Small molecule antimicrobial peptide mimetics and their applications

Abstract

The present invention discloses a small molecule antimicrobial peptide mimetic and application thereof. The small molecule antimicrobial peptide mimetic is based on a cysteine ​​skeleton structure and is obtained by chemically modifying an amino terminal, a carboxyl terminal, or a side chain thiol group. The general formula thereof is: The small molecule antimicrobial peptide mimetic of the present invention can cause cell membrane depolarization, destroy cell membrane integrity, quickly kill bacteria, inhibit biofilm formation, have certain biofilm removal activity, and are not prone to inducing bacterial resistance. It has good activity against Gram-positive bacteria and exhibits good antimicrobial activity against resistant bacteria such as MRSA and VRE. It can solve the current problem of lack of drugs against resistant bacteria, has the function of replacing or assisting current clinical antibiotics in treating infectious diseases caused by various resistant bacteria, can be used for the treatment of infectious diseases caused by sensitive bacteria and resistant bacteria, has high-efficiency antimicrobial activity, low toxicity, and is not affected by traditional resistance mechanisms.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to a small molecule antimicrobial peptide mimetic and its application in the treatment of infectious diseases. Background Art

[0002] Antibiotics are the main drugs currently used to treat diseases caused by bacterial infections. However, with the widespread use and abuse of antibiotics in medicine, animal husbandry and food industries, more and more bacteria have become resistant to conventional antibiotics, which has become one of the most serious problems facing world health today. At present, there is a lack of drugs against resistant Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) and multidrug-resistant Gram-negative bacteria (Pseudomonas aeruginosa), which seriously threaten human health and life safety. However, in the past four decades, few innovative structural antibiotics have entered clinical practice, so the development of new structural antibacterial drugs is of great significance.

[0003] In recent years, cationic antimicrobial peptides have shown certain prospects as potential antimicrobial drugs. Antimicrobial peptides are sentinels of innate immunity in most species and are the first line of defense against any infection. Unlike most antibiotics that work by targeting intracellular organelles, antimicrobial peptides and their mimics exert their antibacterial effects mainly by causing cell membrane lysis. Therefore, unlike conventional antibiotics that can lose their activity due to point mutations in bacteria, it is difficult for bacteria to develop resistance to antimicrobial peptides and their mimics. However, problems such as poor stability, high production costs, and high systemic toxicity limit the clinical application of cationic antimicrobial peptides. Therefore, many research groups are committed to developing new strategies to solve these problems faced by antimicrobial peptides. Developing antimicrobial peptide mimics by simulating the structure and mechanism of action of antimicrobial peptides is one of the important strategies, which has attracted the attention of more and more researchers.

[0004] Although there are currently a large number of macromolecular mimetics of antimicrobial peptides, there are not many cases of successful development of small molecule mimetics of antimicrobial peptides. This is mainly because the design of these compounds focuses more on the structure, while ignoring the limitations of their application due to their structural complexity, numerous synthesis steps, high production costs, and the presence of peptide bonds that cannot be degraded by proteases. Therefore, antimicrobial peptide mimetics with simple structures, easy synthesis, low production costs, and low inducing bacterial resistance have better clinical application potential. Summary of the invention

[0005] The object of the present invention is to overcome at least one deficiency of the prior art and to provide small molecule antimicrobial peptide mimetics and applications thereof.

[0006] The technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides: a small molecule antimicrobial peptide mimetic, which is based on a cysteine ​​backbone structure and is obtained by chemically modifying the amino terminal, carboxyl terminal, or side chain thiol group. The general structural formula of the small molecule antimicrobial peptide mimetic is as follows:

[0008] Where:

[0009] R 1 Selected from One of;

[0010] R 2 Selected from

[0011] One of;

[0012] R 3 Selected from One of them.

[0013] In some examples of small molecule antimicrobial peptide mimetics, R 1 Selected from One of;

[0014] R 2 Selected from

[0015] One of;

[0016] R 3 Selected from

[0017] In some examples of small molecule antimicrobial peptide mimetics, the structural formula is one of Formulas 1, 5-12, 15-18, 20, 23, 25, 27-30, 32-35:

[0018]

[0019]

[0020]

[0021] The second aspect of the present invention provides: a composition, the active ingredient of which includes at least one of the small molecule antimicrobial peptide mimetics described in the first aspect of the present invention.

[0022] In some examples of the composition, an acceptable carrier is also included.

[0023] In some examples of the composition, the dosage form is an oral preparation, an injection, a mucosal administration preparation, or an external preparation.

[0024] The third aspect of the present invention provides: use of the small molecule antimicrobial peptide mimetic according to the first aspect of the present invention in the preparation of anti-infective drugs.

[0025] In some embodiments of the invention, the infection is caused by bacteria or fungi.

[0026] In some application examples, the bacteria or fungi are drug-resistant bacteria or fungi.

[0027] In some application examples, the drug-resistant bacteria or fungi are selected from methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus.

[0028] The beneficial effects of the present invention are:

[0029] The small molecule antimicrobial peptide mimetics of some examples of the present invention can cause cell membrane depolarization, destroy cell membrane integrity, quickly kill bacteria, inhibit biofilm formation, have certain biofilm removal activity, are not easy to induce bacterial resistance, have good activity against Gram-positive bacteria, and show good antibacterial activity against resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). They can solve the current problem of lack of drugs against resistant bacteria, have the ability to replace or assist current clinical antibiotics in the treatment of infectious diseases caused by various resistant bacteria, can be used for the treatment of infectious diseases caused by sensitive bacteria and resistant bacteria, have high antibacterial activity, low toxicity, and are not affected by traditional resistance mechanisms.

[0030] The synthesis method of the small molecule antimicrobial peptide mimetics of some examples of the present invention is simple, the raw materials are easy to obtain, and the operation method is simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The results of the depolarization experiment on Staphylococcus aureus membrane by compound 1-35 at a concentration of 4×MIC.

[0032] Figure 2 The results are as follows: The positive rate of propidium iodide (PI) in Staphylococcus aureus was measured after compound 1-35 with a concentration of 4×MIC was incubated with Staphylococcus aureus for 30 minutes and then incubated in the dark for 15 minutes.

[0033] Figure 3 The results of the bactericidal kinetics experiment of compound 1-35 against Staphylococcus aureus were shown in Figure 1. 0.9% saline and vancomycin were used as negative control and positive control, respectively.

[0034] Figure 4The results of the biofilm inhibition experiment. Compounds 1-35 at a concentration of 4×MIC were incubated with Staphylococcus aureus to remove the surface planktonic bacteria, and then washed and fixed and stained with crystal violet solution. After removing the floating color, 95% ethanol was added to dissolve the crystal violet in the biofilm. Finally, the OD was measured. 570 Measure the light absorption value at the location and record the result.

[0035] Figure 5 The results of the bacterial resistance experiment are shown in Figure 2. Staphylococcus aureus was co-cultured with representative compounds 10, 28 and 33, ampicillin and ciprofloxacin at 1 / 2×MIC. After 16 hours, the bacterial solution with the highest drug concentration where bacteria grew was taken and cultured to the logarithmic phase, and then the staged MIC was determined. A total of 21 generations were determined.

[0036] Figure 6 The results of lung changes in the control group and mice after drug treatment. Experimental mice were selected and divided into six groups: model group, vancomycin (Vanc) group, compound 10, 28 and 33 administration group and blank control group. After treatment, the mice in different groups were dissected and their lung changes were observed.

[0037] Figure 7 The in vivo antibacterial results of compounds 10, 28 and 33. The results of colony counts after 24 hours of lung grinding and plating culture in the compound 10, 28 and 33 treatment groups, vancomycin treatment control group, blank control group, and non-treatment model group (MD). DETAILED DESCRIPTION

[0038] The embodiments of the present invention are described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0039] Instruments used in the present invention:

[0040] The high performance liquid chromatograph was a Delta 600 provided by Waters Company of the United States. The preparative column used was a reversed-phase C18 column (XBridge BEH 130Prep), 19 mm*250 mm; the analytical column was a reversed-phase C18 column, 4.6 mm*250 mm; the mass spectrometer was a Bruker Maxis 4G mass spectrometer; and the nuclear magnetic resonance spectrometer was a Bruker AVANCE III HD 400. The reagents included: NA-Fmoc-protected amino acids, N-hydroxybenzotriazole (HOBt), oxy-benzotriazole-N,N,N',N'-tetramethylurea-hexafluorophosphate (HBTU), diisopropylethylamine (DIEA) and triisopropylsilane (TIS), all purchased from Shanghai Jier Biochemical Co., Ltd.; dichloromethane, N,N-dimethylformamide (DMF), hexahydropyridine (piperidine), methanol, trifluoroacetic acid (TFA), phenol and pyridine were all purchased from Tianjin Second Reagent Factory. All other instruments and reagents used without indicating the manufacturer are conventional products that can be obtained through commercial purchase. If no specific conditions are indicated in the examples, conventional conditions or conditions recommended by the manufacturer were followed.

