2Hz,1H),1.98–1.87(m,1H),1.86–1.84(m,1H),1.83–1.68(m,6H),1.65–1.53(m,3H),1.48(dt,J=13.2,4.7Hz,1H),1.43–1.30(m,2H),1.11(t,J=1.4Hz,3H),1.05(d,J=1.2Hz,3H),0.95(dd,J=5.8,1.5Hz,3H),0.91(d,J=6.3Hz,3H).

[0105] 13C NMR (151MHz, DMSO-d6) δ218.98, 171.18, 166.07, 163.35 (d, J = 305.0Hz), 148.36 (d, J = 10.6Hz ),146.01,135.56(d,J=10.6Hz),112.70,110.90(d,J=3.0Hz),107.46(d,J=24.2Hz),107.05 (d,J=24.2Hz),80.87,76.56,72.67,58.36,46.70,46.52,46.43,46.01,44.33,43.24,37.35 ,37.26,36.19,34.46,33.29,33.12,32.25,28.73,26.71,26.47,20.56,17.23,17.05,12.92.

[0106] (6) Preparation of intermediate VI-1

[0107]

[0108] Dissolve 2,3-dimethoxybenzoic acid (0.45 g, 2.45 mmol) in 5 mL of DMF and stir until the system is clear. Add EDCI (0.54 g, 2.80 mmol) and DMAP (0.34 g, 2.80 mmol), stir and react for 2 h, and monitor the carboxyl activation process by TLC. Then add intermediate V-1 (1.50 g, 2.33 mmol), continue to stir and react for 4 h, and monitor the reaction process by TLC. After the reaction is completed, add saturated sodium chloride aqueous solution to the system, extract, collect and concentrate the organic phase, and separate by rapid column chromatography (eluent is dichloromethane: methanol = 18:1) to obtain 1.00 g of intermediate VI-1 with a yield of 53.05%.

[0109] 1H NMR(600MHz,DMSO-d6)δ9.91(s,1H),8.13(t,J=2.2Hz,1H),7.69(dt,J=7.9,1.9Hz,1H),7.57–7.34(m,3H),7.21–6.95(m,2H),5.64(tdq,J=11.2,1.9,0.9Hz,1H),5.17(dd,J=10.2,2.4Hz,1H),5.05(tq,J=5.4,1.5Hz,1H),4.93(dd,J=10.5,2.4Hz,1H),4.07–3.91(m,2H),3.84(d,J=5.5Hz,6H),3.43(tdd,J=7.2,3.1,1.6Hz,1H),3.38(s,2H),3.18(d,J=4.4Hz,1H),2.94(dt,J=6.6,4.2Hz,1H),2.85(d,J=6.6Hz,1H),2.38(d,J=3.3Hz,1H),2.33(t,J=4.7Hz,2H),2.14(dd,J=11.8,5.1Hz,1H),2.01(dt,J=12.6,6.6Hz,1H),1.96–1.80(m,2H),1.80–1.68(m,6H),1.65–1.55(m,3H),1.51(dt,J=12.0,4.3Hz,1H),1.42–1.31(m,2H),1.12(t,J=1.3Hz,3H),1.06(d,J=1.4Hz,3H),0.96(dd,J=6.0,1.5Hz,3H),0.92(d,J=8.2Hz,3H).

[0110] 13C NMR (151MHz, DMSO-d6) δ218.50, 171.48, 167.86, 165.07, 163.10 (d, J = 303.5Hz), 150.93, 148.98, 146.84, 139. 44(d,J=9.1Hz),135.16(d,J=10.6Hz),122.93,122.85,122.08,116.05(d,J=4.5Hz),115.49,112.38,111.24( d,J=24.2Hz),110.35(d,J=24.2Hz),80.01,76.27,72.78,61.96,58.70,55.91,46.93,46.77,46.42,46.13,44 .69,43.26,37.62,37.11,36.54,34.57,33.43,33.06,32.79,28.00,26.97,26.24,20.02,17.59,17.25,12.27.

[0111] (7) Preparation of Compound 1

[0112]

[0113] Dissolve intermediate VI-1 (0.80 g, 0.99 mmol) in 5 mL of DCM and stir until the system is clear. Add anhydrous aluminum chloride (0.40 g, 2.97 mmol) to the system, heat to reflux, react for 3 h, and monitor the reaction progress by TLC. After the reaction is completed, the reaction solution is concentrated under reduced pressure to remove the solvent, and the resulting solid concentrated state is hydrolyzed with dilute hydrochloric acid aqueous solution. At this time, the crude compound 1 is precipitated. Recrystallize with 80% ethanol aqueous solution to obtain 0.67 g of compound 1 with a yield of 86.66%.

[0114] 1H NMR(600MHz,DMSO-d6)δ10.43(s,1H),10.12(s,1H),8.38(s,1H),8.01(t,J=2.0Hz,1H),7.65(dt,J=7.8,2.1Hz,1H),7.53(dt,J=8.3,2.2Hz,1H),7.47(d,J=7.6Hz,1H),7.29(dd,J=6.2,3.1Hz,1H),6.85–6.76(m,2H),5.44(tdq,J=16.6,1.8,1.0Hz,1H),5.07(dd,J=17.3,2.4Hz,1H),5.00(tq,J=5.7,1.5Hz,1H),4.97(dd,J=15.8,2.4Hz,1H),4.07–3.93(m,2H),3.53(dddd,J=8.2,6.6,3.1,1.5Hz,1H),3.42(s,2H),3.13(d,J=4.4Hz,1H),2.99(dt,J=5.2,4.0Hz,1H),2.81(d,J=6.6Hz,1H),2.34(d,J=3.4Hz,1H),2.29(t,J=4.6Hz,2H),2.11(dd,J=12.5,5.1Hz,1H),2.01(dt,J=12.3,6.2Hz,1H),1.98–1.87(m,1H),1.85–1.81(m,1H),1.80–1.77(m,1H),1.76–1.75(m,1H),1.74(d,J=2.2Hz,1H),1.73–1.63(m,3H),1.61–1.50(m,3H),1.47(dt,J=11.8,4.7Hz,1H),1.46–1.36(m,1H),1.35–1.33(m,1H),1.11(t,J=1.3Hz,3H),1.10(d,J=1.5Hz,3H),0.95(dd,J=6.2,1.5Hz,3H),0.91(d,J=7.1Hz,3H).

[0115] 13C NMR(151MHz,DMSO-d6)δ218.10,171.29,167.36,166.06,163.48(d,J=303.5Hz),149.61,146.17,145.90,1 39.04(d,J=9.1Hz),135.34(d,J=10.6Hz),120.61,120.13,118.65,116.96,116.86(d,J=4.5Hz),112.75,1 11.32(d,J=24.2Hz),110.12(d,J=24.2Hz),80.49,76.44,72.16,58.68,46.93,46.75,46.61,46.42,44.40 ,43.68,37.70,37.02,36.37,34.87,33.76,33.57,32.00,28.07,26.23,26.11,20.95,17.88,17.12,12.93.

[0116] Example 2

[0117] Compound 2: Preparation of (3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propylcyclopenta[8]annul-5-yl 2-((4-(3-(2-chloro-3,4-dihydroxybenzamido)-5-fluorobenzamido)-2-hydroxycyclohexyl)thio)acetate

[0118]

[0119] The preparation method is similar to that of Example 1, except that the corresponding raw materials are replaced. The yield of the obtained compound 2 is 61.25%.

[0120] 1H NMR(600MHz,DMSO-d6)δ10.35(s,1H),8.49(s,1H),8.06(t,J=2.2Hz,1H),7.70(dt,J=8.5,2.2Hz,1H),7.57(t,J=2.5Hz,1H),7.50(d,J=2.0Hz,2H),7.46(d,J=7.8Hz,1H),6.76(d,J=8.6Hz,1H),5.68(tdq,J=10.1,1.9,0.9Hz,1H),5.10(dd,J=10.8,2.4Hz,1H),5.01(tq,J=5.4,1.5Hz,1H),4.96(dd,J=11.6,2.4Hz,1H),4.00–3.91(m,2H),3.46(tdd,J=7.2,3.1,1.6Hz,1H),3.44(s,2H),3.19(d,J=4.4Hz,1H),2.96(dt,J=6.0,4.8Hz,1H),2.92(d,J=6.6Hz,1H),2.36(d,J=4.0Hz,1H),2.31(t,J=4.7Hz,2H),2.13(dd,J=12.5,5.3Hz,1H),2.00(dt,J=12.6,6.2Hz,1H),1.95–1.88(m,1H),1.87–1.80(m,1H),1.80–1.76(m,1H),1.76–1.68(m,5H),1.61–1.52(m,3H),1.49(dt,J=12.5,4.2Hz,1H),1.44–1.33(m,2H),1.08(t,J=1.2Hz,3H),1.07(d,J=1.6Hz,3H),0.97(dd,J=5.8,1.2Hz,3H),0.94(d,J=7.5Hz,3H).

[0121] 13C NMR(151MHz,DMSO-d6)δ218.73,171.06,167.36,165.31,163.15(d,J=303.5Hz),148.52,146.28,144.05,1 39.71(d,J=9.1Hz),135.56(d,J=10.6Hz),123.96,121.99,121.42,116.05(d,J=4.5Hz),115.04,112.82,1 11.32(d,J=24.2Hz),110.85(d,J=24.2Hz),80.44,76.85,72.55,58.68,46.84,46.62,46.51,46.01,44.93 ,43.30,37.76,37.19,36.51,34.05,33.69,33.37,32.86,28.80,26.69,26.43,20.98,17.55,17.46,12.58.

