Pleuromutilin derivative with amino acid fragment as well as preparation method and application of pleuromutilin derivative

Abstract

The invention belongs to the field of pharmacy, and relates to pleuromutilin derivatives with amino acid fragments as shown in a general formula (I) as well as a preparation method and application of the pleuromutilin derivatives. The pleuromutilin derivative with the amino acid fragment has a structure as shown in a formula (I) or a pharmaceutically acceptable salt of the pleuromutilin derivative. The compound provided by the invention has excellent antibacterial activity when being combined with a medicine, and can be used as an active substance for treating infectious diseases. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of pharmacy, and particularly relates to a class of pleuromutilin derivatives with amino acid fragments, and a preparation method and use thereof. Background Art

[0002] Pleuromutilin is a tricyclic diterpenoid antibiotic isolated from the higher fungi Basidiomycetes Pleurotus mutilis and Pleurotus passeckeranius. Its main skeleton is composed of a five-membered ring, a six-membered ring and an eight-membered ring, with a tricyclic rigid structure. Pleuromutilin and its derivatives mainly have good inhibitory activities against Gram-positive bacteria and mycoplasma pathogens, and have good application prospects in veterinary and human antibiotics. Pleuromutilin antibiotics mainly act antibacterial by selectively binding to the ribosomes of prokaryotic microorganisms and inhibiting the activity of peptidyl transferase, thereby hindering the protein synthesis of prokaryotic cells. Due to the specific binding of pleuromutilin to bacterial ribosomes, it has high antibacterial activity, high selectivity, and is less likely to produce drug resistance compared with other types of antibiotics. Currently, no cross-resistance of pleuromutilin to mupirocin, β-lactam antibiotics, macrolide antibiotics or quinolones has been found.

[0003] Pleuromutilin-type antibiotics have been on the market for nearly 60 years. The marketed veterinary pleuromutilin antibiotics are tiamulin (approved in 1979) and valnemulin (approved in the EU in 1999), which are mainly used for the prevention and treatment of swine dysentery and have a broad antibacterial spectrum; retapamulin is a 1% ointment for topical use (trade name Altabax) developed by GlaxoSmithKline, mainly used for the treatment of skin infectious diseases such as impetigo. It was approved for marketing by the US FDA in 2007 and was approved by the European Medicines Agency (EMA) for the short-term treatment of secondary epidermal infections in the same year. Retapamulin is the first new topical prescription antibiotic; Lefamulin (BC-3781) is a semi-synthetic antibiotic developed by Austrian Nabriva Therapeutics for the treatment of community-acquired pneumonia (CABP). It was approved for marketing by the FDA in 2019. This new drug is an antibiotic with an innovative mechanism of action approved by the FDA in the past 20 years, providing a new treatment option for the treatment of CABP.

[0004]

[0005] The tricyclic diterpene nucleus of pleuromutilin is the key part for its antibacterial activity. However, due to its poor water solubility and low oral bioavailability, it cannot be directly used as a drug and needs to be structurally modified. (Cui Ge. Synthesis of Novel Derivatives of Pleuromutilin [D]. Hebei University of Science and Technology, 2021. DOI: 10.27107 / d.cnki.ghbku.2021.000190.) During the process of structurally modifying pleuromutilin, researchers found that the C14 side chain of pleuromutilin is the main site for chemical modification, and its ester group structure is usually an essential group for activity. Domestic and foreign researchers usually modify the C14 side chain while retaining the ester group, and introduce various groups to improve its antibacterial activity and bioavailability. (Wang Qian, Xia Yuxiang. Patent Analysis of the Side Chain Improvement of Pleuromutilin Drugs [J]. Science & Technology Vision, 2019(20): 27-28. DOI: 10.19694 / j.cnki.issn2095-2457.2019.20.011.)

[0006]

[0007] The clinical compound under investigation, BC-7013, introduced a phenylthioether fragment at the C22 position and showed high activity against common Gram-positive bacterial pathogens, especially Staphylococcus spp. In addition, Fang Binghu from South China Agricultural University (Chem Biol Drug Des. 2015; 86: 239-245), Tang Youzhi (Chem Biol Drug Des. 2018; 92: 1627-1637; European Journal of Medicinal Chemistry 181(2019)111594; European Journal of Medicinal Chemistry 204(2020)112604), etc. also found that the introduction of the phenylthioether fragment at the C22 position can enhance the anti-positive bacterial activity of pleuromutilin compounds through the extension of the phenylthioether fragment at the C22 position, but its anti-negative bacterial activity is lost.

[0008] In addition to being able to serve as a pharmacodynamic fragment to provide rich interaction forces for the molecular binding domain with the target, amino acid fragments are also important druggability fragments that can effectively regulate the physicochemical properties such as hydrophilicity and lipophilicity of the molecule. In some drugs, such as melphalan and valacyclovir, amino acid fragments have been found to be an important tool fragment for promoting drug transmembrane transport and improving the transmembrane ability of the molecule. The purpose of the present invention is to enhance the anti-Gram-positive and anti-Gram-negative bacterial activities of pleuromutilin derivatives by introducing amino acid fragments. Summary of the Invention

[0009] To solve the deficiencies of the prior art, the main object of the present invention is to provide a pleuromutilin derivative with an amino acid fragment.

[0010] Another object of the present invention is to provide a preparation method of the above-mentioned pleuromutilin derivative with an amino acid fragment.

[0011] Still another object of the present invention is to provide a pharmaceutical combination, which comprises a therapeutically effective amount of one or more pleuromutilin derivatives with an amino acid fragment or a pharmaceutically acceptable salt thereof as the main active ingredient, and a pharmaceutically acceptable excipient.

[0012] Yet another object of the present invention is to provide the use of a pleuromutilin derivative with an amino acid fragment or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating infectious diseases, especially infectious diseases caused by Staphylococcus aureus, drug-resistant Staphylococcus aureus, Staphylococcus epidermidis, drug-resistant Staphylococcus epidermidis, Streptococcus pneumoniae, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa or Mycoplasma, or infectious diseases caused by multi-drug resistant bacteria.

[0013] The object of the present invention is achieved by the following technical solutions:

[0014] A pleuromutilin derivative with an amino acid fragment, the derivative has a structure shown in formula (I):

[0015]

[0016] Wherein,

[0017] is selected from

[0018] is selected from

[0019] n is selected from one of 0, 1, 2, 3;

[0020] R is selected from hydrogen, methyl, isopropyl, amino, carbamoyl, guanidino.

[0021] More preferably, the pleuromutilin derivative with an amino acid fragment is selected from the following:

[0022]

[0023]

[0024] The preparation method of the above-mentioned pleuromutilin derivative with an amino acid fragment includes the following steps: 1) React pleuromutilin with p-toluenesulfonyl chloride to obtain intermediate (II);

[0025]

[0026] (2) Using intermediate (II) as a raw material, reacting with o / m / p-aminothiophenol to obtain intermediate (III);

[0027]

[0028] (3) Using intermediate (III) as a raw material, in the presence of HATU condensing agent and triethylamine, reacting with various N-Boc-amino acids for 3 h to obtain intermediate (IV);

[0029]

[0030] (4) Using intermediate (IV) as a raw material, removing the protecting group in 4M HCl 1,4-dioxane solution to obtain the pleuromutilin derivative with an amino acid fragment having the structure shown in formula (I).

[0031]

[0032] The reaction described in step (1) uses dichloromethane as a solvent, triethylamine as an acid-binding agent, reacts at room temperature for 8 h, and the molar ratio of p-toluenesulfonyl chloride to pleuromutilin is 1.1:1;

[0033] The reaction described in step (2) uses N,N-dimethylformamide as a solvent, adds intermediate (II) and o / m / p-aminothiophenol, uses potassium carbonate as an acid-binding agent, reacts at 60 °C for 4 h, and the molar ratio of intermediate (II) to o / m / p-aminothiophenol is 1:1.3;

[0034] The reaction described in step (3) uses dichloromethane as a solvent, adds various N-Boc-amino acids, and reacts with intermediate (III) at room temperature for 6 h under the action of HATU condensing agent, triethylamine and a catalytic amount of DMAP. The molar ratio of intermediate (III) to N-Boc-amino acids is 1:1.2;

[0035] The reaction described in step (4) uses dichloromethane as a solvent, adds 4M HCl 1,4-dioxane solution, and reacts with intermediate (IV) at room temperature for 4 h. The molar ratio of intermediate (IV) to hydrochloric acid is 1:4, and the target product having the structure shown in formula (I) is obtained, which can be purified by recrystallization or column chromatography.