[0041] Example 1: Synthesis of Compound 1-16

[0042] The synthetic route of compound 1-13 is as follows:

[0043]

[0044] In a typical reaction, commercially available amino acid Fmoc-Cys(R)-OH (0.5mmol, 1eq), HOBT (0.6mmol, 81.08mg, 1.2eq), HBTU (0.6mmol, 227.54mg, 1.2eq) and DIEA (1.2mmol, 155.1mg, 2.4eq) were added to 30mL of DMF, stirred and dissolved, and N-Boc-Ethylenediamine (0.6mmol, 96.13mg, 1.2eq) was added and stirred at room temperature for 3h. After the reaction was completed, 60mL of water was added, and 60mL of ethyl acetate was shaken and washed, and the organic phase was retained, and then the organic layer was washed with brine, dried with anhydrous sodium sulfate, and then the organic phase was evaporated under reduced pressure to obtain the intermediate product with a yield of more than 95%. The intermediate product obtained does not need further treatment. Trifluoroacetic acid (8 mL), dichloromethane (20 mL) and TIS (200 μL) are added and stirred for 3 h (TIS is not added when compound 8k is treated). The solvent is removed under reduced pressure, and glacial ether is added to precipitate the target product. The final compound 1-11 is obtained by further HPLC purification. Part of the product obtained in this step (0.3 mmol, 1 eq) and 1H-pyrazole-1-carboxamidine hydrochloride (0.36 mmol, 1.2 eq) are dissolved in 10 ml DMF, DIEA (0.6 mmol, 2 eq) is added dropwise at room temperature, the reaction is carried out for 3 h, dilute hydrochloric acid is added to quench, and after dilution with water, it is purified by HPLC and freeze-dried to obtain compounds 12 and 13.

[0045] The synthetic routes of compounds 14-16 are as follows:

[0046]

[0047] fmoc-cys(Trt)-OH (1.7mmol, 1g, 1eq) was dissolved in dichloromethane (40mL), trifluoroacetic acid (10mL) and TIS (500μL) were added, and the mixture was stirred at room temperature for 2h. After the reaction was completed, the solvent was removed under reduced pressure, and icy ether was added for precipitation to obtain an intermediate product. The compound was directly dissolved in dichloromethane (50mL) without further treatment, DIEA (2.5mL) and Br-R (3.4mmol, 2eq) were added, and the reaction progress was monitored by thin layer chromatography after stirring for 16h. After the reaction was completed, the solvent was removed by reduced pressure, and the mixture was purified by silica gel column to obtain an intermediate product. 20% TFA (15mL) was added to obtain the final compound 14-16.

[0048] Example 2: Synthesis of Compounds 17-26

[0049] The synthetic route of compound 17-26 is as follows:

[0050]

[0051] Fmoc-cys(trt)-OH (292 mg, 0.5 mmol, 1 eq), HOBT (0.6 mmol, 81.08 mg, 1.2 eq), HBTU (0.6 mmL, 227.54 mg, 1.2 eq) and DIEA (1 mmol, 2 eq, 130 mg) were added to 30 mL of DMF, stirred and dissolved, and an amine compound (0.6 mmol, 1.2 eq) was added, and stirred at room temperature for two hours. After the reaction was completed, 60 mL of water was added, and 60 mL of ethyl acetate was shaken and washed, and the organic phase was retained. Then, the organic layer was washed with brine, dried over anhydrous sodium sulfate, and then the organic phase was evaporated under reduced pressure to obtain the intermediate product and the final product 17 with a yield of more than 95%. The product obtained in the previous step (0.479 mmol) was dissolved in 15 mL of tetrahydrofuran, piperidine (5 mL) was added, and stirred at room temperature for 1 h. After the reaction was completed, the solvent was removed under reduced pressure, and ice ether was added for precipitation to obtain the product 21 and the intermediate product. The intermediate product (0.4mmol, 290mg, 1eq) was dissolved in DMF (30mL), and HOBT (0.48mmol, 64.89mg, 1.2eq), HBTU (0.48mmol, 181.92mg, 1.2eq), DIEA (0.96mmol, 124.07mg, 2.4eq), 4-Biphenylacetic acid (0.48mmol, 101.87mg, 1.2eq) were added and stirred at room temperature for 3h. After the reaction was completed, 60mL of water was added, and 60mL of ethyl acetate was shaken and washed, and the organic phase was retained, and then the organic layer was washed with brine, dried with anhydrous sodium sulfate, and the organic phase was evaporated under reduced pressure. The obtained compound was added with trifluoroacetic acid (8 mL) and dichloromethane (20 mL) without further treatment, and stirred for 3 h. The solvent was removed under reduced pressure, and glacial ether was added to precipitate the target product. The final compound 25 was obtained by further HPLC purification. Compound 25 was obtained by the same steps as in Scheme 1a to obtain compound 26. The intermediate product obtained in the first step was treated with 20% TFA for 3 h to obtain compounds 18-20. Compound 21 (0.35 mmol, 1 eq), HOBT, HBTU, DIEA (0.42 mmol, 1.2 eq) were dissolved in 20 ml DMF with BOC-LYS(BOC)-OH and BOC-SER-OH (0.4 mmol), respectively, and reacted for 3 h. After treatment according to the experimental steps of compound 25, compounds 22 and 24 were obtained.

[0052] Example 3: Synthesis of Compounds 27-35

[0053] The synthetic route of compound 27-35 is as follows:

[0054]

[0055] In a needle tube synthesizer, 0.5 degree of substitution dichlororesin (1 g) was added, and the resin was swollen with dichloromethane (10 mL) for 5 min. The solvent was removed by suction filtration, and dichloromethane (10 mL), fmoc-cys(Dpm)-OH (763.5 mg, 1.5 mmol, 3 eq) and DIEA (516.96 mg, 4 mmol, 8 eq) were added. The reaction was continued for 2 h, and the solvent was removed by suction filtration. The mixture was washed with DMF for 3 times. 20% piperidine (10 mL) was added and the reaction was continued for 30 min. The solvent was removed by suction filtration, and DMF was added and washed for 4 times. DMF (10 mL) was added and the mixture was reacted with biphenylcarboxylic acid, biphenylacetic acid, piperazinecarboxylic acid, theophyllineacetic acid, ferrocenecarboxylic acid (1 mmol, 2 eq), HOBT (135.12 mg, 1 mmol, 2 eq), HBTU (379.25 mg, 1 mmol, 2 eq), and DIEA (258.48 mg, 2 mmol, 4 eq) for 1 h. The solvent was removed by filtration, washed with DMF three times, dichloroethane twice, methanol twice, and dichloroethane once, 20% trifluoroethanol (10 mL) was added to react for 2 h, the solvent was collected, and the solvent was removed under reduced pressure. The intermediate product with R group being felbinac did not need further treatment and was respectively reacted with 3-(2-aminoethyl)pentane-1,5-diamine (1.25 mmol, 183.17 mg, 3 eq) 2,2'-oxybis(ethan-1-amine) (120 mg), 2,2'-(ethane-1,2-di ylbis(oxy))bis(ethan-1-amine)(160mg) was dissolved in DMF(30mL), HOBT(0.5mmol, 67.56mg, 1.2eq), HBTU(0.5mmol, 189.62mg, 1.2eq), DIEA(1.5mmol, 193.9mg, 3.6eq) and the product of the fourth step (0.417mmol, 199.74mg, 1eq) were dissolved in DMF(10mL), and added dropwise to the above amine solution over 1h, and the reaction was continued for 2h. After the reaction was completed, the solvent was removed under reduced pressure, and purified by HPLC to obtain compounds 27-29. Compounds 30-35 were obtained by a similar reaction process.

