Small-molecule antibacterial peptide mimetic and use thereof

Abstract

Disclosed in the present invention are a small-molecule antibacterial peptide mimetic and the use thereof. The small-molecule antibacterial peptide mimetic is obtained by means of chemical modification at the amino terminus, the carboxyl terminus or on a side-chain sulfydryl group on the basis of a cysteine skeleton structure, and has general formula (I). The small-molecule antibacterial peptide mimetic can cause cell membrane depolarization, destroy cell membrane integrity, rapidly kill bacteria and inhibit biofilm formation, has certain biofilm clearance activity, and is less likely to induce bacterial drug resistance. The small-molecule antibacterial peptide mimetic has good activity against Gram-positive bacteria, shows good antibacterial activity against drug-resistant bacteria such as MRSA and VRE, and therefore can solve the problem of the current shortage of drugs against drug-resistant bacteria, has the potential to replace or assist currently used clinical antibiotics in treating infectious diseases caused by various drug-resistant bacteria, and can be used for the treatment of infectious diseases caused by both sensitive bacteria and drug-resistant bacteria, The small-molecule antibacterial peptide mimetic has efficient antibacterial activity and low toxicity, and is not influenced by traditional drug-resistant mechanisms.

Description

Small molecule antimicrobial peptide mimetics and their applications Technical Field

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

[0002] Antibiotics are currently the main drugs used to treat diseases caused by bacterial infections. However, with the widespread use and abuse of antibiotics in medicine, animal husbandry, and the food industry, more and more bacteria have become resistant to conventional antibiotics, and this has become one of the most serious problems facing world health today. Currently, there is a lack of drugs against drug-resistant Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE), as well as 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 promise as promising antimicrobial agents. Antimicrobial peptides serve as sentinels of innate immunity in most species and constitute the first line of defense against any infection. Unlike most antibiotics, which act by targeting intracellular organelles, antimicrobial peptides and their mimetics primarily exert their antimicrobial effects by inducing cell membrane lysis. Therefore, unlike conventional antibiotics, which can be inactivated by point mutations in bacteria, it is difficult for bacteria to develop resistance to antimicrobial peptides and their mimetics. However, poor stability, high production costs, and significant systemic toxicity limit the clinical application of cationic antimicrobial peptides. Consequently, numerous research groups are dedicated to developing novel strategies to address these challenges facing antimicrobial peptides. The development of antimicrobial peptide mimetics, which mimic the structure and mechanism of action of antimicrobial peptides, is one such strategy and has attracted increasing attention.

[0004] Despite the current abundance of macromolecular mimetics of antimicrobial peptides, the successful development of small-molecule mimetics is relatively rare. This is primarily due to the fact that the design of these compounds has focused primarily on structure, while ignoring the limitations of their application due to their structural complexity, numerous synthetic steps, high production costs, and the presence of peptide bonds that are resistant to protease degradation. Therefore, antimicrobial peptide mimetics with simple structures, ease of synthesis, low production costs, and a low likelihood of inducing bacterial resistance hold greater potential for clinical application. 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 terminus, carboxyl terminus, or side chain thiol group. The general structural formula of the small molecule antimicrobial peptide mimetic is as follows:

[0008] Where:

[0009] R1 is selected from One of the following;

[0010] R2 is selected from One of the following;

[0011] R3 is selected from One of them.

[0012] In some examples of small molecule antimicrobial peptide mimetics, R1 is selected from One of the following;

[0013] R2 is selected from One of the following;

[0014] R3 is selected from

[0015] 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:

[0016] 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.

[0017] In some examples of the composition, an acceptable carrier is also included.

[0018] In some examples of the composition, the dosage form is an oral preparation, an injection, a mucosal administration preparation, or an external preparation.

[0019] 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.

[0020] In some embodiments, the infection is caused by bacteria or fungi.

[0021] In some application examples, the bacteria or fungi are drug-resistant bacteria or fungi.

[0022] In some application examples, the drug-resistant bacteria or fungi are selected from methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci.

[0023] The beneficial effects of the present invention are:

[0024] 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, and are not easy to induce bacterial resistance. They 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, and have the potential to replace or assist current clinical antibiotics in treating infectious diseases caused by various resistant bacteria. They can be used to treat infectious diseases caused by sensitive bacteria and resistant bacteria, have high antibacterial activity, low toxicity, and are not affected by traditional resistance mechanisms.

[0025] The synthesis methods of the small molecule antimicrobial peptide mimetics of some examples of the present invention are simple, the raw materials are easily available, and the operation methods are simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG1 shows the results of the depolarization experiment on Staphylococcus aureus membrane by compound 1-35 at a concentration of 4×MIC.

