Honokiol-antibacterial peptide mimic conjugate containing oxazolethione structure as well as preparation method and antibacterial application of honokiol-antibacterial peptide mimic conjugate

Abstract

The invention discloses a series of honokiol-antibacterial peptide mimic conjugates containing an oxazolethione structure as well as a preparation method and antibacterial application of the honokiol-antibacterial peptide mimic conjugates. The series of compounds take a natural product honokiol as a parent structure, a nitrogen-containing heterocyclic ring is introduced, and then the compound is connected with a hydrophilic small-molecule peptidomimetic fragment, so that a series of honokiol-antibacterial peptide mimic conjugates containing an oxazolethione structure are prepared, and the structural general formula of the honokiol-antibacterial peptide mimic conjugates is shown as a formula (1). The series of compounds have relatively strong in vivo and in vitro antibacterial activity on Staphylococcus aureus ATCC 29213 and clinically isolated methicillin-resistant Staphylococcus aureus (Methicillin-Resistance S. aureus, MRSA), and can be used for preparing the antibacterial agent for treating the staphylococcus aureus, the antibacterial agent for treating the staphylococcus aureus, the antibacterial agent for treating the methicillin-resistant Staphylococcus aureus and the antibacterial agent for treating the methicillin-resistant Staphylococcus aureus, the antibacterial agent for treating the staphylococcus aureus, the antibacterial agent for treating the staphylococcus aureus and the antibacterial agent for treating the staphylococcus aureus. Through structural optimization, the water solubility and antibacterial activity of the compound are enhanced, and the compound has relatively low in-vivo and in-vitro toxicity, hemolytic activity and low drug resistance. Therefore, the honokiol-antibacterial peptide mimic conjugates containing the oxazolethione structure have the potential of being further developed into novel antibacterial drugs. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemistry, and in particular relates to a honokiol-antimicrobial peptide mimetic conjugate containing an oxazolethione structure, a preparation method thereof and antibacterial application thereof. Background Art

[0002] Antibiotics are mainly used to treat various bacterial infections, but due to overuse and misuse of antibiotics, bacteria have developed resistance to currently approved drugs (A. Asadi, S. Razavi, M. Talebi, M. Gholami, A review on anti-adhesion therapies of bacterial diseases [J]. Infection, 2019, 07: 13-23.). Staphylococcus aureus (S. aureus) is a round Gram-positive bacterium that can cause a variety of diseases, ranging from skin and soft tissue infections to severe pneumonia, sepsis and meningitis. At present, more than 60% of Staphylococcus aureus isolates are resistant to methicillin (methicillin-resistant Staphylococcus aureus / MRSA). The drug resistance of MRSA is due to the gene encoding PBP2a in the body. PBP2a has ultra-low β-lactam affinity and can synthesize cell walls at normally lethal β-lactam concentrations to complete bacterial reproduction and growth. Therefore, MRSA is also known as a superbug (N. Koyama, J. Inokoshi, H. Tomoda, Anti-infectious agents against MRSA [J]. Molecules, 2012, 18: 204-224.). Therefore, we urgently need to develop new antibacterial drugs with potential therapeutic effects on MRSA infections.

[0003] Antimicrobial peptides (AMPs), also known as host defense peptides, are small molecule peptides produced by various organisms to protect hosts from other pathogens. They have attracted widespread interest due to their broad-spectrum activity and low drug resistance. However, AMPs have shortcomings such as poor stability, weak antimicrobial activity and high cost, which seriously limit their clinical application (Su M, Xia D, Teng P, et al. Membrane-Active Hydantoin Derivatives as Antibiotic Agents [J]. Journal of Medicinal Chemistry, 2017, 60 (20): 8456-8465.). Honokiol is a bisphenol widely found in Magnolia officinalis Rehd. et Wils. and Magnolia officinalis var. biloba Rehd. et Wils., both of the Magnoliaceae family. It has anti-inflammatory, antioxidant, and antibacterial activities (Cheng CH, Zhang QW, Yang YE, et al. Honokiol: A naturally occurring lignan with pleiotropic bioactivities [J]. Chinese Journal of Natural Medicines, 2021, 19 (7): 481-490.). However, honokiol is limited by its poor water solubility and bioavailability, making it difficult to directly become an antibacterial drug. Summary of the invention

[0004] Purpose of the invention: In view of the above technical problems, the present invention provides a mimetic conjugate of honokiol-antimicrobial peptide containing an oxazolethione structure, a preparation method thereof and an antibacterial application thereof. The present invention adopts the concept of learning from each other's strengths and making up for each other's weaknesses, and draws on the structural and functional characteristics of antimicrobial peptides. The natural product honokiol is used as a matrix, and its structure is modified to introduce a nitrogen-containing heterocycle, and then connected with a cationic antimicrobial peptide simulation fragment, in order to obtain a mimetic conjugate of honokiol-antimicrobial peptide containing an oxazolethione structure with high activity and low toxicity. The obtained mimetic conjugate of honokiol-antimicrobial peptide containing an oxazolethione structure, a preparation method thereof and an antibacterial application thereof have a good antibacterial effect on Staphylococcus aureus (S. aureus) ATCC 29213, clinically isolated MRSA strains and MRSA standard strain N315, and solves the problem of large toxicity difference.

[0005] Technical solution: In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is as follows:

[0006] The structure of the magnolol-antimicrobial peptide mimetic conjugate containing an oxazolethione structure is shown in the following formula (1):

[0007]

[0008] Wherein, R1, R2 and R3 are independently selected from H, halogen, C1-C3 alkoxy or CF3.

[0009] As a preferred embodiment, R1, R2 and R3 are one of the following combinations:

[0010] (1) R1=R2=R3=H; (2) R1=R2=H, R3=2-F; (3) R1=R2=H, R3=4-F;

[0011] (4) R1=R2=H, R3=2-Cl; (5) R1=R2=H, R3=4-Cl; (6) R1=R2=H, R3=2-Br;

[0012] (7) R1=R2=H, R3=3-Br; (8) R1=R2=H, R3=4-Br; (9) R1=H, R2=2-F, R3=4-Cl;

[0013] (10) R1=H, R2=2-F, R3=5-Br; (11) R1=H, R2=3-F, R3=4-F;

[0014] (12) R1=H, R2=3-F, R3=4-Cl; (13) R1=H, R2=2-Cl, R3=4-F;

[0015] (14) R1=H, R2=3-Cl, R3=4-F; (15) R1=H, R2=2-Cl, R3=3-Cl;

[0016] (16) R1=H, R2=2-Cl, R3=4-Cl; (17) R1=H, R2=2-Cl, R3=6-Cl;

[0017] (18) R1=H, R2=3-Cl, R3=4-Cl; (19) R1=H, R2=3-Cl, R3=5-Cl;

[0018] (20) R1=H, R2=2-Br, R3=4-Cl; (21) R1=H, R2=3-Br, R3=4-F;

[0019] (22) R1=H, R2=3-Br, R3=4-Cl; (23) R1=H, R2=2-Br, R3=4-Br;

[0020] (24) R1=H, R2=3-Br, R3=5-Br; (25) R1=3-F, R2=4-F, R3=5-F;

[0021] (26) R1=R2=H, R3=3-OCH3; (27) R1=R2=H, R3=4-OCH3; (28) R1=R2=H, R3=4-CF3.

[0022] The above specific selections represent compounds 1-28 in the following examples (each compound corresponds to a combination of the same serial numbers as above).

[0023] The present invention also provides a method for preparing the honokiol-antimicrobial peptide mimetic conjugate containing an oxazolethione structure, comprising the following steps:

[0024] (1) Using honokiol as a substrate, it reacts with iodine to convert the ortho-phenolic hydroxyl group and the propenyl group into an oxygen-containing five-membered ring to obtain intermediate 2;

[0025] (2) intermediate 2 reacts with concentrated nitric acid to introduce a nitro group to synthesize intermediate 3;

[0026] (3) Intermediate 3 is then reacted with a reducing agent to reduce the nitro group to an amino group to obtain intermediate 4;

[0027] (4) intermediate 4 is subjected to the action of a catalyst to open the oxygen-containing five-membered ring to generate a phenolic hydroxyl group and a propenyl group, thereby obtaining intermediate 5;

[0028] (5) Intermediate 5 is then reacted with benzaldehyde containing different substituents to generate intermediate 6;

[0029] (6) Intermediate 6 reacts with a reducing agent to generate intermediate 7;

[0030] (7) Intermediate 7 reacts with carbon disulfide under alkaline conditions to generate intermediate 8

[0031] (8) Intermediate 8 reacts with 1,3-dibromopropane under alkaline conditions to generate intermediate 9;

[0032] (9) The intermediate 9 then reacts with a small molecule peptide mimetic 10 to generate the honokiol-antimicrobial peptide mimetic conjugate containing an oxazolethione structure, and the reaction formula is as follows:

[0033]

[0034]

[0035] Wherein, R1, R2 and R3 are the same as those described in claim 1 or 2.

[0036] As a specific implementation scheme, in step (1), the reaction molar ratio of honokiol to iodine is 1:(1.0-1.2), the reaction temperature is 45-55°C, and the reaction solvent is 9:1-7:3 of ethanol and ultrapure water;

[0037] As a specific implementation scheme, in step (2), the reaction molar ratio of the intermediate 2 to concentrated nitric acid is 1:(2.5-3.5), the reaction temperature is 0±5°C, and the reaction solvent is dichloromethane.

[0038] As a specific embodiment, in step (3), the reducing agent is selected from stannous chloride dihydrate, the molar ratio of the intermediate 3 to the reducing agent is 1:(9-11), the reaction temperature is 75-80°C, and the reaction solvent is ethyl acetate;

[0039] As a specific implementation scheme, in step (4), the catalyst is selected from zinc; the molar ratio of the intermediate 4 to the catalyst is 1:(4-6), the reaction temperature is 75-80°C, and the reaction solvent is anhydrous ethanol.