[0056] Example 4. Structural characterization of compounds

[0057] Weigh the crude product after desalting (about 40 mg) and dissolve it in 20% acetonitrile / water solution (5 mL). Filter it with a filter and then load it. The column used in the purification process is a μ-bondapaktm C18 reverse column (19 mm × 300 mm). Use 20%-80% acetonitrile / water solution for gradient elution. Observe the absorption value of the UV detector at 220 nm, collect the main peak, and place the collected liquid in a 50 mL beaker at -80 ° C overnight, and then vacuum freeze-dry to obtain pure compound powder. The structure and NMR characterization of the synthesized compound are as follows:

[0058] NMR spectrum analysis data of compound 1: 1 H NMR (400 MHz, DMSO-d 6 )δ8.32(dt,J=16.2,5.7Hz,1H),7.90(q,J=7.6,7.0Hz,5H),7.78–7.64(m,3H),7.47–7.26(m,4H),4.44– 4.11(m,4H),3.65(s,2H),3.31(dt,J=13.0,6.4Hz,2H),2.96–2.86(m,2H),2.84(dd,J=9.1,4.6Hz,1H); 13 C NMR (101 MHz, DMSO-d 6 )δ170.61,156.00,143.71,140.70,127.64,127.06,125.25,120.10,65.73,57.28,46.62,38.36,36.52,25.93; Compound molecular weight data: theoretical molecular weight C 20 H 23 N 3 O 3 S[M+H] + =386.1460, and the molecular weight determined by mass spectrometry was 386.1562. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 93.92%.

[0059] NMR spectrum analysis data of compound 2: 1 H NMR (400 MHz, DMSO-d 6)δ8.62(t,J=6.4Hz,1H),8.27(t,J=5.7Hz,1H),8.00–7.87(m,5H),7.75(d,J=7.5Hz,2H) ,7.70(d,J=8.3Hz,1H),7.43(td,J=7.5,1.1Hz,2H),7.34(td,J=7.5,1.2Hz,2H),4.39–4. 28(m,2H),4.24(t,J=4.1Hz,2H),4.23–4.17(m,2H),3.34(tp,J=13.7,6.7Hz,2H),2.97( dd,J=13.8,4.9Hz,1H),2.88(h,J=6.3Hz,2H),2.74(dd,J=13.8,9.5Hz,1H),1.87(s,3H); 13 C NMR (101MHz, DMSO) δ171.08, 155.98, 143.78, 140.70, 127.63, 127.04, 125.28, 120.08, 65.72, 54.01, 46.61, 38.32, 36.52, 35.56, 14.98; Compound molecular weight data: theoretical molecular weight C 21 H 25 N 3 O 3 S[M+H] + =400.1617, the molecular weight determined by mass spectrometry was 400.1672. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 97.56%.

[0060] NMR spectrum analysis data of compound 3: 1 H NMR (400 MHz, DMSO-d 6 )δ8.82(t,J=6.3Hz,1H),8.20(t,J=5.7Hz,1H),7.90(d,J=7.5Hz,5H),7.72( dd,J=17.1,7.9Hz,3H),7.42(td,J=7.5,1.1Hz,2H),7.37–7.18(m,7H),4.40 –4.27(m,2H),4.27–4.16(m,4H),3.47(s,2H),3.37–3.26(m,2H),2.94(dd,J =13.8,4.9Hz,1H),2.85(q,J=7.1,6.7Hz,2H),2.73(dd,J=13.8,9.5Hz,1H); 13C NMR (101MHz, DMSO) δ171.00, 170.61, 155.99, 143.76, 140.69, 135.96, 128.96, 128.22, 127.63, 127.05, 126.42, 125.33, 120.09, 65.79, 54.64, 46.57, 42.22, 40.31, 38.31, 36.50, 32.38; Compound molecular weight data: theoretical molecular weight C 24 H 31 N 3 O 3 S[M+H] + =442.2086, the molecular weight determined by mass spectrometry was 442.2144. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 98.30%.

[0061] NMR spectrum analysis data of compound 4: 1 H NMR (400 MHz, DMSO-d 6 )δ8.62(t,J=6.4Hz,1H),8.27(t,J=5.7Hz,1H),7.95–7.87(m,4H),7.73(d d,J=17.2,7.9Hz,3H),7.43(td,J=7.5,1.1Hz,2H),7.34(td,J=7.5,1.2Hz, 2H),4.36–4.21(m,6H),3.34(tp,J=13.7,6.7Hz,2H),2.97(dd,J=13.8,4. 9Hz, 1H), 2.88 (h, J = 6.3Hz, 2H), 2.74 (dd, J = 13.8, 9.5Hz, 1H), 1.87 (s, 3H); 13 C NMR (101MHz, DMSO) δ171.02, 169.67, 155.98, 143.75, 140.69, 127.63, 127.05, 125.33, 120.09, 65.78, 54.67, 46.57, 38.32, 36.51, 32.41, 22.55; Compound molecular weight data: theoretical molecular weight C 23 H 28 N 4 O 4 S[M+H] + =457.1831, the molecular weight determined by mass spectrometry was 457.1929. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 97.50%.

[0062] NMR spectrum analysis data of compound 5: 1H NMR (400 MHz, DMSO-d 6 )δ8.62(t,J=6.4Hz,1H),8.27(t,J=5.7Hz,1H),7.92(d,J=6.2Hz,4H),7.73(dd,J=17.2,7.9Hz,3H),7.43(td,J=7.5,1.1Hz ,2H),7.34(td,J=7.5,1.2Hz,2H),4.41–4.13(m,8H),3.35(ddt,J=19.3,13.0,6.7Hz,2H),2.99–2.71(m,4H),1.87(s,3H); 13 C NMR (101MHz, DMSO) δ171.02, 155.98, 143.75, 140.69, 127.63, 127.05, 125.33, 120.09, 65.78, 54.67, 46.57, 40.18, 38.32, 36.51, 32.41, 22.55; Compound molecular weight data: theoretical molecular weight C 29 H 32 N 4 O 4 S[M+H] + =533.2144, the molecular weight determined by mass spectrometry was 533.2086. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 97.42%.

[0063] NMR spectrum analysis data of compound 6: 1 H NMR (400 MHz, DMSO-d 6 )δ8.38(t,J=5.7Hz,1H),7.90(d,J=7.5Hz,4H),7.88(s,1H),7.75(dd,J=7.8,3.7 Hz,3H),7.66(d,J=8.1Hz,2H),7.54(d,J=8.0Hz,2H),7.42(t,J=7.5Hz,2H),7.32( tt,J=7.5,1.6Hz,2H),4.44–4.33(m,1H),4.26–4.17(m,3H),3.85(s,2H),3.34(h ,J=6.7Hz,2H),2.84(ddd,J=23.8,13.0,5.8Hz,3H),2.61(dd,J=13.6,9.0Hz,1H); 13 C NMR (101 MHz, DMSO-d 6)δ171.04,155.95,143.74(d,J=4.0Hz),140.69,134.84,127.63,127.03(d,J=3.4Hz),125.31(d,J=6.7Hz),120.24,120.08,65.79,54.54,46.59,38.32,36.50,32.99,29.01,25.34,17.47; Compound molecular weight data: theoretical molecular weight C 28 H 28 F 3 N 3 O 3 S[M+H] + =544.1803, the molecular weight determined by mass spectrometry was 544.1840. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 95.51%.

[0064] NMR spectrum analysis data of compound 7: 1 H NMR (400 MHz, DMSO-d 6 )δ8.36(t,J=5.7Hz,1H),7.89(t,J=8.1Hz,5H),7.74(dd,J=12.0,7.9Hz,3H),7.42( td,J=7.5,1.1Hz,2H),7.37–7.28(m,6H),7.24(ddt,J=8.5,5.1,2.8Hz,1H),4.45–4. 34(m,1H),4.24(pd,J=8.6,7.9,4.0Hz,3H),3.77(d,J=2.2Hz,2H),3.42–3.24(m,2H ),2.87(q,J=6.3Hz,2H),2.81(dd,J=13.6,5.5Hz,1H),2.61(dd,J=13.6,8.8Hz,1H); 13 C NMR (101 MHz, DMSO-d 6 )δ170.95,155.99,143.75,140.70,138.28,128.84,128.33,127.63,127.06,127.02,126.82,125.35,125.26,120.09,65.80,54.27,46.61,38.36,36.51,35.23,33.07; Compound molecular weight data: theoretical molecular weight C 27 H 29 N 3 O 3 S[M+H] +=476.1930, and the molecular weight determined by mass spectrometry was 476.2038. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 94.06%.