[0122] Example 3

[0123] Compound 3: Preparation of 3-((3-fluoro-5-((3-hydroxy-4-((2-(((3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propylcyclopenta[8]annul-5-yl)oxy)-2-oxoethyl)thio)cyclohexyl)carbamoyl)phenyl)carbamoyl)-1,2-benzenediacetate

[0124] (1) Preparation of Intermediate Ⅰ, Intermediate Ⅱ-1, Intermediate Ⅲ-1, Intermediate Ⅳ-1 and Intermediate Ⅴ-1

[0125]

[0126] The preparation methods of intermediates I, II-1, III-1, IV-1 and V-1 refer to Example 1.

[0127] (2) Preparation of Compound 3

[0128]

[0129] Dissolve 2,3-diacetoxybenzoic acid (0.58 g, 2.45 mmol) in 5 mL of DMF and stir until the system is clear. Add EDCI (0.54 g, 2.80 mmol) and DMAP (0.34 g, 2.80 mmol), stir and react for 2 h, and monitor the carboxyl activation process by TLC. Then add intermediate V-1 (1.50 g, 2.33 mmol), continue to stir and react for 4 h, and monitor the reaction process by TLC. After the reaction is completed, add saturated sodium chloride aqueous solution to the system, extract, collect and concentrate the organic phase, separate by rapid column chromatography (eluent is dichloromethane: methanol = 18:1), and obtain 1.40 g of compound 3 with a yield of 69.46%.

[0130] 1 H NMR (600MHz, DMSO-d6) δ9.88 (s, 1H), 8.05 (t, J = 3.2Hz, 1H), 7.68 (dt, J = 8.0, 2.1Hz, 1H), 7.62 (dd, J = 8. 6,1.3Hz,1H),7.51(dt,J=8.1,2.2Hz,1H),7.47(d,J=7.2Hz,1H),7.37(dd,J=8.8,1.3Hz,1H),7.23(t,J =8.4Hz,1H),5.89(tdt,J=10.6,1.9,1.0Hz,1H),5.21(dd,J=9.8,2.0Hz,1H),5.13(tq,J=5.9,1.5Hz,1 H),4.88(dd,J=11.8,2.4Hz,1H),4.03–3.91(m,2H),3.52(tdd,J=6.6,3.1,1.6Hz,1H),3.48(s,2H),3.2 4(d,J=4.4Hz,1H),2.99(dt,J=5.8,4.8Hz,1H),2.94(d,J=6.2Hz,1H),2.39(s,3H),2.37(s,3H),2.34( s,1H),2.32(t,J=4.8Hz,2H),2.09(dd,J=13.6,5.7Hz,1H),2.01(dt,J=12.7,6.2Hz,1H),1.97–1.88(m, 1H),1.86–1.81(m,1H),1.80–1.65(m,6H),1.63–1.50(m,3H),1.48(dt,J=12.6,4.7Hz,1H),1.46–1.31( m,2H),1.05(t,J=1.6Hz,3H),1.01(d,J=1.1Hz,3H),0.93(dd,J=7.2,1.5Hz,3H),0.87(d,J=6.3Hz,3H).

[0131] 13 C NMR(151MHz,DMSO-d6)δ219.01,171.45,168.87,168.48,167.72,164.99,164.20(d,J=303.5Hz),146.19,143.27, 142.71,139.52(d,J=9.1Hz),135.80(d,J=10.6Hz),126.73,123.79,123.17,123.01,116.07(d,J=4.5Hz),112.77, 111.69(d,J=24.2Hz),109.31(d,J=24.2Hz),80.13,76.83,72.04,58.93,46.96,46.56,46.22,46.00,44.26,43.90 ,37.49,37.20,36.98,34.50,33.64,33.45,32.79,28.98,26.95,26.35,20.52,20.43,20.07,17.34,17.01,11.91.

[0132] Example 4

[0133] Compound 4: Preparation of (3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propylcyclopenta[8]annul-5-yl 2-((4-(3-chloro-5-(2,3-dihydroxybenzamido)benzamido)-2-hydroxycyclohexyl)thio)acetate

[0134]

[0135] The preparation method is similar to that of Example 1, except that the corresponding raw materials are replaced. The yield of the obtained compound 4 is 54.08%.

[0136] 1H NMR(600MHz,DMSO-d6)δ10.67(s,1H),10.40(s,1H),8.82(s,1H),8.23(t,J=2.5Hz,1H),7.89(t,J=2.2Hz,1H),7.71(t,J=1.8Hz,1H),7.48(d,J=6.9Hz,1H),7.24(dd,J=6.8,3.1Hz,1H),6.88–6.84(m,2H),5.55(tdt,J=16.7,1.8,0.9Hz,1H),5.26(dd,J=16.6,2.4Hz,1H),5.13(tq,J=5.8,1.5Hz,1H),4.81–4.78(m,1H),3.99–3.90(m,2H),3.49(dddd,J=6.9,5.2,3.0,1.6Hz,1H),3.41(s,2H),3.12(d,J=4.6Hz,1H),3.02(dd,J=5.9,4.2Hz,1H),2.81(d,J=6.6Hz,1H),2.33(d,J=3.8Hz,1H),2.27(t,J=4.2Hz,2H),2.11(dd,J=12.6,5.3Hz,1H),1.99(dt,J=12.3,6.1Hz,1H),1.93–1.90(m,1H),1.89–1.84(m,1H),1.83–1.71(m,6H),1.59–1.50(m,3H),1.45(dt,J=13.8,5.1Hz,1H),1.43–1.26(m,2H),1.18(t,J=1.4Hz,3H),1.13(d,J=1.2Hz,3H),0.99(dd,J=6.9,1.5Hz,3H),0.96(d,J=6.0Hz,3H).

[0137] 13 C NMR(151MHz,DMSO-d6)δ218.52,171.33,167.40,167.28,149.30,146.87,145.11,139.23,135.28,133.27,124.89,122.21,120.25,120.05,118.71,117.26,116.29,112.46,80.32,76.29,72.56,58.39,46.89,46.35,46.32,46.17,44.58,43.21,37.74,37.16,36.40,34.64,33.93,33.50,32.19,28.95,26.54,26.49,20.59,17.83,17.77,12.90.

[0138] Example 5

[0139] Compound 5: Preparation of (3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propylcyclopenta[8]annul-5-yl 2-((4-(3-chloro-5-(2-chloro-3,4-dihydroxybenzamide)benzamide)-2-hydroxycyclohexyl)thio)acetate

[0140]

[0141] The preparation method is similar to that of Example 1, except that the corresponding raw materials are replaced. The yield of the obtained compound 5 is 56.67%.

[0142] 1 H NMR(600MHz,DMSO-d6)δ9.90(s,1H),8.63(s,1H),8.17(t,J=2.6Hz,1H),8.01(t,J=1.8Hz,1H),7 .81(t,J=2.0Hz,1H),7.48(d,J=7.8Hz,1H),7.31(d,J=9.6Hz,2H),6.63(d,J=9.3Hz,1H),5.77(td t,J=10.2,1.4,1.1Hz,1H),5.29(dd,J=10.8,2.4Hz,1H),5.07(tq,J=5.0,1.3Hz,1H),4.83(dd,J =11.8,2.2Hz,1H),4.13–3.97(m,2H),3.56(tdd,J=6.3,3.2,1.4Hz,1H),3.49(s,2H),3.25(d,J=4 .8Hz,1H),3.00(dt,J=5.6,4.3Hz,1H),2.96(d,J=6.8Hz,1H),2.41(d,J=3.4Hz,1H),2.36(t,J=4 .8Hz,2H),2.19(dd,J=12.2,5.7Hz,1H),2.03(dt,J=12.6,6.4Hz,1H),1.97–1.88(m,1H),1.88–1. 83(m,1H),1.82–1.65(m,6H),1.64–1.55(m,3H),1.50(dt,J=12.5,4.9Hz,1H),1.46–1.29(m,2H) ,1.16(t,J=1.4Hz,3H),1.11(d,J=1.3Hz,3H),1.00(dd,J=6.6,1.1Hz,3H),0.98(d,J=8.0Hz,3H).

[0143] 13 C NMR(151MHz,DMSO-d6)δ218.92,171.02,167.63,165.81,148.55,146.75,144.94,139 .45,135.88,133.57,124.78,123.39,122.17,121.63,121.04,117.38,115.24,112.77 ,80.74,76.50,72.27,58.36,46.90,46.79,46.67,46.04,44.68,43.85,37.89,37.59,36.05,34.74,33.92,33.70,32.75,28.32,26.82,26.40,20.31,17.83,17.74,12.19.

[0144] Example 6

[0145] Compound 6: Preparation of 3-((3-chloro-5-((3-hydroxy-4-((2-(((3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propylcyclopenta[8]annul-5-yl)oxy)-2-oxoethyl)thio)cyclohexyl)carbamoyl)phenyl)carbamoyl)-1,2-benzenediacetate

[0146]

[0147] The preparation method is similar to that of Example 3, except that the corresponding raw materials are replaced. The yield of the obtained compound 6 is 51.98%.