[0040] As a specific implementation scheme, in step (5), the molar ratio of the intermediate 5 to the benzaldehyde containing different substituents is 1:(2.5-3.5), the reaction temperature is 40-50°C, and the reaction solvent is methanol;

[0041] As a specific embodiment, in step (6), the reducing agent is selected from sodium borohydride, the molar ratio of intermediate 6 to the reducing agent is 1:(5-7), the reaction temperature is 0±5°C, and the reaction solvent is dichloromethane;

[0042] As a specific implementation scheme, in step (7), the base in the alkaline condition is selected from KOH, the molar ratio of the intermediate 7 to carbon disulfide is 1:(3-5), the reaction temperature is 45-55°C, and the reaction solvent is anhydrous ethanol.

[0043] As a specific implementation scheme, in step (8), the molar ratio of the intermediate 8 to 1,3-dibromopropane is 1:(2.5-3.5), the reaction temperature is 45-55°C, and the reaction solvent is anhydrous acetone.

[0044] As a specific implementation scheme, in step (9), the method for preparing the small molecule peptide mimetic 10 comprises the following steps: bromoacetyl bromide and mono-n-butylamine undergo a substitution reaction under alkaline conditions to generate bromoacetamide, and then bromoacetamide continues to undergo a substitution reaction with dimethylamine under alkaline conditions to generate a small molecule peptide mimetic:

[0045]

[0046] Preferably, the molar ratio of the intermediate 9 to the small molecule peptide mimetic 10 is 1:(2.5-3.5), the reaction temperature is 75-85° C., and the reaction solvent is anhydrous acetonitrile.

[0047] Furthermore, as a preferred solution:

[0048] In step (1), the reaction molar ratio of honokiol to iodine is 1:1.1, the reaction temperature is 50° C., and the reaction solvent is 9:1 ethanol and ultrapure water.

[0049] In step (2), the reaction molar ratio of the intermediate 2 to concentrated HNO3 is 1:3, the reaction temperature is 0°C, and the reaction solvent is dichloromethane.

[0050] In step (3), the molar ratio of the intermediate 3 to stannous chloride dihydrate is 1:10, the reaction temperature is 77° C., and the reaction solvent is ethyl acetate.

[0051] In step (4), the molar ratio of the intermediate 4 to zinc is 1:5, the reaction temperature is 78° C., and the reaction solvent is anhydrous ethanol.

[0052] In step (5), the molar ratio of the intermediate 5 to various substituted benzaldehydes is 1:3, the reaction temperature is 45° C., and the reaction solvent is methanol.

[0053] In step (6), the molar ratio of the intermediate 6 to sodium borohydride is 1:6, the reaction temperature is 0°C, and the reaction solvent is dichloromethane.

[0054] In step (7), the molar ratio of the intermediate 7 to carbon disulfide is 1:4, the reaction temperature is 50° C., and the reaction solvent is anhydrous ethanol.

[0055] In step (8), the molar ratio of the intermediate 8 to 1,3-dibromopropane is 1:3, the reaction temperature is 50° C., and the reaction solvent is anhydrous acetone.

[0056] In step (9), the molar ratio of the intermediate 9 to the small molecule peptide mimetic 10 is 1:3, the reaction temperature is 80° C., and the reaction solvent is anhydrous acetonitrile.

[0057] The preparation method of the small molecule peptide mimetic 10 is as follows:

[0058]

[0059] The molar ratio of mono-n-butylamine to bromoacetyl bromide is 1:1.5, the base used in the reaction is K2CO3, the reaction temperature is 0°C to room temperature, the reaction time is 12 hours, and the reaction solvent is anhydrous dichloromethane; the molar ratio of bromoamide to dimethylamine is 1:1.5, the reaction temperature is room temperature, the reaction time is 12 hours, and the reaction solvent is anhydrous acetone.

[0060] Finally, the present invention provides the use of the honokiol-antimicrobial peptide mimetic conjugate containing an oxazolethione structure in the preparation of an antibacterial drug. Preferably, the antibacterial drug can inhibit Staphylococcus aureus or methicillin-resistant Staphylococcus aureus. For example, it can inhibit Staphylococcus aureus ATCC 29213 and various clinical methicillin-resistant Staphylococcus aureus MRSA.

[0061] The compound of the present invention effectively connects the natural product honokiol with the antimicrobial peptide simulation fragment, takes advantage of the strengths and makes up for the weaknesses, draws on the structure and function of the antimicrobial peptide, and designs and synthesizes a series of honokiol-antimicrobial peptide simulation conjugates containing oxazolethione structure. The parent structure honokiol in the conjugate as a hydrophobic part is conducive to the compound inserting into the bacterial phospholipid bilayer membrane, and the positively charged cationic small molecule peptide mimetic part is conducive to interacting with the negatively charged bacterial cell membrane, thereby causing bacterial death. The present invention evaluates the in vitro antibacterial activity of all target compounds, and finds that all target compounds show good in vitro antibacterial activity against S. aureus ATCC 29213 and clinically isolated MRSA strains, with a minimum inhibitory concentration (MIC≤128μg / mL). In particular, the preferred compound 3 has the most outstanding in vitro and in vitro anti-MRSA activity, and the in vivo antibacterial effect is equivalent to vancomycin at the same dose. In addition, the target compound has low hemolytic activity, low in vivo toxicity, and plasma stability. Therefore, this type of compound provides certain inspiration for the study of new anti-MRSA drugs and has broad application prospects.

[0062] Technical effect: The magnolol-antimicrobial peptide mimetic conjugate containing an oxazolethione structure prepared by the present invention has good in vivo and in vitro antibacterial effects on S. aureus ATCC 29213 and various clinical MRSA strains, and has low biological toxicity and high yield, and is expected to be further developed into a potential clinical antibacterial drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 This is the dynamic bactericidal curve of compound 3.

[0064] Figure 2 These are the blood routine test and blood biochemical indexes of compound 3 in vivo.

[0065] Figure 3 The changes in bacterial load on mouse skin in response to compound 3.

[0066] Figure 4 For compound 3 1 H-NMR spectrum.

[0067] Figure 5 For compound 3 13 C-NMR spectrum. DETAILED DESCRIPTION

[0068] The present invention is further described in detail below through examples.

[0069] Example 1 Preparation of Intermediate 2

[0070] Honokiol (1 mmol) was dissolved in anhydrous ethanol and ultrapure water in a ratio of 9:1, I2 (1.1 mmol) was added, and then refluxed with stirring at 50°C for 6 h. After the completion of the reaction monitored by thin layer chromatography (TLC), extraction was performed, and the mixture was dried over Na2SO4 and concentrated under reduced pressure. The concentrate was purified by column chromatography using petroleum ether and ethyl acetate (12:1) to obtain light yellow transparent oily intermediate 2.

[0071] Example 2 Preparation of Intermediate 3

[0072] After dissolving the intermediate 2 (1 mmol) with an appropriate amount of CH2Cl2, an appropriate amount of CH3COOH was added, and after stirring at 0°C for 5 min, concentrated nitric acid (3 mmol) was slowly added. The reaction was carried out for 1 h in an ice bath. After the reaction was completed by TLC monitoring, the reaction solution was mixed with ice water, and the pH was adjusted to 7 with a saturated Na2CO3 aqueous solution, extracted, dried with Na2SO4, and concentrated under reduced pressure. The concentrate was purified by column chromatography using petroleum ether and ethyl acetate (12:1) to obtain a brown oily intermediate 3.

[0073] Example 3 Preparation of Intermediate 4

[0074] After the intermediate 3 (1 mmol) was dissolved by stirring with an appropriate amount of ethyl acetate, stannous chloride dihydrate (10 mmol) was added, and the mixture was refluxed and stirred at 77°C for 2 h under nitrogen protection. After the reaction was completed by TLC monitoring, the pH of the reaction solution was adjusted to 7 with a saturated aqueous Na2CO3 solution, filtered, and the filtrate was extracted, dried with Na2SO4, and concentrated under reduced pressure to obtain a white oily intermediate 4.

[0075] Example 4 Preparation of Intermediate 5

[0076] The intermediate 4 (1 mmol) was dissolved by stirring with an appropriate amount of ethanol, and zinc powder (5 mmol) and an appropriate amount of CH3COOH were added. The mixture was refluxed at 78°C and reacted for 3 h. After the reaction was completed by TLC monitoring, zinc was filtered out and the mixture was concentrated under reduced pressure and purified by column chromatography using petroleum ether and ethyl acetate (2:1) to obtain a dark brown solid intermediate 5.

[0077] Example 5 Preparation of Intermediate 6

[0078] Intermediate 5 (1 mmol) was dissolved in methanol solution, and the corresponding benzaldehyde (3 mmol) was added. The mixture was refluxed and stirred at 45°C for 45 min. After the reaction was completed as monitored by TLC, the mixture was concentrated under reduced pressure to obtain intermediate 6 as a dark brown oil.

[0079] Example 6 Preparation of Intermediate 7

[0080] NaBH4 (6 mmol) was added to a CH2Cl2 solution of intermediate 6 (1 mmol), and the mixture was reacted for 30 min in an ice bath. The reaction was detected by TLC until the reaction was complete. The reaction solution was poured into ice water, extracted with dichloromethane, and concentrated to obtain intermediate 7.

[0081] Example 7 Preparation of Intermediate 8

[0082] To an ethanol solution of intermediate 7 (1 mmol), KOH (0.5 mmol) and CS2 (4 mmol) were added, and the mixture was refluxed at 50°C with stirring for 12 h. The reaction was completed by TLC detection. The reaction solution was mixed with water, extracted, concentrated under reduced pressure, and separated and purified by silica gel column chromatography to obtain intermediate 8.

[0083] Example 8 Preparation of Intermediate 9

[0084] 1,3-Dibromopropane (3 mmol) was added to an acetone solution of intermediate 8 (1 mmol), and the mixture was stirred at 50°C for 12 h. After the reaction was completed as monitored by TLC, K2CO3 was removed by filtration, and the filtrate was concentrated and purified by PTLC to obtain intermediate 9.

