Pleuromutilin derivatives, processes for their preparation and use thereof

Abstract

The present application relates to a pleuromutilin derivative with the structure shown in formula (I) and its preparation method and application. The pleuromutilin derivative of the present application shows very excellent antibacterial activity to clinically common gram-positive bacteria (including methicillin-resistant Staphylococcus aureus) and gram-negative bacteria (including Klebsiella pneumoniae), and has the excellent characteristics of broad-spectrum antibacterial, most of the compounds of the present application have stronger antibacterial activity than the marketed drug lefamulin, and are not easy to produce drug resistance. In addition, the pleuromutilin derivative of the present application has no obvious hemolysis and cytotoxicity to mammalian cells, has good safety and drugability, and has great potential for clinical application. (I)

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to antibacterial drugs, specifically to truncated pleurotin derivatives, their preparation methods, and their applications in antibacterial applications. Background Technology

[0002] Community-acquired bacterial pneumonia (CABP) is a significant clinical and economic burden among infectious diseases. With increasing resistance to existing antimicrobial agents in bacteria such as Staphylococcus aureus, there is a need to develop novel drugs with entirely new chemical structures and mechanisms of action to treat these infections.

[0003] Pleuromutilin, a tricyclic diterpenoid antibacterial drug isolated in the 1950s from the higher fungi *Pleurotus mutilus* and *Pleurotus passeckerianus*, possesses a novel chemical structure, a unique mechanism of action, and is less prone to cross-resistance with other classes of antibacterial drugs, offering hope for the treatment of drug-resistant bacteria. However, pleuromutilin antibiotics have low antibacterial activity and poor water solubility. For many years, researchers have continuously modified its structure, resulting in several optimized pleuromutilin derivatives that not only improve antibacterial activity but also enhance its water solubility and pharmacokinetic properties. Since its discovery, four derivatives have been approved for marketing by the US FDA: tiamulin, vornimulin, retamorin, and lefamorin.

[0004] Tiamulin, a truncated pleurotin derivative developed in 1974, is a veterinary antibacterial drug used to treat gastrointestinal and respiratory infections in pigs. Valnemulin, the second truncated pleurotin derivative developed in 1984, has 10-100 times greater antibacterial activity than tiamulin and is mainly used for the prevention or treatment of swine dysentery, swine endemic pneumonia, and swine spirochetosis. Retapamulin, the first truncated pleurotin drug approved for topical human use by the US FDA and Europe in 2007, is mainly used to treat skin impetigo, skin infections, and infections of small skin lesions, and is limited to topical application. Lefamorin was approved for marketing in 2019, primarily for the treatment of community-acquired bacterial pneumonia (CABP) in adults aged 18 years and older. Lefamorin is the first novel semi-synthetic truncated pleurotin drug that can be administered systemically in humans. It can effectively combat common pathogenic Gram-positive bacteria, Gram-negative bacteria, and atypical pathogens associated with CABP. After more than 70 years of unremitting efforts, the modification of truncated pleurotin has not only enabled it to leap from veterinary antibacterial drugs to human use, but also from local administration to systemic administration, indicating that truncated pleurotin has great application prospects in the field of new drug development.

[0005] Nevertheless, the number of antimicrobial drugs currently available to combat drug-resistant bacteria remains very limited, and the development process for antimicrobial drugs is extremely slow. Some antimicrobial compounds in clinical trials are terminated in preclinical stages due to toxicity issues, and the number of new antimicrobial drugs approved for marketing is very small. Summary of the Invention

[0006] Based on this, the purpose of this invention is to develop a novel truncated pleurotin derivative to obtain an antibacterial compound with stronger antibacterial activity, better safety, lower tendency to develop drug resistance, and the ability to combat drug-resistant bacteria.

[0007] The technical solutions for achieving the above objectives include the following.

[0008] In a first aspect, the present invention provides a truncated pleurotin derivative having the structure shown in formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0009]

[0010] (I)

[0011] Where R is -L-R1;

[0012] L is selected from: C1-C6 alkylene, C1-C6 alkylene-O-, C1-C6 alkylene-S-;

[0013] R1 is selected from: one or more R2-substituted phenyl groups;

[0014] Each R2 is independently selected from: cyano, nitro, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkyl acyl, halogen-substituted C1-C6 alkyl, and at least one R2 is cyano or nitro.

[0015] In some of these embodiments, L is selected from: C1-C3 alkylene, C1-C3 alkylene-O-, C1-C3 alkylene-S-.

[0016] In some of these embodiments, L is selected from: -CH2-, -CH2-CH2-, -CH2-O-, -CH2-CH2-O-.

[0017] In some of these embodiments, R2 is selected from: cyano, nitro, halogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkyl acyl, halogen-substituted C1-C3 alkyl.

[0018] In some of these embodiments, R2 is selected from: cyano, nitro, fluorine, chlorine, bromine, methyl, ethyl, methoxy, ethoxy, acetyl, and trifluoromethyl.

[0019] In some embodiments, R1 is selected from:

[0020]

[0021] Each n is independently selected from: 0, 1, 2, preferably 0 or 1;

[0022] Each m is independently selected from: 0, 1, 2;

[0023] Each of R3 and R4 is independently selected from: cyano, nitro, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkyl acyl, and halogen-substituted C1-C6 alkyl.

[0024] In some of these embodiments, each of R3 and R4 is independently selected from: cyano, nitro, halogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkyl acyl, halogen-substituted C1-C3 alkyl.

[0025] In some of these embodiments, each of R3 and R4 is independently selected from: cyano, nitro, fluorine, chlorine, bromine, methyl, ethyl, methoxy, ethoxy, acetyl, and trifluoromethyl.

[0026] In some of these embodiments, each R3 is independently selected from: fluorine, chlorine, bromine, methoxy, and ethoxy.

[0027] In some of these embodiments, each R4 is independently selected from: nitro, fluorine, chlorine, bromine, methyl, ethyl, methoxy, ethoxy, and acetyl.

[0028] In some embodiments, R1 is selected from:

[0029]

[0030] .

[0031] Secondly, the present invention provides a method for preparing the truncated pleurotin derivative, comprising the following steps:

[0032] The pleurotin derivative is obtained by reacting lefamoline acetate with the compound of formula (II).

[0033] The reaction formula is as follows:

[0034]

[0035] Where X is -Br or -Cl; R is as described above.

[0036] In some embodiments, the reaction is carried out in an organic solvent, preferably acetonitrile.

[0037] In some embodiments, the molar ratio of the lefamoline acetate to the compound of formula (II) is 1:1.2-2.5.

[0038] In some embodiments, the reaction is carried out in the presence of potassium carbonate and potassium iodide.

[0039] In some embodiments, the molar ratio of lefamoline acetate, potassium carbonate, and potassium iodide is preferably 1:1.5-2.5:1.5-2.5.

[0040] In some of these embodiments, the reaction temperature is 80°C-90°C.

[0041] In some embodiments, the reaction time is 2 to 8 hours.

[0042] Thirdly, the present invention provides the use of the truncated pleurotin derivative or its stereoisomer or its pharmaceutically acceptable salt in the preparation of medicaments for killing bacteria, inhibiting bacterial growth, preventing bacterial infection, or treating bacterial infection.

[0043] In some embodiments, the bacteria are Gram-positive and / or Gram-negative bacteria.

[0044] In some embodiments, the bacteria are Staphylococcus or Enterobacter.

[0045] In some embodiments, the bacteria are Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, or Klebsiella pneumoniae.

[0046] Fourthly, the present invention provides an antibacterial drug prepared from an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises the truncated pleurotin derivative or its stereoisomer or its pharmaceutically acceptable salt as described in the present invention.

[0047] The present invention has the following beneficial effects:

[0048] This invention prepares a series of truncated pleurotin derivatives with novel chemical structures, modified with nitroaryl or cyanoaryl side chains. These truncated pleurotin derivatives exhibit excellent in vivo and in vitro antibacterial activity against common clinical Gram-positive bacteria (including methicillin-resistant Staphylococcus aureus) and Gram-negative bacteria (including Klebsiella pneumoniae), demonstrating broad-spectrum antibacterial properties. Most of the compounds in this invention exhibit stronger antibacterial activity than the marketed drug lefamoline and are less prone to inducing drug resistance. Furthermore, the truncated pleurotin derivatives of this invention do not show significant hemolysis or cytotoxicity in mammalian cells, exhibiting good safety and drug-likeness, and possessing significant potential for clinical application. Attached Figure Description

[0049] Figure 1 The data represent the cytotoxicity of compounds LM43 (A) and lefamoline (B) on mouse fibroblasts; data represent the mean ± SD of two independent experiments.

[0050] Figure 2 Cytotoxicity of compounds LM43 (A) and lefamoline (B) to human embryonic kidney cells; data represent mean ± SD of two independent experiments.

[0051] Figure 3 The results show the drug resistance test results of compounds LM43, lefamoline, and norfloxacin against Staphylococcus aureus.

[0052] Figure 4 The in vivo antibacterial effect of compound LM43 in a mouse lung infection model is given by: A represents the efficacy of intravenous administration (15 mg / kg, n = 5); B represents the efficacy of oral administration (50 mg / kg, n = 5); ** P < 0.01, *** P < 0.001, **** P < 0.0001, and data represent mean ± SD. Detailed Implementation

[0053] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0054] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0055] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0056] Furthermore, as used herein, the term "or" is an inclusive "or" sign and is equivalent to the term "and / or" unless the context clearly specifies otherwise. The term "based on" is not exclusive and allows for basing on other factors not described unless the context clearly specifies otherwise. Additionally, throughout the specification, the meanings of "an," "a," and "the" include plural indicators. The meaning of "in" includes both "in" and "on."

[0057] In the compounds of this invention, when any variable (e.g., R2, etc.) appears more than once in any component, the definition of each occurrence is independent of the definitions of other occurrences. Similarly, combinations of substituents and variables are permitted, provided such combinations stabilize the compound. It will be understood that those skilled in the art can select the substituents and substitution patterns of the compounds of this invention to provide chemically stable compounds that can be easily synthesized from readily available starting materials using techniques in the art and the methods described below. If a substituent is itself substituted by more than one group, it should be understood that these groups can be on the same carbon atom or on different carbon atoms, as long as structural stability is achieved.

[0058] The term "substituted" refers to the replacement of a hydrogen group in a specific structure with a specified substituent.