[0036]

[0037] Beneficial technical effects

[0038] The present invention provides a novel class of pleuromutilin compounds with a structure as shown in formula (I). By connecting aminoacylaminophenylthiol to the 22-position of pleuromutilin, the antibacterial activities against Gram-positive and Gram-negative bacteria of this class of compounds are enhanced. Such pleuromutilin derivatives having an amino acid fragment and their pharmaceutically acceptable salts can be used as novel antibacterial drugs for the treatment of bacterial infections in animals or humans. Detailed implementation manners

[0039] The present invention will be further described below in conjunction with examples, but the implementation manners of the present invention are not limited thereto.

[0040] In all examples, 1 1H-NMR was recorded using a Bruker AVANCE III 400 nuclear magnetic resonance spectrometer, a Bruker AVANCEIII 500 nuclear magnetic resonance spectrometer or a QOne 400 nuclear magnetic resonance spectrometer, and the chemical shift was expressed in δ (ppm); low-resolution mass spectrometry was determined using a Waters H-Class-SQD type mass spectrometer.

[0041] Unless otherwise specified, the experimental materials and reagents used in the following experimental examples and examples can be obtained from commercial channels.

[0042] The numbering of the pleuromutilin parent nucleus described in the present invention is as follows:

[0043]

[0044] Experimental example 1

[0045] Preparation of intermediate (II)

[0046]

[0047] Weigh 10 g of pleuromutilin (26 mmol) and dissolve it in 60 mL of dichloromethane solution. Add 5.5 g of p-toluenesulfonyl chloride (28.9 mmol) and 7.3 mL of triethylamine (98.8 mmol). After stirring at room temperature for 12 h, add 100 mL of saturated ammonium chloride aqueous solution to quench the reaction solution. Separate and recover the organic phase. Extract the aqueous phase three times with 30 mL of dichloromethane each time. Combine the organic phases, dry them over anhydrous sodium sulfate, and purify them by column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain 12 g of intermediate II with a yield of 85%. ESI-MS (m / z): 533.69 [M+H] + . 11H-NMR (500 MHz, Chloroform-d) δ 7.85 (d, J = 8.2 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 6.45 (dd, J = 17.4, 11.0 Hz, 1H), 5.81 (d, J = 8.5 Hz, 1H), 5.37 (d, J = 11.0 Hz, 1H), 5.23 (d, J = 17.4 Hz, 1H), 4.52 (s, 2H), 3.38 (s, 1H), 2.49 (s, 3H), 2.35 - 2.17 (m, 3H), 2.15 - 2.04 (m, 2H), 1.79 (dq, J = 14.9, 3.0 Hz, 1H), 1.73 - 1.61 (m, 3H), 1.45 (s, 3H).

[0048] Preparation of Intermediate Ⅲ-A

[0049]

[0050] Weigh 2 g of Intermediate Ⅱ (3.8 mmol) and dissolve it in 10 mL of N,N-dimethylformamide. Add 612 mg of 2-aminothiophenol (4.9 mmol) and 1.6 g of potassium carbonate (11.3 mmol). After stirring at room temperature for 12 h, add 15 mL of saturated ammonium chloride aqueous solution to quench the reaction solution. Extract the aqueous phase three times with 15 mL of ethyl acetate. Combine the organic phases, dry over anhydrous sodium sulfate, and purify by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain 1.5 g of Intermediate Ⅲ-A with a yield of 83%. ESI-MS (m / z): 486.26 [M + H] + . 11H NMR (500 MHz, Chloroform-d) δ 7.43 (dd, J = 7.7, 1.5 Hz, 1H), 7.17 (td, J = 7.7, 1.6 Hz, 1H), 6.99 (dd, J = 7.7, 1.6 Hz, 1H), 6.80 (td, J = 7.6, 1.5 Hz, 1H), 6.44 (dd, J = 17.4, 11.0 Hz, 1H), 5.69 (d, J = 8.4 Hz, 1H), 5.37 (dd, J = 11.0, 1.6 Hz, 1H), 5.18 (dd, J = 17.4, 1.6 Hz, 1H), 3.46 (d, J = 15.5 Hz, 1H), 3.40 (d, J = 15.5 Hz, 1H), 3.32 (d, J = 6.5 Hz, 1H), 2.30 (m, 1H), 2.26 - 2.13 (m, 2H), 2.05 (s, 1H), 1.98 (dd, J = 16.1, 8.5 Hz, 1H), 1.75 (dd, J = 14.8, 3.3 Hz, 1H), 1.68 - 1.57 (m, 2H), 1.55 - 1.40 (m, 2H), 1.38 (s, 3H), 1.34 - 1.24 (m, 2H), 1.14 (s, 3H), 1.09 (m, 1H), 0.87 (d, J = 6.9 Hz, 3H), 0.57 (d, J = 7.0 Hz, 3H).

[0051] Preparation of Intermediate Ⅲ-B

[0052]

[0053] Weigh 2 g of Intermediate Ⅱ (3.8 mmol) and dissolve it in 10 mL of N,N-dimethylformamide. Add 612 mg of 3-aminobenzenethiol (4.9 mmol) and 1.6 g of potassium carbonate (11.3 mmol). After stirring at room temperature for 12 h, add 15 mL of saturated ammonium chloride aqueous solution to quench the reaction solution. Extract the aqueous phase three times with 15 mL of ethyl acetate. Combine the organic phases, dry over anhydrous sodium sulfate, and purify by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain 1.6 g of Intermediate Ⅲ-B with a yield of 86%. ESI-MS (m / z): 486.33 [M + H] + . 11H NMR (400 MHz, Chloroform-d) δ 7.18 (t, J = 7.9 Hz, 1H), 7.06 (t, J = 8.0 Hz, 1H), 7.02 (dd, J = 7.9, 2.1 Hz, 1H), 6.90 (dt, J = 8.0, 1.3 Hz, 1H), 6.40 (dd, J = 17.4, 11.0 Hz, 1H), 5.72 (d, J = 8.4 Hz, 1H), 5.30 (dd, J = 11.0, 1.6 Hz, 1H), 5.15 (dd, J = 17.4, 1.6 Hz, 1H), 3.58 (s, 2H), 3.33 (d, J = 6.4 Hz, 1H), 2.34 - 2.15 (m, 3H), 2.10 - 1.95 (m, 2H), 1.75 (dd, J = 14.3, 3.1 Hz, 1H), 1.68 - 1.56 (m, 2H), 1.54 - 1.46 (m, 2H), 1.41 (s, 3H), 1.38 - 1.16 (m, 3H), 1.12 (s, 3H), 0.86 (d, J = 7.1 Hz, 3H), 0.69 (d, J = 6.9 Hz, 3H).

[0054] Preparation of Intermediate Ⅲ-C

[0055]

[0056] Weigh 2 g of Intermediate Ⅱ (3.8 mmol) and dissolve it in 10 mL of N,N-dimethylformamide. Add 612 mg of 4-aminothiophenol (4.9 mmol) and 1.6 g of potassium carbonate (11.3 mmol). After stirring at room temperature for 12 h, add 15 mL of saturated ammonium chloride aqueous solution to quench the reaction solution. Extract the aqueous phase three times with 15 mL of ethyl acetate. Combine the organic phases, dry over anhydrous sodium sulfate, and purify by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain 1.4 g of Intermediate Ⅲ-C with a yield of 79%. ESI-MS (m / z): 486.35 [M + H] + . 11H NMR (500 MHz, Chloroform-d) δ 7.25 (d, J = 7.9 Hz, 2H), 6.57 (d, J = 8.1 Hz, 2H), 6.45 (dd, J = 17.4, 11.0 Hz, 1H), 5.71 (d, J = 8.4 Hz, 1H), 5.34 (d, J = 11.0 Hz, 1H), 5.19 (d, J = 17.4 Hz, 1H), 3.41 (d, J = 14.7 Hz, 1H), 3.36 (d, J = 14.5 Hz, 1H), 3.33 (d, J = 6.6 Hz, 1H), 2.31 (m, 1H), 2.28 - 2.12 (m, 2H), 2.06 (s, 1H), 1.99 (m, 1H), 1.75 (m, 1H), 1.68 - 1.58 (m, 2H), 1.56 - 1.41 (m, 2H), 1.39 (s, 3H), 1.35 (m, 1H), 1.18 (d, J = 16.0 Hz, 1H), 1.14 (s, 3H), 1.10 (m, 1H), 0.86 (d, J = 7.0 Hz, 3H), 0.66 (d, J = 6.9 Hz, 3H).