[0085] Example 9 Preparation of small molecule peptide mimetics

[0086] K2CO3 (1.5 mmol) was added to a solution of n-butylamine (1 mmol) in redissolved CH2Cl2, and bromoacetyl bromide was slowly added in an ice bath. After reacting at low temperature for 30 min, the mixture was transferred to room temperature and stirred for 12 h. After the reaction was completed as monitored by TLC, the mixture was extracted, dried with Na2SO4, and dried under reduced pressure to obtain a light yellow oily intermediate bromoacetamide.

[0087] After bromoacetamide (1 mmol) was dissolved in acetone, 1.5 mmol of anhydrous K2CO3 and aqueous dimethylamine solution were added respectively, and the reaction was carried out at room temperature for 12 h. After TLC monitoring of the completion of the reaction, the mixture was concentrated under reduced pressure, extracted, dried with Na2SO4, concentrated under reduced pressure, and purified by column chromatography with dichloromethane and methanol (20:1) to obtain a small molecule peptide mimetic.

[0088] Example 11 Compound 1

[0089] The intermediate 9 (1 mmol) was dissolved in anhydrous acetonitrile, and the small molecule peptide fragment (3 mmol) was added. The reaction was carried out under pressure at 80°C for 12 h. After the reaction was completed by TLC monitoring, the target compound was obtained by preparative thin layer chromatography (dichloromethane: methanol = 10:1).

[0090] The physicochemical properties of compound 1 are as follows:

[0091] 1) Yellow-brown oily substance;

[0092] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0093] With CDCl3 as solvent, each peak is attributed to: 1 H NMR(400MHz CDCl3)δ:8.82(s,1H,-NH-),7.656(dd,J=8.4Hz,2Hz,1H,-Ph),7.51(d,J=2Hz,1H,-Ph),7.42(d,J=6.4Hz,2H,-Ph),7.38-7.31(m ,3H,-Ph),7.15(s,1H,-Ph),6.92(d,J=8.8Hz,1H,-Ph),6.72(s,1H,-Ph),6.02-5.87(m,2H,-CH=),5.43(s,2H,=CH2),5.10-5.01 (m,4H,=CH2,-CH2-),4.49(s,2H,-CH2-),4.15(s,2H,-CH2-),3.90(s,2H,-CH2-),3.41(s,8H,-CH2-,-CH3),3.26(d,J=5.6Hz,2H HRMS(ESI)C 37 H 46 BrN3O3S[M+H]calcd=692.7570; found=612.3267.

[0094] Example 12 Compound 2

[0095] Compound 2 was synthesized by the method described in Example 9. The physicochemical properties of compound 2 are as follows:

[0096] 1) Yellow-brown oily substance;

[0097] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0098] With CDCl3 as solvent, each peak is attributed to: 1HNMR(400MHz CDCl3)δ:8.89(s,1H,-NH-),7.66(dd,J=8.4Hz,2.4Hz,1H,-Ph),7.51(d,J=2.4Hz,1H,-Ph),7.34-7.28(m,1H,-Ph),7.17-7.08(m,4H,- Ph),6.92(d,J=8.4Hz,1H,-Ph),6.84(s,1H,-Ph),6.04-5.87(m,2H,-CH=),5.48(s,2H,=CH2),5.12-5.01(m,4H,=CH2,-CH2-),4.48(s, 2H,-CH2-),4.14(t,J=5.2Hz,2H,-CH2-),3.92-3.88(m,2H,-CH2-),3.43(s,2H,-CH2-),3.41(s,6H,-CH3),3.24(dd,J=12.8Hz,6.8Hz, 2H,-CH2-),2.38(d,J=4Hz,2H,-CH2-),1.55-1.51(m,2H,-CH2-),1.35(d,J=7.6Hz,2H,-CH2-),1.24(s,2H,-CH2-),0.89(s,3H,-CH3); 13 C NMR(100MHz CDCl3)δ:181.0,162.7,156.1,142.9,138.0,136.8,136.6,132.3,130.1,128.9,127.1,124.1,12 1.1,116.7,115.6,111.5,64.3,63.0,52.3,40.0,39.6,34.5,30.9,29.6,20.1,13.7; HRMS(ESI)C 37 H 45 BrFN3O3S[M+H]calcd=710.7474; found=630.3153.

[0099] Example 13 Compound 3

[0100] Compound 3 was synthesized by the method described in Example 9. The physicochemical properties of compound 3 are as follows:

[0101] 1) Yellow-brown oily substance;

[0102] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0103] With CDCl3 as solvent, the peaks are attributed to: 1H NMR(400MHz CDCl3)δ:9.04(s,1H,-NH-),7.63-7.61(m,1H,-Ph),7.49(d,J=2Hz,1H,-Ph),7.44(dd,J=8.4Hz,5.3Hz,2H,-Ph),7.16(s,1H,-Ph), 7.05-7.01(m,1H,-Ph),6.93(d,J=8.4Hz,1H,-Ph),6.73(s,1H,-Ph),6.03-5.85(m,2H,-CH=),5.38(s,2H,=CH2),5.11-5.01(m,4H,= CH2,-CH2-),4.61(s,2H,-CH2-),4.15(s,2H,-CH2-),3.92(s,2H,-CH2-),3.45(s,6H,-CH3),3.43(s,2H,-CH2-),3.26(s,2H,-CH2-) ,2.41(s,2H,-CH2-),1.57-1.54(m,2H,-CH2-),1.35(t,J=7.2Hz,2H,-CH2-),1.27-1.23(m,2H,-CH2-),0.90(t,J=7.2Hz,3H,-CH3); 13 C NMR(100MHz CDCl3)δ:180.7,162.7,156.2,142.9,136.6,132.2,129.7,129.0,126.9,116.7,115.8 ,111.5,107.9,64.5,63.1,52.5,48.7,40.0,39.5,34.4,31.0,20.2,13.7; HRMS(ESI)C 37 H 45 BrFN3O3S[M+H]calcd=710.7474; found=630.3171.

[0104] Example 14 Compound 4

[0105] Compound 4 was synthesized by the method described in Example 9. The physicochemical properties of compound 4 are as follows:

[0106] 1) Yellow-brown oily substance;

[0107] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0108] With CDCl3 as solvent, each peak is attributed to: 1H NMR(400MHz CDC13)δ:9.25(s,1H,-NH-),7.69-7.66(m,1H,-Ph),7.54(d,J=2Hz,1H,-Ph),7.46(d,J=8.8Hz,1H,-Ph),7.29(d,J=2Hz,1H,-Ph),7.25( d,J=4Hz,1H,-Ph),7.19-7.16(m,2H,-Ph),6.95(d,J=8.8Hz,1H,-Ph),6.70(s,1H,-Ph),6.05-5.84(m,2H,-CH=),5.55(s,1H,=CH2),5.1 4-5.02(m,5H,=CH2,-CH2-),4.68(s,2H,-CH2-),4.17(s,2H,-CH2-),3.90(s,2H,-CH2-),3.45(s,8H,-CH2-,-CH3),3.27(s,2H,-CH2-), 2.42(s,2H,-CH2-),1.57(t,J=7.2Hz,2H,-CH2-),1.38(d,J=7.2Hz,2H,-CH2-),1.28-1.24(m,2H,-CH2-),0.91(s,3H,-CH3); HRMS(ESI)C 37 H 45 BrClN3O3S[M+H]calcd=727.1990; found=646.2877.

[0109] Example 15 Compound 5

[0110] Compound 5 was synthesized by the method described in Example 4. The physicochemical properties of compound 5 are as follows:

[0111] 1) Yellow-brown oily substance;

[0112] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0113] With CDCl3 as solvent, each peak is attributed to: 1H NMR(400MHz CDCl3)δ:9.17(s,1H,-NH-),7.64(d,J=8.4Hz,1H,-Ph),7.50(d,J=1.6Hz,1H,-Ph),7.36(dd,J=15.6Hz,8.4Hz,4H,-Ph),7.17 (s,1H,-Ph),6.92(d,J=8.8Hz,1H,-Ph),6.71(s,1H,-Ph),6.04-5.86(m,2H,-CH=),5.38(s,2H,=CH2),5.11-5.01(m,4H,=CH2 ,-CH2-),4.61(s,2H,-CH2-),4.15(s,2H,-CH2-),3.90(s,2H,-CH2-),3.43(s,6H,-CH3),3.42(s,2H,-CH2-),3.26(s,2H,-CH 2-),2.41(s,2H,-CH2-),1.56(t,J=7.2Hz,2H,-CH2-),1.37-1.33(m,2H,-CH2-),1.24(s,2H,-CH2-),0.92-0.88(m,3H,-CH3); 13 C NMR(100MHz CDCl3)δ:180.9,162.6,156.1,142.9,138.0,136.5,134.4,132.4,132.2,129.2,129.1,129.0,127.0,124. 2,116.8,115.9,111.5,107.8,64.4,63.1,52.4,48.7,40.0,39.6,34.5,31.0,29.6,20.2,13.7; HRMS(ESI)C 37 H 45 BrClN3O3S[M+H]calcd=727.1990; found=646.2878.

[0114] Example 16 Compound 6

[0115] Compound 6 was synthesized by the method described in Example 4. The physicochemical properties of compound 6 are as follows:

[0116] 1) Yellow-brown oily substance;

[0117] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0118] With CDCl3 as solvent, each peak is attributed to: 1H NMR(400MHz CDCl3)δ:8.96(s,1H,-NH-),7.69-7.63(m,2H,-Ph),7.54(d,J=2Hz,1H,-Ph),7.23-7.20(m,1H,-Ph),7.19-7.13(m,2H,-Ph),7. 10-7.08(m,1H,-Ph),6.94(d,J=8.4Hz,1H,-Ph),6.69(s,1H,-Ph),6.02-5.85(m,2H,-CH=),5.53(s,2H,=CH2),5.10-5.02(m,4H, =CH2,-CH2-),4.51(s,2H,-CH2-),4.16(s,2H,-CH2-),3.93-3.89(m,2H,-CH2-),3.42(s,8H,-CH2-,-CH3),3.26(d,J=6Hz,2H,- CH2-),2.39(s,2H,-CH2-),1.56-1.52(m,2H,-CH2-),1.36(d,J=7.6Hz,2H,-CH2-),1.24(s,2H,-CH2-),0.92-0.88(m,3H,-CH3); 13 C NMR(100MHz CDCl3)δ:181.2,162.7,156.1,142.9,138.1,136.8,132.9,132.3,130.1,128.9,127.0,124.2,122.7,1 16.8,115.8,111.6,108.1,64.4,63.0,52.3,49.2,39.6,34.5,31.0,29.7,22.7,20.1,13.7; HRMS(ESI)C 37 H 45 Br2N3O3S[M+H]calcd=771.6530; found=690.2375.