[0065] NMR spectrum analysis data of compound 8: 1 H NMR (400 MHz, DMSO-d 6 )δ8.37(t,J=5.7Hz,1H),7.89(t,J=6.9Hz,5H),7.74(dd,J=10.7,8.0Hz,3H),7.46–7.38(m,2H) ,7.32(tt,J=7.5,1.6Hz,2H),7.19(d,J=7.9Hz,2H),7.10(d,J=7.8Hz,2H),4.44–4.33(m,1H),4. 33–4.23(m,2H),4.20(dt,J=8.6,4.3Hz,1H),3.72(d,J=2.3Hz,2H),3.40–3.24(m,J=6.6Hz,2H) ,2.87(q,J=5.9Hz,2H),2.79(dd,J=13.6,5.5Hz,1H),2.59(dd,J=13.6,8.8Hz,1H),2.26(s,3H); 13 C NMR (101 MHz, DMSO-d 6 )δ170.96,155.98,143.74,140.69,135.91,135.12,128.90,128.76,127.64,127.04 (d, J = 4.1 Hz), 125.31 (d, J = 8.6 Hz), 120.10,65.80,54.24,46.59,38.34,36.49,34.90,32.95,20.63; Compound molecular weight data: theoretical molecular weight C 28 H 31 N 3 O 3 S[M+H] + =490.2086, the molecular weight determined by mass spectrometry was 490.2218. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 96.96%.

[0066] NMR spectrum analysis data of compound 9: 1 H NMR (400 MHz, DMSO-d 6)δ8.39(t,J=5.8Hz,1H),7.91(dd,J=12.4,6.0Hz,5H),7.79–7.70(m,3H),7.42(td, J=7.5,1.1Hz,2H),7.32(tt,J=7.5,1.5Hz,2H),7.28–7.17(m,2H),6.89–6.81(m,2H ),4.44–4.34(m,1H),4.26–4.19(m,3H),3.71(d,J=2.4Hz,5H),3.35(h,J=6.7Hz,2H ),2.88(h,J=6.2Hz,2H),2.80(dd,J=13.7,5.5Hz,1H),2.60(dd,J=13.6,8.8Hz,1H); 13 C NMR (101 MHz, DMSO-d 6 )δ170.98,158.13,155.99,143.75,140.69,129.97,127.63,127.06,125.28,120.09,118.36,115.39,114.47,113.72,65.80,54.96,54.27,46.60,38.33,36.50,34.60,32.94; Compound molecular weight data: theoretical molecular weight C 28 H 31 N 3 O 4 S[M+H] + =506.2035, the molecular weight determined by mass spectrometry was 506.2170. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 91.33%.

[0067] NMR spectrum analysis data of compound 10: 1 H NMR (400 MHz, DMSO-d 6 )δ8.36(t,J=5.8Hz,1H),7.89(t,J=8.0Hz,5H),7.81–7.71(m,3H),7.45– 7.39(m,6H),7.35–7.28(m,6H),7.26–7.21(m,2H),5.38(s,1H),4.42–4.3 2(m,1H),4.23(td,J=7.1,6.2,4.2Hz,3H),3.32(t,J=6.5Hz,2H),2.85(p, J=6.1Hz,2H),2.72(dd,J=13.4,5.6Hz,1H),2.57(dd,J=13.4,8.7Hz,1H); 13 C NMR (101 MHz, DMSO-d 6)δ170.79,155.93,143.73,141.37,140.68,128.52,128.03,127.97,127.63,127.09,125.34,125.28,120.09,118.45,115.48,65.85,54.18,52.76,46.57,38.31,36.48,33.68; Compound molecular weight data: theoretical molecular weight C 33 H 33 N 3 O 3 S[M+H] + =552.7050, the molecular weight determined by mass spectrometry was 552.2386. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 92.49%.

[0068] NMR spectrum analysis data of compound 11: 1 H NMR (400 MHz, DMSO-d 6 )δ8.19(t,J=5.8Hz,1H),7.89(dd,J=12.1,6.7Hz,5H),7.76(dd,J=8.2,2.9Hz,2H),7.41(t,J=7.6Hz,2H),7.37–7.21(m,18H) ,4.43–4.33(m,2H),4.27–4.19(m,2H),4.03(q,J=7.5Hz,1H),3.30–3.21(m,2H),2.82(h,J=6.3Hz,2H),2.45(d,J=7.2Hz,1H); 13 C NMR (101MHz, DMSO) δ170.38, 155.65, 144.27, 143.64, 140.68, 129.07, 128.01, 127.63, 127.04, 126.75, 125.34, 125.28, 120.08, 118.58, 115.60, 65.95, 65.84, 53.79, 46.57, 38.28, 36.49, 33.72; Compound molecular weight data: theoretical molecular weight C 39 H 37 N 3 O 3 S[M+H] + =628.2556, and the molecular weight determined by mass spectrometry was 628.2636. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 98.35%.

[0069] NMR spectrum analysis data of compound 12: 1H NMR(400MHz,DMSO-d6)δ7.82(dd,J=8.0,1.2Hz,2H),7.62(dd,J=7.7,1.4Hz,2H),7.50(t d,J=7.7,1.2Hz,2H),7.41(td,J=7.9,1.5Hz,2H),7.34–7.21(m,11H),6.92(s,1H),6.42( d,J=8.2Hz,1H),6.00(t,J=3.5Hz,1H),5.89(d,J=6.6Hz,1H),5.83(d,J=6.6Hz,1H),5.4 4–5.39(m,1H),4.99(s,1H),4.47–4.40(m,3H),3.43–3.26(m,4H),2.99–2.88(m,2H).13C NMR (101MHz, DMSO-d6) δ171.58, 158.57, 156.10, 143.62, 141.66, 141.39, 128.66, 128.01, 127.89, 127.40, 127.17, 125.13, 120.00, 66.74, 61.68, 54.96, 46.70, 40.19, 39.56, 33.16. Compound molecular weight data: theoretical molecular weight C 27 H 31 N 7 O 4 S[M+H] + =594.2641, mass spectrometry determined molecular weight 594.2702. Reverse phase high performance liquid chromatography (RP-HPLC) determined the purity of the compound to be 98.56%.

[0070] NMR spectrum analysis data of compound 13: 1H NMR (400MHz, DMSO-d6) δ7.82(dd,J=8.0,1.2Hz,2H),7.62(dd,J=7.7,1.4Hz,2H),7.50(td,J=7.7,1.2Hz ,2H),7.41(td,J=7.8,1.5Hz,2H),7.36–7.23(m,16H),6.92(s,1H),6.37(d,J=8.0Hz,1H),6.00(t,J=3. 5Hz,1H),5.89(d,J=6.6Hz,1H),5.83(d,J=6.6Hz,1H),5.44–5.39(m,1H),4.46(dt,J=7.9,4.2Hz,1H),4 .42(d,J=4.8Hz,2H),3.43–3.26(m,4H),2.91(dd,J=14.9,4.3Hz,1H),2.70(dd,J=14.9,4.3Hz,1H).13C NMR (101MHz, DMSO-d6) δ171.61, 158.57, 156.10, 144.35, 143.62, 141.39, 128.68, 128.36, 128.03, 127.40, 127.17, 125.13, 120.00, 67.55, 66.74, 54.46, 46.70, 40.19, 39.56, 32.51. Compound molecular weight data: theoretical molecular weight C 27 H 31 N 7 O 4 S[M+H] + =670.2774, and the molecular weight determined by mass spectrometry was 670.2812. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 99.11%.

[0071] NMR spectrum analysis data of compound 14: 1 H NMR (400 MHz, DMSO-d 6)δ8.36(t,J=5.7Hz,1H),7.92(dd,J=21.1,6.4Hz,5H),7.75(d,J=7.5Hz,2H),7.69(d,J=8.3Hz, 1H),7.43(t,J=7.4Hz,3H),7.33(tt,J=7.5,1.6Hz,2H),5.18(td,J=7.8,3.9Hz,1H),4.41–4.31 (m,2H),4.28–4.21(m,2H),4.15(td,J=8.6,5.5Hz,1H),3.41–3.30(m,2H),3.17(d,J=7.9Hz,2H ),2.86(ddd,J=13.6,10.4,5.8Hz,3H),2.63(dd,J=13.6,8.8Hz,1H),1.67(s,2H),1.60(s,2H); 13 C NMR (101 MHz, DMSO-d 6 )δ171.04,155.95,143.74(d,J=4.0Hz),140.69,134.84,127.63,127.03(d,J=3.4Hz),125.31(d,J=6.7Hz),120.24,120.08,65.79,54.54,46.59,38.32,36.50,32.99,29.01,25.34,17.47; Compound molecular weight data: theoretical molecular weight C 25 H 31 N 3 O 3 S[M+H] + =454.2086, and the molecular weight determined by mass spectrometry was 454.2162. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 97.45%.