[0057] Example 1: Preparation of 14-O-[2-(2-Aminoacetamido)phenyl]thioacetyl Moxilin (Compound 1)

[0058]

[0059] Step 1: Preparation of Intermediate 1A

[0060] Weigh 70 mg of N-(tert-Butoxycarbonyl)-glycine (0.45 mmol) and 203 mg of HATU (0.53 mmol), dissolve them in 5 mL of dichloromethane, stir at room temperature for 30 min, add 200 mg of Intermediate III-A (0.41 mmol) and 85 μL of triethylamine (0.62 mmol). After stirring at room temperature for 2 h, add 10 mL of saturated ammonium chloride aqueous solution to quench the reaction solution, separate and recover the organic phase, extract the aqueous phase three times with 5 mL of dichloromethane each time, combine the organic phases, dry over anhydrous sodium sulfate, and purify by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain 223 mg of Intermediate 1A with a yield of 85%. ESI-MS (m / z): 643.43 [M + H] + .

[0061] Step 2: Preparation of Compound 1

[0062] Intermediate 1A was dissolved in 2 mL of dichloromethane. 0.5 mL of 4 M hydrochloric acid 1,4-dioxane was added dropwise to the above solution. After stirring at room temperature for 2 h, the organic solvent was removed by rotary evaporation under vacuum. Purification was carried out using reverse-phase column chromatography. After freeze-drying, 151 mg of off-white solid compound 1 was obtained with a yield of 80%. ESI-MS (m / z): 543.28 [M+H] + . 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.22 (dd, J = 7.8, 1.5 Hz, 1H), 7.52 (dd, J = 7.9, 1.5 Hz, 1H), 7.30 (td, J = 7.8, 1.6 Hz, 1H), 7.05 (td, J = 7.6, 1.4 Hz, 1H), 6.04 (dd, J = 17.6, 11.3 Hz, 1H), 5.48 (d, J = 8.2 Hz, 1H), 5.06 - 4.88 (m, 2H), 4.50 (d, J = 6.1 Hz, 1H), 3.77 (d, J = 15.5 Hz, 1H), 3.69 (d, J = 15.6 Hz, 1H), 3.38 (t, J = 5.9 Hz, 1H), 3.32 (s, 2H), 2.36 (d, J = 2.6 Hz, 1H), 2.24 - 2.12 (m, 1H), 2.16 - 1.89 (m, 3H), 1.69 - 1.54 (m, 2H), 1.44 (m, 1H), 1.39 - 1.30 (m, 1H), 1.29 (s, 3H), 1.27 - 1.11 (m, 2H), 1.06 (d, J = 15.9 Hz, 1H), 1.00 (s, 3H), 0.96 (m, 1H), 0.80 (d, J = 6.9 Hz, 3H), 0.53 (d, J = 6.9 Hz, 3H).

[0063] Example 2: Preparation of 14-O-[2-((2R)-2-aminopropionamido)phenyl]thioacetyl moxilin (Compound 2)

[0064]

[0065] Step 1: Preparation of Intermediate 2A

[0066] Weigh 85 mg of N-(tert-butoxycarbonyl)-D-alanine (0.45 mmol), dissolve 203 mg of HATU (0.53 mmol) in 5 mL of dichloromethane, stir at room temperature for 30 min, add 200 mg of intermediate Ⅲ-A (0.41 mmol), and 85 μL of triethylamine (0.62 mmol). After stirring at room temperature for 2 h, add 10 mL of saturated ammonium chloride aqueous solution to quench the reaction solution, separate and recover the organic phase, extract the aqueous phase three times with 5 mL of dichloromethane each time. Combine the organic phases, dry over anhydrous sodium sulfate, and purify by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain 245 mg of intermediate 2A with a yield of 91%. ESI-MS (m / z): 657.35 [M+H] + .

[0067] Step 2 Preparation of Compound 2:

[0068] Dissolve intermediate 2A in 2 mL of dichloromethane, add 0.5 mL of 4 M hydrochloric acid in 1,4-dioxane dropwise to the above solution, stir at room temperature for 2 h, then remove the organic solvent by rotary evaporation under vacuum, purify by reverse-phase column chromatography, and obtain 122 mg of off-white solid compound 2 after freeze-drying with a yield of 59%. ESI-MS (m / z): 557.30 [M+H] + . 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.20 (ddd, J = 8.2, 4.6, 1.4 Hz, 1H), 7.51 (dt, J = 7.8, 1.4 Hz, 1H), 7.28 (ddd, J = 8.6, 7.5, 1.5 Hz, 1H), 7.03 (td, J = 7.6, 1.4 Hz, 1H), 6.03 (dd, J = 17.5, 11.4 Hz, 1H), 5.47 (d, J = 8.2 Hz, 1H), 5.00 - 4.90 (m, 2H), 4.49 (dd, J = 6.3, 2.0 Hz, 1H), 3.80 - 3.64 (m, 2H), 3.47 (q, J = 7.0 Hz, 1H), 3.38 (t, J = 5.9 Hz, 1H), 2.35 (s, 1H), 2.17 (m, 1H), 2.11 - 1.88 (m, 3H), 1.68 - 1.53 (m, 2H), 1.39 (m, 2H), 1.29 (s, 3H), 1.27 (d, J = 3.6 Hz, 3H), 1.25 - 1.15 (m, 2H), 1.04 (m, 1H), 0.99 (s, 3H), 0.96 (m, 1H), 0.79 (d, J = 6.9 Hz, 3H), 0.51 (d, J = 6.9 Hz, 3H).

[0069] Example 3: Preparation of 14-O-[2-((2R)-2,5-diaminopentanamido)phenyl]thioacetyl moxilin (Compound 3)

[0070]

[0071] Step 1: Preparation of Intermediate 3A

[0072] Weigh 150 mg of (2R)-2,5-bis[(tert-butoxycarbonyl)amino]pentanoic acid (0.45 mmol) and 203 mg of HATU (0.53 mmol), dissolve them in 5 mL of dichloromethane, stir at room temperature for 30 min, add 200 mg of Intermediate III-A (0.41 mmol) and 85 μL of triethylamine (0.62 mmol). After stirring at room temperature for 2 h, add 10 mL of saturated ammonium chloride aqueous solution to quench the reaction solution. Separate and recover the organic phase, extract the aqueous phase three times with 5 mL of dichloromethane each time. Combine the organic phases, dry over anhydrous sodium sulfate, and purify by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain 249 mg of Intermediate 3A with a yield of 76%. ESI-MS (m / z): 800.45 [M+H] + .

[0073] Step 2: Preparation of Compound 3

[0074] Dissolve Intermediate 3A in 2 mL of dichloromethane, add 1 mL of 4 M hydrochloric acid in 1,4-dioxane dropwise to the above solution, stir at room temperature for 2 h, then remove the organic solvent by rotary evaporation under vacuum. Purify by reverse-phase column chromatography and freeze-dry to obtain 142 mg of off-white solid Compound 3 with a yield of 76%. ESI-MS (m / z): 600.24 [M+H] + . 1 H NMR (400 MHz, DMSO-d 6)δ8.03 - 7.95(m, 1H), 7.47(dd, J = 7.9, 1.4Hz, 1H), 7.27(td, J = 7.8, 1.5Hz, 1H), 7.12 - 7.03(m, 1H), 6.12 - 5.99(m, 1H), 5.49(d, J = 8.2Hz, 1H), 5.02 - 4.90(m, 2H), 3.77(d, J = 15.7Hz, 1H), 3.69(d, J = 15.6Hz, 1H), 3.39(d, J = 5.8Hz, 1H), 2.81(q, J = 7.0Hz, 2H), 2.36(d, J = 2.5Hz, 1H), 2.23 - 2.11(m, 1H), 2.11 - 1.93(m, 3H), 1.92 - 1.83(m, 1H), 1.74(dd, J = 10.7, 5.1Hz, 2H), 1.66 - 1.58(m, 3H), 1.50 - 1.30(m, 2H), 1.29(s, 3H), 1.27 - 1.15(m, 2H), 1.10(d, J = 15.8Hz, 1H), 1.00(s, 3H), 0.96(dd, J = 13.5, 4.2Hz, 1H), 0.79(d, J = 6.9Hz, 3H), 0.52(d, J = 6.9Hz, 3H).