[0027] FIG2 shows the positive rate of Staphylococcus aureus after compound 1-35 at a concentration of 4×MIC was co-incubated with Staphylococcus aureus for 30 minutes and then incubated in the dark for 15 minutes with propidium iodide (PI).

[0028] Figure 3 shows the bactericidal kinetics of compound 1-35 against Staphylococcus aureus. 0.9% saline and vancomycin were used as negative and positive controls, respectively.

[0029] Figure 4 shows the results of the biofilm inhibition experiment. Compounds 1-35 at a concentration of 4×MIC were incubated with Staphylococcus aureus, and then surface planktonic bacteria were removed. After washing and fixation, crystal violet solution was added for staining. 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 point and record the result.

[0030] Figure 5 shows the results of a bacterial resistance experiment. Staphylococcus aureus was incubated with representative compounds 10, 28, and 33 at 1 / 2× their MIC, ampicillin, and ciprofloxacin. After 16 hours, the bacterial suspension with the highest drug concentration showing bacterial growth was sampled and cultured to the logarithmic phase, whereupon the MIC was determined. A total of 21 passages were tested.

[0031] Figure 6 shows the lung changes in control and drug-treated mice. Experimental mice were divided into six groups: a model group, a vancomycin (Vanc) group, groups treated with compounds 10, 28, and 33, and a blank control group. Following treatment, the mice in each group were dissected and their lungs observed for changes.

[0032] Figure 7 shows the in vivo antibacterial results of compounds 10, 28, and 33. Lung abrasions from the compound 10, 28, and 33 treatment groups, vancomycin treatment control group, blank control group, and untreated model group (MD) were followed by colony counts after 24-hour plate culture. DETAILED DESCRIPTION

[0033] The embodiments of the present invention are described in detail below with reference to the examples. However, 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.

[0034] Instruments used in the present invention:

[0035] The high performance liquid chromatograph was a Delta 600 model provided by Waters Corporation in the United States. The preparative column used was a reversed-phase C18 column (XBridge BEH 130 Prep), 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. Reagents included: NA-Fmoc-protected amino acids, N-hydroxybenzotriazole (HOBt), oxybenzotriazole-N,N,N',N'-tetramethyluronium-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 purchased from Tianjin Second Reagent Factory. All other instruments and reagents used, unless their manufacturers are indicated, are commercially available. In the examples, if specific conditions are not indicated, conventional conditions or those recommended by the manufacturer were followed.

[0036] Example 1: Synthesis of Compound 1-16

[0037] The synthetic route of compound 1-13 is as follows:

[0038] In a typical reaction, the commercially available amino acid Fmoc-Cys(R)-OH (0.5 mmol, 1 eq), HOBT (0.6 mmol, 81.08 mg, 1.2 eq), HBTU (0.6 mmol, 227.54 mg, 1.2 eq), and DIEA (1.2 mmol, 155.1 mg, 2.4 eq) were added to 30 mL of DMF and stirred to dissolve. N-Boc-Ethylenediamine (0.6 mmol, 96.13 mg, 1.2 eq) was then added and stirred at room temperature for 3 hours. After the reaction, 60 mL of water was added and 60 mL of ethyl acetate was shaken and washed. The organic phase was retained and then washed with brine, dried over anhydrous sodium sulfate, and then evaporated under reduced pressure to obtain the intermediate product with a yield of over 95%. The intermediate product was prepared without further treatment. Trifluoroacetic acid (8 mL), dichloromethane (20 mL), and TIS (200 μL) were added and stirred for 3 h (TIS was not added when treating compound 8k). The solvent was removed under reduced pressure, and the target product was precipitated by adding glacial ether. The product was further purified by HPLC to obtain the final compound 1-11. A portion of the product obtained in this step (0.3 mmol, 1 eq) was dissolved with 1H-pyrazole-1-carboxamidine hydrochloride (0.36 mmol, 1.2 eq) in 10 ml of DMF. DIEA (0.6 mmol, 2 eq) was added dropwise at room temperature and reacted for 3 h. The mixture was quenched by adding dilute hydrochloric acid, diluted with water, purified by HPLC, and lyophilized to obtain compounds 12 and 13.

[0039] The synthetic routes of compounds 14-16 are as follows:

[0040] fmoc-cys(Trt)-OH (1.7 mmol, 1 g, 1 eq) was dissolved in dichloromethane (40 mL), trifluoroacetic acid (10 mL) and TIS (500 μL) were added, and the mixture was stirred at room temperature for 2 h. After the reaction was completed, the solvent was removed under reduced pressure, and glacial ether was added to precipitate to obtain an intermediate product. The compound was directly dissolved in dichloromethane (50 mL) without further treatment, DIEA (2.5 mL) and Br-R (3.4 mmol, 2 eq) were added, and the reaction progress was monitored by thin layer chromatography after stirring for 16 h. After the reaction was completed, the solvent was removed under reduced pressure, and the mixture was purified by silica gel column to obtain an intermediate product. 20% TFA (15 mL) was added to obtain the final compound 14-16.