[0072] NMR spectrum analysis data of compound 15: 1 H NMR (400 MHz, DMSO-d 6 )δ8.41(t,J=5.7Hz,1H),7.90(d,J=7.5Hz,5H),7.76(dd,J=8.1,2.1Hz,3H),7.66–7.56(m,4H),7.48–7.35(m,7H),7.32(td,J=7.5,1.2Hz, 2H),4.45–4.35(m,1H),4.31–4.21(m,3H),3.87–3.79(m,2H),3.43–3.26(m,J=6.6Hz,2H),2.92–2.81(m,3H),2.65(dd,J=13.6,8.9Hz,1H);13 C NMR (101 MHz, DMSO-d 6 )δ170.96,156.01,143.75,140.70,139.76,138.67,137.58,129.46,128.89,127.64,127.35,127.07,126.62,126.51,125.36,120.10,118.59,115.62,65.81,54.27,46.61,38.35,36.52,34.85,33.09; Compound molecular weight data: theoretical molecular weight C 33 H 33 N 3 O 3 S[M+H] + =552.2243, the molecular weight determined by mass spectrometry was 552.2383. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 95.30%.

[0073] NMR spectrum analysis data of compound 16: 1 H NMR (400 MHz, DMSO-d 6 )δ8.41(t,J=5.7Hz,1H),7.93–7.73(m,11H),7.54–7.45(m,3H),7.45–7.37(m,2H),7.32(t,J=7.4Hz,2H),4.45–4.35(m,1H),4.32–4.21( m,3H),3.94(d,J=2.7Hz,2H),3.33(dq,J=11.3,6.9Hz,2H),2.92–2.87(m,1H),2.82(dd,J=13.6,5.4Hz,2H),2.62(dd,J=13.7,8.9Hz,1H); 13 C NMR (101MHz, DMSO) δ 170.33, 170.15, 141.32, 141.30, 139.92, 138.25, 135.45, 129.66, 128.88, 128.51, 128.48, 128.04, 127.97, 127.26, 127.09, 127.06, 126.47, 126.40, 52.77, 52.35, 51.89, 50.70, 41.55, 36.55, 36.16, 33.64; Compound molecular weight data: theoretical molecular weight C 31 H 31 N 3 O 3 S[M+H] +=526.2086, the molecular weight determined by mass spectrometry was 526.2224. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 99.36%.

[0074] NMR spectrum analysis data of compound 17: 1 H NMR (400 MHz, DMSO-d 6 )δ7.88(dd,J=15.8,6.5Hz,3H),7.75(dd,J=7.5,2.1Hz,2H),7.69(d,J=8.7Hz,1H),7.44–7.22(m,19H),4.37–4.28(m, 1H),4.28–4.18(m,2H),4.05(td,J=8.3,6.1Hz,1H),3.37(d,J=12.5Hz,2H),3.10(q,J=6.1Hz,2H),2.51–2.22(m,3H); 13 C NMR (101MHz, DMSO) δ169.81, 155.58, 144.28, 143.68, 140.67, 129.08, 128.03, 127.62, 127.04, 126.74, 125.37, 120.07, 65.87, 65.81, 59.55, 53.79, 46.56, 41.50, 34.08; Compound molecular weight data: theoretical molecular weight C 39 H 36 N 2 O 4 S[M+Na]+=629.2396, and the molecular weight determined by mass spectrometry was 629.2490. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 95.61%.

[0075] NMR spectrum analysis data of compound 18: 1 H NMR (400 MHz, DMSO-d 6 )δ7.98(t,J=5.7Hz,1H),7.90(d,J=7.5Hz,2H),7.78(d,J=8.6Hz,2H),7 .80–7.68(m,5H),7.41(td,J=7.4,2.6Hz,2H),7.35–7.23(m,16H),4.40– 4.27(m,1H),4.27–4.15(m,2H),4.02(td,J=8.4,6.0Hz,1H),3.02(d,J= 6.6Hz,2H),2.76(h,J=6.0Hz,2H),2.46–2.32(m,2H),1.54–1.35(m,4H); 13C NMR (101MHz, DMSO) δ169.72, 155.58, 144.27, 143.68, 140.67, 129.07, 128.02, 127.62, 127.03, 126.74, 125.37, 120.07, 65.91, 65.79, 53.86, 46.55, 38.39, 37.96, 34.01, 25.80, 24.31; Compound molecular weight data: theoretical molecular weight C 41 H 41 N 3 O 3 S[M+H] + =656.2869, and the molecular weight determined by mass spectrometry was 656.2957. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 98.03%.

[0076] NMR spectrum analysis data of compound 19: 1 H NMR (400 MHz, DMSO-d 6 )δ8.14(t,J=5.7Hz,1H),8.00–7.49(m,12H),7.50–6.87(m,19H),4.29–4.09(m,4H),3.91(s,2H),3.27(qd,J=6.8, 3.0Hz,2H),2.81(q,J=6.6Hz,2H),2.68(h,J=6.4Hz,2H),2.50–2.34(m,2H),1.60–1.44(m,3H),1.30–1.13(m,2H); 13 C NMR (101MHz, DMSO) δ171.59, 169.67, 155.66, 144.22, 140.68, 129.06, 128.04, 127.65, 127.06, 126.76, 125.30, 120.11, 65.89, 65.81, 53.81, 52.23, 46.53, 38.59, 38.24, 36.37, 33.72, 31.16, 26.57, 21.96; Compound molecular weight data: theoretical molecular weight C 45 H 49 N 5 O 4 S[M+H] + =756.3505, the molecular weight determined by mass spectrometry was 756.3611. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 89.00%.

[0077] NMR spectrum analysis data of compound 20: 1 H NMR (400 MHz, DMSO-d6 )δ8.37(t,J=6.0Hz,1H),7.99(d,J=8.1Hz,1H),7.89(s,2H),7.75(dd,J=11.1,5.9Hz, 5H),7.41(t,J=7.5Hz,2H),7.35–7.15(m,23H),4.37–4.28(m,1H),4.22(ddt,J=17.7,9 .4,4.0Hz,5H),4.07(td,J=8.3,5.8Hz,1H),2.69(h,J=6.4Hz,2H),2.43(qd,J=11.9,7 .1Hz,3H),1.75–1.62(m,1H),1.60–1.49(m,2H),1.49–1.44(m,1H),1.33–1.15(m,2H); 13 C NMR (101MHz, DMSO) δ170.97, 169.70, 155.60, 144.20, 143.65, 140.67, 139.04, 129.05, 128.19, 128.03, 127.63, 127.03, 126.95, 126.76, 125.31, 120.09, 117.70, 114.77, 65.90, 65.82, 53.76, 52.35, 46.52, 41.94, 38.59, 33.75, 31.30, 26.51, 22.02; Compound molecular weight data: theoretical molecular weight C 50 H 50 N 4 O 4 S[M+H] + =803.3553, and the molecular weight determined by mass spectrometry was 803.3648. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 98.87%.

[0078] NMR spectrum analysis data of compound 21: 1 H NMR (400 MHz, DMSO-d 6 )δ9.06(t,J=5.9Hz,1H),8.55–8.28(m,3H),7.40–7.20(m,21H),4.49(dd,J=15.2,6.3Hz,1H),4.29(dd,J=15.2,5.3Hz,1H),2.51–2.38(m,3H); 13C NMR (101MHz, DMSO) δ166.75, 143.66, 138.20, 128.93, 128.22, 128.17, 127.42, 127.03, 126.97, 66.16, 50.97, 42.38, 32.22; Compound molecular weight data: theoretical molecular weight C 29 H 28 N 2 OS[M+H] + =453.1922, the molecular weight determined by mass spectrometry was 453.2123. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 96.46%.

[0079] NMR spectrum analysis data of compound 22: 1 H NMR (400 MHz, DMSO-d 6 )δ9.00–8.69(m,2H),8.22(d,J=5.4Hz,2H),7.92(q,J=8.2,5.7Hz,3H),7.4 0–7.05(m,21H),4.49(p,J=8.0Hz,1H),4.24(dd,J=15.6,5.8Hz,1H),3.78(q ,J=5.8Hz,2H),2.68(tt,J=11.1,5.2Hz,2H),2.37(ddd,J=33.2,11.4,7.4H z,2H),1.88–1.60(m,2H),1.50(p,J=7.7Hz,2H),1.30(q,J=7.5,6.9Hz,2H); 13 C NMR (101MHz, DMSO) δ169.24, 168.33, 144.08, 138.87, 138.83, 129.00, 128.09, 126.94, 121.42, 118.46, 115.49, 112.52, 65.93, 65.85, 51.98, 51.71, 42.03, 38.81, 38.42, 38.38, 33.46, 30.44, 26.37, 20.97; Compound molecular weight data: theoretical molecular weight C 35 H 40 N 4 O 2 S[M+H] + =581.2872, the molecular weight determined by mass spectrometry was 581.2957. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 91.70%.