[0059] The term "alkyl" refers to both branched and straight-chain saturated aliphatic hydrocarbon groups having a specific number of carbon atoms. For example, the definition of "C1-C6" in "C1-C6 alkyl" includes groups having 1, 2, 3, 4, 5, or 6 carbon atoms arranged in a straight or branched chain. Specifically, "C1-C6 alkyl" includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl.

[0060] The term "alkoxy" refers to a group in which an alkyl group is directly attached to an oxygen group, that is, a group with an -O-alkyl structure, such as -OCH3, -OCH2CH3, -OCH2CH2CH3, -OCH2CH(CH3)2, -OCH2CH2CH2CH3, -O-CH(CH3)2, etc.

[0061] This invention includes the free form of compounds of Formula I, as well as their pharmaceutically acceptable salts and stereoisomers. Certain exemplary compounds in this invention are amine compounds in non-salt form, i.e., "free form". Pharmaceutically acceptable salts of this invention can be synthesized from compounds of this invention containing a basic or acidic moiety using conventional chemical methods. Typically, salts of basic compounds are prepared by ion-exchange chromatography or by reacting a free base with a stoichiometric or excess amount of the desired salt form of an inorganic or organic acid in a suitable solvent or a combination of solvents. Similarly, salts of acidic compounds are formed by reacting with a suitable inorganic or organic base.

[0062] Therefore, pharmaceutically acceptable salts of the compounds of the present invention include conventional non-toxic salts of the compounds of the present invention formed by reacting an alkaline compound of the present invention with an inorganic or organic acid. For example, conventional non-toxic salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, nitric acid, etc., and also include salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pyric acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, hydroxyethylsulfonic acid, trifluoroacetic acid, etc.

[0063] If the compounds of this invention are acidic, then a suitable "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable non-toxic alkali, including inorganic and organic bases. Salts derived from inorganic bases include aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganese salts, manganese salts, potassium salts, sodium salts, zinc salts, etc. Ammonium salts, calcium salts, magnesium salts, potassium salts, and sodium salts are particularly preferred. Salts derived from pharmaceutically acceptable organic non-toxic bases, including salts of primary, secondary, and tertiary amines, wherein substituted amines include naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, hydroxycobalamin, isopropylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine, guanidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, aminobutanetriol, etc.

[0064] The stereoisomers described in this invention are (depending on their structure) enantiomers, diastereomers, syn- / anti-isomers, cis- / trans-isomers, epimers, and (E)- / (Z)-isomers. Compounds of Formula I can be used in the context of this invention as pure stereoisomers or as any mixture of stereoisomers, with racemic mixtures preferred in the latter case.

[0065] The antibacterial drug provided by the present invention is prepared from an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises the truncated pleurotin derivative or its stereoisomer or its pharmaceutically acceptable salt as described in the present invention.

[0066] The amount of active ingredients contained therein is within the safe and effective range. "Safe and effective range" means that the amount of active ingredients is sufficient to significantly improve the condition without causing serious side effects.

[0067] "Pharmaceutical acceptable excipients" refer to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity.

[0068] "Compatibility" here refers to the ability of the components in the composition or formulation to interact with and incorporate with the active ingredient of the present invention without significantly reducing the efficacy of the active ingredient.

[0069] Examples of pharmaceutically acceptable excipients include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween®), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0070] The administration method of the active ingredient, pharmaceutical composition, or pharmaceutical preparation of the present invention is not particularly limited. Representative administration methods include, but are not limited to, oral, transdermal, rectal, and parenteral (intravenous, intramuscular, or subcutaneous) administration. That is, the dosage form of the pharmaceutical preparation includes, but is not limited to, capsules, granules, tablets, pills, powders, drops, ointments, patches, liniments, sprays, powders, suppositories, sustained-release preparations, and injections.

[0071] Solid dosage forms for oral administration include capsules, granules, tablets, pills, and powders. In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or excipient or carrier), such as sodium citrate or dicalcium phosphate, or with the following components:

[0072] (a) Fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol and silica;

[0073] (b) Adhesives, such as hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic;

[0074] (c) Moisturizers, such as glycerin;

[0075] (d) Disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginate, certain complex silicates, and sodium carbonate;

[0076] (e) Slow solvents, such as paraffin;

[0077] (f) Absorption accelerators, for example, quaternary ammonium compounds;

[0078] (g) Wetting agents, such as cetyl alcohol and glyceryl monostearate;

[0079] (h) Adsorbents, such as kaolin; and

[0080] (i) Lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain a buffer.

[0081] The solid dosage form can also be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active ingredient from this composition can be delayed in a portion of the digestive tract. Examples of suitable encapsulating components are polymers and waxes.

[0082] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active ingredient, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. Besides these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0083] In addition to the active ingredient, the suspension may contain suspending agents, such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0084] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0085] The present invention will be further described in detail below with reference to specific embodiments.

[0086] The synthetic reaction formula for the truncated pleurotin derivative LM13-46 in the following examples is as follows:

[0087]

[0088] Where X is -Br or -Cl; R is:

[0089]

[0090]

[0091] Example 1: Preparation of compound LM13

[0092]

[0093] Lefamoline acetate (100.0 mg, 176.1 µmol) was weighed and dissolved completely in 6 mL of anhydrous acetonitrile. Then, 4-bromomethyl-3-chlorobenzonitrile (60.9 mg, 264.2 µmol), potassium carbonate (48.7 mg, 352.3 µmol), and potassium iodide (58.5 mg, 352.3 µmol) were added sequentially. The mixture was refluxed at 85 °C and reacted magnetically for 4 hours. After the reaction was completed, 100 mL of water was added to quench the reaction. The mixture was extracted three times with an equal volume of ethyl acetate. The organic phase was concentrated under reduced pressure and purified using a silica gel column (ethyl acetate / petroleum ether, 3:1, v / v). After vacuum drying, the target product LM13 (47.1 mg, 41%) was obtained as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.63 (s, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.53 (d, J = 7.9 Hz, 1H), 6.45 (dd, J = 17.3, 11.0 Hz, 1H), 5.75 (d, J =8.4 Hz, 1H), 5.33 (d, J = 11.0 Hz, 1H), 5.18 (d, J = 17.4 Hz, 1H), 3.97 –3.89 (m, 2H), 3.44 – 3.37 (m, 1H), 3.34 (d, J = 6.4 Hz, 1H), 3.23 (dd, J =37.3, 15.2 Hz, 2H), 2.66 – 2.54 (m, 2H), 2.35 – 2.18 (m, 4H), 2.15 – 1.91 (m,5H), 1.75 (d, J = 14.5 Hz, 1H), 1.68 – 1.35 (m, 11H), 1.30 – 1.22 (m, 3H), 1.16 – 1.09 (m, 4H), 0.86 (d, J = 6.8 Hz, 3H), 0.69 (d, J = 6.8 Hz, 3H). 13 CNMR (100 MHz, CDCl3) δ 217.21, 170.03, 139.07, 134.43, 132.76, 130.69,130.50, 117.59, 117.41, 112.32, 100.00, 74.66, 71.31, 69.85, 61.86, 58.23,54.20, 48.24, 45.53, 44.91, 43.97, 41.83, 36.79, 36.08, 34.55, 33.47, 30.48,26.91, 26.44, 24.92, 16.93, 14.97, 11.66. HRMS (ESI+): calculated forC 36 H 49 ClN2O5S [M + H] + 656.3051, found 657.3120.

[0094] Example 2 Preparation of compound LM14

[0095]

[0096] The starting materials used were lefamoline acetate (100.0 mg, 176.1 µmol) and 3-bromo-4-(bromomethyl)benzonitrile (72.6 mg, 264.2 µmol), and the synthesis followed the method for LM13. The target product LM14 (77.7 mg, 63%) was obtained as a white solid after vacuum drying. 1 H NMR (400 MHz, CD3OD) δ 7.99 (d, J = 1.5 Hz, 1H), 7.76 – 7.66 (m,2H), 6.32 (dd, J = 17.2, 11.5 Hz, 1H), 5.74 (d, J = 8.3 Hz, 1H), 5.22 – 5.18(m, 1H), 5.18 – 5.14 (m, 1H), 3.94 (s, 2H), 3.52 – 3.45 (m, 2H), 3.44 – 3.33(m, 1H), 3.29 – 3.24 (m, 1H), 2.66 – 2.55 (m, 2H), 2.39 – 1.98 (m, 8H), 1.85– 1.55 (m, 3H), 1.51 – 1.41 (m, 4H), 1.39 – 1.17 (m, 6H), 1.16 – 1.10 (m,4H), 0.93 (d, J = 7.0 Hz, 3H), 0.73 (d, J = 6.6 Hz, 3H). 13 C NMR (100 MHz, CD3OD) δ 219.66, 171.39, 146.26, 141.13, 136.98, 132.46, 131.76, 125.15,118.26, 116.67, 113.57, 75.45, 74.28, 71.21, 59.27, 55.65, 52.39, 51.20,46.80, 46.04, 45.23, 43.10, 38.14, 37.69, 35.29, 34.65, 33.10, 31.51, 30.47,28.22, 28.04, 25.84, 17.11, 15.44, 11.81. HRMS (ESI+): calculated forC 36 H 49 BrN2O5S [M + H] + 700.2546, found 701.2678.

[0097] Example 3 Preparation of compound LM15

[0098]

[0099] The starting materials used were lefamoline acetate (100.0 mg, 176.1 µmol) and 4-cyano-2-fluorobenzyl bromide (75.4 mg, 352.3 µmol), and the synthesis followed the method of LM13. After vacuum drying, the target product LM15 (45.4 mg, 40%) was obtained as a yellow solid. 1 H NMR (400 MHz, CDCl3) δ 7.54 – 7.48 (m, 1H), 7.45 – 7.40 (m, 1H), 7.34– 7.30 (m, 1H), 6.45 (dd, J = 17.4, 11.0 Hz, 1H), 5.84 – 5.71 (m, 1H), 5.33(dd, J = 11.0, 1.5 Hz, 1H), 5.23 – 5.13 (m, 1H), 3.89 (s, 2H), 3.44 – 3.32(m, 2H), 3.22 (dd, J = 36.9, 15.7 Hz, 2H), 2.66 – 2.54 (m, 2H), 2.37 – 1.91(m, 10H), 1.78 – 1.59 (m, 3H), 1.51 – 1.32 (m, 8H), 1.30 – 1.21 (m, 3H), 1.15– 1.07 (m, 4H), 0.86 (d, J = 7.0 Hz, 3H), 0.69 (d, J = 7.0 Hz, 3H). 13 C NMR(100 MHz, CDCl3) δ 217.22, 170.05, 161.71, 159.22, 139.04, 131.34, 128.48,119.27, 119.01, 117.48, 100.00, 74.67, 71.25, 69.87, 58.23, 53.99, 45.54,44.90, 43.98, 41.84, 36.80, 36.08, 34.56, 33.52, 30.48, 26.91, 26.41, 24.93,16.95, 14.97, 11.67. HRMS (ESI+): calculated for C 36 H 49 FN2O5S [M + H] + 640.3346, found 641.3418.