[0148] 1H NMR(600MHz,DMSO-d6)δ9.77(s,1H),8.21(t,J=1.8Hz,1H),7.99(t,J=2.4Hz,1H),7.83(t,J=2.3Hz,1H),7.79(dd,J=7.9,1.2Hz,1H),7.61(d,J=7.6Hz,1H),7.40(dd,J=8.6,1.2Hz,1H),7.29(t,J=9.2Hz,1H),5.79(tdq,J=12.8,3.9,1.9Hz,1H),5.33–5.17(m,1H),5.09(tq,J=5.6,1.5Hz,1H),4.98(dd,J=12.4,2.8Hz,1H),4.13–3.94(m,2H),3.53(tdd,J=6.9,3.6,1.2Hz,1H),3.46(s,2H),3.22(d,J=4.8Hz,1H),2.98(dt,J=5.2,3.2Hz,1H),2.90(d,J=6.6Hz,1H),2.43(s,3H),2.37(s,3H),2.33(s,1H),2.25(t,J=4.4Hz,2H),2.10(dd,J=12.5,5.8Hz,1H),2.03(dt,J=12.9,6.3Hz,1H),2.00–1.94(m,1H),1.93–1.87(m,1H),1.86–1.70(m,6H),1.69–1.58(m,3H),1.53(dt,J=12.5,4.7Hz,1H),1.49–1.37(m,2H),1.13(t,J=2.1Hz,3H),1.09(d,J=1.5Hz,3H),0.99(dd,J=6.8,1.2Hz,3H),0.93(d,J=7.0Hz,3H).

[0149] 13C NMR(151MHz,DMSO-d6)δ218.39,171.80,168.69,168.08,167.58,164.75,146.00,143.46,142 .56,139.11,135.96,133.15,126.45,124.04,123.97,123.62,123.01,122.67,117.77,112.0 0,80.96,76.54,72.70,58.18,46.49,46.33,46.21,46.03,44.84,43.03,37.72,37.24,36.42,34.29,33.52,33.38,32.45,28.15,26.31,26.07,20.82,20.75,20.26,17.84,17.16,12.49.

[0150] Example 7

[0151] Compound 7: Preparation of (3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propylcyclopenta[8]annul-5-yl 2-((4-(3-bromo-5-(2,3-dihydroxybenzamido)benzamido)-2-hydroxycyclohexyl)thio)acetate

[0152]

[0153] The preparation method is similar to that of Example 1, except that the corresponding raw materials are replaced. The yield of the obtained compound 7 is 58.27%.

[0154] 1H NMR(600MHz,DMSO-d6)δ10.37(s,1H),10.15(s,1H),8.85(s,1H),8.43(dt,J=14.2,2.0Hz,2H),8.10(t,J=2.0Hz,1H),7.79(d,J=7.8Hz,1H),7.48(dd,J=7.1,3.2Hz,1H),6.99–6.86(m,2H),5.74(tdt,J=16.6,1.8,0.9Hz,1H),5.32(dd,J=16.2,2.3Hz,1H),5.17(tq,J=5.9,1.5Hz,1H),5.09(dd,J=16.4,2.4Hz,1H),4.12–3.95(m,2H),3.63(tdd,J=6.6,3.1,1.6Hz,1H),3.52(s,2H),3.29(d,J=4.4Hz,1H),3.07(dt,J=5.6,4.2Hz,1H),3.00(d,J=6.6Hz,1H),2.38(d,J=3.4Hz,1H),2.29(t,J=4.6Hz,2H),2.20(dd,J=12.6,5.8Hz,1H),2.13(dt,J=12.8,6.0Hz,1H),2.03–1.95(m,1H),1.94–1.88(m,1H),1.87–1.69(m,6H),1.66–1.54(m,3H),1.50(dt,J=12.9,4.8Hz,1H),1.47–1.35(m,2H),1.12(t,J=1.3Hz,3H),1.03(d,J=1.6Hz,3H),0.95(dd,J=6.0,1.4Hz,3H),0.90(d,J=7.8Hz,3H).

[0155] 13 C NMR(151MHz,DMSO-d6)δ218.17,171.70,167.39,166.68,149.60,146.11,145.34,139.56,135.30,126.49,126.23,122.39,120.35,120.11,118.57,118.35,116.16,112.54,80.57,76.27,72.14,58.95,46.60,46.35,46.21,46.01,44.34,43.52,37.90,37.46,36.22,34.87,33.76,33.32,32.10,28.39,26.72,26.56,20.54,17.33,17.05,11.39.

[0156] Example 8

[0157] Compound 8: Preparation of (3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propylcyclopenta[8]annul-5-yl 2-((4-(3-bromo-5-(2-chloro-3,4-dihydroxybenzamide)benzamide)-2-hydroxycyclohexyl)thio)acetate

[0158]

[0159] The preparation method is similar to that of Example 1, except that the corresponding raw materials are replaced. The yield of the obtained compound 8 is 60.03%.

[0160] 1 H NMR (600MHz, DMSO-d6) δ10.33(s,1H),8.68(s,1H),8.32(dt,J=8.6,2.2Hz,2H),7.99(t,J=2. 2Hz,1H),7.61(d,J=7.9Hz,1H),7.54(d,J=9.5Hz,2H),6.89(d,J=9.1Hz,1H),5.81(tdt,J=10. 1,1.9,1.0Hz,1H),5.33(dd,J=10.8,2.8Hz,1H),5.20(tq,J=5.8,1.2Hz,1H),4.97–4.92(m,1H ),4.13–3.97(m,2H),3.61(tdd,J=6.9,3.1,1.6Hz,1H),3.56(s,2H),3.22(d,J=4.4Hz,1H),2. 91(dt,J=6.0,4.8Hz,1H),2.83(d,J=6.8Hz,1H),2.30(d,J=3.4Hz,1H),2.27(t,J=4.7Hz,2H) ,2.09(dd,J=12.8,5.7Hz,1H),2.03(dt,J=12.7,6.2Hz,1H),1.98–1.90(m,1H),1.89–1.83(m, 1H),1.81–1.70(m,6H),1.69–1.57(m,3H),1.52(dt,J=12.5,4.4Hz,1H),1.48–1.36(m,2H),1. 12(t,J=1.3Hz,3H),1.04(d,J=1.2Hz,3H),0.98(dd,J=7.5,1.8Hz,3H),0.92(d,J=6.8Hz,3H).

[0161] 13C NMR(151MHz,DMSO-d6)δ218.45,171.25,166.89,165.19,148.73,146.55,144.74,139 .75,135.09,126.87,126.26,123.43,122.15,121.36,121.29,118.34,115.28,112.44 ,80.96,76.40,72.17,58.77,46.85,46.67,46.39,46.03,44.95,43.84,37.38,37.14,36.89,34.42,33.46,33.10,32.28,28.51,26.81,26.03,20.84,17.96,17.43,12.35.

[0162] Example 9

[0163] Compound 9: Preparation of 3-((3-bromo-5-((3-hydroxy-4-((2-(((3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propylcyclopenta[8]annul-5-yl)oxy)-2-oxoethyl)thio)cyclohexyl)carbamoyl)phenyl)carbamoyl)-1,2-benzenediacetate

[0164]

[0165] The preparation method is similar to that of Example 3, except that the corresponding raw materials are replaced. The yield of the obtained compound 9 is 59.29%.

[0166] 1H NMR(600MHz,DMSO-d6)δ10.03(s,1H),8.47(t,J=2.1Hz,1H),8.22(t,J=2.2Hz,1H),7.97(t,J=2.2Hz,1H),7.73(dd,J=8.5,1.3Hz,1H),7.61(d,J=7.1Hz,1H),7.47(dd,J=8.6,1.3Hz,1H),7.32(t,J=8.7Hz,1H),5.72(tdq,J=10.2,1.6,0.9Hz,1H),5.23(dd,J=10.4,2.4Hz,1H),5.17(tq,J=5.8,1.5Hz,1H),4.82(dd,J=11.8,2.2Hz,1H),4.13–3.94(m,2H),3.56(dddd,J=6.6,5.9,3.3,1.6Hz,1H),3.53(s,2H),3.24(d,J=4.7Hz,1H),3.11(dt,J=5.6,4.3Hz,1H),2.96(d,J=6.8Hz,1H),2.39(s,3H),2.37(s,3H),2.33(s,1H),2.27(t,J=4.6Hz,2H),2.19(dd,J=12.8,5.9Hz,1H),2.07(dt,J=12.6,6.4Hz,1H),1.99–1.90(m,1H),1.89–1.83(m,1H),1.82–1.65(m,6H),1.60–1.51(m,3H),1.47(dt,J=12.8,4.5Hz,1H),1.42–1.31(m,2H),1.14(t,J=1.1Hz,3H),1.05(d,J=1.9Hz,3H),0.94(dd,J=6.9,1.2Hz,3H),0.91(d,J=8.6Hz,3H).

[0167] 13C NMR(151MHz,DMSO-d6)δ218.20,171.14,168.86,168.52,166.05,164.67,146.15,143.49,142 .28,139.14,135.50,126.88,126.63,126.35,123.93,123.54,123.20,122.44,118.98,112.8 3,80.12,76.71,72.95,58.99,46.87,46.55,46.10,46.06,44.07,43.85,37.83,37.36,36.53,34.33,33.55,33.12,32.10,28.83,26.92,26.84,20.99,20.41,20.26,17.84,17.05,12.79.

[0168] Example 10

[0169] Compound 10: Preparation of (3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propylcyclopenta[8]annul-5-yl 2-((4-(5-(2,3-dihydroxybenzamide)nicotinamide)-2-hydroxycyclohexyl)thio)acetate

[0170]

[0171] The preparation method is similar to that of Example 1, except that the corresponding raw materials are replaced. The yield of the obtained compound 10 is 60.90%.