[0080] NMR spectrum analysis data of compound 23: 1 H NMR (400 MHz, DMSO-d6 )δ8.90(t,J=5.7Hz,1H),8.44(s,2H),8.08(d,J=5.7Hz,3H),7.53–7.24(m,16H),3.77(t,J=6.5Hz,1H),3.48(dt ,J=13.6,6.8Hz,1H),3.33(ddt,J=13.5,7.9,5.6Hz,1H),2.91(qd,J=12.4,11.8,5.7Hz,2H),2.51–2.44(m,2H); 13 C NMR (101MHz, DMSO) δ167.39, 143.65, 128.94, 128.17, 126.98, 66.24, 51.09, 38.01, 36.47, 31.81; Compound molecular weight data: theoretical molecular weight C 24 H 27 N 3 OS[M+H] + =406.1875, the molecular weight determined by mass spectrometry was 406.2052. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 91.91%.

[0081] NMR spectrum analysis data of compound 24: 1 H NMR (400 MHz, DMSO-d 6 )δ8.89(dd,J=23.3,8.3Hz,1H),8.68(dt,J=15.1,6.0Hz,1H),8.17(d,J=5.2Hz,3H),7.39–7.20(m,20H),4.55–4.45(m,1H),4.34(dt,J=1 3.7,6.8Hz,1H),4.20(ddd,J=14.8,8.9,5.6Hz,1H),3.87(ddd,J=14.9,11.3,5.3Hz,1H),3.67(dd,J=11.3,6.5Hz,1H),2.52–2.36(m,3H); 13 C NMR (101MHz, DMSO) δ169.05, 166.69, 144.11, 138.79, 129.02, 128.16, 128.07, 126.99, 126.96, 126.81, 65.90, 60.35, 53.99, 51.92, 42.04, 33.73; Compound molecular weight data: theoretical molecular weight C 32 H 33 N 3 O 3 S[M+H] +=540.2243, mass spectrometry determined the molecular weight to be 540.2456. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 99.02%.

[0082] NMR spectrum analysis data of compound 25: 1 H NMR (400 MHz, DMSO-d 6 )δ8.54(d,J=8.1Hz,1H),8.25(t,J=5.7Hz,1H),7.86(s,2H),7.70–7.53(m,5H),7.50–7.24(m,20H),4.33(t d,J=7.8,6.0Hz,1H),3.72–3.45(m,2H),3.30(dq,J=12.8,6.7Hz,2H),2.89–2.76(m,2H),2.44–2.35(m,2H); 13 C NMR (101MHz, DMSO) δ172.63, 170.27, 170.11, 144.22, 139.97, 139.93, 138.28, 135.40, 129.68, 129.02, 128.87, 128.01, 127.24, 126.72, 126.49, 126.39, 118.09, 115.15, 65.76, 51.62, 41.60, 38.25, 36.43, 33.86; Compound molecular weight data: theoretical molecular weight C 38 H 37 N 3 O 2 S[M+H] + =600.2606, the molecular weight determined by mass spectrometry was 600.2710. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 98.54%.

[0083] NMR spectrum analysis data of compound 26: 1H NMR(400MHz, DMSO-d6)δ7.95(d,J=7.7Hz,1H),7.62–7.58(m,2H),7.58–7.52(m,2H),7.48–7.41(m,2H),7.4 1–7.36(m,1H),7.36–7.31(m,8H),7.31–7.23(m,10H),6.92(s,1H),6.00(t,J=3.5Hz,1H),5.89(d,J=6.6Hz ,1H),5.83(d,J=6.6Hz,1H),4.50(dt,J=7.7,4.4Hz,1H),3.46(t,J=1.0Hz,2H),3.42–3.36(m,2H),3.34(dd d,J=9.8,4.9,1.3Hz,1H),3.32–3.26(m,1H),3.17(dd,J=14.8,4.4Hz,1H),3.11(dd,J=14.8,4.6Hz,1H).13C NMR (101MHz, DMSO-d6) δ172.32, 171.71, 158.57, 144.35, 140.94, 139.06, 134.71, 130.09, 129.09, 128.68, 128.36, 128.13, 128.03, 127.90, 127.27, 67.55, 54.79, 41.47, 40.19, 39.56, 32.53. Compound molecular weight data: theoretical molecular weight C 27 H 31 N 7 O 4 S[M+H] + =642.2824, and the molecular weight determined by mass spectrometry was 642.2890. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 96.56%.

[0084] NMR spectrum analysis data of compound 27: 1 H NMR (400 MHz, DMSO-d 6 )δ8.59(d,J=8.1Hz,1H),8.20(t,J=5.7Hz,1H),7.82(s,6H),7.69–7.54(m,4H),7.51–7.17(m,15H),5.34(s,1 H),4.46(td,J=8.0,6.1Hz,1H),3.56(q,J=14.1Hz,2H),3.19(dhept,J=19.8,6.5Hz,2H),2.88–2.45(m,12H); 13C NMR (101MHz, DMSO) δ169.24, 168.33, 144.08, 138.87, 138.83, 129.00, 128.09, 126.94, 121.42, 118.46, 115.49, 112.52, 65.93, 65.85, 51.98, 51.71, 42.03, 38.81, 38.42, 38.38, 33.46, 30.44, 26.37, 20.97; Compound molecular weight data: theoretical molecular weight C 36 H 43 N 5 O 2 S[M+H] + =610.3137, and the molecular weight determined by mass spectrometry was 610.3359. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 98.87%.

[0085] NMR spectrum analysis data of compound 28: 1 H NMR (400MHz, DMSO-d6) δ8.58(d,J=8.5Hz,1H),8.34(t,J=5.6Hz,1H),8.04(s,3H),7.68–7. 61(m,2H),7.59–7.53(m,2H),7.50–7.43(m,2H),7.43–7.34(m,7H),7.34–7.26(m,4H),7.2 6–7.19(m,2H),5.38(s,1H),4.54(td,J=8.1,6.0Hz,1H),3.62–3.50(m,4H),3.43(t,J=5.7 Hz,2H),3.26(p,J=5.7Hz,2H),2.92(t,J=5.2Hz,2H),2.68–2.60(m,1H),2.59–2.52(m,1H). 13 C NMR (101MHz, DMSO) δ 170.15, 170.07, 141.39, 139.95, 138.20, 135.53, 129.68, 128.88, 128.48, 128.45, 128.08, 128.00, 127.24, 127.05, 127.01, 126.48, 126.39, 68.72, 66.25, 52.59, 52.22, 41.59, 40.11, 39.95, 39.90, 39.69, 39.48, 39.27, 39.07, 38.86, 38.48, 33.96. Compound molecular weight data: theoretical molecular weight C 27 H 31 N 7 O 4S[M+H] + =568.2556, the molecular weight determined by mass spectrometry was 568.2611. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 97.10%.

[0086] NMR spectrum analysis data of compound 29: 1 H NMR(400MHz, DMSO-d6)δ8.02(d,J=8.0Hz,1H),7.62–7.52(m,4H),7.48–7.41(m,2H), 7.40–7.36(m,1H),7.34–7.27(m,10H),7.27–7.21(m,3H),4.99(s,1H),4.47(dt,J=8. 1,4.9Hz,1H),3.63–3.53(m,6H),3.50(t,J=3.9Hz,2H),3.46(t,J=1.0Hz,2H),3.44–3 .30(m,2H),3.08–2.97(m,2H),2.84(tt,J=7.0,3.9Hz,2H),2.27(t,J=7.0Hz,2H).13C NMR (101MHz, DMSO-d6) δ172.32, 171.74, 141.66, 140.94, 139.06, 134.71, 130.09, 129.09, 128.66, 128.13, 128.01, 127.90, 127.89, 127.27, 72.88, 69.93, 69.65, 69.61, 61.66, 55.19, 41.61, 41.47, 40.36, 33.17. Compound molecular weight data: theoretical molecular weight C 27 H 31 N 7 O 4 S[M+H] + =612.2818, the molecular weight determined by mass spectrometry was 612.2896. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 98.44%.

[0087] NMR spectrum analysis data of compound 30: 1 H NMR (400 MHz, DMSO-d 6)δ9.12(s,1H),8.56(q,J=6.1Hz,1H),8.01(s,3H),7.44–7.24(m,10H),5.34(d,J=14.6Hz,1H),4.58–4. 41(m,1H),4.30–4.16(m,1H),3.79(ddd,J=30.4,13.2,3.4Hz,1H),3.52–3.07(m,8H),2.95–2.53(m,5H); 13 C NMR (101MHz, DMSO) δ169.73, 158.75, 141.27, 128.56, 128.00, 127.93, 127.89, 127.17, 53.38, 52.85, 52.82, 51.98, 42.35, 38.07, 36.48, 33.95, 33.24; Compound molecular weight data: theoretical molecular weight C 24 H 33 N 5 O 2 S[M+H] + =442.2198, the molecular weight determined by mass spectrometry was 442.2271. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 96.89%.