[0100] Example 4 Preparation of compound LM16

[0101]

[0102] The starting materials used were lefamoline acetate (100.0 mg, 176.1 µmol) and 4-cyano-3-fluorobenzyl bromide (56.6 mg, 264.2 µmol), and the synthesis followed the method for LM13. After vacuum drying, the target product LM16 (35.5 mg, 31%) was obtained as a yellow solid. 1 H NMR (400 MHz, CDCl3) δ 7.58 – 7.52 (m, 1H), 7.26 – 7.19 (m, 2H), 6.46 (dd, J = 17.4, 11.0 Hz, 1H), 5.76 (d, J = 8.4 Hz, 1H), 5.34 (d, J = 11.0 Hz,1H), 5.19 (d, J = 17.4 Hz, 1H), 3.92 – 3.82 (m, 2H), 3.45 – 3.33 (m, 2H),3.31 – 3.15 (m, 2H), 2.64 – 2.50 (m, 2H), 2.34 – 2.15 (m, 5H), 2.12 – 1.93(m, 5H), 1.79 – 1.58 (m, 4H), 1.48 – 1.38 (m, 5H), 1.31 – 1.24 (m, 5H), 1.18– 1.11 (m, 4H), 0.87 (d, J = 6.9 Hz, 3H), 0.70 (d, J = 7.0 Hz, 3H). 13 C NMR(100 MHz, CDCl3) δ 217.17, 170.06, 164.72, 139.13, 133.49, 124.20, 117.39,115.84, 115.65, 114.16, 99.80, 74.69, 71.40, 69.92, 58.25, 54.27, 50.34,45.55, 44.95, 44.01, 41.86, 36.81, 36.11, 34.55, 33.45, 30.50, 29.80, 29.42,26.93, 26.46, 24.94, 22.79, 16.93, 14.97, 14.24, 11.64. HRMS (ESI+):calculated for C 36 H 49FN2O5S [M + H] + 640.3346, found 641.3404.

[0103] Example 5 Preparation of compound LM17

[0104]

[0105] The starting materials used were lefamoline acetate (100.0 mg, 176.1 µmol) and 2-fluoro-5-(bromomethyl)benzonitrile (56.6 mg, 264.2 µmol), and the synthesis followed the method for LM13. The target product LM17 (40.4 mg, 36%) was obtained as a white solid after vacuum drying. 1 H NMR (400 MHz, CDCl3) δ 7.63 – 7.53 (m, 2H), 7.20 – 7.10 (m, 1H), 6.46 (dd, J = 17.4, 11.0 Hz, 1H), 5.76 (d, J = 8.4 Hz, 1H), 5.34 (dd, J =11.0, 1.5 Hz, 1H), 5.19 (dd, J = 17.4, 1.6 Hz, 1H), 3.87 – 3.73 (m, 2H), 3.47– 3.37 (m, 1H), 3.35 (d, J = 6.5 Hz, 1H), 3.24 (dd, J = 37.0, 15.6 Hz, 2H),2.65 – 2.54 (m, 2H), 2.34 – 2.18 (m, 4H), 2.16 – 1.92 (m, 5H), 1.80 – 1.72(m, 1H), 1.68 – 1.50 (m, 3H), 1.49 – 1.30 (m, 8H), 1.29 – 1.18 (m, 3H), 1.17– 1.07 (m, 4H), 0.87 (d, J = 7.0 Hz, 3H), 0.70 (d, J = 7.0 Hz, 3H). 13C NMR(100 MHz, CDCl3) δ 217.26, 170.06, 163.62, 161.05, 139.06, 134.83, 132.86,117.44, 116.48, 114.10, 101.32, 74.66, 71.35, 69.87, 58.23, 54.15, 49.64,45.53, 44.90, 43.98, 41.83, 36.80, 36.07, 34.55, 33.48, 30.47, 26.91, 26.43,24.92, 16.94, 14.97, 11.67. HRMS (ESI+): calculated for C 36 H 49 FN2O5S [M + H] + 640.3346, found 641.3493.

[0106] Example 6 Preparation of compound LM18

[0107]

[0108] Lefamoline acetate (100.0 mg, 176.1 µmol) was weighed and dissolved completely in 6 mL of anhydrous acetonitrile. Then, 2-cyano-5-fluorobenzyl bromide (56.6 mg, 264.2 µmol), potassium carbonate (48.7 mg, 352.3 µmol), and potassium iodide (58.5 mg, 352.3 µmol) were added sequentially. The mixture was refluxed at 85 °C and reacted magnetically for 4 hours. After the reaction was complete, 100 mL of water was added to quench the reaction. The mixture was extracted three times with an equal volume of n-butanol. The organic phase was concentrated under reduced pressure, and preparative reversed-phase high-performance liquid chromatography (RP-HPLC) was used for separation and purification. After vacuum drying, the target product LM18 (22.9 mg, 20%) was obtained as a white gel. 1H NMR(400 MHz, CD3OD) δ 8.16 – 8.09 (m, 1H), 7.55 – 7.48 (m, 1H), 7.46 – 7.37 (m,1H), 6.38 – 6.29 (m, 1H), 5.75 (d, J = 8.3 Hz, 1H), 5.22 – 5.16 (m, 2H), 4.26 – 4.08 (m, 1H), 3.66 – 3.47 (m, 3H), 3.35 (d, J = 15.1 Hz, 1H), 2.77 – 2.67(m, 1H), 2.41 – 2.30 (m, 3H), 2.28 – 2.10 (m, 5H), 2.08 – 2.02 (m, 1H), 1.86– 1.70 (m, 3H), 1.70 – 1.53 (m, 3H), 1.52 – 1.42 (m, 6H), 1.41 – 1.30 (m,2H), 1.19 – 1.12 (m, 4H), 0.94 (d, J = 7.1 Hz, 3H), 0.75 (d, J = 6.7 Hz, 3H). 13 C NMR (100 MHz, CD3OD) δ 219.74, 171.22, 168.84, 162.02, 141.27, 141.25,126.77, 126.66, 116.63, 101.32, 75.42, 74.08, 71.35, 71.32, 59.30, 53.77,46.81, 46.80, 46.02, 45.96, 45.29, 43.13, 39.80, 38.16, 37.73, 35.30, 31.50,30.71, 30.46, 28.27, 28.04, 25.83, 17.16, 15.43, 11.79. HRMS (ESI+):calculated for C 36 H 49 FN2O5S [M + H] + 640.3346, found 641.3417.

[0109] Example 7 Preparation of compound LM19

[0110]

[0111] The starting materials used were lefamoline acetate (100.0 mg, 176.1 µmol) and 4-(bromomethyl)-3,5-difluorobenzonitrile (61.3 mg, 264.2 µmol), and the synthesis followed the method for LM13. The target product LM19 (12.4 mg, 11%) was obtained as a yellow solid after vacuum drying. 1 H NMR (400 MHz, CDCl3) δ 7.26 – 7.22 (m, 2H), 6.47 (dd, J =17.4, 11.1 Hz, 1H), 5.85 – 5.74 (m, 1H), 5.35 (dd, J = 10.9, 1.0 Hz, 1H), 5.26 – 5.16 (m, 1H), 4.20 – 4.07 (m, 1H), 4.01 – 3.88 (m, 2H), 3.49 – 3.16(m, 3H), 2.69 – 2.57 (m, 2H), 2.35 – 2.21 (m, 4H), 2.18 – 1.95 (m, 6H), 1.81– 1.48 (m, 6H), 1.46 – 1.38 (m, 5H), 1.33 – 1.26 (m, 4H), 1.16 (s, 3H), 0.90 – 0.86 (m, 3H), 0.76 – 0.66 (m, 3H). 13 C NMR (100 MHz, CDCl3) δ 217.22,170.00, 164.43, 162.83, 162.74, 160.33, 160.23, 139.04, 117.45, 115.90,115.60, 74.68, 71.21, 69.86, 60.53, 58.23, 53.57, 45.53, 44.91, 43.98, 41.84,37.78, 36.80, 36.08, 34.55, 33.57, 30.49, 29.42, 26.91, 26.42, 24.93, 21.19,16.94, 14.97, 14.30, 11.66. HRMS (ESI+): calculated for C 36 H 48 F2N2O5S [M + H] + 658.3252, found 659.3327.

[0112] Example 8 Preparation of compound LM20

[0113]

[0114] The starting materials used were lefamoline acetate (100.0 mg, 176.1 µmol) and 4-bromomethylbenzonitrile (51.8 mg, 264.2 µmol), and the synthesis followed the method of LM18. After vacuum drying, the target product LM20 (51.9 mg, 47%) was obtained as a yellow solid. 1 H NMR (400 MHz, CDCl3) δ 7.59 (d, J = 8.1 Hz, 2H), 7.41 (d, J = 8.1 Hz, 2H), 6.45 (dd, J = 17.4, 11.0 Hz, 1H), 5.75 (d, J = 8.4 Hz, 1H), 5.33 (dd, J = 11.0, 1.4 Hz, 1H), 5.18 (dd, J = 17.4, 1.5 Hz, 1H), 3.90 – 3.80 (m, 2H), 3.46 – 3.37 (m, 1H), 3.34 (d, J = 6.5 Hz, 1H), 3.22 (dd, J = 36.4, 15.6 Hz, 2H), 2.70 – 2.51 (m, 3H), 2.34 – 2.28 (m, 1H), 2.26 – 2.17 (m, 3H), 2.15 –1.92 (m, 5H), 1.78 – 1.71 (m, 1H), 1.66 – 1.59 (m, 2H), 1.55 – 1.48 (m, 1H),1.47 – 1.42 (m, 4H), 1.39 – 1.29 (m, 3H), 1.27 – 1.20 (m, 2H), 1.14 (s, 3H),1.12 – 1.06 (m, 1H), 0.86 (d, J = 7.0 Hz, 3H), 0.69 (d, J = 7.0 Hz, 3H). 13CNMR (100 MHz, CDCl3) δ 217.20, 169.98, 145.85, 139.09, 132.40, 128.72,118.97, 117.38, 110.94, 74.66, 71.33, 69.84, 58.23, 54.06, 50.77, 45.53,44.92, 43.98, 41.83, 36.80, 36.08, 34.54, 33.50, 30.48, 26.91, 26.46, 24.92,16.91, 14.96, 11.64. HRMS (ESI+): calculated for C 36 H 50 N₂O₅S [M + H] + 622.3440, found 623.3522.