[0119] Example 17 Compound 7

[0120] Compound 7 was synthesized by the method described in Example 4. The physicochemical properties of compound 7 are as follows:

[0121] 1) Yellow-brown oily substance;

[0122] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0123] With CDCl3 as solvent, the peaks are attributed to: 1H NMR(400MHz CDC13)δ:9.10(s,1H,-NH-),7.66(d,J=6.4Hz,1H,-Ph),7.56(s,1H,-Ph),7.52(d,J=2Hz,1H,-Ph),7.47(d,J=8Hz,1H,-Ph),7.34(d,J=8Hz ,1H,-Ph),7.25-7.18(m,2H,-Ph),6.93(d,J=8.4Hz,1H,-Ph),6.71(d,J=1.2Hz,1H,-Ph),6.04-5.87(m,2H,-CH=),5.39(s,2H,=CH2),5.12- 5.01(m,4H,=CH2,-CH2-),4.61(s,2H,-CH2-),4.17(s,2H,-CH2-),3.90(s,2H,-CH2-),3.44(d,J=6Hz,8H,-CH2-,-CH3),3.27(s,2H,-CH2- ),2.41(s,2H,-CH2-),1.56(d,J=6.8Hz,2H,-CH2-),1.37(d,J=7.2Hz,2H,-CH2-),1.25(s,2H,-CH2-),0.92-0.89(m,3H,-CH3); HRMS(ESI)C 37 H 45 Br2N3O3S[M+H]calcd=771.6530; found=690.2369.

[0124] Example 18 Compound 8

[0125] Compound 8 was synthesized by the method described in Example 4. The physicochemical properties of compound 8 are as follows:

[0126] 1) Yellow-brown oily substance;

[0127] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0128] With CDCl3 as solvent, the peaks are attributed to: 1H NMR(400MHz CDCl3)δ:9.21(s,1H,-NH-),7.65(d,J=8.4Hz,1H,-Ph),7.51-7.47(m,3H,-Ph),7.30(d,J=8.4Hz,2H,-Ph),7.17(s,1H,-P h),6.93(d,J=8.4Hz,1H,-Ph),6.71(s,1H,-Ph),6.04-5.86(m,2H,-CH=),5.37(s,2H,=CH2),5.12-5.01(m,4H,=CH2,-CH2 -),4.61(s,2H,-CH2-),4.16(s,2H,-CH2-),3.90(s,2H,-CH2-),3.44-3.42(m,8H,-CH2-,-CH3),3.27(d,J=5.2Hz,2H,-CH 2-),2.40(s,2H,-CH2-),1.58-1.53(m,2H,-CH2-),1.39-1.34(m,2H,-CH2-),1.24(s,2H,-CH2-),0.92-0.89(m,3H,-CH3); 13 C NMR(100MHz CDCl3)δ:180.9,162.7,156.2,142.9,138.2,137.2,135.6,132.0,129.0,126.1,124.4,116.9 ,115.8,111.6,107.7,107.6,64.4,63.1,48.3,40.0,39.6,34.5,31.0,20.2,13.7; HRMS(ESI)C 37 H 45 Br2N3O3S[M+H]calcd=771.6530; found=692.2365.

[0129] Example 19 Compound 9

[0130] Compound 9 was synthesized by the method described in Example 4. The physicochemical properties of compound 9 are as follows:

[0131] 1) Yellow-brown oily substance;

[0132] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0133] With CDCl3 as solvent, the peaks are attributed to: 1H NMR(400MHz CDCl3)δ:8.61(s,1H,-NH-),7.652(dd,J=8.4Hz,2Hz,1H,-Ph),7.48(t,J=8Hz,2H,-Ph),7.17-7.09(m,3H,-Ph),6.93(d,J= 8.4Hz,1H,-Ph),6.83(s,1H,-Ph),6.02-5.88(m,2H,-CH=),5.44(s,2H,=CH2),5.13-5.01(m,4H,=CH2,-CH2-),4.45(s,2H,- CH2-),4.18-4.16(m,2H,-CH2-),3.93(t,J=8Hz,2H,-CH2-),3.64(s,2H,-CH2-),3.41(s,6H,-CH3),3.26(s,2H,-CH2-),2.3 9(d,J=5.2Hz,2H,-CH2-),1.54(s,2H,-CH2-),1.43-1.38(m,2H,-CH2-),1.25(s,2H,-CH2-),0.90(s,3H,-CH3); HRMS(ESI)C 37 H 44 BrClFN3O3S[M+H]calcd=745.1894; found=664.2775.

[0134] Example 20 Compound 10

[0135] Compound 10 was synthesized by the method described in Example 4. The physicochemical properties of compound 10 are as follows:

[0136] 1) Yellow-brown oily substance;

[0137] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0138] With CDCl3 as solvent, each peak is attributed to: 1H NMR(400MHz CDC13)δ:9.06(s,1H,-NH-),7.66-7.57(m,2H,-Ph),7.52(d,J=2Hz,1H,-Ph),7.45-7.41(m,1H,-Ph),7.19(s,1H,-Ph),7.06-6.70 (m,1H,-Ph),6.93(d,J=8.8Hz,1H,-Ph),6.81(s,1H,-Ph),6.04-5.88(m,2H,-CH=),5.44(s,1H,=CH2),5.13-5.02(m,5H,=CH2,-CH 2-),4.59(s,2H,-CH2-),4.16(s,2H,-CH2-),3.90(s,2H,-CH2-),3.44(d,J=4Hz,8H,-CH2-,-CH3),3.26(s,2H,-CH2-),2.41(s,2H ,-CH2-),1.56(d,J=6.8Hz,2H,-CH2-),1.37(d,J=7.6Hz,2H,-CH2-),1.26(d,J=7.2Hz,2H,-CH2-),0.90(s,3H,-CH3); HRMS(ESI)C 37 H 44 Br2FN3O3S[M+H]calcd=789.6434; found=708.2280.

[0139] Example 21 Compound 11

[0140] Compound 11 was synthesized by the method described in Example 4. The physicochemical properties of compound 11 are as follows:

[0141] 1) Yellow-brown oily substance;

[0142] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0143] CDCl3 is used as solvent, and the peaks are attributed to: 1H NMR(400MHz CDCl3)δ:9.19(s,1H,-NH-),7.652(dd,J=8.4Hz,2Hz,1H,-Ph),7.51(d,J=2Hz,1H,-Ph),7.27(s,1H,-Ph),7.18-7.14(m,3 H,-Ph),6.93(d,J=8.4Hz,1H,-Ph),6.72(s,1H,-Ph),6.04-5.87(m,2H,-CH=),5.37(s,2H,=CH2),5.12-5.01(m,4H,=CH2, -CH2-),4.64(s,2H,-CH2-),4.16(s,2H,-CH2-),3.90(s,2H,-CH2-),3.44(s,8H,-CH2-,-CH3),3.28(d,J=4.8Hz,2H,-CH2 -),2.42(s,2H,-CH2-),1.59-1.55(m,2H,-CH2-),1.43-1.36(m,2H,-CH2-),1.26(s,2H,-CH2-),0.93-0.89(m,3H,-CH3); 13 C NMR(100MHz CDCl3)δ:180.9,162.6,156.2,142.9,138.1,132.1,129.0,127.0,124.3,124.0 ,116.9,115.9,64.3,63.2,52.5,48.4,40.0,39.6,34.5,31.0,20.2; HRMS(ESI)C 37 H 44 BrF2N3O3S[M+H]calcd=728.7378; found=648.3083.

[0144] Example 22 Compound 12

[0145] Compound 12 was synthesized by the method described in Example 4. The physicochemical properties of compound 12 are as follows:

[0146] 1) Yellow-brown oily substance;

[0147] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0148] CDCl3 is used as solvent, and the peaks are attributed to: 1H NMR(400MHz CDCl3)δ:9.09(s,1H,-NH-),7.66-7.64(m,1H,-Ph),7.52(d,J=1.6Hz,1H,-Ph),7.40-7.36(m,1H,-Ph),7.22-7.13(m,3H,-Ph) ),6.93(d,J=8.4Hz,1H,-Ph),6.70(s,1H,-Ph),6.03-5.87(m,2H,-CH=),5.38(s,2H,=CH2),5.13-5.06(m,4H,=CH2,-CH2-),4. 55(s,2H,-CH2-),4.16(s,2H,-CH2-),3.99(s,2H,-CH2-),3.43(s,8H,-CH2-,-CH3),3.27(d,J=4.4Hz,2H,-CH2-),2.40(s,2H ,-CH2-),1.55(t,J=7.2Hz,2H,-CH2-),1.43-1.37(m,2H,-CH2-),1.24(s,2H,-CH2-),0.90(t,J=7.2Hz,3H,-CH3); HRMS(ESI)C 37 H 44 BrClFN3O3S[M+H]calcd=745.1894; found=664.2787.

[0149] Example 23 Compound 13

[0150] Compound 13 was synthesized by the method described in Example 4. The physicochemical properties of compound 13 are as follows:

[0151] 1) Yellow-brown oily substance;

[0152] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0153] CDCl3 is used as solvent, and the peaks are attributed to: 1H NMR(400MHz CDCl3)δ:8.89(s,1H,-NH-),7.67(dd,J=8.4Hz,2Hz,1H,-Ph),7.54(d,J=2Hz,1H,-Ph),7.23-7.19(m,3H,-Ph),6.95- 6.90(m,2H,-Ph),6.71(s,1H,-Ph),6.04-5.88(m,2H,-CH=),5.52(s,2H,=CH2),5.12-5.02(m,4H,=CH2,-CH2-),4.71 (s,2H,-CH2-),4.19(s,2H,-CH2-),3.91(s,2H,-CH2-),3.57(s,2H,-CH2-),3.46(s,6H,-CH3),3.28(s,2H,-CH2-),2 .44(s,2H,-CH2-),1.58-1.56(m,2H,-CH2-),1.38(s,2H,-CH2-),1.26(s,2H,-CH2-),0.92(s,3H,-CH3); HRMS(ESI)C 37 H 44 BrClFN3O3S[M+H]calcd=745.1894; found=664.2785.