[0075] Example 4: Preparation of 14 - O - [2 - ((2S)-2,5 - diamino pentanamido)phenyl]thioacetyl moxilin (Compound 4)

[0076]

[0077] Step 1: Preparation of Intermediate 4A

[0078] Weigh 150 mg of (2S)-2,5 - bis[(tert - butoxycarbonyl)amino]pentanoic acid (0.45 mmol) and 203 mg of HATU (0.53 mmol), dissolve them in 5 mL of dichloromethane, stir at room temperature for 30 min, add 200 mg of Intermediate III - A (0.41 mmol) and 85 μL of triethylamine (0.62 mmol). After stirring at room temperature for 2 h, add 10 mL of saturated ammonium chloride aqueous solution to quench the reaction mixture. Separate and recover the organic phase, extract the aqueous phase three times with 5 mL of dichloromethane each time. Combine the organic phases, dry over anhydrous sodium sulfate, and purify by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain 282 mg of Intermediate 4A with a yield of 86%. ESI - MS (m / z): 800.45 [M + H] + .

[0079] Step 2: Preparation of Compound 4

[0080] Intermediate 4A (152 mg) was dissolved in 2 mL of dichloromethane. 1 mL of 4 M hydrochloric acid in 1,4-dioxane was added dropwise to the above solution. After stirring at room temperature for 2 h, the organic solvent was removed by rotary evaporation under vacuum. Purification was carried out using reverse-phase column chromatography, and 152 mg of a creamy white solid compound 4 was obtained after lyophilization, with a yield of 72%. ESI-MS (m / z): 600.59 [M+H] + . 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.12 (d, J = 8.0 Hz, 1H), 7.49 (dd, J = 7.8, 1.4 Hz, 1H), 7.32 - 7.22 (m, 1H), 7.05 (t, J = 7.5 Hz, 1H), 6.04 (dd, J = 17.6, 11.3 Hz, 1H), 5.48 (d, J = 8.2 Hz, 1H), 5.02 - 4.90 (m, 2H), 3.77 (d, J = 15.6 Hz, 1H), 3.69 (d, J = 15.6 Hz, 1H), 3.43 - 3.33 (m, 2H), 2.79 (m, 2H), 2.36 (s, 1H), 2.17 (m, 1H), 2.02 (m, 3H), 1.91 - 1.80 (m, 1H), 1.78 - 1.51 (m, 4H), 1.50 - 1.24 (m, 2H), 1.29 (s, J = 4.6 Hz, 3H), 1.25 - 1.14 (m, 2H), 1.12 - 1.00 (m, 1H), 1.00 (s, 3H), 0.96 (m, 1H), 0.80 (d, J = 6.7 Hz, 3H), 0.53 (d, J = 6.8 Hz, 3H).

[0081] Example 5: Preparation of 14-O-[3-(2-aminoacetamido)phenyl]thioacetyl moxilin (Compound 5)

[0082] Step 1: Preparation of Intermediate 5A

[0083] 70 mg of N-(tert-butoxycarbonyl)-glycine (0.45 mmol) and 203 mg of HATU (0.53 mmol) were dissolved in 5 mL of dichloromethane and stirred at room temperature for 30 min. 200 mg of Intermediate III-B (0.41 mmol) and 85 μL of triethylamine (0.62 mmol) were added. After stirring at room temperature for 2 h, the reaction solution was quenched with 10 mL of saturated ammonium chloride aqueous solution. The organic phase was separated and recovered, and the aqueous phase was extracted three times with 5 mL of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate and purified by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain 203 mg of Intermediate 5A, with a yield of 77%. ESI-MS (m / z): 643.38 [M+H] + . Step 2: Preparation of Compound 5

[0084] The intermediate 5A was dissolved in 2 mL of dichloromethane. 0.5 mL of 4 M hydrochloric acid 1,4-dioxane was added dropwise to the above solution. After stirring at room temperature for 2 h, the organic solvent was removed by rotary evaporation under vacuum. Purification was carried out using reverse-phase column chromatography. After freeze-drying, 137 mg of off-white solid compound 5 was obtained with a yield of 80%. ESI-MS (m / z): 543.28 [M+H] + . 1H NMR (500 MHz, Methanol-d4) δ 7.74 (t, J = 1.9 Hz, 1H), 7.40 (dd, J = 7.9, 2.1 Hz, 1H), 7.25 (t, J = 8.0 Hz, 1H), 7.14 (dt, J = 8.0, 1.3 Hz, 1H), 6.18 (dd, J = 17.6, 11.2 Hz, 1H), 5.66 (d, J = 8.3 Hz, 1H), 5.10 - 4.98 (m, 2H), 3.71 (d, J = 15.4 Hz, 1H), 3.64 (m, 3H), 3.43 (d, J = 6.2 Hz, 1H), 2.32 - 2.20 (m, 3H), 2.13 (m, 1H), 1.99 (m, 1H), 1.78 (m, 1H), 1.66 (m, 1H), 1.55 (m, 2H), 1.37 (s, 3H), 1.31 (m, 2H), 1.13 (m, 2H), 1.06 (s, 3H), 0.91 (d, J = 6.9 Hz, 3H), 0.66 (d, J = 6.6 Hz, 3H).

[0085] Example 6: Preparation of 14-O-[3-((2R)-2-aminopropionamido)phenyl]thioacetyl moxilin (Compound 6)

[0086]

[0087] Step 1: Preparation of intermediate 6A

[0088] 85 mg of N-(tert-butoxycarbonyl)-D-alanine (0.45 mmol) and 203 mg of HATU (0.53 mmol) were dissolved in 5 mL of dichloromethane. The mixture was stirred at room temperature for 30 min, then 200 mg of intermediate III-B (0.41 mmol) and 85 μL of triethylamine (0.62 mmol) were added. After stirring at room temperature for 2 h, 10 mL of saturated ammonium chloride aqueous solution was added to quench the reaction mixture. The organic phase was separated and recovered. The aqueous phase was extracted three times with 5 mL of dichloromethane each time. The combined organic phases were dried over anhydrous sodium sulfate and purified by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain 237 mg of intermediate 6A with a yield of 88%. ESI-MS (m / z): 657.35 [M+H] + . Step 2: Preparation of Compound 6

[0089] The intermediate 6A was dissolved in 2 mL of dichloromethane. 0.5 mL of 4 M hydrochloric acid 1,4-dioxane was added dropwise to the above solution. After stirring at room temperature for 2 h, the organic solvent was removed by rotary evaporation under vacuum. Purification was carried out using reverse-phase column chromatography. After freeze-drying, 126 mg of off-white solid compound 6 was obtained with a yield of 63%. ESI-MS (m / z): 557.30 [M+H] + .1H NMR (400 MHz, DMSO-d 6 ) δ 7.63 (t, J = 1.9 Hz, 1H), 7.43 (dd, J = 8.4, 2.0 Hz, 1H), 7.29 (t, J = 8.0 Hz, 1H), 7.11 (dt, J = 8.0, 1.3 Hz, 1H), 6.02 (dd, J = 18.3, 10.7 Hz, 1H), 5.49 (d, J = 8.2 Hz, 1H), 4.96 (m, 1H), 4.92 (m, 1H), 4.52 (d, J = 6.0 Hz, 1H), 4.00 (m, 1H), 3.84 (d, J = 15.7 Hz, 1H), 3.77 (d, J = 15.7 Hz, 1H), 3.38 (t, J = 6.0 Hz, 1H), 2.36 (s, 1H), 2.18 (m, 1H), 2.11 - 1.90 (m, 3H), 1.69 - 1.55 (m, 2H), 1.44 (d, J = 7.0 Hz, 3H), 1.51 - 1.32 (m, 2H), 1.31 (s, 3H), 1.28 - 1.2 (m, 2H), 1.12 (d, J = 15.8 Hz, 1H), 1.06 - 1.00 (m, 1H), 0.98 (s, 3H), 0.80 (d, J = 7.0 Hz, 3H), 0.55 (d, J = 6.8 Hz, 3H).

[0090] Example 7: Preparation of 14-O-[3-((2R)-2,5-diaminopentanamido)phenyl]thioacetyl moxilin (Compound 7)

[0091]

[0092] Step 1: Preparation of intermediate 7A

[0093] Weigh 150 mg of (2R)-2,5-bis[(tert-butoxycarbonyl)amino]pentanoic acid (0.45 mmol), and dissolve 203 mg of HATU (0.53 mmol) in 5 mL of dichloromethane. Stir at room temperature for 30 min, add 200 mg of intermediate Ⅲ-B (0.41 mmol), and 85 μL of triethylamine (0.62 mmol). After stirring at room temperature for 2 h, add 10 mL of saturated aqueous ammonium chloride solution to quench the reaction mixture. Separate and recover the organic phase, extract the aqueous phase three times with 5 mL of dichloromethane each time. Combine the organic phases, dry over anhydrous sodium sulfate, and purify by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain 246 mg of intermediate 7A with a yield of 75%. ESI-MS (m / z): 800.45 [M+H] + .