[0115] Example 9 Preparation of compound LM21

[0116]

[0117] The starting materials used were lefamoline acetate (100.0 mg, 176.1 µmol) and 3-cyanobenzyl bromide (51.8 mg, 264.2 µmol), and the synthesis followed the method of LM13. After vacuum drying, the target product LM21 (41.7 mg, 38%) was obtained as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.62 (s, 1H), 7.59 – 7.50 (m, 2H), 7.41 (t, J = 7.7Hz, 1H), 6.44 (dd, J = 17.4, 11.0 Hz, 1H), 5.74 (d, J = 8.3 Hz, 1H), 5.32 (d,J = 10.9 Hz, 1H), 5.17 (d, J = 17.4 Hz, 1H), 3.88 – 3.78 (m, 2H), 3.45 – 3.31(m, 2H), 3.30 – 3.15 (m, 2H), 2.67 – 2.54 (m, 2H), 2.34 – 2.15 (m, 4H), 2.13– 1.92 (m, 5H), 1.75 (d, J = 14.4 Hz, 1H), 1.68 – 1.49 (m, 3H), 1.45 – 1.22(m, 11H), 1.14 – 1.07 (m, 4H), 0.85 (d, J = 6.6 Hz, 3H), 0.69 (d, J = 6.8 Hz, 3H). 13 C NMR (100 MHz, CDCl3) δ 217.23, 170.01, 141.24, 139.07, 132.75,131.74, 131.07, 129.42, 118.90, 117.42, 112.60, 74.66, 72.40, 71.33, 69.85,61.83, 60.52, 58.23, 54.00, 50.20, 45.53, 44.91, 43.98, 41.83, 36.80, 36.08,34.55, 33.50, 30.48, 29.78, 26.91, 26.45, 24.92, 16.93, 14.96, 11.66. HRMS(ESI+): calculated for C 36 H 50 N₂O₅S [M + H] + 622.3440, found 623.3513.

[0118] Example 10 Preparation of compound LM22

[0119]

[0120] The starting materials used were lefamoline acetate (100.0 mg, 176.1 µmol) and 4-(bromomethyl)-3-methoxybenzonitrile (59.7 mg, 264.2 µmol), and the synthesis followed the method of LM18. The target product LM22 (52.0 mg, 45%) was obtained as a yellow solid after vacuum drying. 1 H NMR (400 MHz, CD3OD) δ 7.61 – 7.27 (m, 3H), 6.32 (dd, J =16.9, 11.7 Hz, 1H), 5.75 (d, J = 8.2 Hz, 1H), 5.22 – 5.10 (m, 2H), 4.27 –4.23 (m, 1H), 3.99 – 3.93 (m, 3H), 3.77 – 3.67 (m, 1H), 3.62 – 3.41 (m, 3H), 3.29 – 3.21 (m, 1H), 2.79 – 2.69 (m, 1H), 2.47 – 2.02 (m, 8H), 1.87 – 1.77(m, 1H), 1.74 – 1.27 (m, 13H), 1.20 – 1.11 (m, 4H), 0.94 (d, J = 7.0 Hz, 3H), 0.75 – 0.69 (m, 3H). 13 C NMR (100 MHz, CD3OD) δ 219.65, 171.11, 159.56,158.31, 141.25, 133.32, 129.68, 126.05, 116.61, 115.36, 101.33, 75.40, 73.01,71.30, 59.26, 56.87, 56.36, 51.00, 46.81, 46.01, 45.27, 45.20, 43.12, 38.14,37.73, 37.68, 35.30, 34.77, 31.50, 28.27, 28.04, 25.84, 17.16, 17.13, 15.43,11.84. HRMS (ESI+): calculated for C 37 H 52 N₂O₆S [M + H] + 652.3546, found653.3621.

[0121] Example 11 Preparation of compound LM23

[0122]

[0123] The starting materials used were lefamoline acetate (100.0 mg, 176.1 µmol) and 4-(2-bromoethoxy)benzonitrile (59.7 mg, 264.2 µmol), and the synthesis followed the method for LM13. The target product LM23 (44.5 mg, 39%) was obtained as a white solid after vacuum drying. 1 H NMR (400 MHz, CD3OD) δ 7.71 (d, J = 8.7 Hz, 2H), 7.16 (d, J = 8.8Hz, 2H), 6.32 (dd, J = 17.0, 11.7 Hz, 1H), 5.74 (d, J = 8.3 Hz, 1H), 5.20 (s,1H), 5.18 – 5.15 (m, 1H), 4.36 (s, 2H), 3.57 – 3.48 (m, 5H), 2.75 – 2.67 (m,1H), 2.46 – 2.31 (m, 3H), 2.28 – 2.11 (m, 5H), 1.85 – 1.78 (m, 1H), 1.76 –1.51 (m, 5H), 1.48 – 1.34 (m, 7H), 1.19 – 1.12 (m, 4H), 0.94 (d, J = 7.0 Hz, 3H), 0.74 (d, J = 6.6 Hz, 3H). 13 C NMR (100 MHz, CD3OD) δ 219.65, 171.11,162.74, 141.23, 135.32, 119.79, 116.65, 116.59, 105.87, 105.85, 75.40, 73.14,71.32, 65.19, 59.26, 56.25, 51.12, 46.79, 46.02, 45.29, 45.26, 43.10, 38.12,37.71, 35.29, 34.78, 31.49, 29.10, 28.96, 28.27, 28.02, 25.82, 17.12, 15.43,11.80. HRMS (ESI+): calculated for C 37 H 52 N₂O₆S [M + H] + 652.3546, found653.3609.

[0124] Example 12 Preparation of compound LM31

[0125]

[0126] The starting materials used were lefamoline acetate (100.0 mg, 176.1 µmol) and 1-(5-(bromomethyl)-2-nitrobenzene)acetone (68.2 mg, 264.2 µmol). The synthesis followed the method of LM18. After vacuum drying, the target product LM31 (39.3 mg, 33%) was obtained as a yellow gel. 1 H NMR (400 MHz, CDCl3) δ 8.05 (d, J = 8.4 Hz, 1H), 7.60 –7.50 (m, 1H), 7.37 (d, J = 1.7 Hz, 1H), 6.46 (dd, J = 17.4, 11.0 Hz, 1H), 5.76 (d, J = 8.4 Hz, 1H), 5.34 (dd, J = 11.0, 1.5 Hz, 1H), 5.19 (dd, J =17.4, 1.6 Hz, 1H), 3.99 – 3.84 (m, 2H), 3.45 – 3.15 (m, 4H), 2.67 – 2.49 (m,5H), 2.34 – 1.93 (m, 10H), 1.80 – 1.73 (m, 1H), 1.69 – 1.53 (m, 3H), 1.52 –1.35 (m, 7H), 1.33 – 1.18 (m, 4H), 1.17 – 1.08 (m, 4H), 0.87 (d, J = 7.0 Hz,3H), 0.70 (d, J = 7.0 Hz, 3H). 13 C NMR (100 MHz, CDCl3) δ 217.20, 200.39,170.07, 148.40, 144.43, 139.09, 138.53, 129.72, 126.47, 124.69, 117.41,74.67, 71.40, 69.91, 58.23, 54.47, 50.27, 45.53, 44.91, 43.99, 41.84, 36.80,36.08, 34.54, 33.44, 30.48, 30.43, 26.91, 26.43, 24.92, 16.93, 14.96, 11.64.HRMS (ESI+): calculated for C 37 H 52 N₂O₈S [M + H] +684.3444, found 685.3582.

[0127] Example 13 Preparation of compound LM32

[0128]

[0129] The starting materials used were lefamoline acetate (100.0 mg, 176.1 µmol) and 3-methyl-4-nitrobenzyl bromide (103.9 mg, 352.3 µmol), and the synthesis followed the method of LM18. After vacuum drying, the target product LM32 (60.8 mg, 53%) was obtained as a yellow solid. 1 H NMR (400 MHz, CD3OD) δ 7.97 (d, J = 8.3 Hz, 1H), 7.48 (s, 1H), 7.44 (d, J = 8.4 Hz, 1H), 6.32 (dd, J = 17.1, 11.6 Hz, 1H), 5.74 (d, J = 8.2Hz, 1H), 5.19 (s, 1H), 5.15 (d, J = 7.3 Hz, 1H), 4.00 (s, 2H), 3.54 – 3.40(m, 3H), 3.26 (s, 1H), 2.82 (s, 1H), 2.64 (t, J = 10.4 Hz, 1H), 2.58 (s, 3H),2.38 – 2.23 (m, 4H), 2.21 – 2.03 (m, 4H), 1.81 (d, J = 13.9 Hz, 1H), 1.75 –1.52 (m, 3H), 1.49 – 1.29 (m, 9H), 1.18 – 1.09 (m, 4H), 0.93 (d, J = 6.9 Hz, 3H), 0.73 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz, CD3OD) δ 219.67, 171.27,150.32, 141.19, 134.96, 134.45, 128.71, 126.00, 116.62, 75.42, 73.70, 71.28,59.26, 55.81, 51.77, 49.72, 46.80, 46.02, 45.25, 43.10, 38.13, 37.71, 35.29,34.67, 31.50, 31.05, 29.76, 29.11, 28.25, 28.03, 25.84, 20.27, 17.11, 15.42,11.81. HRMS (ESI+): calculated for C 36 H 52 N₂O₇S [M + H] + 656.3495, found657.3559.