[0154] Example 24 Compound 14

[0155] Compound 14 was synthesized by the method described in Example 4. The physicochemical properties of compound 14 are as follows:

[0156] 1) Yellow-brown oily substance;

[0157] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0158] CDCl3 is used as solvent, and the peaks are attributed to: 1H NMR(400MHz CDCl3)δ:8.90(s,1H,-NH-),7.66(dd,J=8.4Hz,2Hz,1H,-Ph),7.52-7.48(m,2H,-Ph),7.19(s,1H,-Ph),7.15-7.11(m,2H ,-Ph),6.92(d,J=8.4Hz,1H,-Ph),6.71(s,1H,-Ph),6.03-5.89(m,2H,-CH=),5.37(s,2H,=CH2),5.14-5.01(m,4H,=CH2,- CH2-),4.52(s,2H,-CH2-),4.16(s,2H,-CH2-),3.89(s,2H,-CH2-),3.41(s,8H,-CH2-,-CH3),3.27(d,J=5.6Hz,2H,-CH2 -),2.39(s,2H,-CH2-),1.55(s,2H,-CH2-),1.41-1.37(m,2H,-CH2-),1.28(s,2H,-CH2-),0.90(s,3H,-CH3); HRMS(ESI)C 37 H 44 BrClFN3O3S[M+H]calcd=745.1894; found=664.2779.

[0159] Example 25 Compound 15

[0160] Compound 15 was synthesized by the method described in Example 4. The physicochemical properties of compound 15 are as follows:

[0161] 1) Yellow-brown oily substance;

[0162] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0163] CDCl3 is used as solvent, and the peaks are attributed to: 1H NMR(400MHz CDC13)δ:9.14(s,1H,-NH-),7.62-7.59(m,1H,-Ph),7.47(d,J=2Hz,1H,-Ph),7.38(d,J=8Hz,1H,-Ph),7.13(s,1H,-Ph),7.08-7.0 4(m,1H,-Ph),6.91-6.86(m,2H,-Ph),6.60(s,1H,-Ph),5.98-5.78(m,2H,-CH=),5.47(s,2H,=CH2),5.03-4.99(m,4H,=CH2,-CH2- ),4.60(s,2H,-CH2-),4.11(s,2H,-CH2-),3.83(s,2H,-CH2-),3.38(m,6H,-CH3),3.36(s,2H,-CH2-),3.21(d,J=3.6Hz,2H,-CH2- ),2.35(s,2H,-CH2-),1.52-1.49(m,2H,-CH2-),1.33-1.29(m,2H,-CH2-),1.21-1.18(m,2H,-CH2-),0.84(t,J=7.2Hz,3H,-CH3); 13 C NMR(100MHz CDCl3)δ:180.1,161.6,155.1,141.9,137.2,132.8,132.5,131.2,130.1,129.1,127.9,12 6.0,123.3,115.9,114.8,63.3,62.1,51.4,46.2,39.0,33.5,30.0,19.2,12.7; HRMS(ESI)C 37 H 44 BrCl2N3O3S[M+H]calcd=761.6410; found=680.2487.

[0164] Example 26 Compound 16

[0165] Compound 16 was synthesized by the method described in Example 4. The physicochemical properties of compound 16 are as follows:

[0166] 1) Yellow-brown oily substance;

[0167] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0168] CDCl3 is used as solvent, and the peaks are attributed to: 1H NMR(400MHz CDCl3)δ:8.80(s,1H,-NH-),7.63-7.66(m,1H,-Ph),7.53(d,J=2Hz,1H,-Ph),7.48(d,J=2Hz,1H,-Ph),7.19-7.11(m,3H,-P h),6.95(d,J=8.4Hz,1H,-Ph),6.69(s,1H,-Ph),6.03-5.86(m,2H,-CH=),5.50(s,2H,=CH2),5.12-5.01(m,4H,=CH2,-CH2- ),4.53(s,2H,-CH2-),4.18(s,2H,-CH2-),3.91(d,J=6.8Hz,2H,-CH2-),3.43(s,8H,-CH2-,-CH3),3.27(d,J=4.8Hz,2H,-C H2-),2.41(s,2H,-CH2-),1.56(s,2H,-CH2-),1.43-1.39(m,2H,-CH2-),1.25(s,2H,-CH2-),0.90(s,3H,-CH3); HRMS(ESI)C 37 H 44 BrCl2N3O3S[M+H]calcd=761.6410; found=680.2491.

[0169] Example 27 Compound 17

[0170] Compound 17 was synthesized by the method described in Example 4. The physicochemical properties of compound 17 are as follows:

[0171] 1) Yellow-brown oily substance;

[0172] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0173] CDCl3 is used as solvent, and the peaks are attributed to: 1H NMR(400MHz CDCl3)δ:9.30(s,1H,-NH-),7.64(dd,J=8.4Hz,2Hz,1H,-Ph),7.49(d,J=2Hz,1H,-Ph),7.40(d,J=7.6Hz,2H,-Ph),7.32-7.29(m,1H,-P h),7.10(d,J=0.8Hz,1H,-Ph),6.92(d,J=8.4Hz,1H,-Ph),6.37(s,1H,-Ph),6.04-5.94(m,1H,-CH=),5.84-5.73(m,3H,-CH=,=CH2),5. 08-4.98(m,4H,=CH2,-CH2-),4.64(s,2H,-CH2-),4.16(s,2H,-CH2-),3.89(s,2H,-CH2-),3.43(s,8H,-CH2-,-CH3),3.32(d,J=6.8Hz, 2H,-CH2-),2.41(s,2H,-CH2-),1.59-1.55(m,2H,-CH2-),1.40-1.36(m,2H,-CH2-),1.28-1.26(m,2H,-CH2-),0.93-0.89(m,3H,-CH3); 13 C NMR(100MHz CDCl3)δ:180.6,162.6,156.0,142.8,137.5,136.8,136.4,131.9,130.7,130.1,129.0,128.8,128.0,127.2,12 3.8,123.5,116.7,115.8,111.5,108.2,64.3,63.1,52.4,46.7,39.9,39.6,34.5,31.0,20.2,13.7; HRMS(ESI)C 37 H 44 BrCl2N3O3S[M+H]calcd=761.6410; found=680.2487.

[0174] Example 28 Compound 18

[0175] Compound 18 was synthesized by the method described in Example 4. The physicochemical properties of compound 18 are as follows:

[0176] 1) Light yellow-brown oil;

[0177] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0178] CDCl3 is used as solvent, and the peaks are attributed to: 1 H NMR(400MHz CDCl3)δ:8.93(s,1H,-NH-),7.66-7.64(m,1H,-Ph),7.52(s,2H,-Ph),7.44(d,J=8.4Hz,1H,-Ph),7.26-7.24(m,1H,-Ph),7.19(s ,1H,-Ph),6.92(d,J=8.4Hz,1H,-Ph),6.71(s,1H,-Ph),6.04-5.87(m,2H,-CH=),5.37(s,2H,=CH2),5.13-5.01(m,4H,=CH2,-CH2- ),4.53(s,2H,-CH2-),4.15(s,2H,-CH2-),3.90(s,2H,-CH2-),3.44(s,2H,-CH2),3.42(s,6H,-CH3),3.26(d,J=4.8Hz,2H,-CH2- ),2.37(s,2H,-CH2-),1.52(d,J=7.2Hz,2H,-CH2-),1.41-1.38(m,2H,-CH2-),1.24(s,2H,-CH2-),0.90(s,3H,-CH3); HRMS(ESI)C 37 H 44 BrCl2N3O3S[M+H]calcd=761.6410; found=680.2487.

[0179] Example 29 Compound 19

[0180] Compound 19 was synthesized by the method described in Example 4. The physicochemical properties of compound 19 are as follows:

[0181] 1) Yellow-brown oily substance;

[0182] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0183] CDCl3 is used as solvent, and the peaks are attributed to: 1H NMR(400MHz CDCl3)δ:9.21(s,1H,-NH-),7.65(d,J=8Hz,1H,-Ph),7.52(s,1H,-Ph),7.32(s,1H,-Ph),7.28(s,2H,-Ph),7.20(s,1H,-Ph), 6.94(d,J=8.4Hz,1H,-Ph),6.72(s,1H,-Ph),6.04-5.87(m,2H,-CH=),5.36(s,2H,=CH2),5.13-5.02(m,4H,=CH2,-CH2-),4.6 7(s,2H,-CH2-),4.16(s,2H,-CH2-),3.99(s,2H,-CH2-),3.47(s,6H,-CH3),3.44(s,2H,-CH2-),3.27(d,J=5.6Hz,2H,-CH2-) ,2.42(s,2H,-CH2-),1.60-1.53(m,2H,-CH2-),1.40-1.34(m,2H,-CH2-),1.29-1.24(m,2H,-CH2-),0.92-0.89(m,3H,-CH3); 13 C NMR(100MHz CDCl3)δ:180.9,162.6,156.1,142.9,138.0,136.8,133.0,129.4,128.9,127.0,124.2,122.5 ,116.8,115.8,111.6,107.9,64.3,63.1,52.4,48.8,40.0,34.5,31.0,20.2,13.7; HRMS(ESI)C 37 H 44 BrCl2N3O3S[M+H]calcd=761.6410; found=680.2488.