[0094] Step 2 Preparation of compound 7:

[0095] Dissolve intermediate 7A in 2 mL of dichloromethane, add 0.5 mL of 4 M hydrochloric acid in 1,4-dioxane dropwise to the above solution, stir at room temperature for 2 h, then remove the organic solvent by rotary evaporation under vacuum. Purify by reverse-phase column chromatography and obtain 129 mg of off-white solid compound 7 after freeze-drying with a yield of 70%. ESI-MS (m / z): 600.34 [M+H] + . 1H NMR (400 MHz, DMSO-d 6 ) δ 7.76 (t, J = 1.9 Hz, 1H), 7.52 (dd, J = 8.4, 2.0 Hz, 1H), 7.27 (t, J = 7.9 Hz, 1H), 7.08 (dt, J = 8.0, 1.3 Hz, 1H), 6.03 (dd, J = 18.3, 10.7 Hz, 1H), 5.50 (d, J = 8.1 Hz, 1H), 5.01 - 4.92 (m, 2H), 4.17 (m, 1H), 3.90 - 3.73 (m, 2H), 3.39 (d, J = 5.9 Hz, 1H), 2.84 (m, 2H), 2.37 (s, 1H), 2.21 - 2.10 (m, 2H), 2.10 - 1.82 (m, 4H), 1.77 - 1.57 (m, 4H), 1.50 - 1.31 (m, 2H), 1.32 (s, 3H), 1.30 - 1.14 (m, 3H), 1.00 (s, 3H), 0.99 - 0.88 (m, 1H), 0.80 (d, J = 6.9 Hz, 3H), 0.56 (d, J = 6.8 Hz, 3H).

[0096] Example 8 Preparation of 14-O-[3-((2S)-2,5-diaminopentanamido)phenyl]thioacetylmurin (Compound 8)

[0097]

[0098] Step 1: Preparation of Intermediate 8A

[0099] Weigh 150 mg of (2S)-2,5-bis[(tert-butoxycarbonyl)amino]pentanoic acid (0.45 mmol) and 203 mg of HATU (0.53 mmol), dissolve them in 5 mL of dichloromethane, stir at room temperature for 30 min, add 200 mg of Intermediate Ⅲ-B (0.41 mmol), and 85 μL of triethylamine (0.62 mmol). After stirring at room temperature for 2 h, quench the reaction mixture with 10 mL of saturated ammonium chloride aqueous solution, separate and recover the organic phase. Extract the aqueous phase three times with 5 mL of dichloromethane each time. Combine the organic phases, dry over anhydrous sodium sulfate, and purify by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain 282 mg of Intermediate 8A with a yield of 86%. ESI-MS (m / z): 800.37 [M+H] + .

[0100] Step 2: Preparation of Compound 8

[0101] Dissolve Intermediate 8A in 2 mL of dichloromethane, add 0.5 mL of 4 M hydrochloric acid in 1,4-dioxane dropwise to the above solution, stir at room temperature for 2 h, then remove the organic solvent by rotary evaporation under reduced pressure. Purify by reverse-phase column chromatography and obtain 156 mg of off-white solid Compound 8 after freeze-drying with a yield of 74%. ESI-MS (m / z): 600.33 [M+H] + . 1 H NMR (400 MHz, DMSO-d 6)δ 7.77 (t, J = 1.9 Hz, 1H), 7.52 (dd, J = 8.1, 2.0 Hz, 1H), 7.27 (t, J = 7.9 Hz, 1H), 7.08 (dt, J = 8.0, 1.3 Hz, 1H), 6.04 (dd, J = 17.7, 11.3 Hz, 1H), 5.50 (d, J = 8.2 Hz, 1H), 5.01 - 4.95 (m, 1H), 4.94 (s, 1H), 4.55 (d, J = 5.8 Hz, 1H), 4.16 (t, J = 6.6 Hz, 1H), 3.84 (d, J = 15.7 Hz, 1H), 3.77 (d, J = 16.1 Hz, 1H), 3.40 (m, 1H), 2.83 (m, 2H), 2.37 (s, 1H), 2.16 (m, 1H), 2.09 - 1.85 (m, 5H), 1.73 (m, 2H), 1.63 (m, 2H), 1.52 - 1.31 (m, 2H), 1.32 (s, 3H), 1.30 - 1.09 (m, 3H), 1.09 - 0.92 (m, 1H), 1.00 (s, 3H), 0.80 (d, J = 6.9 Hz, 3H), 0.56 (d, J = 6.8 Hz, 3H).

[0102] Example 9: Preparation of 14 - O - [4 - (2 - aminoacetamido)phenyl]thioacetyl moxalactam (Compound 9)

[0103]

[0104] Step 1: Preparation of Intermediate 9A

[0105] Weigh 70 mg of N - (tert - butoxycarbonyl) - glycine (0.45 mmol), dissolve 203 mg of HATU (0.53 mmol) in 5 mL of dichloromethane, stir at room temperature for 30 min, add 200 mg of Intermediate III - C (0.41 mmol), and 85 μL of triethylamine (0.62 mmol). After stirring at room temperature for 2 h, quench the reaction mixture with 10 mL of saturated ammonium chloride aqueous solution, separate and recover the organic phase. Extract the aqueous phase three times with 5 mL of dichloromethane each time. Combine the organic phases, dry over anhydrous sodium sulfate, and purify by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain 190 mg of Intermediate 9A with a yield of 72%. ESI - MS (m / z): 643.33 [M + H] + .

[0106] Step 2: Preparation of Compound 9

[0107] Intermediate 9A (2 mL) was dissolved in dichloromethane, and 0.5 mL of 4 M hydrochloric acid 1,4-dioxane was added dropwise to the above solution. After stirring at room temperature for 2 h, the organic solvent was removed by rotary evaporation under vacuum. Purification was performed using reverse-phase column chromatography, and 107 mg of off-white solid compound 9 was obtained after freeze-drying, with a yield of 67%. ESI-MS (m / z): 543.29 [M+H] + . 1 H NMR (400 MHz, Methanol-d4) δ 7.52 (d, J = 8.7 Hz, 2H), 7.36 (d, J = 8.7 Hz, 2H), 6.18 (dd, J = 17.6, 11.2 Hz, 1H), 5.64 (d, J = 8.3 Hz, 1H), 5.06 (d, J = 11.1 Hz, 1H), 5.01 (d, J = 17.6 Hz, 1H), 3.81 (s, 2H), 3.61 (d, J = 15.2 Hz, 1H), 3.54 (d, J = 15.3 Hz, 1H), 3.41 (d, J = 6.2 Hz, 1H), 2.31 - 2.16 (m, 3H), 2.12 (m, 1H), 1.98 (m, 1H), 1.69 - 1.59 (m, 2H), 1.56 - 1.46 (m, 2H), 1.34 (s, 3H), 1.30 - 1.23 (m, 2H), 1.16 - 1.07 (m, 2H), 1.05 (s, 3H), 0.88 (d, J = 7.0 Hz, 6H), 0.62 (d, J = 6.7 Hz, 3H).

[0108] Example 10: Preparation of 14-O-[4-((2R)-2-aminopropionamido)phenyl]thioacetyl moxilin (Compound 10)

[0109]

[0110] Step 1: Preparation of Intermediate 10A

[0111] 85 mg of N-(tert-butoxycarbonyl)-D-alanine (0.45 mmol) and 203 mg of HATU (0.53 mmol) were dissolved in 5 mL of dichloromethane and stirred at room temperature for 30 min. 200 mg of Intermediate III-C (0.41 mmol) and 85 μL of triethylamine (0.62 mmol) were added. After stirring at room temperature for 2 h, 10 mL of saturated ammonium chloride aqueous solution was added to quench the reaction mixture. The organic phase was separated and recovered, and the aqueous phase was extracted three times with 5 mL of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate and purified by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain 242 mg of Intermediate 10A, with a yield of 90%. ESI-MS (m / z): 657.25 [M+H] + .