[0130] Example 14 Preparation of compound LM33

[0131]

[0132] The starting materials used were lefamoline acetate (50.0 mg, 88.1 µmol) and 4,5-dimethoxy-2-nitrobenzyl bromide (48.6 mg, 176.1 µmol). The synthesis followed the method of LM18. After vacuum drying, the target product LM33 (33.9 mg, 55%) was obtained as a brown solid. 1H NMR (400 MHz, CDCl3) δ 7.60 (s, 1H), 7.19 – 6.90 (m, 1H), 6.45 (dd, J = 17.4, 11.0 Hz, 1H), 5.80 – 5.72 (m, 1H), 5.36 – 5.30 (m, 1H), 5.24 – 5.15 (m, 1H), 4.08 – 3.85 (m, 7H), 3.51 – 3.41 (m, 1H), 3.37 – 3.14 (m, 3H), 2.75 – 2.66 (m, 1H), 2.64 – 2.54 (m, 1H), 2.37 – 1.95 (m, 9H), 1.78 – 1.51(m, 5H), 1.46 – 1.19 (m, 11H), 1.18 – 1.09 (m, 4H), 0.86 (d, J = 6.9 Hz, 3H), 0.70 (d, J = 6.9 Hz, 3H). 13 C NMR (100 MHz, CD3OD) δ 219.71, 171.17, 155.25,151.34, 142.73, 141.21, 129.01, 122.80, 116.65, 109.66, 75.42, 73.23, 71.30,59.26, 57.21, 56.99, 56.93, 56.82, 51.23, 46.80, 46.02, 45.25, 45.20, 43.10,43.05, 38.14, 37.70, 37.65, 35.29, 34.79, 31.49, 28.24, 28.03, 25.83, 17.13,15.43, 11.81. HRMS (ESI+): calculated for C 37 H 54 N₂O₉S [M + H] + 702.3550, found703.3624.

[0133] Example 15 Preparation of compound LM34

[0134]

[0135] The starting materials used were lefamoline acetate (100.0 mg, 176.1 µmol) and 3-nitrobenzyl bromide (76.1 mg, 352.3 µmol), and the synthesis followed the method of LM18. After vacuum drying, the target product LM34 (11.5 mg, 10%) was obtained as a yellow gel. 1 HNMR (400 MHz, CDCl3) δ 8.19 (s, 1H), 8.11 (dd, J = 8.2, 1.3 Hz, 1H), 7.66 (d,J = 7.7 Hz, 1H), 7.49 (t, J = 7.9 Hz, 1H), 6.47 (dd, J = 17.4, 11.0 Hz, 1H), 5.77 (d, J = 8.4 Hz, 1H), 5.35 (dd, J = 11.0, 1.4 Hz, 1H), 5.20 (dd, J =17.4, 1.5 Hz, 1H), 3.92 (q, J = 13.8 Hz, 2H), 3.47 – 3.40 (m, 1H), 3.35 (d, J= 6.5 Hz, 1H), 3.24 (dd, J = 36.3, 15.6 Hz, 1H), 2.71 – 2.64 (m, 1H), 2.63 –2.52 (m, 1H), 2.34 – 2.20 (m, 4H), 2.16 – 2.06 (m, 3H), 2.03 – 1.97 (m, 1H), 1.80 – 1.72 (m, 1H), 1.67 – 1.53 (m, 3H), 1.48 – 1.43 (m, 4H), 1.32 – 1.24(m, 8H), 1.16 (s, 4H), 0.89 – 0.83 (m, 4H), 0.71 (d, J = 7.0 Hz, 3H). 13C NMR(100 MHz, CDCl3) δ 217.21, 170.05, 148.51, 142.36, 139.09, 134.33, 129.55,122.99, 122.38, 117.47, 74.72, 71.38, 69.90, 58.27, 54.20, 50.42, 45.57,44.96, 44.02, 41.87, 36.84, 36.12, 34.58, 33.52, 30.52, 29.82, 29.45, 26.95,26.44, 24.96, 22.81, 16.96, 15.00, 14.26, 11.67. HRMS (ESI+): calculated forC 35 H 50 N₂O₇S [M + H] + 642.3339, found 643.3413.

[0136] Example 16 Preparation of compound LM35

[0137]

[0138] The starting materials used were lefamoline acetate (100.0 mg, 176.1 µmol) and 4-nitrobenzyl bromide (76.1 mg, 352.3 µmol), and the synthesis followed the method of LM18. After vacuum drying, the target product LM35 (55.5 mg, 49%) was obtained as a yellow gel. 1HNMR (400 MHz, CDCl3) δ 8.17 (d, J = 8.7 Hz, 2H), 7.49 (d, J = 8.7 Hz, 2H), 6.46 (dd, J = 17.4, 11.0 Hz, 1H), 5.76 (d, J = 8.4 Hz, 1H), 5.34 (d, J = 11.0Hz, 1H), 5.19 (d, J = 17.4 Hz, 1H), 3.91 (q, J = 14.2 Hz, 2H), 3.45 – 3.39(m, 1H), 3.37 – 3.15 (m, 3H), 2.68 – 2.56 (m, 2H), 2.34 – 2.20 (m, 4H), 2.13– 1.97 (m, 5H), 1.79 – 1.73 (m, 1H), 1.68 – 1.51 (m, 4H), 1.47 – 1.36 (m,6H), 1.32 – 1.26 (m, 4H), 1.18 – 1.11 (m, 4H), 0.87 (d, J = 7.0 Hz, 3H), 0.71 (d, J = 7.0 Hz, 3H). 13 C NMR (100 MHz, CDCl3) δ 217.15, 169.98, 147.81,147.17, 139.06, 128.74, 123.80, 117.36, 74.65, 71.32, 69.85, 58.21, 54.12,50.43, 45.51, 44.90, 43.96, 41.82, 36.78, 36.06, 34.52, 33.46, 30.46, 29.76,29.38, 26.89, 26.41, 24.90, 22.76, 16.89, 14.94, 14.21, 11.61. HRMS (ESI+):calculated for C 35 H 50 N₂O₇S [M + H] + 642.3339, found 643.3407.

[0139] Example 17 Preparation of compound LM36

[0140]

[0141] The starting materials used were lefamoline acetate (100.0 mg, 176.1 µmol) and 2-nitrobenzyl bromide (76.1 mg, 352.3 µmol). The synthesis followed the method of LM18. After vacuum drying, the target product LM36 (65.2 mg, 58%) was obtained as a yellow solid. 1 HNMR (400 MHz, CDCl3) δ 7.95 (d, J = 8.0 Hz, 1H), 7.58 (d, J = 4.0 Hz, 2H), 7.45 – 7.39 (m, 1H), 6.47 (dd, J = 17.4, 11.0 Hz, 1H), 5.76 (d, J = 8.4 Hz,1H), 5.35 (d, J = 11.0 Hz, 1H), 5.20 (d, J = 17.4 Hz, 1H), 4.02 (s, 2H), 3.49– 3.42 (m, 1H), 3.38 – 3.14 (m, 3H), 2.72 – 2.58 (m, 2H), 2.36 – 2.31 (m,1H), 2.27 – 2.18 (m, 3H), 2.14 – 1.98 (m, 5H), 1.79 – 1.73 (m, 1H), 1.68 –1.52 (m, 4H), 1.47 – 1.43 (m, 4H), 1.26 (d, J = 16.9 Hz, 6H), 1.17 – 1.10 (m,4H), 0.87 (d, J = 7.0 Hz, 3H), 0.71 (d, J = 7.0 Hz, 3H). 13 C NMR (100 MHz, CDCl3) δ 217.16, 169.94, 149.21, 139.01, 135.15, 133.47, 131.70, 128.35,124.94, 117.46, 74.68, 71.33, 69.79, 58.24, 54.26, 48.33, 45.52, 44.92,43.97, 41.83, 36.80, 36.07, 34.53, 33.51, 30.48, 29.77, 29.39, 26.90, 26.41,24.92, 22.76, 16.90, 14.96, 14.21, 11.62. HRMS (ESI+): calculated forC 35 H 50 N₂O₇S [M + H] +642.3339, found 643.3412.

[0142] Example 18 Preparation of compound LM37

[0143]

[0144] The starting materials used were lefamoline acetate (100.0 mg, 176.1 µmol) and 3,5-dinitrobenzyl chloride (76.3 mg, 352.3 µmol). The synthesis followed the method of LM18. After vacuum drying, the target product LM37 (67.4 mg, 56%) was obtained as a yellow solid. 1 H NMR (400 MHz, CD3OD) δ 8.92 – 8.87 (m, 1H), 8.68 – 8.66 (m, 1H), 8.62– 8.43 (m, 1H), 6.41 – 6.20 (m, 1H), 5.79 – 5.62 (m, 1H), 5.25 – 5.06 (m,2H), 4.78 (s, 1H), 4.10 (s, 2H), 3.53 – 3.40 (m, 3H), 3.30 – 3.23 (m, 1H), 2.77 – 2.60 (m, 1H), 2.40 – 2.23 (m, 4H), 2.19 – 2.03 (m, 4H), 1.85 – 1.56(m, 4H), 1.49 – 1.39 (m, 5H), 1.37 – 1.25 (m, 4H), 1.16 – 1.10 (m, 4H), 0.95 – 0.91 (m, 3H), 0.74 – 0.68 (m, 3H). 13 C NMR (100 MHz, CD3OD) δ 219.67,171.34, 165.88, 149.94, 144.50, 141.15, 129.69, 128.56, 118.50, 116.64,75.45, 74.14, 71.26, 59.28, 57.43, 55.81, 52.29, 50.04, 46.80, 46.04, 45.25,43.10, 38.14, 37.69, 35.30, 34.68, 32.67, 32.64, 31.50, 30.35, 28.22, 28.03,25.83, 17.10, 15.44, 11.79. HRMS (ESI+): calculated for C 35 H49 N3O9S [M + H] + 687.3190, found 688.3261.