[0172] 1H NMR(600MHz,DMSO-d6)δ10.69(s,1H),10.39(s,1H),8.97(t,J=1.8Hz,1H),8.90(t,J=1.9Hz,1H),8.83(s,1H),8.52(t,J=2.0Hz,1H),7.69(d,J=7.7Hz,1H),7.47(dd,J=6.4,3.8Hz,1H),7.00–6.83(m,2H),5.73(tdq,J=16.7,1.8,1.0Hz,1H),5.36(dd,J=16.0,2.4Hz,1H),5.19(tq,J=5.4,1.5Hz,1H),4.98(dd,J=16.8,2.4Hz,1H),4.04–3.96(m,2H),3.56(dddt,J=8.5,6.0,3.1,1.6Hz,1H),3.48(s,2H),3.29(d,J=4.8Hz,1H),3.03(dt,J=6.0,4.4Hz,1H),2.96(d,J=6.9Hz,1H),2.43(d,J=3.8Hz,1H),2.36(t,J=4.7Hz,2H),2.21(dd,J=12.6,5.6Hz,1H),2.09(dt,J=12.3,6.2Hz,1H),1.98–1.85(m,1H),1.84–1.82(m,1H),1.81–1.70(m,6H),1.67–1.58(m,3H),1.52(dt,J=12.8,4.6Hz,1H),1.49–1.37(m,2H),1.15(t,J=1.3Hz,3H),1.09(d,J=1.8Hz,3H),0.99(dd,J=5.8,1.4Hz,3H),0.92(d,J=6.9Hz,3H).

[0173] 13 C NMR(151MHz,DMSO-d6)δ218.66,171.62,167.44,166.45,149.62,146.57,145.10,144.55,144.00,136.14,131.06,121.80,121.03,120.01,118.33,116.24,112.95,80.90,76.64,72.10,58.28,46.87,46.71,46.34,46.02,44.64,43.19,37.64,37.35,36.71,34.43,33.49,33.04,32.05,28.76,26.85,26.38,20.03,17.73,17.22,12.16.

[0174] Embodiment 11

[0175] Compound 11: Preparation of (3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propylcyclopenta[8]annul-5-yl 2-((4-(5-(2-chloro-3,4-dihydroxybenzamide)nicotinamide)-2-hydroxycyclohexyl)thio)acetate

[0176]

[0177] The preparation method is similar to that of Example 1, except that the corresponding raw materials are replaced. The yield of the obtained compound 11 is 51.81%.

[0178] 1 H NMR (600MHz, DMSO-d6) δ10.00(s,1H),9.12(t,J=2.0Hz,1H),8.96(t,J=1.9Hz,1H),8.52(s,1 H),8.44(t,J=2.2Hz,1H),7.63–7.49(m,3H),6.83(d,J=9.6Hz,1H),5.72(tdt,J=11.2,1.8,1. 0Hz,1H),5.16(dd,J=10.6,2.4Hz,1H),5.09(tq,J=5.7,1.5Hz,1H),5.01(dd,J=11.7,2.4Hz,1 H),4.08–3.93(m,2H),3.48(tdd,J=6.9,3.5,1.2Hz,1H),3.40(s,2H),3.25(d,J=4.8Hz,1H),2 .99(dt,J=5.6,4.7Hz,1H),2.95(d,J=6.6Hz,1H),2.37(d,J=3.7Hz,1H),2.33(t,J=4.7Hz,2H) ,2.17(dd,J=12.8,5.6Hz,1H),2.05(dt,J=12.9,6.2Hz,1H),1.97–1.91(m,1H),1.90–1.83(m, 1H),1.82–1.70(m,6H),1.65–1.56(m,3H),1.50(dt,J=12.7,4.8Hz,1H),1.44–1.32(m,2H),1. 13(t,J=1.4Hz,3H), 1.06(d,J=1.6Hz,3H), 0.94(dd,J=6.6,1.2Hz,3H), 0.86(d,J=7.0Hz,3H).

[0179] 13C NMR(151MHz,DMSO-d6)δ218.43,171.76,166.56,165.06,148.38,146.60,144.89,1 44.75,144.47,136.48,131.09,123.19,122.95,121.52,121.39,115.44,112.03,80 .36,76.04,72.65,58.54,46.98,46.77,46.39,46.17,44.35,43.61,37.90,37.24,36.65,34.02,33.69,33.31,32.29,28.58,26.91,26.40,20.22,17.21,17.05,11.98.

[0180] Example 12

[0181] Compound 12: Preparation of 3-((5-((3-hydroxy-4-((2-(((3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propylcyclopenta[8]annulene-5-yl)oxy)-2-oxoethyl)thio)cyclohexyl)carbamoyl)pyridin-3-yl)carbamoyl)-1,2-benzenediacetate

[0182]

[0183] The preparation method is similar to that of Example 3, except that the corresponding raw materials are replaced. The yield of the obtained compound 12 is 58.40%.

[0184] 1H NMR(600MHz,DMSO-d6)δ10.16(s,1H),8.95(t,J=1.8Hz,1H),8.86(t,J=1.9Hz,1H),8.39(t,J=2.0Hz,1H),7.69(dd,J=7.8,1.6Hz,1H),7.57(d,J=7.2Hz,1H),7.35(dd,J=8.6,1.2Hz,1H),7.22(t,J=9.2Hz,1H),5.69(tdq,J=10.2,1.8,0.9Hz,1H),5.17(dd,J=10.4,2.4Hz,1H),5.06(tq,J=5.8,1.5Hz,1H),4.94(dd,J=11.6,2.4Hz,1H),4.06–3.97(m,2H),3.48(tdd,J=6.6,3.1,1.6Hz,1H),3.43(s,2H),3.17(d,J=4.8Hz,1H),2.98(dt,J=6.4,4.5Hz,1H),2.90(d,J=6.6Hz,1H),2.34(s,3H),2.30(s,3H),2.28(s,1H),2.21(t,J=4.3Hz,2H),2.11(dd,J=12.7,5.2Hz,1H),2.02(dt,J=12.2,6.4Hz,1H),1.96–1.87(m,1H),1.87–1.79(m,1H),1.78–1.67(m,6H),1.64–1.52(m,3H),1.48(dt,J=12.7,4.6Hz,1H),1.44–1.31(m,2H),1.09(t,J=1.3Hz,3H),1.02(d,J=1.6Hz,3H),0.94(dd,J=6.3,1.0Hz,3H),0.87(d,J=7.3Hz,3H).

[0185] 13C NMR(151MHz,DMSO-d6)δ218.38,171.41,168.44,168.09,166.69,164.17,146.19,144.99,1 44.07,143.51,142.67,136.40,131.46,126.78,123.93,123.18,122.58,121.75,112.02,8 0.74,76.43,72.50,58.35,46.96,46.67,46.52,46.07,44.62,43.49,37.98,37.70,36.62,34.28,33.59,33.47,32.29,28.13,26.88,26.26,20.93,20.84,20.14,17.45,17.05,12.34.

[0186] Example 13

[0187] Compound 13: Preparation of (3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propylcyclopenta[8]annul-5-yl 2-((3-(3-(2,3-dihydroxybenzamido)-5-fluorobenzamido)phenyl)thio)acetate

[0188]

[0189] The preparation method is similar to that of Example 1, except that the corresponding raw materials are replaced. The yield of the obtained compound 13 is 59.19%.

[0190] 1H NMR(600MHz,DMSO-d6)δ10.63(s,1H),10.32(s,1H),8.81(s,1H),8.33(s,1H),8.21(t,J=2.2Hz,1H),7.91(t,J=2.0Hz,1H),7.76–7.68(m,2H),7.48(dt,J=7.6,2.3Hz,1H),7.32–7.25(m,2H),7.21(ddd,J=7.2,2.0,1.5Hz,1H),6.90–6.81(m,2H),5.52(tdq,J=11.2,1.8,0.9Hz,1H),5.16(dd,J=10.6,2.4Hz,1H),5.01(tq,J=5.8,1.5Hz,1H),4.94(dd,J=11.4,2.4Hz,1H),3.76–3.64(m,2H),3.41(dddd,J=8.7,6.3,3.1,1.6Hz,1H),2.98(d,J=7.2Hz,1H),2.34(d,J=3.4Hz,1H),2.27(t,J=4.7Hz,2H),2.15(dd,J=12.8,5.2Hz,1H),1.79(dt,J=12.4,4.6Hz,1H),1.74–1.65(m,2H),1.63–1.50(m,3H),1.42(dt,J=12.2,4.2Hz,1H),1.40–1.27(m,2H),1.12(t,J=1.1Hz,3H),1.06(d,J=1.6Hz,3H),0.96(dd,J=6.8,1.2Hz,3H),0.89(d,J=7.8Hz,3H).

[0191] 13C NMR(151MHz,DMSO-d6)δ218.63,170.98,167.45,166.85,164.10(d,J=303.5Hz),149.47,146.07,145.63,13 9.88(d,J=10.6Hz),139.22,136.54,135.90(d,J=9.1Hz),129.17,125.95,120.83,120.57,120.38,119.85,1 18.36,116.81,116.00(d,J=4.5Hz),112.89,111.74(d,J=10.6Hz),110.48(d,J=10.6Hz),80.15,76.68,58.2 7,46.69,46.12,44.57,43.02,37.71,37.37,35.64,34.90,33.75,33.15,26.07,20.87,17.79,17.10,12.56.

[0192] Embodiment 14

[0193] Compound 14: Preparation of (3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propylcyclopenta[8]annul-5-yl 2-((3-(3-(2-chloro-3,4-dihydroxybenzamide)-5-fluorobenzamide)phenyl)thio)acetate

[0194]

[0195] The preparation method is similar to that of Example 1, except that the corresponding raw materials are replaced. The yield of the obtained compound 14 is 49.75%.