[0112] Step 2: Preparation of Compound 10

[0113] Dissolve Intermediate 10A in 2 mL of dichloromethane. Add 0.5 mL of 4 M hydrochloric acid 1,4 - dioxane dropwise to the above - mentioned solution. After stirring at room temperature for 2 h, remove the organic solvent by rotary evaporation under vacuum. Purify by reverse - phase column chromatography and obtain 135 mg of off - white solid Compound 10 after freeze - drying, with a yield of 66%. ESI - MS (m / z): 557.40 [M + H] + . 1 H NMR (400 MHz, DMSO - d 6 ) δ 7.59 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.5 Hz, 2H), 6.09 - 6.00 (dd, J = 17.4, 11.0 Hz, 1H), 5.50 (d, J = 8.2 Hz, 1H), 4.98 (m, 1H), 4.95 (s, 1H), 3.88 (q, J = 7.0 Hz, 1H), 3.77 (d, J = 15.5 Hz, 1H), 3.71 (d, J = 15.5 Hz, 1H), 3.32 (m, 1H), 2.36 (s, 1H), 2.18 (m, 1H), 2.11 - 1.95 (m, 3H), 1.68 - 1.55 (m, 2H), 1.48 - 1.35 (m, 3H), 1.31 (s, 3H), 1.27 - 1.24 (m, 1H), 1.22 (d, J = 6.5 Hz, 3H), 1.19 - 1.06 (m, 2H), 1.00 (s, 3H), 0.80 (d, J = 6.9 Hz, 3H), 0.56 (d, J = 6.8 Hz, 3H).

[0114] Example 11: Preparation of 14 - O - [4 - ((2S)-2 - aminopropanamido)phenyl]thioacetyl moxilin (Compound 11)

[0115]

[0116] Step 1: Preparation of Intermediate 11A

[0117] Weigh 85 mg of N-(tert-butoxycarbonyl)-L-alanine (0.45 mmol), dissolve 203 mg of HATU (0.53 mmol) in 5 mL of dichloromethane, stir at room temperature for 30 min, add 200 mg of intermediate Ⅲ-C (0.41 mmol), and 85 μL of triethylamine (0.62 mmol). After stirring at room temperature for 2 h, add 10 mL of saturated ammonium chloride aqueous solution to quench the reaction solution. Separate and recover the organic phase, extract the aqueous phase three times with 5 mL of dichloromethane each time. Combine the organic phases, dry over anhydrous sodium sulfate, and purify by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain 221 mg of intermediate 11A with a yield of 82%. ESI-MS (m / z): 657.24 [M+H] + .

[0118] Step 2 Preparation of Compound 11:

[0119] Dissolve intermediate 11A in 2 mL of dichloromethane, add 0.5 mL of 4 M hydrochloric acid in 1,4-dioxane dropwise to the above solution, stir at room temperature for 2 h, then remove the organic solvent by rotary evaporation under vacuum. Purify by reverse-phase column chromatography and obtain 154 mg of off-white solid compound 11 after freeze-drying, with a yield of 77%. ESI-MS (m / z): 557.13 [M+H] + . 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.59 (d, J = 8.3 Hz, 2H), 7.32 (d, J = 8.3 Hz, 2H), 6.05 (dd, J = 17.6, 11.2 Hz, 1H), 5.50 (d, J = 8.2 Hz, 1H), 4.99 (m, 1H), 4.96 (s, 1H), 4.50 (s, 1H), 3.75 (d, J = 15.4 Hz, 1H), 3.69 (d, J = 15.4 Hz, 1H), 3.50 (q, J = 6.9 Hz, 1H), 3.39 (m, 1H) 2.36 (s, 1H), 2.17 (m, 1H), 2.11 - 1.93 (m, 3H), 1.67 - 1.54 (m, 2H), 1.50 - 1.32 (m, 2H), 1.31 (s, 3H), 1.28 - 1.25 (m, 1H), 1.23 (d, J = 6.8 Hz, 3H), 1.21 - 1.13 (m, 1H), 1.10 (d, J = 15.9 Hz, 1H), 1.00 (s, 3H), 0.98 - 0.92 (m, 1H), 0.80 (d, J = 6.9 Hz, 3H), 0.56 (d, J = 6.8 Hz, 3H).

[0120] Example 12: Preparation of 14-O-[4-((2R)-2-amino-4-methylpentanamido)phenyl]thioacetylmorpholine (Compound 12)

[0121]

[0122] Step 1: Preparation of Intermediate 12A

[0123] Weigh 104 mg of N-(tert-butoxycarbonyl)-D-leucine (0.45 mmol) and 203 mg of HATU (0.53 mmol), dissolve them in 5 mL of dichloromethane, stir at room temperature for 30 min, add 200 mg of Intermediate III-C (0.41 mmol) and 85 μL of triethylamine (0.62 mmol). After stirring at room temperature for 2 h, quench the reaction solution with 10 mL of saturated ammonium chloride aqueous solution, separate and recover the organic phase, extract the aqueous phase three times with 5 mL of dichloromethane each time. Combine the organic phases, dry over anhydrous sodium sulfate, and purify by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain 220 mg of Intermediate 12A with a yield of 77%. ESI-MS (m / z): 699.21 [M+H] + .

[0124] Step 2: Preparation of Compound 12

[0125] Dissolve Intermediate 12A in 2 mL of dichloromethane, add 0.5 mL of 4 M hydrochloric acid in 1,4-dioxane dropwise to the above solution, stir at room temperature for 2 h, then remove the organic solvent by rotary evaporation under vacuum. Purify by reverse-phase column chromatography and obtain 151 mg of off-white solid Compound 12 after freeze-drying with a yield of 80%. ESI-MS (m / z): 599.75 [M+H] + . 1 1H NMR (400 MHz, DMSO) δ 7.61 (d, J = 8.5 Hz, 2H), 7.34 (d, J = 8.7 Hz, 2H), 6.06 (dd, J = 17.9, 10.7 Hz, 1H), 5.52 (d, J = 7.9 Hz, 1H), 5.03 - 4.94 (m, 2H), 4.51 (d, J = 5.9 Hz, 1H), 3.78 (d, J = 15.3 Hz, 1H), 3.71 (d, J = 15.5 Hz, 1H), 3.45 (m, 1H), 2.38 (s, 1H), 2.25 - 1.90 (m, 4H), 1.78 - 1.56 (m, 3H), 1.56 - 1.36 (m, 4H), 1.33 (s, 3H), 1.29 - 1.20 (m, 3H), 1.11 (d, J = 15.9 Hz, 1H), 1.01 (s, 3H), 0.91 (m, 6H), 0.82 (d, J = 7.0 Hz, 3H), 0.58 (d, J = 6.8 Hz, 3H).

[0126] Example 13: Preparation of 14-O-[4-((2S)-2-amino-4-methylpentanamido)phenyl]thioacetyl moxilin (Compound 13)

[0127]

[0128] Step 1: Preparation of Intermediate 13A

[0129] Weigh 104 mg of N-(tert-butoxycarbonyl)-L-leucine (0.45 mmol) and 203 mg of HATU (0.53 mmol), dissolve them in 5 mL of dichloromethane, stir at room temperature for 30 min, add 200 mg of Intermediate III-C (0.41 mmol) and 85 μL of triethylamine (0.62 mmol). After stirring at room temperature for 2 h, add 10 mL of saturated ammonium chloride aqueous solution to quench the reaction solution. Separate and recover the organic phase, extract the aqueous phase three times with 5 mL of dichloromethane each time. Combine the organic phases, dry over anhydrous sodium sulfate, and purify by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain 189 mg of Intermediate 13A with a yield of 66%. ESI-MS (m / z): 699.40 [M+H] + .

[0130] Step 2: Preparation of Compound 13

[0131] Dissolve Intermediate 13A in 2 mL of dichloromethane, add 0.5 mL of 4 M hydrochloric acid 1,4-dioxane dropwise to the above solution, stir at room temperature for 2 h, then remove the organic solvent by rotary evaporation under vacuum. Purify by reverse-phase column chromatography and obtain 76 mg of off-white solid Compound 13 after freeze-drying, with a yield of 47%. ESI-MS (m / z): 599.26 [M+H] + . 1 1H NMR (400 MHz, DMSO-d 6)δ 7.63 - 7.56 (m, 2H), 7.32 (d, J = 8.7 Hz, 2H), 6.10 - 5.98 (m, 1H), 5.50 (d, J = 8.2 Hz, 1H), 5.01 - 4.92 (m, 2H), 4.50 (d, J = 6.0 Hz, 1H), 3.75 (d, J = 15.4 Hz, 1H), 3.69 (d, J = 15.4 Hz, 1H), 3.47 (m, 1H), 3.38 (m, 1H), 2.36 (s, 1H), 2.24 - 2.12 (m, 1H), 2.12 - 1.92 (m, 3H), 1.78 - 1.67 (m, 1H), 1.67 - 1.55 (m, 2H), 1.54 - 1.34 (m, 4H), 1.31 (s, 3H), 1.29 - 1.20 (m, 3H), 1.09 (d, J = 15.9 Hz, 1H), 0.99 (s, 3H), 0.90 (d, J = 6.6 Hz, 3H), 0.88 (d, J = 6.5 Hz, 3H), 0.80 (d, J = 6.9 Hz, 3H), 0.56 (d, J = 6.8 Hz, 3H).