[0184] Example 30 Compound 20

[0185] Compound 20 was synthesized by the method described in Example 4. The physicochemical properties of compound 20 are as follows:

[0186] 1) Yellow-brown oily substance;

[0187] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0188] CDCl3 is used as solvent, and the peaks are attributed to: 1H NMR(400MHz CDC13)δ:9.02(s,1H,-NH-),7.68-7.66(m,2H,-Ph),7.54(d,J=2Hz,1H,-Ph),7.22-7.20(m,2H,-Ph),7.05(d,J=8.4Hz,1H,-Ph) ,6.95(d,J=8.4Hz,1H,-Ph),6.68(d,J=0.8Hz,1H,-Ph),6.04-5.86(m,2H,-CH=),5.48(s,2H,=CH2),5.11-5.02(m,4H,=CH2,-CH 2-),4.59(s,2H,-CH2-),4.17(s,2H,-CH2-),3.91(s,2H,-CH2-),3.45(s,6H,-CH3),3.43(s,2H,-CH2-),3.26(s,2H,-CH2-),2. 42(s,2H,-CH2-),1.57-1.54(m,2H,-CH2-),1.35(t,J=7.2Hz,2H,-CH2-),1.23(d,J=6.4Hz,2H,-CH2-),0.92-0.89(m,3H,-CH3); 13 C NMR(100MHz CDCl3)δ:181.1,162.7,156.2,138.2,134.8,132.8,132.1,131.6,130.1,129.0,126.9,124.3,123.0 ,116.9,115.9,111.6,107.9,64.4,63.1,52.5,48.7,40.0,39.6,34.5,31.0,20.2,13.7; HRMS(ESI)C 37 H 44 Br2ClN3O3S[M+H]calcd=806.0950; found=726.1966

[0189] Example 31 Compound 21

[0190] Compound 21 was synthesized by the method described in Example 4. The physicochemical properties of compound 21 are as follows:

[0191] 1) Yellow-brown oily substance;

[0192] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0193] With CDCl3 as solvent, each peak is attributed to: 1H NMR(400MHz CDCl3)δ:9.24(s,1H,-NH-),7.66-7.60(m,2H,-Ph),7.52(d,J=2Hz,1H,-Ph),7.37-7.34(m,1H,-Ph),7.19(d,J=1.2Hz,1H,-Ph),7.1 2(t,J=8.4Hz,1H,-Ph),6.93(d,J=8.4Hz,1H,-Ph),6.72(s,1H,-Ph),6.04-5.87(m,2H,-CH=),5.37(s,2H,=CH2),5.14-5.01(m,4H,= CH2,-CH2-),4.65(s,2H,-CH2-),4.17(s,2H,-CH2-),3.89(s,2H,-CH2-),3.44(s,8H,-CH2-,-CH3),3.28(dd,J=12Hz,6.8Hz,2H,-CH 2-),2.42(s,2H,-CH2-),1.57(t,J=7.2Hz,2H,-CH2-),1.41-1.35(m,2H,-CH2-),1.27-1.24(m,2H,-CH2-),0.93-0.89(m,3H,-CH3); 13 C NMR(100MHz CDCl3)δ:180.9,162.6,156.1,143.0,136.8,136.5,133.0,132.1,132.1 131.4,130.1,127.1,124.3,116.9,115.8,111.5,109.8,64.3,63.1,52.5,48.1,40.0,39.6,34.5,31.0,20.2; HRMS(ESI)C 37 H 44 Br2FN3O3S[M+H]calcd=789.6434; found=708.2282.

[0194] Example 32 Compound 22

[0195] Compound 22 was synthesized by the method described in Example 4. The physicochemical properties of compound 22 are as follows:

[0196] 1) Yellow-brown oily substance;

[0197] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0198] With CDCl3 as solvent, each peak is attributed to: 1H NMR(400MHz CDCl3)δ:8.76(s,1H,-NH-),7.69-7.64(m,2H,-Ph),7.52(d,J=2Hz,1H,-Ph),7.44(d,J=8.4Hz,1H,-Ph),7.19(s,2H, -Ph),6.93(d,J=8.4Hz,1H,-Ph),6.70(s,1H,-Ph),6.03-5.89(m,2H,-CH=),5.37(s,2H,=CH2),5.14-5.06(m,4H,=CH2 ,-CH2-),4.47(s,2H,-CH2-),4.16(s,2H,-CH2-),3.91(s,2H,-CH2-),3.41(s,8H,-CH2-,-CH3),3.25(s,2H,-CH2-), 2.38(s,2H,-CH2-),1.52(s,2H,-CH2-),1.43-1.41(m,2H,-CH2-),1.25(s,2H,-CH2-),0.88(s,3H,-CH3); HRMS(ESI)C 37 H 44 Br2ClN3O3S[M+H]calcd=806.0950; found=726.1974.

[0199] Example 33 Compound 23

[0200] Compound 23 was synthesized by the method described in Example 4. The physicochemical properties of compound 23 are as follows:

[0201] 1) Light yellow-brown oil;

[0202] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0203] CDCl3 is used as solvent, and the peaks are attributed to: 1H NMR(400MHz CDC13)δ:9.08(s,1H,-NH-),7.82(d,J=2Hz,1H,-Ph),7.69-7.67(m,1H,-Ph),7.54(d,J=2Hz,1H,-Ph),7.37-7.35(m,1H,-Ph),7.20( s,1H,-Ph),6.96(dd,J=18.4Hz,8.4Hz,2H,-Ph),6.68(s,1H,-Ph),6.03-5.86(m,2H,-CH=),5.47(s,2H,=CH2),5.12-5.07(m,4H,=CH 2,-CH2-),4.55(s,2H,-CH2-),4.17(s,2H,-CH2-),3.90(s,2H,-CH2-),3.43(s,6H,-CH3),3.421(s,2H,-CH2-),3.26(s,2H,-CH2-), 2.40(d,J=5.2Hz,2H,-CH2-),1.57-1.54(m,2H,-CH2-),1.43-1.37(m,2H,-CH2-),1.24(s,2H,-CH2-),0.91(s,3H,-CH3); HRMS(ESI)C 37 H 44 Br3N3O3S[M+H]calcd=850.5490; found=770.1469.

[0204] Example 34 Compound 24

[0205] Compound 24 was synthesized by the method described in Example 4. The physicochemical properties of compound 24 are as follows:

[0206] 1) Yellow-brown oily substance;

[0207] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0208] CDCl3 is used as solvent, and the peaks are attributed to: 1H NMR(400MHz CDCl3)δ:9.04(s,1H,-NH-),7.67-7.64(m,2H,-Ph),7.53(s,1H,-Ph),7.48(s,2H,-Ph),7.20(s,2H,-Ph),6.94(d, J=9.2Hz,1H,-Ph),6.70(s,1H,-Ph),6.05-5.88(m,2H,-CH=),5.36(s,2H,=CH2),5.14-5.03(m,4H,=CH2,-CH2-),4 .54(s,2H,-CH2-),4.18(s,2H,-CH2-),3.90(s,2H,-CH2-),3.57(s,2H,-CH2),3.45(s,6H,-CH3),3.27(s,2H,-CH2 -),2.49(s,2H,-CH2-),1.55(s,2H,-CH2-),1.37(s,2H,-CH2-),1.26(s,2H,-CH2-),0.92(s,3H,-CH3); HRMS(ESI)C 37 H 44 Br3N3O3S[M+H]calcd=850.5490; found=770.1465.

[0209] Example 35 Compound 25

[0210] Compound 25 was synthesized by the method described in Example 4. The physicochemical properties of compound 25 are as follows:

[0211] 1) Yellow-brown oily substance;

[0212] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0213] With CDCl3 as solvent, the peaks are attributed to: 1H NMR(400MHz CDCl3)δ:9.15(s,1H,-NH-),7.65(dd,J=8.4Hz,2.4Hz,1H,-Ph),7.52(d,J=2Hz,1H,-Ph),7.21(d,J=1.2Hz,1H,-Ph),7.08-7.04( m,2H,-Ph),6.94(d,J=8.4Hz,1H,-Ph),6.71(s,1H,-Ph),6.05-5.88(m,2H,-CH=),5.34(s,2H,=CH2),5.14-5.01(m,4H,=CH2,-CH 2-),4.66(s,2H,-CH2-),4.17(s,2H,-CH2-),3.91(s,2H,-CH2-),3.45(s,8H,-CH2-,-CH3),3.28(dd,J=12.4Hz,6.8Hz,2H,-CH2- ),2.42(s,2H,-CH2-),1.59-1.57(m,2H,-CH2-),1.41-1.36(m,2H,-CH2-),1.29-1.24(m,2H,-CH2-),0.91(t,J=7.2Hz,3H,-CH3); 13 C NMR(100MHz CDCl3)δ:180.9,162.6,156.2,142.9,138.2,136.7,136.5,132.0,130.1,128.9,128.0,127.0,124.5,124.1 ,117.0,115.8,112.2,112.0,111.6,63.1,52.5,48.1,40.0,39.6,34.5,31.0,23.5,20.2,13.7; HRMS(ESI)C 37 H 43 BrF3N3O3S[M+H]calcd=746.7282; found=666.2986.

[0214] Example 36 Compound 26

[0215] Compound 26 was synthesized by the method described in Example 4. The physicochemical properties of compound 26 are as follows:

[0216] 1) Yellow-brown oily substance;

[0217] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0218] With CDCl3 as solvent, the peaks are attributed to: 1H NMR(400MHz CDCl3)δ:9.04(s,1H,-NH-),7.65(dd,J=8.4Hz,2Hz,1H,-Ph),7.51(d,J=2Hz,1H,-Ph),7.25(s,1H,-Ph),7.16(s,1H,-Ph),6.98-6.97(m ,2H,-Ph),6.92(d,J=8.8Hz,1H,-Ph),6.86-6.84(m,1H,-Ph),6.74(s,1H,-Ph),6.04-5.86(m,2H,-CH=),5.39(s,2H,=CH2),5.10-5.01(m ,4H,=CH2,-CH2-),4.53(s,2H,-CH2-),4.15(s,2H,-CH2-),3.91(s,2H,-CH2-),3.78(s,3H,-OCH3),3.42(s,8H,-CH2-,-CH3),3.26(d,J =4Hz,2H,-CH2-),2.39(s,2H,-CH2-),1.55(t,J=7.2Hz,2H,-CH2-),1.36(d,J=7.6Hz,2H,-CH2-),1.24(s,2H,-CH2-),0.90(s,3H,-CH3); 13 C NMR(100MHzCDCl3)δ:180.9,162.7,160.1,156.1,143.0,137.8,135.5,132.4,130.1,128.9, 127.1,124.1,116.7,113.4,63.1,52.4,40.0,39.6,34.5,30.9,29.6,20.2,13.7; HRMS(ESI)C 38 H 48 BrN3O4S[M+H]calcd=722.7830; found=642.3367.