[0196] 1H NMR(600MHz,DMSO-d6)δ9.89(s,1H),8.49(s,1H),8.42(s,1H),8.15(t,J=2.0Hz,1H),7.80(t,J=2.2Hz,1H),7.71–7.62(m,2H),7.55(dt,J=8.2,2.1Hz,1H),7.49(d,J=9.6Hz,2H),7.28(dd,J=7.4,6.2Hz,1H),7.23(ddd,J=6.9,2.3,1.1Hz,1H),6.76(d,J=9.8Hz,1H),5.68(tdq,J=10.2,1.7,0.6Hz,1H),5.10(dd,J=10.4,2.8Hz,1H),4.99(tq,J=5.6,1.8Hz,1H),4.82(d,J=2.2Hz,1H),3.93–3.74(m,2H),3.51(dddd,J=8.0,6.6,3.1,1.6Hz,1H),2.87(d,J=6.6Hz,1H),2.40(d,J=3.3Hz,1H),2.34(t,J=4.8Hz,2H),2.10(dd,J=12.6,5.3Hz,1H),1.79(dt,J=12.4,4.0Hz,1H),1.74–1.67(m,2H),1.63–1.53(m,3H),1.50(dt,J=12.5,4.4Hz,1H),1.46–1.34(m,2H),1.10(t,J=1.3Hz,3H),1.04(d,J=1.6Hz,3H),0.95(dd,J=6.8,1.6Hz,3H),0.87(d,J=8.0Hz,3H).

[0197] 13C NMR(151MHz,DMSO-d6)δ218.19,170.79,166.10,165.05,164.11(d,J=303.5Hz),148.91,146.37,144.38,13 9.83(d,J=10.6Hz),139.09,136.34,135.83(d,J=9.1Hz),129.77,125.58,123.63,121.99,121.61,120.39,1 19.97,116.71(d,J=4.5Hz),115.94,112.36,111.72(d,J=10.6Hz),110.24(d,J=10.6Hz),80.37,76.71,58.1 6,46.59,46.15,44.83,43.12,37.93,37.45,35.61,34.72,33.80,33.39,26.01,20.63,17.65,17.04,12.25.

[0198] Embodiment 15

[0199] Compound 15: Preparation of 3-((3-fluoro-5-((3-((2-(((3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propylcyclopenta[8]annulene-5-yl)oxy)-2-oxoethyl)thio)phenyl)carbamoyl)phenyl)carbamoyl)-1,2-benzenediacetate

[0200]

[0201] The preparation method is similar to that of Example 3, except that the corresponding raw materials are replaced. The yield of the obtained compound 15 is 52.82%.

[0202] 1H NMR(600MHz,DMSO-d6)δ9.93(s,1H),8.51(s,1H),8.25(t,J=1.8Hz,1H),7.91(t,J=2.2Hz,1H),7.76(dt,J=7.2,2.1Hz,1H),7.70(ddd,J=7.9,2.3,1.3Hz,1H),7.65(dd,J=8.3,1.2Hz,1H),7.58(dt,J=7.8,2.4Hz,1H),7.42(dd,J=8.6,1.1Hz,1H),7.29–7.24(m,1H),7.22–7.19(m,2H),5.72(tdq,J=11.2,1.8,0.9Hz,1H),5.26(dd,J=10.8,2.2Hz,1H),4.93(ddt,J=5.7,3.9,1.2Hz,1H),4.84(dd,J=11.2,2.4Hz,1H),3.90–3.73(m,2H),3.53(dddd,J=8.6,6.2,3.2,1.6Hz,1H),2.98(d,J=6.6Hz,1H),2.41(s,3H),2.38(s,3H),2.36(s,1H),2.30(t,J=4.6Hz,2H),2.11(dd,J=12.8,5.3Hz,1H),1.81(dt,J=12.4,4.2Hz,1H),1.76–1.69(m,2H),1.64–1.51(m,3H),1.47(dt,J=12.2,4.1Hz,1H),1.42–1.30(m,2H),1.13(t,J=1.4Hz,3H),1.07(d,J=1.6Hz,3H),0.97(dd,J=6.0,1.8Hz,3H),0.89(d,J=7.1Hz,3H).

[0203] 13C NMR(151MHz,DMSO-d6)δ219.28,170.08,168.72,168.44,165.48,163.59,163.01(d,J=303.5Hz),146.27,143.63,14 1.16,140.62(d,J=10.6Hz),140.02,136.24,134.72(d,J=9.1Hz),129.22,125.98,125.85,124.57,122.10,121.26, 120.37, 119.15, 116.94 (d, J = 4.5Hz), 112.62, 112.00 (d, J = 10.6Hz), 110.13 (d, J = 10.6Hz), 80.26, 76.55, 58.05, 48. 18,47.65,44.73,42.98,40.37,38.75,37.84,36.62,34.29,32.97,26.79,21.19,20.33,20.04,19.76,18.16,13.18.

[0204] Example 16

[0205] Compound 16: Preparation of (3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propylcyclopenta[8]annul-5-yl 2-((3-(3-chloro-5-(2,3-dihydroxybenzamido)benzamido)phenyl)thio)acetate

[0206]

[0207] The preparation method is similar to that of Example 1, except that the corresponding raw materials are replaced. The yield of the obtained compound 16 is 58.05%.

[0208] 1H NMR(600MHz,DMSO-d6)δ10.88(s,1H),10.27(s,1H),8.80(s,1H),8.57(s,1H),8.21(t,J=2.1Hz,1H),7.90(t,J=2.2Hz,1H),7.85(t,J=1.8Hz,1H),7.71(t,J=1.9Hz,1H),7.62(ddd,J=7.8,2.3,1.3Hz,1H),7.40–7.27(m,2H),7.16(ddd,J=7.0,2.3,1.3Hz,1H),6.86–6.78(m,2H),5.72(tdq,J=10.6,1.8,0.9Hz,1H),5.15(dd,J=10.3,2.4Hz,1H),4.97(tq,J=5.8,1.5Hz,1H),4.92(dd,J=11.3,2.1Hz,1H),3.87–3.62(m,2H),3.39(dddd,J=8.6,6.6,3.1,1.6Hz,1H),2.90(d,J=6.6Hz,1H),2.37(d,J=3.4Hz,1H),2.28(t,J=4.8Hz,2H),2.14(dd,J=12.9,5.1Hz,1H),1.80(dt,J=12.8,4.4Hz,1H),1.76–1.71(m,2H),1.65–1.54(m,3H),1.48(dt,J=12.8,4.6Hz,1H),1.42–1.31(m,2H),1.12(t,J=1.2Hz,3H),1.09(d,J=1.7Hz,3H),0.99(dd,J=6.5,1.5Hz,3H),0.95(d,J=7.6Hz,3H).

[0209] 13 C NMR(151MHz,DMSO-d6)δ218.46,170.25,167.84,166.58,149.04,146.16,145.72,139.49,139.07,136.10,134.90,133.41,129.52,126.79,125.54,122.47,120.77,120.64,120.43,119.05,118.60,118.48,116.79,112.93,80.77,76.48,58.15,46.97,46.06,44.90,43.12,37.92,37.20,35.29,34.30,33.98,33.61,26.33,20.52,17.80,17.26,12.11.

[0210] Embodiment 17

[0211] Compound 17: Preparation of (3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propylcyclopenta[8]annul-5-yl 2-((3-(3-chloro-5-(2-chloro-3,4-dihydroxybenzamide)benzamide)phenyl)thio)acetate

[0212]

[0213] The preparation method is similar to that of Example 1, except that the corresponding raw materials are replaced. The yield of the obtained compound 17 is 60.03%.

[0214] 1 H NMR (600MHz, DMSO-d6) δ9.87(s,1H),8.61(s,1H),8.43(s,1H),8.17(t,J=2.2Hz,1H),7.90(t,J=1. 8Hz,1H),7.82(t,J=1.9Hz,1H),7.74(t,J=2.1Hz,1H),7.62(ddd,J=8.2,2.3,1.3Hz,1H),7.50(d,J =8.6Hz,2H),7.38(dd,J=7.2,6.0Hz,1H),7.21(ddd,J=8.3,2.2,1.3Hz,1H),6.78(d,J=9.6Hz,1H), 5.74(tdq,J=10.4,1.9,0.9Hz,1H),5.12(dd,J=11.3,2.4Hz,1H),4.95(tq,J=5.4,1.5Hz,1H),4.84 (dd,J=11.8,2.4Hz,1H),3.81–3.64(m,2H),3.53(dddd,J=8.2,6.6,3.2,1.6Hz,1H),2.86(d,J=6.9 Hz, 1H), 2.38 (d, J = 3.4Hz, 1H), 2.33 (t, J = 4.7Hz, 2H), 2.15 (dd, J = 12.2, 4.3Hz, 1H), 1.79 (dt, J = 12. 6,4.2Hz,1H),1.75–1.66(m,2H),1.61–1.49(m,3H),1.42(dt,J=12.8,4.2Hz,1H),1.38–1.29(m,2H ),1.10(t,J=1.3Hz,3H),1.06(d,J=1.5Hz,3H),0.96(dd,J=6.8,1.4Hz,3H),0.91(d,J=7.2Hz,3H).

[0215] 13 C NMR(151MHz,DMSO-d6)δ218.10,170.91,166.35,165.98,148.06,146.58,144.25,139.5 1,139.04,136.77,134.58,133.46,129.69,126.78,125.58,123.02,122.22,121.99,12 1.41,120.97,119.36,118.65,115.11,112.96,80.28,76.16,58.47,46.32,46.15,44.76,43.50,37.62,37.16,35.28,34.53,33.98,33.63,26.37,20.79,17.94,17.44,12.73.

[0216] Embodiment 18

[0217] Compound 18: Preparation of 3-((3-chloro-5-((3-((2-(((3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propylcyclopenta[8]annulene-5-yl)oxy)-2-oxoethyl)thio)phenyl)carbamoyl)phenyl)carbamoyl)-1,2-benzenediacetate

[0218]

[0219] The preparation method is similar to that of Example 3, except that the corresponding raw materials are replaced. The yield of the obtained compound 18 is 58.94%.