[0132] Example 14: Preparation of 14 - O - [4 - ((2R)-2,5 - diamino - pentanamido)phenyl]thioacetyl - moxilin (Compound 14)

[0133]

[0134] Step 1: Preparation of Intermediate 14A

[0135] Weigh 150 mg of (2R)-2,5 - bis[(tert - butoxycarbonyl)amino]valeric acid (0.45 mmol) and 203 mg of HATU (0.53 mmol), dissolve them in 5 mL of dichloromethane, stir at room temperature for 30 min, add 200 mg of Intermediate III - C (0.41 mmol) and 85 μL of triethylamine (0.62 mmol). After stirring at room temperature for 2 h, add 10 mL of saturated ammonium chloride aqueous solution to quench the reaction solution. Separate and recover the organic phase, extract the aqueous phase three times with 5 mL of dichloromethane each time. Combine the organic phases, dry over anhydrous sodium sulfate, and purify by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain 278 mg of Intermediate 14A with a yield of 85%. ESI - MS (m / z): 800.62 [M + H] + .

[0136] Step 2: Preparation of Compound 14

[0137] Dissolve intermediate 14A in 2 mL of dichloromethane. Add 0.5 mL of 4 M hydrochloric acid in 1,4-dioxane dropwise to the above solution. After stirring at room temperature for 2 h, remove the organic solvent by rotary evaporation under vacuum. Purify using reverse-phase column chromatography and obtain 130 mg of off-white solid compound 14 after freeze-drying, with a yield of 62%. ESI-MS (m / z): 600.55 [M+H] + . 1 H NMR (500 MHz, Methanol-d4) δ 7.60 (d, J = 8.3 Hz, 2H), 7.40 (d, J = 8.3 Hz, 2H), 6.21 (dd, J = 17.6, 11.2 Hz, 1H), 5.68 (d, J = 8.4 Hz, 1H), 5.10 (d, J = 11.0 Hz, 1H), 5.06 (d, J = 17.6 Hz, 1H), 4.07 (m, 1H), 3.64 (d, J = 15.3 Hz, 1H), 3.58 (d, J = 15.2 Hz, 1H), 3.45 (d, J = 6.1 Hz, 1H), 2.99 (m, 2H), 2.36 - 2.19 (m, 3H), 2.16 - 1.99 (m, 4H), 1.86 - 1.76 (m, 3H), 1.73 - 1.40 (m, 4H), 1.37 (s, 3H), 1.33 - 1.22 (m, 2H), 1.22 - 1.13 (m, 2H), 1.10 (s, 3H), 0.91 (d, J = 7.0 Hz, 3H), 0.66 (d, J = 6.6 Hz, 3H).

[0138] Example 15: Preparation of 14-O-[4-((2S)-2,5-diaminopentanamido)phenyl]thioacetyl moxilin (Compound 15)

[0139]

[0140] Step 1: Preparation of intermediate 15A

[0141] Weigh 150 mg of (2S)-2,5-bis[(tert-butoxycarbonyl)amino]valeric acid (0.45 mmol) and 203 mg of HATU (0.53 mmol) and dissolve them in 5 mL of dichloromethane. Stir at room temperature for 30 min, add 200 mg of intermediate III-C (0.41 mmol) and 85 μL of triethylamine (0.62 mmol). After stirring at room temperature for 2 h, quench the reaction mixture with 10 mL of saturated ammonium chloride aqueous solution. Separate and recover the organic phase, extract the aqueous phase three times with 5 mL of dichloromethane each time. Combine the organic phases, dry over anhydrous sodium sulfate, and purify by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain 242 mg of intermediate 15A, with a yield of 74%. ESI-MS (m / z): 800.07 [M+H] + .

[0142] Step 2 Preparation of Compound 15:

[0143] Dissolve intermediate 15A in 2 mL of dichloromethane. Add 0.5 mL of 4 M hydrochloric acid in 1,4 - dioxane dropwise to the above - mentioned solution. After stirring at room temperature for 2 h, remove the organic solvent by rotary evaporation under vacuum. Purify by reverse - phase column chromatography and obtain 125 mg of off - white solid compound 15 after freeze - drying, with a yield of 69%. ESI - MS (m / z): 600.45[M + H] + .

[0144] Example 16: Preparation of 14 - O - [4 - ((2R)-2,5 - diamino - 1,5 - dioxopentanamido)phenyl]thioacetylmurin (Compound 16)

[0145]

[0146] Step 1 Preparation of Intermediate 16A:

[0147] Weigh 111 mg of N - (tert - butoxycarbonyl)-D - glutamine (0.45 mmol) and 203 mg of HATU (0.53 mmol), dissolve them in 5 mL of dichloromethane, stir at room temperature for 30 min, add 200 mg of intermediate III - C (0.41 mmol) and 85 μL of triethylamine (0.62 mmol). After stirring at room temperature for 2 h, add 10 mL of saturated ammonium chloride aqueous solution to quench the reaction mixture. Separate and recover the organic phase, extract the aqueous phase three times with 5 mL of dichloromethane each time. Combine the organic phases, dry over anhydrous sodium sulfate and purify by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain 213 mg of intermediate 16A, with a yield of 73%. ESI - MS (m / z): 714.37[M + H] + . Step 2 Preparation of Compound 16:

[0148] Dissolve intermediate 16A in 2 mL of dichloromethane. Add 0.5 mL of 4 M hydrochloric acid in 1,4 - dioxane dropwise to the above - mentioned solution. After stirring at room temperature for 2 h, remove the organic solvent by rotary evaporation under vacuum. Purify by reverse - phase column chromatography and obtain 158 mg of off - white solid compound 16 after freeze - drying, with a yield of 86%. ESI - MS (m / z): 614.51[M + H] + . 1H NMR (400 MHz, DMSO - d 6)δ 7.64 - 7.54 (m, 2H), 7.32 (d, J = 8.8 Hz, 2H), 6.05 (dd, J = 17.0, 11.1 Hz, 1H), 5.50 (d, J = 8.5 Hz, 1H), 5.03 - 4.95 (m, 1H), 4.95 (s, 1H), 4.50 (d, J = 6.0 Hz, 1H), 3.76 (d, J = 15.4 Hz, 1H), 3.69 (d, J = 15.4 Hz, 1H), 3.47 (t, J = 6.5 Hz, 1H), 3.38 (d, J = 5.7 Hz, 1H), 2.37 (s, 1H), 2.24 - 2.11 (m, 3H), 2.10 - 1.95 (m, 3H), 1.75 (m, 1H), 1.68 - 1.54 (m, 2H), 1.50 - 1.33 (m, 2H), 1.31 (s, 3H), 1.26 - 1.18 (m, 3H), 1.16 - 1.06 (m, 1H), 1.01 (s, 3H), 0.80 (d, J = 6.9 Hz, 3H), 0.56 (d, J = 7.2 Hz, 3H).