[0145] Example 19 Preparation of compound LM38

[0146]

[0147] The starting materials used were lefamoline acetate (100.0 mg, 176.1 µmol) and 4-bromo-3-nitrobenzyl bromide (103.9 mg, 352.3 µmol). The synthesis followed the method for LM18. After vacuum drying, the target product LM38 (103.4 mg, 81%) was obtained as a yellow gel. 1 H NMR (400 MHz, CDCl3) δ 7.83 (s, 1H), 7.66 (d, J = 8.2 Hz, 1H), 7.42 –7.38 (m, 1H), 6.47 (dd, J = 17.4, 11.0 Hz, 1H), 5.76 (d, J = 8.4 Hz, 1H),5.36 – 5.30 (m, 1H), 5.24 – 5.14 (m, 1H), 3.85 (q, J = 14.2 Hz, 2H), 3.43 –3.16 (m, 4H), 2.68 – 2.52 (m, 2H), 2.35 – 2.19 (m, 5H), 2.12 – 1.94 (m, 5H), 1.80 – 1.45 (m, 8H), 1.33 – 1.24 (m, 6H), 1.19 – 1.09 (m, 4H), 0.87 (d, J =7.0 Hz, 3H), 0.71 (d, J = 7.0 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 217.15,170.05, 149.90, 139.06, 135.04, 132.78, 125.01, 117.41, 112.69, 74.67, 71.36,69.88, 58.22, 54.22, 49.69, 45.52, 44.91, 43.97, 41.83, 36.79, 36.07, 34.53,33.41, 30.47, 29.78, 29.40, 26.90, 26.40, 24.91, 22.77, 16.91, 14.95, 14.22,11.62. HRMS (ESI+): calculated for C 35 H 49 BrN2O7S [M + H] + 720.2444, found721.2510.

[0148] Example 20 Preparation of compound LM39

[0149]

[0150] The starting materials used were lefamoline acetate (50.0 mg, 88.1 µmol) and 1-bromo-2-bromomethyl-3-nitrobenzene (51.9 mg, 176.2 µmol). The synthesis followed the method of LM18. After vacuum drying, the target product LM39 (34.4 mg, 54%) was obtained as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.96 – 7.92 (m, 1H), 7.88 – 7.83 (m, 1H), 7.42 (t, J = 8.1 Hz, 1H), 6.30 (dd, J = 17.2, 11.5 Hz, 1H), 5.73 (d, J = 8.3Hz, 1H), 5.18 (s, 1H), 5.16 – 5.13 (m, 1H), 4.11 (s, 2H), 3.51 – 3.39 (m,3H), 3.27 (s, 1H), 2.74 – 2.65 (m, 1H), 2.64 – 2.56 (m, 1H), 2.37 – 2.21 (m, 4H), 2.17 – 2.03 (m, 3H), 1.98 (d, J = 12.0 Hz, 1H), 1.79 (d, J = 14.4 Hz,1H), 1.72 – 1.51 (m, 3H), 1.47 – 1.41 (m, 4H), 1.37 – 1.08 (m, 9H), 0.91 (d,J = 7.0 Hz, 3H), 0.71 (d,J = 6.6 Hz, 3H). 13 C NMR (100 MHz, CD3OD) δ 219.67,171.34, 153.08, 141.10, 138.69, 133.76, 131.17, 128.04, 125.15, 116.71,75.45, 74.16, 71.21, 59.27, 56.52, 52.17, 47.86, 46.80, 46.02, 45.23, 43.10,38.14, 37.68, 35.29, 34.71, 31.50, 28.21, 28.04, 25.84, 17.11, 15.44, 11.82.HRMS (ESI+): calculated for C 35 H 49 BrN2O7S [M + H] + 720.2444, found 721.2500.

[0151] Example 21 Preparation of compound LM40

[0152]

[0153] Weigh 100.0 mg (176.1 µmol) of lefamoline acetate and dissolve it completely in 8 mL of anhydrous acetonitrile. Then add 2-bromomethyl-4-chloro-1-nitrobenzene (88.2 mg, 352.3 µmol), potassium carbonate (48.7 mg, 352.3 µmol), and potassium iodide (58.5 mg, 352.3 µmol) in sequence. The mixture is refluxed at 85 °C and reacted magnetically for 4 hours. After the reaction is complete, 100 mL of water is added to quench the reaction. The mixture is extracted three times with an equal volume of ethyl acetate. The organic phase is concentrated under reduced pressure to remove the organic solvent. The mixture is purified by silica gel chromatography (ethyl acetate / petroleum ether, 3:1, v / v) and dried under vacuum to obtain a yellow solid product LM40 (50.2 mg, 42%). 1 H NMR (400 MHz, CDCl3) δ 7.95 – 7.88 (m, 1H), 7.69 –7.62 (m, 1H), 7.39 – 7.33 (m, 1H), 6.51 – 6.36 (m, 1H), 5.83 – 5.71 (m, 1H),5.37 – 5.29 (m, 1H), 5.24 – 5.13 (m, 1H), 4.07 – 3.99 (m, 2H), 3.45 – 3.24(m, 3H), 3.23 – 3.14 (m, 1H), 2.65 – 2.48 (m, 3H), 2.34 – 2.17 (m, 4H), 2.13– 1.93 (m, 5H), 1.79 – 1.55 (m, 4H), 1.47 – 1.39 (m, 5H), 1.38 – 1.21 (m,5H), 1.16 – 1.08 (m, 4H), 0.87 – 0.82 (m, 3H), 0.73 – 0.67 (m, 3H). 13 C NMR(100 MHz, CDCl3) δ 217.26, 170.10, 147.13, 140.10, 139.00, 131.55, 128.53,126.57, 126.54, 117.56, 74.68, 71.35, 69.86, 58.24, 54.76, 47.92, 45.54,45.51, 44.91, 43.98, 41.84, 36.81, 36.08, 34.56, 33.44, 30.49, 26.92, 26.39,24.93, 16.96, 14.98, 11.68. HRMS (ESI+): calculated for C35 H 49 ClN2O7S [M + H] + 676.2949, found 677.3009.

[0154] Example 22 Preparation of compound LM41

[0155]

[0156] The starting materials used were lefamoline acetate (50.0 mg, 88.1 µmol) and 4-fluoro-2-nitrobenzyl bromide (41.2 mg, 176.1 µmol), and the synthesis followed the method of LM18. After vacuum drying, the target product LM41 (31.0 mg, 53%) was obtained as a yellow solid. 1 H NMR (400 MHz, CDCl3) δ 7.73 – 7.67 (m, 1H), 7.66 – 7.60 (m, 1H), 7.38 –7.29 (m, 1H), 6.47 (dd, J = 17.4, 11.0 Hz, 1H), 5.77 (d, J = 8.4 Hz, 1H),5.37 – 5.33 (m, 1H), 5.21 (d, J = 17.4 Hz, 1H), 4.02 (s, 1H), 3.48 – 3.40 (m,1H), 3.39 – 3.15 (m, 3H), 2.69 (s, 1H), 2.63 – 2.54 (m, 1H), 2.37 – 2.18 (m,5H), 2.11 – 1.97 (m, 5H), 1.77 (d, J = 14.6 Hz, 1H), 1.70 – 1.51 (m, 5H), 1.49 – 1.38 (m, 6H), 1.32 – 1.29 (m, 3H), 1.19 – 1.09 (m, 4H), 0.88 (d, J =6.9 Hz, 3H), 0.71 (d, J = 7.0 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 217.16,169.98, 139.03, 130.10, 129.81, 120.81, 120.60, 117.47, 112.66, 112.40,74.68, 71.32, 69.84, 58.24, 54.46, 47.75, 45.53, 44.92, 43.98, 41.84, 36.81,36.08, 34.54, 33.49, 31.99, 30.49, 29.78, 29.41, 26.91, 26.41, 24.92, 22.77,16.92, 14.96, 11.63. HRMS (ESI+): calculated for C 35 H 49 FN2O7S [M + H] + 660.3245, found 661.3325.

[0157] Example 23 Preparation of compound LM42

[0158]

[0159] The starting materials used were lefamoline acetate (100.0 mg, 176.1 µmol) and 4-fluoro-3-nitrobenzyl bromide (82.4 mg, 352.3 µmol). The synthesis followed the method of LM18. After vacuum drying, the target product LM42 (55.5 mg, 48%) was obtained as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.23 ​​(dd, J = 7.1, 2.2 Hz, 1H), 7.85 – 7.75 (m, 1H), 7.47 (dd, J = 10.9, 8.6 Hz, 1H), 6.32 (dd, J = 17.0, 11.7 Hz, 1H), 5.74 (d, J = 8.3 Hz, 1H), 5.20 (s, 1H), 5.16 (dd, J = 7.1, 1.6 Hz, 1H), 4.10 (s, 2H), 3.54 – 3.44 (m, 3H), 3.30 – 3.26 (m, 1H), 3.00 – 2.90 (m, 1H), 2.71– 2.61 (m, 1H), 2.40 – 2.31 (m, 3H), 2.30 – 2.22 (m, 1H), 2.21 – 2.09 (m,4H), 1.82 (d, J = 17.0 Hz, 1H), 1.74 – 1.51 (m, 3H), 1.50 – 1.45 (m, 4H), 1.43 – 1.32 (m, 5H), 1.16 (s, 4H), 0.94 (d, J = 7.1 Hz, 3H), 0.73 (d, J = 6.7Hz, 3H). 13 C NMR (100 MHz, CD3OD) δ 219.67, 171.32, 157.23, 154.63, 141.13,137.28, 127.22, 119.60, 119.39, 116.65, 75.42, 74.09, 71.19, 59.23, 55.53,52.12, 46.79, 45.98, 45.22, 43.08, 38.12, 37.68, 35.28, 34.67, 32.13, 31.49,30.19, 28.23, 28.03, 25.83, 17.12, 15.42, 11.84. HRMS (ESI+): calculated forC 35 H 49 FN2O7S [M + H] + 660.3245, found 661.3310.