[0220] 1H NMR(600MHz,DMSO-d6)δ10.12(s,1H),8.71(s,1H),8.34(t,J=2.1Hz,1H),8.13(t,J=2.2Hz,1H),7.95(t,J=2.1Hz,1H),7.84(t,J=2.2Hz,1H),7.63(ddd,J=8.3,2.3,1.3Hz,1H),7.57(dd,J=8.6,1.2Hz,1H),7.35(dd,J=8.8,1.3Hz,1H),7.30–7.22(m,3H),5.56(tdq,J=11.2,1.7,0.8Hz,1H),5.19(dd,J=9.6,2.4Hz,1H),4.98(tq,J=5.1,1.5Hz,1H),4.90(dd,J=11.9,2.4Hz,1H),3.78–3.64(m,2H),3.41(dddd,J=8.6,6.6,3.2,1.6Hz,1H),2.90(d,J=6.8Hz,1H),2.39(s,3H),2.35(s,3H),2.33(s,1H),2.28(t,J=5.2Hz,2H),2.14(dd,J=12.0,5.8Hz,1H),1.78(dt,J=12.4,4.6Hz,1H),1.70–1.65(m,2H),1.58–1.50(m,3H),1.43(dt,J=12.8,4.9Hz,1H),1.39–1.31(m,2H),1.08(t,J=1.2Hz,3H),1.04(d,J=1.8Hz,3H),0.93(dd,J=7.2,2.3Hz,3H),0.85(d,J=7.6Hz,3H).

[0221] 13C NMR (151MHz, DMSO-d6) δ218.34,170.81,168.82,168.46,166.32,164.86,146.69,143.35,142. 23,139.90,139.73,136.43,135.03,129.66,128.80,126.95,126.38,125.59,123.45,123.34,1 23.08,122.60,120.81,119.93,119.23,112.12,80.65,76.90,58.73,46.95,46.43,44.03,43.66,37.80,37.38,35.59,34.56,33.45,33.08,26.01,20.67,20.39,20.22,17.96,17.54,12.28.

[0222] Embodiment 19

[0223] Compound 19: Preparation of (3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propylcyclopenta[8]annul-5-yl 2-((3-(3-bromo-5-(2,3-dihydroxybenzamide)benzamide)phenyl)thio)acetate

[0224]

[0225] The preparation method is similar to that of Example 1, except that the corresponding raw materials are replaced. The yield of the obtained compound 19 is 57.82%.

[0226] 1H NMR(600MHz,DMSO-d6)δ10.49(s,1H),10.16(s,1H),8.77(s,1H),8.64(s,1H),8.28(t,J=2.1Hz,1H),8.17(t,J=2.2Hz,1H),7.93(t,J=1.8Hz,1H),7.88(t,J=2.0Hz,1H),7.61(ddd,J=7.3,2.0,1.6Hz,1H),7.32–7.23(m,2H),7.18(ddd,J=10.2,2.4,1.3Hz,1H),6.85–6.74(m,2H),5.72(tdq,J=11.2,1.8,0.9Hz,1H),5.23(dd,J=10.6,2.2Hz,1H),4.96(tq,J=5.6,1.5Hz,1H),4.89(dd,J=10.8,2.4Hz,1H),3.83–3.67(m,2H),3.39(dddd,J=8.6,6.6,3.1,1.4Hz,1H),2.90(d,J=6.8Hz,1H),2.37(d,J=3.4Hz,1H),2.30(t,J=4.5Hz,2H),2.17(dd,J=12.3,5.2Hz,1H),1.80(dt,J=12.8,4.4Hz,1H),1.76–1.69(m,2H),1.61–1.52(m,3H),1.47(dt,J=12.3,4.6Hz,1H),1.42–1.28(m,2H),1.11(t,J=1.2Hz,3H),1.06(d,J=1.6Hz,3H),0.96(dd,J=7.0,1.3Hz,3H),0.92(d,J=6.9Hz,3H).

[0227] 13 C NMR(151MHz,DMSO-d6)δ218.43,170.11,167.97,166.36,149.38,146.67,145.97,139.76,139.27,136.22,135.84,129.95,128.81,126.36,125.32,122.81,120.76,120.47,120.03,119.52,119.36,118.16,116.99,112.87,80.03,76.83,58.16,46.54,46.02,44.80,43.57,37.61,37.14,35.52,34.17,33.79,33.10,26.07,20.49,17.62,17.09,12.71.

[0228] Embodiment 20

[0229] Compound 20: Preparation of (3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propylcyclopenta[8]annul-5-yl 2-((3-(3-bromo-5-(2-chloro-3,4-dihydroxybenzamide)benzamide)phenyl)thio)acetate

[0230]

[0231] The preparation method is similar to that of Example 1, except that the corresponding raw materials are replaced. The yield of the obtained compound 20 is 52.92%.

[0232] 1 H NMR (600MHz, DMSO-d6) δ10.08(s,1H),8.69(s,1H),8.48(s,1H),8.26(t,J=1.9Hz,1H),8.13(t,J=1 .8Hz,1H),7.93(t,J=2.1Hz,1H),7.79(t,J=2.0Hz,1H),7.64(ddd,J=7.9,2.5,1.8Hz,1H),7.46(d, J=8.3Hz,2H),7.23(dd,J=8.8,5.3Hz,1H),7.15(ddd,J=6.6,2.0,1.4Hz,1H),6.74(d,J=9.6Hz,1H) ,5.61(tdq,J=10.2,1.8,0.6Hz,1H),5.16(dd,J=10.2,2.6Hz,1H),4.97(tq,J=5.3,1.6Hz,1H),4.9 0(dd,J=11.2,2.6Hz,1H),3.82–3.66(m,2H),3.45(dddd,J=8.6,6.8,3.0,1.2Hz,1H),2.96(d,J=6. 6Hz,1H),2.34(d,J=3.6Hz,1H),2.29(t,J=4.0Hz,2H),2.15(dd,J=12.3,5.1Hz,1H),1.79(dt,J=12 .4,4.6Hz,1H),1.73–1.65(m,2H),1.60–1.49(m,3H),1.43(dt,J=12.1,4.0Hz,1H),1.41–1.29(m,2 H), 1.13 (t, J = 1.3Hz, 3H), 1.08 (d, J = 1.6Hz, 3H), 0.99 (dd, J = 7.0, 2.5Hz, 3H), 0.96 (d, J = 6.6Hz, 3H).

[0233] 13 C NMR (151MHz, DMSO-d6) δ218.68,170.97,166.03,165.39,148.28,146.30,144.76,139.61,139.33,136.78,135.48 129.81,128.30,126.59,125.02,123.45,122.23,121.98,121.22,120.64,119.65,119.53,115.34,112.75,80.93,76.79,58.24,46.70,46.09,44.37,43.25,37.85,37.59,35.78,34.96,33.71,33.03,26.82,20.37,17.63,17.38,12.99.

[0234] Embodiment 21

[0235] Compound 21: Preparation of 3-((3-bromo-5-((3-((2-(((3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propylcyclopenta[8]annulene-5-yl)oxy)-2-oxoethyl)thio)phenyl)carbamoyl)phenyl)carbamoyl)-1,2-benzenediacetate

[0236]

[0237] The preparation method is similar to that of Example 3, except that the corresponding raw materials are replaced. The yield of the obtained compound 21 is 58.67%.

[0238] 1H NMR(600MHz,DMSO-d6)δ9.77(s,1H),8.58(s,1H),8.32(t,J=2.2Hz,1H),7.94(t,J=2.2Hz,1H),7.78(t,J=2.3Hz,1H),7.73(t,J=1.8Hz,1H),7.61(ddd,J=7.4,2.6,1.2Hz,1H),7.57(dd,J=8.3,1.1Hz,1H),7.31(dd,J=8.2,1.3Hz,1H),7.28–7.21(m,1H),7.17–7.10(m,1H),7.01(t,J=1.6Hz,1H),5.63(tdq,J=11.2,1.7,1.0Hz,1H),5.14(dd,J=10.6,2.2Hz,1H),4.98(tq,J=5.6,1.3Hz,1H),4.93–4.89(m,1H),3.87–3.69(m,2H),3.50(dddd,J=8.8,6.2,3.4,1.6Hz,1H),2.91(d,J=6.6Hz,1H),2.40(s,3H),2.38(s,3H),2.36(s,1H),2.30(t,J=4.7Hz,2H),2.14(dd,J=12.2,5.8Hz,1H),1.78(dt,J=12.3,4.6Hz,1H),1.72–1.68(m,2H),1.62–1.51(m,3H),1.49(dt,J=12.3,4.2Hz,1H),1.43–1.32(m,2H),1.11(t,J=1.2Hz,3H),1.07(d,J=1.2Hz,3H),0.98(dd,J=6.2,1.3Hz,3H),0.94(d,J=7.4Hz,3H).

[0239] 13C NMR (151MHz, DMSO-d6) δ218.30,170.86,168.64,168.59,166.91,164.25,146.17,143.68,142. 96,139.70,139.22,136.23,134.43,133.48,129.26,126.74,126.25,125.40,123.61,123.36,1 23.04,122.23,120.83,119.42,118.85,112.29,80.73,76.02,58.62,46.43,46.03,44.10,43.04,37.73,37.25,35.94,34.62,33.47,33.01,26.95,20.83,20.77,20.12,17.67,17.04,12.94.

[0240] Embodiment 22

[0241] Compound 22: Preparation of (3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propylcyclopenta[8]annul-5-yl 2-((3-(5-(2,3-dihydroxybenzamide)nicotinamide)phenyl)thio)acetate

[0242]

[0243] The preparation method is similar to that of Example 1, except that the corresponding raw materials are replaced. The yield of the obtained compound 22 is 59.11%.