[0088] NMR spectrum analysis data of compound 31: 1 H NMR (400 MHz, DMSO-d 6 )δ8.84(d,J=8.0Hz,1H),8.28(t,J=5.7Hz,1H),8.03(s,1H),7.89(s,2H),7.42(tt,J=8 .0,1.4Hz,4H),7.33(q,J=7.5Hz,4H),7.28–7.19(m,2H),5.36(s,1H),5.11(d,J=16.6Hz ,1H),5.03(d,J=16.5Hz,1H),4.47(td,J=7.9,5.8Hz,1H),3.45(s,3H),3.33(q,J=6.6Hz ,2H),2.86(h,J=6.6Hz,2H),2.72(dd,J=13.4,5.8Hz,1H),2.58(dd,J=13.4,8.0Hz,1H); 13C NMR (101MHz, DMSO) δ170.18, 166.37, 154.55, 150.93, 148.01, 143.67, 141.27, 128.52, 128.48, 128.04, 128.00, 127.11, 106.43, 52.76, 52.47, 48.19, 38.20, 36.48, 33.37, 29.44, 27.48; Compound molecular weight data: theoretical molecular weight C 27 H 31 N 7 O 4 S[M+H] + =550.2158, the molecular weight determined by mass spectrometry was 550.2242. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 97.97%.

[0089] NMR spectrum analysis data of compound 32: 1 H NMR (400MHz, DMSO-d6) δ8.39(d,J=8.0Hz,1H),7.93–7.87(m,2H),7.66–7.56(m,4H),7.48–7.35(m,3H),7.34–7.21(m,10H),7.18(t,J= 13C NMR (101MHz, DMSO-d6) δ 172.14, 166.98, 144.41, 141.66, 139.46, 134.60, 129.08, 128.74, 128.66, 128.01, 127.91, 127.90, 127.89, 127.71, 61.65, 55.16, 42.27, 41.66, 33.13. Compound molecular weight data: theoretical molecular weight C 27 H 31 N 7 O 4 S[M+H] + =510.2137, the molecular weight determined by mass spectrometry was 510.2188. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 96.35%.

[0090] NMR spectrum analysis data of compound 33: 1 H NMR (400 MHz, DMSO-d 6)δ8.56(d,J=8.1Hz,1H),8.40(t,J=5.7Hz,1H),7.90(d,J=5.9Hz,3H),7.68–7.62(m,2H),7.58–7.56(m,1H),7.51–7.17(m,16H),5.33(s,1H), 4.50(td,J=8.0,5.9Hz,1H),3.65–3.52(m,2H),3.34(t,J=6.4Hz,2H),2.86(q,J=5.9Hz,2H),2.70(dd,J=13.3,5.9Hz,1H),2.57–2.51(m,1H); 13 CNMR (101MHz, DMSO)δ171.08,155.98,143.78,143.73,140.70,127.63,127.04,127.02,125.34,125.28,120.08,65.72,54.01,46.61,38.32,36.52,35.56; Compound molecular weight data: theoretical molecular weight C 32 H 33 N 3 O 2 S[M+H] + =524.2293, the molecular weight determined by mass spectrometry was 524.2380. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 98.80%.

[0091] NMR spectrum analysis data of compound 34: 1 H NMR (400 MHz, DMSO-d 6 )δ8.29(t,J=5.6Hz,1H),8.04(d,J=7.9Hz,1H),7.89(s,2H),7.46(dd,J=17.3,6.9Hz,4H),7.29(dq,J=34.9,7.0,6.2Hz,7H),5.44(s,1H),4.88 (d,J=9.3Hz,2H),4.62(q,J=7.4Hz,1H),4.37(s,2H),4.17(s,5H),3.32(d,J=6.2Hz,2H),2.83(d,J=15.0Hz,3H),2.67(dd,J=13.1,8.9Hz,1H); 13C NMR (101MHz, DMSO) δ171.14, 169.41, 141.45, 141.36, 128.52, 128.03, 127.98, 127.09, 75.66, 70.11, 69.39, 68.65, 68.17, 52.69, 52.10, 38.31, 36.49, 33.25; Compound molecular weight data: theoretical molecular weight C 30 H 25 FeN 3 O 2 S[M+H] + =534.0860, and the molecular weight determined by mass spectrometry was 534.1123. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 96.53%.

[0092] NMR spectrum analysis data of compound 35: 1 H NMR(400MHz,DMSO-d6)δ8.02(d,J=8.0Hz,1H),7.59(dd,J=8.1,1.5Hz,2H),7.58 –7.52(m,2H),7.48–7.41(m,2H),7.41–7.21(m,14H),6.92(s,1H),6.00(t,J=3. 5Hz,1H),5.89(d,J=6.6Hz,1H),5.83(d,J=6.6Hz,1H),4.99(s,1H),4.47(dt,J= 8.1,4.9Hz,1H),3.46(t,J=1.0Hz,2H),3.43–3.26(m,4H),3.08–2.97(m,2H).13C NMR (101MHz, DMSO-d6) δ172.32, 171.69, 158.57, 141.66, 140.94, 139.06, 134.71, 130.09, 129.09, 128.66, 128.13, 128.01, 127.90, 127.89, 127.27, 61.66, 55.19, 41.47, 40.19, 39.56, 33.17. Compound molecular weight data: theoretical molecular weight C 27 H 31 N 7 O 4 S[M+H] + =566.2511, the molecular weight determined by mass spectrometry was 566.2560. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 97.25%.

[0093] Example 5. Determination of the in vitro antibacterial activity of the compounds

[0094] The strains used for the determination of the in vitro antibacterial activity of compounds 1-28 and control antibiotics are: Escherichia coli (E.coli, ATCC25922), Staphylococcus aureus (S.aureus, ATCC 29213), Bacillus subtilis (ATCC 23857), Staphylococcus epidermidis (ATCC 12228), Candida albicans (C.albicans, ATCC 14053), Enterococcus faecalis (E.faecalis, ATCC 19433), Enterococcus gallinarum (E.gallinarum, ATCC 51299), all of which are standard strains from the American Type Culture Collection. In addition, clinically isolated drug-resistant strains MSRA S2, MSRA S3, and MSRA S4 were provided by the First Hospital of Lanzhou University.

[0095] The minimum inhibitory concentration (MIC) values ​​of the compounds for the tested strains were determined using the standard two-fold dilution method recommended by the American Clinical and Laboratory Standards Association (NCCLS). Simply, take an appropriate amount of frozen bacterial or fungal solution into fresh MH / SD medium, place the bacterial solution on a 37°C shaker, and culture it overnight at 180rpm. The above bacterial solution was transferred twice and continued to be cultured on a shaker for 4-5 hours to obtain bacteria in the logarithmic growth phase. Then take 1×105CFU / mL bacterial solution and add it to a 96-well plate, 100μL per well, and then dilute it by two times to add 100μL of different concentrations of polypeptide (1-256μM) twice the final concentration per well. The negative control is fresh culture medium, and three parallels are made for each concentration. After adding the drug, place the 96-well plate in a 37°C constant temperature and humidity incubator and culture it for 12 hours to observe the results. The compound concentration of the first transparent well after the turbid well visible to the naked eye is recorded as the minimum inhibitory concentration of the compound, that is, its MIC value for the bacteria. The results are shown in Table 1.

[0096] Table 1: Minimum inhibitory concentration MIC (μg / mL) of compounds 1-35 against test bacteria

[0097]

[0098]

[0099] As shown in Table 1, compounds 1-35 all showed good antibacterial activity against the test bacteria, and their minimum inhibitory concentrations were between 1.56 μg / mL and 125 μg / mL. In particular, compounds 10, 25, 28, 29, and 32 all showed good inhibitory effects against a variety of test bacteria, and are expected to be developed as broad-spectrum antibacterial agents; compounds 5, 11, 12, 17, 23, and 35 all showed good inhibitory effects against a variety of test bacteria, but had poor inhibitory effects against a few test bacteria, and are expected to be developed as inhibitors against a variety of test bacteria; compounds 16, 6, 7, 8, 9, 15, 18, 27, 33, and 34 also showed good inhibitory effects against a variety of test bacteria, but had poor inhibitory effects against a few test bacteria, and are expected to be developed as inhibitors against a variety of test bacteria.