[0160] Example 24 Preparation of compound LM43

[0161]

[0162] The starting materials used were lefamoline acetate (100.0 mg, 176.1 µmol) and 4-nitro-3-fluorobenzyl chloride (76.3 mg, 352.3 µmol). The synthesis followed the method of LM18. After vacuum drying, the target product LM43 (67.4 mg, 56%) was obtained as a yellow solid. 1 H NMR (400 MHz, CDCl3) δ 8.05 – 7.99 (m, 1H), 7.32 (d, J = 11.9 Hz, 1H), 7.24 (s, 1H), 6.47 (dd, J = 17.4, 11.0 Hz, 1H), 5.76 (d, J = 8.4 Hz, 1H),5.37 – 5.33 (m, 1H), 5.24 – 5.15 (m, 1H), 3.97 – 3.84 (m, 2H), 3.43 – 3.33(m, 2H), 3.24 (dd, J = 38.0, 15.7 Hz, 2H), 2.65 – 2.52 (m, 2H), 2.34 – 2.19(m, 5H), 2.13 – 2.05 (m, 3H), 2.03 – 1.95 (m, 2H), 1.79 – 1.74 (m, 1H), 1.69– 1.54 (m, 4H), 1.47 – 1.43 (m, 4H), 1.39 – 1.29 (m, 5H), 1.17 – 1.11 (m,4H), 0.87 (d, J = 7.0 Hz, 3H), 0.71 (d, J = 7.0 Hz, 3H). 13 C NMR (100 MHz, CDCl3) δ 217.19, 170.09, 157.12, 154.48, 139.05, 126.32, 126.29, 123.70,117.63, 117.40, 74.61, 71.30, 69.88, 58.20, 54.22, 49.94, 45.51, 44.88,43.95, 41.81, 36.77, 36.05, 34.52, 33.39, 31.98, 30.45, 29.40, 26.89, 26.38,24.90, 22.77, 16.92, 14.94, 14.23, 11.63. HRMS (ESI+): calculated forC 35 H 49FN2O7S [M + H] + 660.3245, found 661.3317.

[0163] Example 25 Preparation of compound LM44

[0164]

[0165] The starting materials used were lefamoline acetate (50.0 mg, 88.1 µmol) and 2-fluoro-6-nitrobenzyl bromide (41.2 mg, 176.1 µmol), and the synthesis followed the method of LM18. After vacuum drying, the target product LM44 (20.5 mg, 35%) was obtained as a yellow solid. 1 HNMR (400 MHz, CD3OD) δ 8.14 – 8.03 (m, 1H), 7.84 – 7.44 (m, 2H), 6.39 – 6.25 (m, 1H), 5.79 – 5.70 (m, 1H), 5.23 – 5.11 (m, 2H), 4.90 – 4.75 (m, 1H), 4.42(s, 1H), 3.72 (s, 1H), 3.60 – 3.46 (m, 3H), 3.30 – 3.21 (m, 1H), 2.69 (d, J =9.3 Hz, 1H), 2.49 – 2.31 (m, 3H), 2.30 – 2.04 (m, 5H), 1.82 (d, J = 14.6 Hz,1H), 1.75 – 1.56 (m, 3H), 1.52 – 1.42 (m, 6H), 1.39 – 1.28 (m, 2H), 1.21 –1.11 (m, 4H), 0.94 (d, J = 7.1 Hz, 3H), 0.76 – 0.69 (m, 3H). 13C NMR (100 MHz, CD3OD) δ 219.66, 171.26, 164.06, 161.58, 151.47, 141.17, 132.36, 122.46,122.22, 116.65, 75.44, 73.73, 71.26, 59.27, 56.67, 51.74, 46.80, 46.03,45.25, 43.11, 40.27, 40.22, 38.14, 37.71, 35.29, 34.75, 31.50, 31.09, 28.23,28.04, 25.84, 17.11, 15.52, 15.42, 11.81. HRMS (ESI+): calculated forC 35 H 49 FN2O7S [M + H] + 660.3245, found 661.3309.

[0166] Example 26 Preparation of compound LM45

[0167]

[0168] The starting materials used were lefamoline acetate (100.0 mg, 176.1 µmol) and 1-(bromomethyl)-2-fluoro-3-nitrobenzene (82.4 mg, 352.3 µmol). The synthesis followed the method of LM18. After vacuum drying, the target product LM45 (56.3 mg, 48%) was obtained as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.10 – 7.98 (m, 1H), 7.83 (t, J = 6.9Hz, 1H), 7.45 – 7.31 (m, 1H), 6.32 (dd, J = 16.9, 11.8 Hz, 1H), 5.74 (d, J =8.3 Hz, 1H), 5.25 – 5.10 (m, 2H), 4.81 – 4.59 (m, 1H), 4.06 (s, 2H), 3.52 –3.42 (m, 3H), 3.30 – 3.24 (m, 1H), 2.90 – 2.74 (m, 1H), 2.71 – 2.58 (m, 1H),2.40 – 2.23 (m, 4H), 2.20 – 2.02 (m, 4H), 1.88 – 1.55 (m, 4H), 1.49 – 1.30 (m, 9H), 1.17 – 1.09 (m, 4H), 0.97 – 0.90 (m, 3H), 0.78 – 0.69 (m, 3H). 13 CNMR (100 MHz, CD3OD) δ 219.67, 171.32, 156.23, 153.62, 141.18, 137.57,126.63, 125.78, 125.73, 116.64, 75.46, 73.95, 71.32, 59.30, 55.82, 52.10,46.81, 46.06, 45.27, 43.76, 43.71, 43.12, 38.15, 37.71, 35.30, 34.71, 31.94,31.51, 28.23, 28.04, 25.84, 17.09, 15.44, 11.77. HRMS (ESI+): calculated forC 35 H 49 FN2O7S [M + H] + 660.3245, found 661.3303.

[0169] Example 27 Preparation of compound LM46

[0170]

[0171] The starting materials used were lefamoline acetate (100.0 mg, 176.1 µmol) and 2-nitro-4-(trifluoromethyl)benzyl bromide (100.1 mg, 352.3 µmol), and the synthesis followed the method of LM18. After vacuum drying, the target product LM46 (42.0 mg, 34%) was obtained as a brown solid. 1 H NMR (400 MHz, CD3OD) δ 8.30 (s, 1H), 7.97 (dd, J = 23.6,8.1 Hz, 2H), 6.32 (dd, J = 17.1, 11.4 Hz, 1H), 5.75 (d, J = 8.3 Hz, 1H), 5.25– 5.11 (m, 2H), 4.15 (s, 2H), 3.59 – 3.36 (m, 3H), 3.30 – 3.21 (m, 1H), 2.73– 2.53 (m, 2H), 2.40 – 2.22 (m, 4H), 2.20 – 1.97 (m, 4H), 1.82 (d, J = 13.5Hz, 1H), 1.75 – 1.52 (m, 3H), 1.48 – 1.39 (m, 5H), 1.37 – 1.25 (m, 3H), 1.22– 1.08 (m, 5H), 0.93 (d, J = 6.9 Hz, 3H), 0.73 (d, J = 6.3 Hz, 3H). 13 C NMR(100 MHz, CD3OD) δ 219.65, 171.36, 150.56, 141.10, 140.27, 133.89, 131.79,130.73, 125.83, 122.95, 116.68, 75.44, 74.23, 71.20, 59.26, 56.13, 52.34,48.17, 46.79, 46.02, 45.22, 43.09, 38.13, 37.67, 35.29, 34.67, 32.93, 31.50,30.43, 28.21, 28.03, 25.83, 17.11, 15.44, 11.82. HRMS (ESI+): calculated forC 36 H 49 F3N2O7S [M + H] + 710.3213, found 711.3274.

[0172] Example 28: In vitro antibacterial activity and in vitro hemolytic activity

[0173] (1) The in vitro antibacterial activity of the truncated pleurotin derivatives synthesized in Examples 1-27 above against bacteria such as Staphylococcus aureus (including MRSA) and Escherichia coli was determined according to the micro-broth dilution method established by the Clinical Laboratory Standards Institute (CLSI). The antibacterial activity was represented by the lowest concentration (MIC) of the compound required to inhibit bacterial growth.

[0174] The strains used in this embodiment, *Staphylococcus aureus* ATCC29213 and *Klebsiella pneumoniae* Kneumoniae ATCC10031, were purchased from Guangdong Huankai Microbial Technology Co., Ltd. Methicillin-resistant *Staphylococcus aureus* MRSA N315 and *Methicillin-resistant *Staphylococcus aureus* MRSA NCTC 10442 were provided by the Affiliated Qingyuan Hospital of Guangzhou Medical University.

[0175] The specific procedure is as follows: The compound to be tested is dissolved in DMSO and ultrapure water to prepare a stock solution of 1 μg / μL, and then diluted with MHB to the initial concentration for testing. 100 μL of MHB is added to each well of a 96-well plate beforehand. 100 μL of the prepared initial concentration of the compound is added to the first row, and serial dilutions are performed using a two-fold dilution method. After dilution to the last row, the 100 μL liquid is discarded to obtain a series of test samples at various concentrations. The bacteria used for testing are cultured on MHA plates in a 37 ℃ oven for 18-24 h, and the bacterial concentration is adjusted to approximately 1×10⁻⁶ with PBS. 6 CFU / mL. Dilute the prepared bacterial suspension 100-fold with MHB, add 100 μL of bacterial suspension to each well of a 96-well plate and mix with the test compound, then incubate at 37 °C for 22–24 h. Measure the absorbance at 600 nm using a microplate reader. The lowest concentration at which 90% bacterial growth is inhibited is the MIC of the compound. The MIC experiment should be performed at least twice and should be biologically reproducible.

[0176] (2) The concentration of the compound required to lyse 50% of rabbit erythrocytes (HC) 50The in vitro hemolytic activity of the truncated pleurotin derivatives synthesized in Examples 1-27 against rabbit erythrocytes (RBCs) was evaluated. The test compound was prepared at 40 μg / μL with DMSO, and 10 μL was diluted in 990 μL of PBS to prepare a stock solution. A series of desired test concentrations were obtained using a two-fold dilution method in 96-well plates. Rabbit RBCs were centrifuged at 800 rpm for 4 min, washed three times with fresh PBS, and a suspension containing 8% (v / v) rabbit RBCs was prepared. 100 μL of the rabbit RBC suspension was added to each well of a 96-well plate and mixed with the test compound, and incubated at 37 °C for 1 h. The mixture in the 96-well plate was centrifuged at 2500 rpm for 5 min, and 100 μL of the supernatant was transferred to a clean 96-well plate. OD was measured using a microplate reader. 576 Triton X-100 was used as a positive control, and PBS as a negative control. Hemolytic activity was calculated using the following formula: Hemolytic activity (%) = [(Abs)] 化合物- –Abs 阴性对照 ) / (Abs 阳性对照 – Abs 阴性对照 )] × 100. The hemolysis experiment must be performed at least twice and be biologically reproducible.