[0244] 1H NMR(600MHz,DMSO-d6)δ10.19(s,1H),10.00(s,1H),9.13(s,1H),9.01(t,J=1.8Hz,1H),8.88(t,J=1.9Hz,1H),8.78(s,1H),8.47(t,J=2.0Hz,1H),7.85(t,J=2.2Hz,1H),7.63(ddd,J=7.9,2.3,1.3Hz,1H),7.38–7.27(m,2H),7.21(ddd,J=6.8,2.2,1.3Hz,1H),6.84–6.73(m,2H),5.62(tdq,J=10.8,1.9,0.9Hz,1H),5.09(dd,J=10.8,2.4Hz,1H),5.01(tq,J=5.7,1.5Hz,1H),4.96(dd,J=11.0,2.4Hz,1H),3.95–3.64(m,2H),3.47(dddd,J=8.2,6.6,3.1,1.6Hz,1H),2.94(d,J=6.6Hz,1H),2.37(d,J=3.7Hz,1H),2.31(t,J=4.7Hz,2H),2.14(dd,J=12.5,5.7Hz,1H),1.75(dt,J=12.4,4.6Hz,1H),1.70–1.63(m,2H),1.59–1.50(m,3H),1.47(dt,J=12.5,4.7Hz,1H),1.44–1.31(m,2H),1.11(t,J=1.3Hz,3H),1.06(d,J=1.5Hz,3H),0.95(dd,J=6.3,1.5Hz,3H),0.86(d,J=7.7Hz,3H).

[0245] 13 C NMR(151MHz,DMSO-d6)δ218.91,170.60,167.09,166.93,149.23,146.79,145.88,145.35,144.69,139.71,136.63,136.32,130.45,129.94,125.80,122.47,121.65,120.73,120.16,119.02,118.87,116.62,112.36,80.54,76.37,58.64,46.73,46.17,44.12,43.32,37.90,37.67,35.12,34.65,33.78,33.13,26.49,20.95,17.72,17.58,12.42.

[0246] Embodiment 23

[0247] Compound 23: Preparation of (3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propylcyclopenta[8]annul-5-yl 2-((3-(5-(2-chloro-3,4-dihydroxybenzamide)nicotinamide)phenyl)thio)acetate

[0248]

[0249] The preparation method is similar to that of Example 1, except that the corresponding raw materials are replaced. The yield of the obtained compound 23 is 61.71%.

[0250] 1 H NMR (600MHz, DMSO-d6) δ10.24(s,1H),9.13(s,1H),9.04(t,J=2.0Hz,1H),8.87(t,J=1.9Hz,1H),8. 43(s,1H),8.39(t,J=2.2Hz,1H),7.82(t,J=2.3Hz,1H),7.69(ddd,J=8.6,2.4,1.2Hz,1H),7.51(d, J=9.2Hz,2H),7.29(dd,J=7.2,6.6Hz,1H),7.24(ddd,J=7.2,2.4,1.6Hz,1H),6.81(d,J=10.6Hz,1H ),5.54(tdq,J=11.8,1.2,0.8Hz,1H),5.12(dd,J=10.2,2.2Hz,1H),4.97(tq,J=5.9,1.4Hz,1H),4.9 0(dd,J=11.4,2.6Hz,1H),3.85–3.61(m,2H),3.44(dddd,J=8.0,6.6,2.9,1.5Hz,1H),2.91(d,J=6. 6Hz, 1H), 2.34 (d, J = 3.4Hz, 1H), 2.28 (t, J = 5.2Hz, 2H), 2.17 (dd, J = 12.3, 5.4Hz, 1H), 1.78 (dt, J = 12 .0,4.6Hz,1H),1.75–1.67(m,2H),1.64–1.52(m,3H),1.50(dt,J=12.1,5.0Hz,1H),1.46–1.31(m,2 H), 1.09 (t, J = 1.0Hz, 3H), 1.04 (d, J = 1.4Hz, 3H), 0.93 (dd, J = 6.0, 1.3Hz, 3H), 0.82 (d, J = 7.3Hz, 3H).

[0251] 13 C NMR(151MHz,DMSO-d6)δ218.28,170.51,166.67,165.80,148.69,146.75,145.61,144 .65,144.12,139.29,136.84,136.69,130.04,129.02,125.87,123.63,122.81,122.5 3,121.60,120.23,119.13,115.56,112.36,80.64,76.97,58.66,46.38,46.03,44.90,43.22,37.19,37.04,35.61,34.72,33.81,33.09,26.35,20.04,17.91,17.54,12.47.

[0252] Embodiment 24

[0253] Compound 24: Preparation of 3-((5-((3-((2-(((3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propylcyclopenta[8]annulene-5-yl)oxy)-2-oxoethyl)thio)phenyl)carbamoyl)pyridin-3-yl)carbamoyl)-1,2-benzenediacetate

[0254]

[0255] The preparation method is similar to that of Example 3, except that the corresponding raw materials are replaced. The yield of the obtained compound 24 is 59.60%.

[0256] 1H NMR(600MHz,DMSO-d6)δ10.27(s,1H),9.10(s,1H),8.97(t,J=1.8Hz,1H),8.82(t,J=1.8Hz,1H),8.43(t,J=2.0Hz,1H),7.84(t,J=2.2Hz,1H),7.61(ddd,J=8.3,2.1,1.2Hz,1H),7.53(dd,J=8.6,1.2Hz,1H),7.39(dd,J=8.2,1.3Hz,1H),7.32–7.25(m,1H),7.24–7.20(m,2H),5.80(tdq,J=10.5,1.4,0.6Hz,1H),5.17(dd,J=10.2,2.0Hz,1H),4.97(tq,J=5.4,1.3Hz,1H),4.89(dd,J=11.8,2.6Hz,1H),3.85–3.63(m,2H),3.43(dddd,J=8.6,6.6,3.4,1.7Hz,1H),2.91(d,J=7.2Hz,1H),2.38(s,3H),2.35(s,3H),2.33(s,1H),2.25(t,J=3.8Hz,2H),2.14(dd,J=11.8,5.0Hz,1H),1.79(dt,J=12.3,4.8Hz,1H),1.76–1.68(m,2H),1.64–1.53(m,3H),1.50(dt,J=11.2,4.4Hz,1H),1.46–1.31(m,2H),1.09(t,J=1.2Hz,3H),1.03(d,J=1.6Hz,3H),0.94(dd,J=6.4,1.5Hz,3H),0.83(d,J=7.8Hz,3H).

[0257] 13C NMR(151MHz,DMSO-d6)δ218.55,170.17,168.60,168.52,166.43,164.38,146.14,145.98,14 4.57,143.88,142.48,139.30,136.72,136.42,130.76,129.11,126.42,125.87,124.34,123. 19,122.73,122.20,120.80,119.97,112.41,80.91,76.60,58.39,46.69,46.45,44.08,43.12,37.86,37.18,35.44,34.13,33.59,33.39,26.70,20.95,20.27,20.04,17.85,17.70,12.51.

[0258] 2. In vitro Antimicrobial Activity Assay

[0259] According to the method of the Clinical and Laboratory Standards Institute (CLSI), the minimum inhibitory concentration (MIC) of the pleuromutilin-siderophore antibacterial conjugate compound was determined by the broth microdilution method with Retapamulin (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) and Cefiderocol (purchased from Jiangsu Yaoze Pharmaceutical Technology Co., Ltd.) as positive controls.

[0260] The experimental strains include Gram-positive bacteria: Methicillin-resistant S.aureus ATCC 33591, Methicillin-resistant S.aureus ATCC 43300, S.aureus ATCC 29213 or Methicillin-resistant S.epidermidis ATCC 51625; Gram-negative bacteria: A.baumannii ATCC 19606, S.enterica ATCC 14028, E.coli ATCC 25922 or E.coli CMCC 44103.

[0261] The experimental strains were provided by Huashan Hospital Affiliated to Fudan University (Institute of Antibiotics, Fudan University) and used after identification by conventional methods.

[0262] The specific steps are as follows:

[0263] (1) Culture medium preparation

[0264] ①CAMHB (cation-adjusted Miller-Hinton broth): Prepare 1000 mL according to the label prescription, sterilize at 121 degrees Celsius for 15 minutes;

[0265] ②ID-CAMHB (Miller-Hinton Broth Medium Adjusted with Iron Deficiency): Add 100 g of Chelex 100 resin (Bio-Rad, 100-200 mesh, sodium form) to 1L CAMHB, sterilize at 121 degrees Celsius for 15 minutes, stir at room temperature for 6 hours to remove the polyvalent cationic metal ion medium, and use a 0.2μm filter to remove the resin. Check and adjust the pH value to 7.3. Then, add a final concentration of 20-25μg / mL calcium (calcium chloride), 10-12.5μg / mL magnesium (magnesium chloride), and 0.5-1.0μg / mL zinc (zinc sulfate) to the culture medium at the same time, adjust the pH value to 7.2-7.4 with 1M hydrochloric acid, and finally filter sterilize with a 0.2μm filter.

[0266] (2) Preparation of bacterial suspension

[0267] ① Thaw the glycerol cryovial of the experimental strain, draw 200 μL, add it to 50 mL of CAMHB and ID-CAMHB culture medium that have been sterilized at high temperature and cooled to room temperature, and culture at 37°C with constant temperature shaking for 16 h to obtain CAMHB culture medium containing the experimental strain and ID-CAMHB culture medium containing the experimental strain;

[0268] ② Take a small amount of each of the above bacterial solutions in a small test tube and dilute it to 0.5 McFarland turbidimetric tube (1.5*10 8 CFU / mL), and then diluted 100 times to obtain a concentration of 1*10 6 CFU / mL bacterial suspension stock solution.