[0219] Example 37 Compound 27

[0220] Compound 27 was synthesized by the method described in Example 4. The physicochemical properties of compound 27 are as follows:

[0221] 1) Yellow-brown oily substance;

[0222] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0223] CDCl3 is used as solvent, and the peaks are attributed to: 1H NMR(400MHz CDC13)δ:9.23(s,1H,-NH-),7.64(d,J=8Hz,1H,-Ph),7.49(s,1H,-Ph),7.39(d,J=8.4Hz,2H,-Ph),7.14(s,1H,-Ph),6.92-6.86(m,3H ,-Ph),6.76(s,1H,-Ph),6.03-5.86(m,2H,-CH=),5.35(s,2H,=CH2),5.11-5.01(m,4H,=CH2,-CH2-),4.64(s,2H,-CH2-),4.15(s,2H,- CH2-),3.93-3.89(m,2H,-CH2-),3.78(s,3H,-OCH3),3.45(s,6H,-CH3),3.43(s,2H,-CH2-),3.26(dd,J=12.4Hz,8.4Hz,2H,-CH2-),2. 41(d,J=3.2Hz,2H,-CH2-),1.56(t,J=7.2Hz,2H,-CH2-),1.38-1.34(m,2H,-CH2-),1.28-1.24(m,2H,-CH2-),0.92-0.89(m,3H,-CH3); 13 C NMR(100MHz CDCl3)δ:180.7,162.7,159.6,156.1,142.9,137.8,136.6,132.4,129.3,129.0,127.1,126.0,124.0,116. 7,115.8,114.3,111.5,108.2,64.4,63.1,55.2,52.5,49.0,40.0,39.5,34.4,31.0,20.2,13.7; HRMS(ESI)C 38 H 48 BrN3O4S[M+H]calcd=722.7830; found=642.3371.

[0224] Example 38 Compound 28

[0225] Compound 28 was synthesized by the method described in Example 4. The physicochemical properties of compound 28 are as follows:

[0226] 1) Yellow-brown oily substance;

[0227] 2) The NMR spectrum of the compound ( 1 H / 13 C NMR, 400MHz) characteristics:

[0228] CDCl3 is used as solvent, and the peaks are attributed to:1 H NMR(400MHz CDC13)δ:9.01(s,1H,-NH-),7.69(d,J=6.8Hz,1H,-Ph),7.63(d,J=8Hz,2H,-Ph),7.53(d,J=8.8Hz,3H,-Ph),7.18(s,1H,-Ph), 7.06(d,J=6.4Hz,1H,-Ph),6.71(s,1H,-Ph),6.01-5.85(m,2H,-CH=),5.48(s,2H,=CH2),5.11-4.94(m,4H,=CH2,-CH2-),4.76 (s,2H,-CH2-),4.59(s,2H,-CH2-),4.28(s,2H,-CH2-),3.56(s,6H,-CH3),3.43(d,J=6.4Hz,4H,-CH2-),3.29(s,2H,-CH2-),1 .58(d,J=7.6Hz,2H,-CH2-),1.41-1.37(m,2H,-CH2-),1.29(d,J=13.6Hz,2H,-CH2-),0.92(t,J=7.2Hz,3H,-CH3); HRMS(ESI)C 38 H 45 BrF3N3O3S[M+H]calcd=760.7552; found=680.3153.

[0229] Application Example 1: In vitro antibacterial activity assay

[0230] 1. Test bacteria:

[0231] Staphylococcus aureus (Staphylococcus aureus ATCC 29213); Escherichia coli (EscherichiacoliATCC25922); Methicillin-resistant Staphylococcus aureus (MRSA).

[0232] 2. Samples and reagents:

[0233] The samples are: honokiol, vancomycin, meropenem and compound 1-28 prepared in the examples.

[0234] 3. Test method:

[0235] According to the standards of the Clinical Laboratory Standards Institute (CLSI), the in vitro antibacterial activities of honokiol, compounds 1-28 of the present invention and the clinical antibacterial drug vancomycin were tested using the broth microdilution method, and the minimum drug concentration of completely clear wells observed by naked eye was taken as the MIC value.

[0236] Table 1. MIC values ​​of the oxazolethione structure-containing magnolol-antimicrobial peptide mimetic conjugates 1-28 of the present invention against 10 MRSA clinical isolates

[0237]

[0238] Sa a .:S.aureusATCC 29213;M11-23 b :10 clinical MRSA strains; Ec c .:E.coli ATCC 25922; SI d :Selectivity coefficient (HC 50 / MICs of S.aureus); ND e : Not detected; Van f : Vancomycin; MEM g :Meropenem. The experiment was repeated at least 3 times.

[0239] It can be concluded from Table 1 that most of the magnolol-antimicrobial peptide mimetic conjugates containing oxazolethione structure prepared by the present invention have enhanced anti-Gram-positive bacteria activity compared with the substrate magnolol, and only a few conjugates have a downward trend in antibacterial effect. The synthesized conjugates have no obvious antibacterial effect on E. coli. Conjugate 3 is the compound with the most significant anti-Gram-positive bacteria activity among the 28 magnolol-antimicrobial peptide mimetic conjugates containing oxazolethione, and the MIC value is between 2-4 μg / mL. And conjugate 3 has a lower hemolytic activity (HC 50 =157.3) and good membrane selectivity and safety (SI=95.096).

[0240] Application Example 2: Time-killing kinetics experiment of compound 3:

[0241] 1. Test bacteria:

[0242] S. aureus ATCC 29213; MRSA-12 (clinical isolate).

[0243] 2. Samples and reagents:

[0244] The samples were: vancomycin and compound 3 prepared in Example.

[0245] 3. Test method:

[0246] Monoclonal colonies of S. aureus ATCC 29213 and MRSA-20 were inoculated into 1 mL LB broth for overnight culture. 50 μL of bacterial solution was placed in 50 mL LB broth for expansion culture for 2.5 h (37 °C, 200 rpm). After the culture was completed, the bacterial concentration was determined to be 1×10 7CFU / mL. Four groups of experiments were set up, namely 4×MIC / 8×MIC compound group, 8×MIC vancomycin group, and Control group. Compound 3 solution dissolved in DMSO and vancomycin solution were added to the cultured bacterial solution, and an equal amount of DMSO was added to the Control group. Then, the culture was continued in a shaker, and 100 μL of bacterial solution was taken at 0, 0.5, 1, 2, 4, 6, and 8 h. After washing and resuspending, gradient dilution was performed in a 96-well plate. 10 μL of sample dilutions at different concentrations were aspirated and dropped onto the MHA solid culture dish, dried, inverted in a 37°C incubator for 12-16 h, and the number of colonies at different time points was read and a sterilization curve was drawn. Three parallel controls were set for each concentration. The experiment was repeated three times. The results are as follows Figure 1 As shown in A and B.

[0247] Figure 1 A and B show that the bactericidal rate of compound 3 against MRSA-12 and S.aureus ATCC29213 is higher than that of vancomycin at the same 8×MIC concentration. For compound 3, within 4h of 8×MIC and within 8h of 4×MIC, the CFU / mL of MRSA-12 was reduced by more than 3 logarithms (killing 99.9% of bacteria). In addition, under the action of 4×MIC concentration of compound 3, bacterial growth was also significantly inhibited. Compound 3 showed rapid bactericidal activity against S.aureusATCC29213, and was able to completely kill bacteria within 6 hours at 8×MIC. These results show that compound 3 has rapid bactericidal activity against Gram-positive bacteria MRSA-12 and S.aureusATCC29213.

[0248] Application Example 3: Drug resistance induction experiment of compound 3:

[0249] After determining the initial MIC values ​​of target compound 3 and norfloxacin, the S. aureus ATCC 29213 monoclonal colonies were placed in two tubes of 1 mL MHB liquid culture medium, and then the test compound and norfloxacin (final concentration of both was 1 / 2 MIC) were added respectively and cultured for 12 hours. The bacterial solution was dipped with a bacterial loop and inoculated onto an MHA solid culture dish using the three-zone streak method. After the monoclonal colonies appeared, the MIC values ​​were re-determined and recorded, which were the first-generation MIC values. After that, the monoclonal colonies were picked and inoculated onto MHA solid culture dishes containing sub-inhibitory concentrations of the test compound and norfloxacin, and cultured in a constant temperature incubator at 37°C for 12 hours. After the monoclonal colonies grew out, the MIC values ​​were re-determined and recorded, which were the second-generation MIC values. Repeat the above steps, and the sub-inhibitory concentration was adjusted according to the MIC value of each generation. A total of 20 generations were cultured. See the results. Figure 1 C.

[0250] from Figure 1C The results showed that in the presence of sub-inhibitory concentration (1 / 2MIC) of compound 3, S. aureus ATCC29213 failed to produce resistant mutants after continuous passage within 20 days, indicating that the target was non-specific. After 6 passages, the MIC of norfloxacin began to rise and drug resistance appeared. After 16 passages, the MIC of norfloxacin increased by 128 times. This shows that the low drug resistance of compound 3 is beneficially affected by its rapid bactericidal activity. .

[0251] Application Example 4: In vivo safety evaluation experiment

[0252] 1. Reagents:

[0253] Compound 3 prepared in Example, 0.9% NaCl.

[0254] 2. Test animals

[0255] SPF grade KM mice (purchased from the Animal Experiment Center of Zhengzhou University, weighing 20-22 g, 7-8 weeks old) were used.