[0177] (3) The results of in vitro antibacterial and hemolytic activity assays are shown in Table 1. Most of the truncated pleurotin derivatives synthesized in this invention exhibit excellent antibacterial activity. Most compounds show superior antibacterial activity against both Gram-positive and Gram-negative bacteria compared to lefamoline, and exhibit very low hemolytic activity against rabbit erythrocytes. HC 50 All values ​​were greater than 200 µg / mL. Among them, compounds LM23 and LM43 showed the best antibacterial activity, with MIC values ​​as low as 0.006-0.012 µg / mL against Gram-positive bacteria, which is 8-16 times higher than the marketed drug lefamoline, a truncated pleurotin derivative, and significantly superior to lefamoline; against the Gram-negative bacterium Klebsiella pneumoniae ATCC10031, its antibacterial activity was increased by 4-8 times (MICs = 0.024-0.048 µg / mL).

[0178] Table 1. In vitro MIC and HC values ​​of truncated pleurotin derivative LM13-46 50 value.

[0179]

[0180]

[0181]

[0182] Example 29 Cytotoxicity

[0183] Two mammalian cell lines, human embryonic kidney cells (Human Embryonic Kidney 293 Cells) and mouse fibroblasts (NCTC clone 929), were selected to evaluate the in vitro cytotoxicity of the compounds. The compounds were dissolved in DMSO and MEM or DMEM medium containing 10% (v / v) FBS. Test cells were seeded in 96-well plates and cultured at 37 °C for 24 hours in an oven containing 5% CO2, and then mixed with 100 μL of a series of different concentrations of the compound. The mixture in the 96-well plates was incubated at 37 °C for another 24 hours. After adding 10 μL of enhanced CCK-8 solution, the mixture was incubated at 37 °C for 1 hour. OD was measured using a Biotek microplate reader. 450 Simple culture medium and cell suspension without added compounds served as the blank group and control group, respectively. Cytotoxicity was calculated using the following formula: Cell viability (%) = [(Abs)] compounds -Abs blank ) / (Abs control - Abs blank [× 100.] Cytotoxicity assays should be performed at least twice and should be biologically reproducible.

[0184] The results are as follows Figure 1 and Figure 2 As shown: Compound LM43, at concentrations up to 1024 × MIC, still maintained a cell viability greater than 50% in mouse fibroblasts. Figure 1 (A in the text), and the cell survival rate of famolin at this concentration is less than 10% ( Figure 1 (B in the text); Compound LM43, at concentrations up to 1024 × MIC, showed a survival rate of 52.3% in human embryonic kidney cells ( ). Figure 2 (A) and famoline at this concentration resulted in a cell survival rate of 36.6% ( Figure 2 (B) indicates that the truncated pleurotin derivative of the present invention has very low toxicity to mammalian cells and has higher safety compared to the marketed drug lefamoline.

[0185] Example 30 Drug Resistance

[0186] In this embodiment, Staphylococcus aureus ATCC29213 was selected to test the resistance development trend of the test compounds. The initial MIC (minimal inhibitory concentration) values ​​of compounds LM43, lefamoline, and norfloxacin were measured using the broth microdilution method established in clinical and laboratory standard research. Then, bacterial cells of compounds LM43, lefamoline, and norfloxacin were taken from 96-well plates at a sub-inhibitory concentration (0.5 × MIC) to prepare bacterial suspensions (approximately 1 × 10⁻⁶). 6 The concentration of CFU / mL will be used for the next determination of the MIC value. The experimental period is 21 days.

[0187] The results are as follows Figure 3 As shown, resistance to the control drug norfloxacin develops very rapidly; its MIC value against Staphylococcus aureus increases eightfold after only 3-5 passages, reaching a maximum of 256 times. Lefamoline showed a 16-fold increase in MIC value after multiple passages, indicating resistance. In contrast, compound LM43 maintained a very stable MIC value after 20 consecutive passages, with an increase not exceeding four times. These results demonstrate that the truncated pleurotin derivative of this invention can overcome the development of resistance.

[0188] Example 31: In vivo antibacterial efficacy

[0189] The in vivo animal efficacy evaluation experiment was approved by the Experimental Animal Center of South China Agricultural University and followed the relevant policies formulated by the National Health Commission. This example used 6-8 week old C57BL / 6 female mice, weighing approximately 18 g, to test the in vivo antibacterial effect of compound LM43 using a Staphylococcus aureus-induced mouse lung infection model. Mice were randomly divided into three groups: a compound treatment group, a positive control group, and a negative control group. The test compound was dissolved in 5% glucose injection solution, with an oral administration concentration of 50 mg / kg and an intravenous administration concentration of 15 mg / kg. 10 μL (OD) was administered to mice via nasal drops. 600 = 1.4) A mouse pneumonia model was established using Staphylococcus aureus bacterial suspension. Treatment of each group (compound, control drug, 5% glucose injection) was started 1 hour after infection, twice a day. After 3 days of treatment, lung tissue of mice was taken and colony count was recorded using the plate count method.

[0190] The results are as follows Figure 4As shown, compared with the control group, the bacterial load in the lung tissue of pneumonia-infected mice treated with compound LM43 and lefamolin was significantly reduced (p < 0.01). At an intravenous dose of 15 mg / kg (A), compound LM43 showed superior efficacy compared to lefamolin, reducing the bacterial load in lung tissue by 4.2-log (**** P < 0.0001), while lefamolin reduced it by 3.4-log (**** P < 0.0001). At an oral dose of 50 mg / kg (B), compound LM43 showed superior efficacy compared to lefamolin, reducing the bacterial load in lung tissue by 3.2-log (*** P < 0.001), while lefamolin reduced it by 2.2-log (** P < 0.01). These results indicate that compound LM43 exhibits a stronger in vivo antibacterial effect than lefamolin and has good application prospects.

[0191] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A truncated pleurotin derivative having the structure shown in formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, （I） in, R is -L-R1; L is selected from: C1-C6 alkylene, C1-C6 alkylene-O-, C1-C6 alkylene-S-; R1 is selected from: one or more R2-substituted phenyl groups; Each R2 is independently selected from: cyano, nitro, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkyl acyl, halogen-substituted C1-C6 alkyl, and at least one R2 is cyano or nitro.

2. The truncated pleurotin derivative or its stereoisomer or its pharmaceutically acceptable salt according to claim 1, characterized in that, L is selected from: C1-C3 alkylene, C1-C3 alkylene-O-, C1-C3 alkylene-S-.

3. The truncated pleurotin derivative or its stereoisomer or its pharmaceutically acceptable salt according to claim 2, characterized in that, L is selected from: -CH2-, -CH2-CH2-, -CH2-O-, -CH2-CH2-O-.

4. The truncated pleurotin derivative or its stereoisomer or its pharmaceutically acceptable salt according to any one of claims 1-3, characterized in that, R2 is selected from: cyano, nitro, halogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkyl acyl, halogen-substituted C1-C3 alkyl.

5. The truncated pleurotin derivative or its stereoisomer or its pharmaceutically acceptable salt according to claim 4, characterized in that, R2 is selected from: cyano, nitro, fluorine, chlorine, bromine, methyl, ethyl, methoxy, ethoxy, acetyl, trifluoromethyl.

6. The truncated pleurotin derivative or its stereoisomer or its pharmaceutically acceptable salt according to any one of claims 1-3, characterized in that, R1 is selected from: Each n is independently selected from: 0, 1, and 2; Each m is independently selected from: 0, 1, 2; Each of R3 and R4 is independently selected from: cyano, nitro, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkyl acyl, and halogen-substituted C1-C6 alkyl.

7. The truncated pleurotin derivative or its stereoisomer or its pharmaceutically acceptable salt according to claim 6, characterized in that, Each of R3 and R4 is independently selected from: cyano, nitro, halogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkyl acyl, halogen-substituted C1-C3 alkyl.

8. The truncated pleurotin derivative or its stereoisomer or its pharmaceutically acceptable salt according to claim 7, characterized in that, Each R3 is independently selected from: fluorine, chlorine, bromine, methoxy, and ethoxy; Each R4 group is independently selected from: nitro, fluorine, chlorine, bromine, methyl, ethyl, methoxy, ethoxy, and acetyl.

9. The truncated pleurotin derivative or its stereoisomer or its pharmaceutically acceptable salt according to any one of claims 1-3, characterized in that, R1 is selected from: 。 10. The truncated pleurotin derivative or its stereoisomer or its pharmaceutically acceptable salt according to claim 1, characterized in that, The truncated pleurotin derivative is selected from the following compounds: 。 11. A method for preparing a truncated pleurotin derivative according to any one of claims 1-10, characterized in that, Includes the following steps: The pleurotin derivative is obtained by reacting lefamoline acetate with the compound of formula (II). The reaction formula is as follows: Wherein, X is -Br or -Cl; R is as described in any one of claims 1-10.

12. The preparation method according to claim 11, characterized in that, The reaction is carried out in an organic solvent; And / or, the molar ratio of the lefamoline acetate to the compound of formula (II) is 1:1.2-2.5; And / or, the reaction is carried out in the presence of potassium carbonate and potassium iodide; And / or, the temperature of the reaction is 80℃-90℃; And / or, the reaction time is 2 to 8 hours.

13. The preparation method according to claim 12, characterized in that, The reaction is carried out in acetonitrile solvent; And / or, the reaction is carried out in the presence of potassium carbonate and potassium iodide, wherein the molar ratio of lefamoline acetate, potassium carbonate and potassium iodide is 1:1.5-2.5:1.5-2.

5.

14. The use of the truncated pleurotin derivative or its stereoisomer or its pharmaceutically acceptable salt as described in any one of claims 1-10 in the preparation of a bactericidal drug, a drug for inhibiting bacterial growth, a drug for preventing bacterial infection, or a drug for treating bacterial infection; wherein the bacteria are Gram-positive and / or Gram-negative bacteria.

15. The application according to claim 14, characterized in that, The bacteria mentioned are Staphylococcus and Enterobacter.

16. The application according to claim 15, characterized in that, The bacteria mentioned are Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Klebsiella pneumoniae.

17. An antibacterial drug, characterized in that, It is prepared from an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises a truncated pleurotin derivative or its stereoisomer or a pharmaceutically acceptable salt thereof as described in any one of claims 1-10.

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