[0269] (3) Preparation of experimental drug solution - two-fold dilution method

[0270] The samples to be tested (compounds 1 to 24 synthesized by the present invention, Retapamulin and Cefiderocol) were weighed and dissolved in DMSO solution to prepare a sample solution with a concentration of 10.24 mg / mL.

[0271] (4) Determination of MIC by two-fold microdilution method

[0272] Take a sterile 96-well plate, add 10 μL of the test compound, and perform serial dilutions by the 2-fold dilution method. At the same time, set up a negative control group without drug. Next, add 190 μL of diluted bacterial solution to each well and incubate in a 37°C constant temperature and humidity chamber for 24 hours.

[0273] (5) Cultivation and Observation

[0274] The bacterial growth in each well of the 96-well plate after culture was recorded to determine the MIC value of the compound. The changes in the MIC value of each compound under iron-deficient and normal iron conditions were compared to analyze the effect of iron carriers on bacterial growth.

[0275] Table 1 Minimum inhibitory concentration of the tested drugs (μg / mL)

[0276]

[0277]

[0278] The test results are Figure 1 to Figure 3 As shown in Table 1, it can be seen that the MIC of the truncated pleuromutilin drug retapamulin in ID-CAMHB medium remains unchanged or increases. This is because the iron carrier is not modified in structure. The bacteria may enter a low metabolic state in an iron-deficient environment, reducing the growth rate and division frequency, thereby increasing the MIC. The iron carrier antibacterial coupling compound cefiderocol has poor antibacterial activity against Gram-positive bacteria Methicillin-resistant S.aureus ATCC 33591, Methicillin-resistant S.aureus ATCC 43300, S.aureus ATCC 29213 and Methicillin-resistant S.epidermidisATCC 51625. The compounds 1 to 24 synthesized by the method have strong inhibitory effects on gram-positive bacteria Methicillin-resistant S. aureus ATCC 33591, Methicillin-resistant S. aureus ATCC 43300, S. aureus ATCC 29213, Methicillin-resistant S. epidermidis ATCC 51625 and gram-negative bacteria A. baumannii ATCC 19606, S. enterica ATCC 14028, E. coli ATCC 25922 and E. coli CMCC 44103 all have antibacterial effects, and the antibacterial effects of compounds 1 and 12 are better than those of retapamulin and cefiderocol. This is because the compounds synthesized by this method contain iron carrier modifications in their structures. Bacteria will activate the iron uptake system (such as TonB-dependent transporter) to compensate for iron deficiency in an iron-deficient environment. At the same time, pleuromutilin antibiotics can effectively locate and act on bacteria under the guidance of iron carriers, jointly enhancing the antibacterial effects of the compounds on Gram-positive bacteria and Gram-negative bacteria, thereby making the antibacterial spectrum of the compounds synthesized by this method broader.

[0279] 3. In vitro safety evaluation

[0280] CCK-8 (Cell Counting Kit-8) method was used to evaluate the toxicity of the compounds to cells:

[0281] (1) Preparation: 10 mM PBS buffer: 8.00 g NaCl, 0.20 g KCl, 1.44 g Na 2 HPO 4 and 0.24 g KH 2 PO 4 Dissolve in 800 mL of distilled water and adjust the pH to 7.4. Then, dilute to 1 L with distilled water and sterilize for 40 minutes before use.

[0282] (2) Preparation of bottom agar medium: Take 6.00 g of agar powder and dissolve it in 400 mL of distilled water. Stir well and then autoclave for 40 minutes to ensure the sterility of the medium. Then, when the solution cools to 60-70°C, add 8 mL of vitamin solution and 8 mL of glucose solution. After adding each solution, mix thoroughly to ensure uniform distribution. Finally, pour the mixed agar solution into a plate to prepare the bottom agar medium.

[0283] (3) Preparation of top agar medium: First, weigh 1.20 g agar powder and 1.00 g NaCl, and add 200 mL distilled water. After mixing, autoclave for 40 min to ensure the sterility of the medium. Next, when the solution cools to 60°C, add 1.00 mL of HBT solution and mix thoroughly. Finally, take 2 mL of the mixture and dispense it into sterile test tubes. This part of the medium needs to be kept warm until use, usually maintained at 45°C in a water bath.

[0284] (4) Cell culture: HepG2, HEK293 and A549 cells were evenly dispersed in culture medium, seeded into 96-well plates, and cultured under appropriate environmental conditions to keep the cells completely attached to the well walls.

[0285] (5) Compound treatment: Add different concentrations of compound 1 and 2 to each well (the concentrations are 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 μM, and perform 6 parallel experiments). Set up a blank control group and a normal control group. After incubation for 24 hours, observe the cell condition, which may be observed under a microscope.

[0286] (6) CCK-8 solution treatment: Add CCK-8 solution to each well and incubate for 4 h to allow it to react with the cells.

[0287] (7) Absorbance determination: Use a full-wavelength scanning multifunctional reader, place the 96-well plate in the instrument, shake and mix to make the color evenly distributed, and measure the absorbance value at 490 nm.

[0288] (8) Data analysis: Based on the measured absorbance values, the cell survival rate at different concentrations can be calculated. 50 ) were used to evaluate the cytotoxicity of the compounds to A549, HEK293 and HepG2 cells. The experimental results are shown in Tables 2 and Figure 4 .

[0289] Table 2 Cytotoxicity test results of compounds 1, 12, retapamulin and cefiderocol

[0290]

[0291] From Table 2 and Figure 4 It can be seen that in the safety evaluation experiment, at a concentration of 100 μM, compounds 1 and 2 showed low toxicity to the tested cells. The tested cells included: HepG2 cells, HEK293 cells and A549 cells. Compared with the control drugs retapamulin and cefiderocol, compounds 1 and 12 showed lower toxicity to the tested cells.

[0292] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A pleuromutilin-siderophore antibacterial coupling compound, characterized in that: It is a compound of formula I, or a stereoisomer, tautomer, enantiomer, diastereomer, homolog, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof of the compound of formula I; Wherein, R1 is selected from any one of aliphatic hydrocarbons substituted with -OH, aliphatic hydrocarbons substituted with -NH2, aliphatic hydrocarbons substituted with -OH and -NH2, aromatic rings substituted with -OH, aromatic rings substituted with -NH2, and aromatic rings substituted with -OH and -NH2; Linker is selected from aromatic heterocyclic rings containing amides, or aromatic rings substituted with amides and halogens; at least two adjacent substituents among R2, R3, R4, R5 and R6 are taken from -OH or -OAc, and the substituents at the remaining positions are independently selected from -H or -Cl.

2. The pleuromutilin-siderophore antibacterial coupling compound according to claim 1, characterized in that: R1 is fragment 1 or fragment 2:

3. The pleuromutilin-siderophore antibacterial coupling compound according to claim 1, characterized in that: Linker is Linker 1, Linker 2, Linker 3, or Linker 4:

4. The pleuromutilin-siderophore antibacterial coupling compound according to claim 1, characterized in that: R2 and R3 are -OH, R4, R5 and R6 are -H; or R2 and R3 are -OAc, R4, R5 and R6 are -H; or R3 and R4 are -OH, R2, R5 and R6 are -H.

5. The pleuromutilin-siderophore antibacterial coupling compound according to claim 1, characterized in that: The pleuromutilin-siderophore antibacterial coupling compound is selected from the following compounds:

6. The pleuromutilin-siderophore antibacterial coupling compound according to any one of claims 1 to 5, characterized in that: The pharmaceutically acceptable salt is a salt formed by the compound represented by formula I and hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid or aspartic acid.

7. The method for preparing a pleuromutilin-siderophore antibacterial coupling compound according to any one of claims 1 to 6, characterized in that: The following steps are involved: 1) Using pleuromutilin and p-toluenesulfonyl chloride as raw materials, the intermediate I is obtained by reaction; Among them, intermediate I is 2) using 3-amino-5-halogenated benzoic acid and di-tert-butyl dicarbonate as raw materials, or using 5-aminonicotinic acid and di-tert-butyl dicarbonate as raw materials, to react to obtain intermediate II; Among them, intermediate II is X is F, Cl or Br; 3) using 5-amino-2-mercaptocyclohexane-1-ol and the intermediate I obtained in step 1) as raw materials, or using 3-aminothiophenol and the intermediate I obtained in step 1) as raw materials, to react to obtain intermediate III; Among them, intermediate III is 4) using the intermediate II obtained in step 2) and the intermediate III obtained in step 3) as raw materials, reacting to obtain intermediate IV; Among them, intermediate IV is X is F, Cl or Br; 5) Using the intermediate IV obtained in step 4) as a raw material, removing the tert-butyloxycarbonyl group to obtain intermediate V; Among them, intermediate V is X is F, Cl or Br; 6) when at least two adjacent substituents among R2, R3, R4, R5 and R6 are taken from -OAc, 2,3-diacetoxybenzoic acid and the intermediate V obtained in step 5) are used as raw materials to react to obtain a pleuromutilin-siderophore antibacterial coupling compound; When at least two adjacent substituents among R2, R3, R4, R5 and R6 are taken from -OH, 2,3-dimethoxybenzoic acid and the intermediate V obtained in step 5) are used as raw materials for reaction, and then aluminum chloride is added to continue the reaction to obtain a truncated pleuromutilin-siderophore antibacterial coupling compound.

8. Use of the pleuromutilin-siderophore antibacterial coupling compound according to any one of claims 1 to 6 in the preparation of drugs for treating infectious diseases.

9. The use according to claim 8, characterized in that: The infectious disease is a disease caused by infection with pathogenic microorganisms, and the pathogenic microorganisms are Gram-positive bacteria or Gram-negative bacteria.

10. A pharmaceutical composition, characterized in that The pleuromutilin-siderophore antibacterial coupling compound according to any one of claims 1 to 6 is used as an active ingredient.

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