[0256] 3. Test methods

[0257] Compound 3 was prepared into test compound solutions with concentrations of 5, 10, 20, 40, and 80 mg / kg using normal saline. 30 healthy female KM mice of uniform size and weight were selected and randomly divided into 6 groups after depilation of the back, with 5 mice in each group. 60 μL of compound solutions of different concentrations were subcutaneously injected into the back of the mice in the drug-treated group, and 60 μL of normal saline was subcutaneously injected into the back of the mice in the blank group. After injection, the survival status of the mice and whether there was an irritation reaction on the skin were observed. After 24 hours, blood was collected from the eyeballs of the mice at the maximum injection concentration without death and irritation, and blood routine, liver function, and kidney function tests were performed to observe whether there were significant changes compared with the blank group.

[0258] By injecting different concentrations of the compound subcutaneously into mice, it was found that when the concentration of compound 3 was ≤20 mg / kg, the mouse skin did not show any adverse reactions (such as redness, swelling, hardening, ulceration, etc.). Therefore, routine blood tests and blood biochemical index tests were performed on the mice at this dosage. Figure 2 The results showed that blood samples were subjected to routine blood tests and blood biochemistry analysis, and the test items included white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), platelet count (PLT), albumin (ALB), urea (UREA) and creatinine (CREA). The statistical results showed that compared with the Control group (0.9% NaCl), after subcutaneous injection of compound 3 (20 mg / kg) into KM mice, there was no significant difference in the results, indicating that compound 3 has a certain in vivo safety.

[0259] Application Example 5: In vivo anti-MRSA infection activity experiment of compound 3

[0260] 1. Test bacteria:

[0261] MRSA-12 (clinical isolate)

[0262] 2. Samples and reagents:

[0263] The samples were: vancomycin, compound 3 prepared in Example, and 0.9% NaCl.

[0264] 3. Test animals:

[0265] SPF grade KM mice (purchased from the Animal Experiment Center of Zhengzhou University, weighing 20-22 g, 7-8 weeks old) were used.

[0266] 4. Test method:

[0267] Thirty healthy female KM mice of the same size and weight were depilated from their backs and randomly divided into a blank group, a control group (normal saline), a vancomycin group (5 mg / kg), a high-dose compound group (10 mg / kg), and a low-dose compound group (5 mg / kg), with 6 mice in each group. After the backs of the mice were disinfected, mice in the other groups except the blank group were subcutaneously injected with 60 μL of a bacterial concentration of 1×10 8 CFU / mL of MRSA-20 bacterial suspension. 2 hours later, 60 μL of different amounts of compound, vancomycin, and saline were injected in situ. The survival and skin infection of each group of mice were recorded. 24 hours after administration, the mice in each group were killed by dislocation, and the skin of the infected area was taken and ground into a broken state using a tissue grinder. The number of colonies was read using the drop plate counting method. The skin tissue grinding liquid of the infected area was aspirated, and the results were as follows: Figure 3 shown.

[0268] The results showed that: Figure 3 As shown, the bacterial load in the skin tissue of mice in the Control group was about 6.7 log 10 CFU / g, while after treatment with low-dose 3 (5 mg / kg) and vancomycin (5 mg / kg), the number of MRSA cells in the mouse skin was significantly reduced (P<0.0001), and the bacterial load was reduced by 1.4 log 10 cfu / g, showing a moderate therapeutic effect. After 3 (10 mg / kg) treatments, the skin bacterial load decreased by about 4.1 log 10cfu / g, the therapeutic effect was the most significant. Therefore, it was confirmed that the magnolol-antimicrobial peptide mimetic conjugate 17 containing oxazolethione structure had a good therapeutic effect on mouse skin abscesses infected with MRSA. The therapeutic effect was the same as that of the control drug vancomycin at the same dose. Compound 3 is expected to be developed into a new anti-MRSA drug.

Claims

1. A magnolol-antimicrobial peptide mimetic conjugate containing an oxazolethione structure, the structure of which is shown in the following formula (1): in, R1, R2 and R3 are independently selected from H, halogen, C1-C3 alkoxy or CF3.

2. The oxazolethione structure-containing honokiol-antimicrobial peptide mimetic conjugate according to claim 1, characterized in that: The R is one of the following substitutions: (1) R1=R2=R3=H; (2) R1=R2=H, R3=2-F; (3) R1=R2=H, R3=4-F; (4) R1=R2=H, R3=2-Cl; (5) R1=R2=H, R3=4-Cl; (6) R1=R2=H, R3=2-Br; (7) R1=R2=H, R3=3-Br; (8) R1=R2=H, R3=4-Br; (9) R1=H, R2=2-F, R3=4-Cl; (10) R1=H, R2=2-F, R3=5-Br; (11) R1=H, R2=3-F, R3=4-F; (12) R1=H, R2=3-F, R3=4-Cl; (13) R1=H, R2=2-Cl, R3=4-F; (14) R1=H, R2=3-Cl, R3=4-F; (15) R1=H, R2=2-Cl, R3=3-Cl; (16) R1=H, R2=2-Cl, R3=4-Cl; (17) R1=H, R2=2-Cl, R3=6-Cl; (18) R1=H, R2=3-Cl, R3=4-Cl; (19) R1=H, R2=3-Cl, R3=5-Cl; (20) R1=H, R2=2-Br, R3=4-Cl; (21) R1=H, R2=3-Br, R3=4-F; (22) R1=H, R2=3-Br, R3=4-Cl; (23) R1=H, R2=2-Br, R3=4-Br; (24) R1=H, R2=3-Br, R3=5-Br; (25) R1=3-F, R2=4-F, R3=5-F; (26) R1=R2=H, R3=3-OCH3; (27) R1=R2=H, R3=4-OCH3; (28) R1=R2=H, R3=4-CF3.

3. The method for preparing the oxazolethione structure-containing honokiol-antimicrobial peptide mimetic conjugate according to any one of claims 1 to 2, characterized in that: The following steps are involved: (1) Using honokiol as a substrate, it reacts with iodine to convert the ortho-phenolic hydroxyl group and the propenyl group into an oxygen-containing five-membered ring to obtain intermediate 2; (2) intermediate 2 reacts with concentrated nitric acid to introduce a nitro group to synthesize intermediate 3; (3) Intermediate 3 is then reacted with a reducing agent to reduce the nitro group to an amino group to obtain intermediate 4; (4) intermediate 4 is subjected to the action of a catalyst to open the oxygen-containing five-membered ring to generate a phenolic hydroxyl group and a propenyl group, thereby obtaining intermediate 5; (5) Intermediate 5 is then reacted with benzaldehyde containing different substituents to generate intermediate 6; (6) Intermediate 6 reacts with a reducing agent to generate intermediate 7; (7) Intermediate 7 reacts with carbon disulfide under alkaline conditions to generate intermediate 8 (8) Intermediate 8 reacts with 1,3-dibromopropane under alkaline conditions to generate intermediate 9; (9) The intermediate 9 then reacts with a small molecule peptide mimetic 10 to generate the honokiol-antimicrobial peptide mimetic conjugate containing an oxazolethione structure, and the reaction formula is as follows: Wherein, R1, R2 and R3 are the same as those described in claim 1 or 2.

4. The method for preparing the oxazolethione structure-containing honokiol-antimicrobial peptide mimetic conjugate according to claim 3, characterized in that: In step (1), the reaction molar ratio of honokiol to iodine is 1:(1.0-1.2), the reaction temperature is 45-55°C, and the reaction solvent is 9:1-7:3 of ethanol and ultrapure water; in step (2), the reaction molar ratio of the intermediate 2 to concentrated nitric acid is 1:(2.5-3.5), the reaction temperature is 0±5°C, and the reaction solvent is dichloromethane.

5. The method for preparing the oxazolethione structure-containing honokiol-antimicrobial peptide mimetic conjugate according to claim 3, characterized in that: In step (3), the reducing agent is selected from stannous chloride dihydrate, the molar ratio of the intermediate 3 to stannous chloride dihydrate is 1:(9-11), the reaction temperature is 75-80°C, and the reaction solvent is ethyl acetate; in step (4), the catalyst is selected from zinc; the molar ratio of the intermediate 4 to the catalyst is 1:(4-6), the reaction temperature is 75-80°C, and the reaction solvent is anhydrous ethanol.

6. The method for preparing the oxazolethione structure-containing honokiol-antimicrobial peptide mimetic conjugate according to claim 3, characterized in that: In step (5), the molar ratio of the intermediate 5 to the benzaldehyde containing different substituents is 1:(2.5-3.5), the reaction temperature is 40-50°C, and the reaction solvent is methanol; in step (6), the reducing agent is selected from sodium borohydride, the molar ratio of the intermediate 6 to sodium borohydride is 1:(5-7), the reaction temperature is 0±5°C, and the reaction solvent is dichloromethane; in step (7), the base in the alkaline condition is selected from KOH, the molar ratio of the intermediate 7 to carbon disulfide is 1:(3-5), the reaction temperature is 45-55°C, and the reaction solvent is anhydrous ethanol.

7. The method for preparing the oxazolethione structure-containing honokiol-antimicrobial peptide mimetic conjugate according to claim 3, characterized in that: In step (8), the molar ratio of the intermediate 8 to 1,3-dibromopropane is 1:(2.5-3.5), the reaction temperature is 45-55° C., and the reaction solvent is anhydrous acetone.

8. The method for preparing the oxazolethione structure-containing honokiol-antimicrobial peptide mimetic conjugate according to claim 3, characterized in that: In step (9), the method for preparing the small molecule peptide mimetic 10 comprises the following steps: bromoacetyl bromide and mono-n-butylamine undergo a substitution reaction under alkaline conditions to generate bromoacetamide, and then bromoacetamide continues to undergo a substitution reaction with dimethylamine under alkaline conditions to generate a small molecule peptide mimetic: Preferably, the molar ratio of the intermediate 9 to the small molecule peptide mimetic 10 is 1:(2.5-3.5), the reaction temperature is 75-85° C., and the reaction solvent is anhydrous acetonitrile.

9. Use of the honokiol-antimicrobial peptide mimetic conjugate containing an oxazolethione structure according to any one of claims 1 to 2 in the preparation of antibacterial drugs.

10. The use according to claim 9, characterized in that: The antibacterial drug can inhibit Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

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