[0100] Example 6. Compounds induce depolarization of bacterial cell membranes

[0101] 6.1 Effect of Compound 1-35 on the Membrane Potential of Staphylococcus aureus Cells

[0102] The membrane potential was detected by using the fluorescent dye DiOC2(3). When the membrane potential was high, the dye aggregated to form polymers and excited by light of a certain wavelength to produce red fluorescence; when the membrane potential was low, the dye could not aggregate and excited by light of a certain wavelength to produce green fluorescence. The relative ratio of red and green fluorescence was used to measure the depolarization ratio. Before the test, Staphylococcus aureus was cultured in a constant temperature oscillator at 37°C to the logarithmic phase. The bacteria in the logarithmic phase were centrifuged at 1500rpm for 10 minutes and washed three times with PBS, and resuspended in 0.1% glucose solution to control the bacterial solution OD600 = 0.5 ± 0.02. The bacterial solution was then added to a 96-well black board and incubated with the prepared fluorescent dye DiOC2(3) at 37°C for 15 minutes. The test compound was then quickly added and the fluorescence change was detected using a multifunctional microplate reader (excitation wavelength was 485nm, emission wavelengths were 530nm and 630nm). The negative control was PBS and the positive control was 1% Triton X-100.

[0103] Figure 1 The results showed that compounds 1-35 can produce membrane depolarization on bacterial cell membranes, destroying cell membrane potential and thus exerting a bactericidal effect. Specifically, after the test bacteria were incubated with the fluorescent dye DiOC2(3) for 15 minutes, the drug at a concentration of 4×MIC was added, and fluorescence was detected using a multifunctional microplate reader. It was found that compared with the PBS treatment group, compounds 1-35, like the positive control 1% Triton X-100, could cause a decrease in ( Figure 1 ), showing that compound 1-32 caused cell membrane depolarization and destroyed its membrane potential after interacting with bacteria.

[0104] 6.2 Effects of compounds on bacterial uptake of the fluorescent dye propidium iodide (PI)

[0105] PI is a DNA-binding dye that can penetrate damaged cell membranes, embed into nucleic acids, and release fluorescence. This experiment is used to detect bacterial cell death; compound 1-35 at a concentration of 4×MIC was incubated with logarithmic phase Staphylococcus aureus for 30 minutes, then a 1 mg / mL PI solution (final concentration of 100 μg / mL) was added, incubated at 37°C in the dark for 15 minutes, washed with PBS, and the PI positive rate was detected by flow cytometry. Figure 2 As shown, compound 1-35, like the positive control 1% TritonX-100, can cause PI to enter the cells, which indicates that compound 1-35 can destroy cell integrity, cause leakage of cell contents and subsequently cause bacterial death.

[0106] Example 7. Bactericidal kinetics of compounds 1-35

[0107] The bactericidal kinetics of compounds 1-35 against Staphylococcus aureus ATCC 29213 were determined by colony forming unit (CFU) counting method. 6 CFU / mL of Staphylococcus aureus was added to a 96-well plate, and then compounds 1-35 (final concentration was 2×MIC and 4×MIC) were added respectively, and co-cultured at 37°C. At intervals of 0, 10 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 12 h, an appropriate amount of bacterial solution was taken out and diluted to an appropriate multiple, and then 100 μL of the diluted bacterial solution was evenly spread on the MH solid culture medium with a spreading rod, and the colonies were counted after overnight culture. Figure 3 It shows that compound 1-35 can kill all bacteria within 4 hours at a concentration of 2×MIC, demonstrating the ability to quickly kill bacteria, while the positive control drug vancomycin can kill the test bacteria within 6 hours.

[0108] Example 8. Anti-biofilm effect of compounds 1-35

[0109] The crystal violet method was used to determine the inhibitory effect of compound 1-35 on the biofilm formation of Staphylococcus aureus. 6 CFU / mL of logarithmic phase Staphylococcus aureus was added to a 96-well plate, and then the test compounds 1-35 were added at a concentration of 4×MIC, and incubated in a 37°C constant temperature bacterial incubator. After 24 hours, the 96-well plate was removed to remove the planktonic bacteria on the surface, washed with PBS, fixed with methanol, and stained with crystal violet solution for 15 minutes, and then 95% ethanol was added to dissolve the crystal violet in the biofilm to measure the OD 570 According to the formula, biofilm formation inhibition rate (%) = [1-(experimental group OD 570 - Negative control group OD 570) / (OD of positive control group 570 - Negative control group OD 570 )]×100%. Figure 4 As shown, the inhibition rate of compounds 1-35 on biofilm formation is >50%, and some compounds can completely inhibit the formation of biofilm.

[0110] Example 9. Determination of drug resistance of Staphylococcus aureus to compounds 10, 28 and 33

[0111] The resistance of Staphylococcus aureus to representative compounds 10, 28 and 33 after continuous contact of the compounds with Staphylococcus aureus was determined by continuously detecting the MIC values ​​of the compounds to the bacteria treated with sub-MIC concentrations. Ampicillin and Ciprofloxacin were used as controls. Staphylococcus aureus was incubated with sub-MIC concentrations of compounds 10, 28 and 33 and ampicillin and ciprofloxacin for 16 hours each time, and the bacterial solution was taken to determine the MIC, and a total of 21 generations were measured. Figure 5 It was shown that in the 21-day bacterial resistance experiment, the MIC values ​​of compounds 10, 28 and 33 fluctuated within the range of 1-2 times the MIC, indicating that the bacteria did not develop resistance to them; while the ciprofloxacin and ampicillin control groups developed 640 times the original MIC on the 18th and 17th days of treatment, respectively, showing significant resistance.

[0112] Example 10. In vivo antibacterial activity assay of compounds 10, 28 and 33

[0113] The in vivo antibacterial activity of compounds 10, 28 and 33 was tested in a mouse pneumonia model infected with MRSA. Kunming mice weighing 20 g ± 1 g (purchased from the Animal Center of Lanzhou University) were selected and randomly divided into groups after 1 week of laboratory adaptation. After anesthesia, bacteria (25 μL at a concentration of 6.0×10 9 The mouse pneumonia model of MRSA infection was established by intraperitoneal injection 2h and 12h after the model was established. 0.9% NaCl was used as a negative control and vancomycin was used as a positive control. 24h after the second administration, the mice were dislocated and killed to observe the changes in the lungs. After weighing, the mice were grinded and smeared on the plates aseptically, and then the plates were smeared and cultured at 37℃ for 24h before colony counting. Figure 6 As shown, compared with the uninfected group, the lungs of mice in the infected control group were significantly congested and swollen, while the vancomycin-treated group had local congestion but no obvious swelling. The compound 10, 28 and 33-treated groups only had very slight congestion but no obvious swelling. Figure 7As shown, compared with the lung tissue colony load of the model group, the lung tissue colony load of the vancomycin and compound 10, 28, and 33 (5 mg / kg body weight) treatment groups decreased significantly.

[0114] In summary, the present invention changes the amphiphilicity of the peptide by linking different hydrophobic groups and cationic groups on the amino end, carboxyl end and side chain thiol group of cysteine ​​through the skeleton to obtain a small molecule antimicrobial peptide mimetic capable of treating infectious diseases caused by sensitive bacteria and resistant bacteria.

[0115] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A small molecule antimicrobial peptide mimetic, which is based on a cysteine ​​backbone structure and is obtained by chemically modifying the amino terminal, carboxyl terminal, or side chain thiol group, characterized in that: Its structural formula is one of formula 1, 5-10, 12, 15, 16, 18, 20, 25, 27-30, 32-35: .

2. A composition, characterized in that The active ingredient comprises at least one of the small molecule antimicrobial peptide mimetics described in claim 1.

3. The composition according to claim 2, characterized in that Also included are acceptable carriers.

4. The composition according to claim 2, characterized in that The dosage forms include oral preparation, injection, mucosal administration preparation and external preparation.

5. The use of small molecule antimicrobial peptide mimetics in the preparation of anti-infective drugs, characterized in that: The small molecule antimicrobial peptide mimetic is based on a cysteine ​​skeleton structure and is obtained by chemically modifying the amino end, the carboxyl end, or the side chain thiol group. The characteristic of the small molecule antimicrobial peptide mimetic is that the general structural formula is as follows: , where: R1 is selected from , , , , One of; R2 is selected from , , , , , , , One of; R3 is selected from , , , , , , , , , , One of them.

6. The use according to claim 5, characterized in that: Its structural formula is one of formula 1, 5-12, 15-18, 20, 23, 25, 27-30, 32-35: .

7. The use according to claim 5, characterized in that: The infection is caused by bacteria or fungi.

8. The use according to claim 7, characterized in that: The bacteria or fungi are drug-resistant bacteria or fungi.

9. The use according to claim 8, characterized in that: The drug-resistant bacteria or fungi are selected from methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus.

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