[0149] Example 17: Preparation of 14 - O - [4 - ((2S) - 2,5 - diamino - 1,5 - dioxopentanamido)phenyl]thioacetyl moxalactam (Compound 17)

[0150]

[0151] Step 1: Preparation of Intermediate 17A

[0152] Weigh 111 mg of N - (tert - butoxycarbonyl) - L - glutamine (0.45 mmol) and 203 mg of HATU (0.53 mmol), dissolve them in 5 mL of dichloromethane, stir at room temperature for 30 min, add 200 mg of Intermediate III - C (0.41 mmol) and 85 μL of triethylamine (0.62 mmol). After stirring at room temperature for 2 h, add 10 mL of saturated ammonium chloride aqueous solution to quench the reaction mixture. Separate and recover the organic phase, extract the aqueous phase three times with 5 mL of dichloromethane each time. Combine the organic phases, dry over anhydrous sodium sulfate, and purify by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain 208 mg of Intermediate 17A with a yield of 71%. ESI - MS (m / z): 714.38 [M + H] + . Step 2: Preparation of Compound 17

[0153] Dissolve intermediate 17A in 2 mL of dichloromethane. Add 0.5 mL of 4 M hydrochloric acid 1,4-dioxane dropwise to the above solution. After stirring at room temperature for 2 h, remove the organic solvent by rotary evaporation under vacuum. Purify using reverse-phase column chromatography and obtain 111 mg of off-white solid compound 17 after freeze-drying, with a yield of 62%. ESI-MS (m / z): 614.83 [M+H] + . 1H NMR (400 MHz, DMSO) δ 7.64 (dd, J = 8.8, 3.0 Hz, 2H), 7.37 (dd, J = 8.9, 2.0 Hz, 2H), 6.08 (dd, J = 17.7, 11.2 Hz, 1H), 5.53 (d, J = 8.1 Hz, 1H), 5.14 - 4.91 (m, 2H), 4.54 (d, J = 7.3 Hz, 1H), 3.80 (d, J = 15.6 Hz, 1H), 3.73 (d, J = 15.6 Hz, 1H), 3.41 (t, J = 5.9 Hz, 1H), 2.39 (s, 1H), 2.29 - 2.23 - 1.97 (m, 7H), 1.65 (m, 2H), 1.52 - 1.37 (m, 2H), 1.34 (s, 3H), 1.29 - 1.23 (m, 3H), 1.16 (d, J = 15.6 Hz, 1H), 1.03 (s, 3H), 0.88 (t, J = 6.5 Hz, 2H), 0.82 (d, J = 6.9 Hz, 3H), 0.59 (d, J = 6.8 Hz, 3H).

[0154] In vitro antibacterial experiment

[0155] Experimental method

[0156] The microbroth dilution method was used to evaluate the in vitro antibacterial activity of the compound against different strains. The specific operation steps are as follows: Place the sterilized 96-well plate in the laminar flow hood. Add 200 μL of the bacterial solution diluted with CAMHB broth medium to the first column (about 5×10 5(CFU / mL). 100 μL of the bacterial suspension was added to each well starting from the second column. 4 μL of the test sample solution dissolved in DMSO at a concentration of 6.4 mg / mL was added to each well in the first column in sequence. Meanwhile, a blank control group (without sample addition) and a positive drug group (tiamulin at a concentration of 6.4 mg / mL) were set up, and each sample was repeated three times. After sample addition, a 8-channel micropipette was used to take 100 μL of the sample from the previous column and add it to the next column for 2-fold serial dilution. When adding the sample to the last well, after adding the sample and mixing evenly, 100 μL of the liquid was aspirated again. The compound concentrations in each well after dilution were 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625 μg / mL respectively. The 96-well plate was placed in an incubator at 37 °C for 18 - 20 h, and the growth status of bacteria in each well was observed. For each compound, starting from the well where the sample was added and counting backward, the compound concentration corresponding to the first well where no bacterial growth was seen was the minimum inhibitory concentration (MIC) of the compound. The strains used in the experiment were 8 strains of Staphylococcus aureus, 5 strains of Streptococcus pneumoniae, 6 strains of Staphylococcus epidermidis, Escherichia coli ATCC25922, and Acinetobacter baumannii ATCC19606.

[0157] The following table shows the MIC test results

[0158] Table - 1 In vitro antibacterial data of compounds

[0159]

[0160]

[0161] The pleuromutilin derivatives linked by aminophenylthioether with amino acid fragments generally showed antibacterial activity close to or better than that of the marketed drugs against Staphylococcus aureus, and showed better antibacterial activity against Escherichia coli than other reported C22 - phenylthioether substituted derivatives.

[0162] Table - 2 In vitro antibacterial data of compound 10 and Lefamulin against standard and drug - resistant strains

[0163]

[0164]

[0165] The antibacterial activity of compound 10 against Gram - negative bacteria was comparable to that of the marketed drug Lefamulin, better than other reported C22 - phenylthioether substituted derivatives, and its antibacterial activity against standard and drug - resistant Staphylococcus aureus and standard and drug - resistant Staphylococcus epidermidis was better than that of the marketed drug Lefamulin.

[0166] Determination of compound cytotoxicity by MTT method

[0167] Experimental method

[0168] Use 0.5% trypsin to digest human cells in the logarithmic growth phase, add serum to terminate the digestion reaction, obtain cell precipitate by centrifugation, and pipette and mix with PBS medium to obtain cell suspension. Add 100 μL of bacterial suspension to each well of a sterile 96-well plate to make the density of the cells to be tested 5000 - 10000 / well, and fill the edge wells with sterile PBS. Culture in an incubator with 5% carbon dioxide at 37 °C until the cell monolayer covers the bottom of the well, add a series of compound solutions with gradient dilution, and repeat each sample three times. Culture in an incubator with 5% carbon dioxide at 37 °C for 24 h. After the drug action is completed, add 20 μL of MTT solution (5 mg / mL) to each well, and continue to culture in the cell incubator for 4 - 6 h. Terminate the culture, carefully aspirate the culture medium in the well. Add 150 μL of DMSO to each well, place it on a shaker and shake at a low speed for 10 min to completely dissolve the crystals, and use an enzyme-linked immunosorbent detector to measure the absorbance at OD490nm of each well. At the same time, set up zero-adjustment wells and control wells.

[0169] Table - 5 Cytotoxicity determination of Compound 10

[0170]

[0171]

[0172] The above embodiments are preferred implementation methods of the present invention, but the implementation methods of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A pleuromutilin derivative with an amino acid fragment shown in formula (I) or a pharmaceutically acceptable salt thereof: Wherein, selected from Selected from n is selected from one of 0, 1, 2, 3; R is selected from hydrogen, methyl, isopropyl, amino, carbamoyl, guanidyl.

2. The pleuromutilin derivative with an amino acid fragment or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The selected from; 3. The pleuromutilin derivative with an amino acid fragment according to any one of claims 1-2, characterized in that, the compound is selected from the following: 。 4. A preparation method of the pleuromutilin derivative with an amino acid fragment according to any one of claims 1-3, characterized in that, comprises the following steps: (1) React pleuromutilin with p-toluenesulfonyl chloride to obtain intermediate (II); (2) Use intermediate (II) as a raw material and react with o / m / p-aminothiophenol to obtain intermediate (III); (3) Use intermediate (III) as a raw material and react with various N-Boc-amino acids for 3 h in the presence of HATU condensing agent and triethylamine to obtain intermediate (IV); (4) Use intermediate (IV) as a raw material and remove the protecting group in 4M HCl 1,4-dioxane solution to obtain a pleuromutilin derivative with an amino acid fragment having the structure shown in formula (I); The reaction in step (1) uses dichloromethane as a solvent, triethylamine as an acid-binding agent, and reacts at room temperature for 8 h. The molar ratio of p-toluenesulfonyl chloride to pleuromutilin is 1.1:1; The reaction in step (2) uses N,N-dimethylformamide as a solvent, adds intermediate (II) and o / m / p-aminothiophenol, uses potassium carbonate as an acid-binding agent, and reacts at 60 °C for 4 h. The molar ratio of intermediate (II) to o / m / p-aminothiophenol is 1:1.3; The reaction in step (3) uses dichloromethane as a solvent, adds various N-Boc-amino acids, and reacts with intermediate (III) at room temperature for 6 h under the action of HATU condensing agent, triethylamine and a catalytic amount of DMAP. The molar ratio of intermediate (III) to N-Boc-amino acid is 1:1.2; The reaction in step (4) uses dichloromethane as a solvent, adds 4M HCl 1,4-dioxane solution, and reacts with intermediate (IV) at room temperature for 4 h. The molar ratio of intermediate (IV) to hydrochloric acid is 1:4, to obtain the target product with the structure shown in formula (I), which can be purified by recrystallization or column chromatography. 。 5. A pharmaceutical composition, which contains a therapeutically effective amount of one or more pleuromutilin derivatives with an amino acid fragment according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof as the main active ingredient, and pharmaceutically acceptable excipients.

6. Use of a pleuromutilin derivative having an amino acid fragment or a pharmaceutically acceptable salt thereof according to any one of claims 1-3 in the preparation of a medicament for treating infectious diseases, wherein the infectious diseases are infections caused by Staphylococcus aureus, drug-resistant Staphylococcus aureus, Staphylococcus epidermidis, drug-resistant Staphylococcus epidermidis, Streptococcus pneumoniae, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycoplasma pneumoniae or Chlamydia pneumoniae, or infectious diseases caused by multi-drug resistant bacteria.

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