[0041] Example 2: Synthesis of Compounds 17-26

[0042] The synthetic route of compound 17-26 is as follows:

[0043] 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 and dissolved by stirring. 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 washed with 60 mL of ethyl acetate by shaking. The organic phase was retained and then washed with brine, dried over anhydrous sodium sulfate, and then evaporated under reduced pressure to obtain the intermediate and 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 hour. After the reaction was completed, the solvent was removed under reduced pressure and glacial ether was added for precipitation to obtain product 21 and the intermediate. The intermediate product (0.4 mmol, 290 mg, 1 eq) was dissolved in DMF (30 mL), and HOBT (0.48 mmol, 64.89 mg, 1.2 eq), HBTU (0.48 mmol, 181.92 mg, 1.2 eq), DIEA (0.96 mmol, 124.07 mg, 2.4 eq), and 4-Biphenylacetic acid (0.48 mmol, 101.87 mg, 1.2 eq) were added and stirred at room temperature for 3 h. After the reaction, 60 mL of water was added and 60 mL of ethyl acetate was added for washing by shaking. The organic phase was retained and then washed with brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The obtained compound was prepared without further treatment. Trifluoroacetic acid (8 mL) and dichloromethane (20 mL) were added and stirred for 3 h. The solvent was removed under reduced pressure, and glacial ether was added to precipitate the desired product. The product was further purified by HPLC to obtain the final compound 25. Compound 25 was obtained by the same steps as in Scheme 1a to obtain compound 26. The intermediate 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, and DIEA (0.42 mmol, 1.2 eq) were dissolved in 20 ml of DMF with BOC-LYS(BOC)-OH and BOC-SER-OH (0.4 mmol), respectively. The mixture was reacted for 3 h and then treated according to the experimental steps of compound 25 to obtain compounds 22 and 24.

[0044] Example 3: Synthesis of Compounds 27-35

[0045] The synthetic routes of compounds 27-35 are as follows:

[0046] A 0.5 degree of substitution dichlororesin (1 g) was added to a needle synthesizer, and the resin was swollen with dichloromethane (10 mL) for 5 minutes. The solvent was removed by 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 hours, and the solvent was removed by filtration. The mixture was washed with DMF three times. 20% piperidine (10 mL) was added and the reaction was continued for 30 minutes. The solvent was removed by filtration, and DMF was added and washed four times. DMF (10 mL) was added and the mixture was reacted with biphenylcarboxylic acid, felbinac 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 hour. The solvent was removed by filtration, washed three times with DMF, washed twice with dichloromethane, washed twice with methanol, washed once with dichloromethane, and reacted for 2 h with 20% trifluoroethanol (10 mL). The solvent was collected and removed under reduced pressure. The intermediate product with R group being felbinac was prepared without further treatment and 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 1-ylbis(oxy)bis(ethan-1-amine) (160 mg) was dissolved in DMF (30 mL). HOBT (0.5 mmol, 67.56 mg, 1.2 eq), HBTU (0.5 mmol, 189.62 mg, 1.2 eq), DIEA (1.5 mmol, 193.9 mg, 3.6 eq), and the product from step 4 (0.417 mmol, 199.74 mg, 1 eq) were dissolved in DMF (10 mL). The mixture was added dropwise to the amine solution over 1 h, and the reaction was continued for 2 h. After completion of the reaction, the solvent was removed under reduced pressure, and the mixture was purified by HPLC to obtain compounds 27-29. Compounds 30-35 were obtained via a similar reaction process.

[0047] Example 4. Structural characterization of compounds

[0048] The desalted crude product (approximately 40 mg) was weighed and dissolved in a 20% acetonitrile / water solution (5 mL). The solution was filtered and loaded onto a μ-bondapaktm C18 reverse phase column (19 mm × 300 mm). The column used for the purification process was a 20%-80% acetonitrile / water gradient elution. The absorbance was observed at 220 nm using a UV detector. The main peak was collected and the collected liquid was placed in a 50 mL beaker at -80°C overnight. The resulting solution was then lyophilized under vacuum to obtain a pure compound powder. The structure and NMR characterization of the synthesized compound are as follows:

[0049] NMR spectrum analysis data of compound 1: 1 H NMR(400MHz, DMSO-d6)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-d6) δ 170.61, 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 N3O3S[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%.

[0050] NMR spectrum analysis data of compound 2: 1H NMR (400MHz, DMSO-d6) 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 (101 MHz, DMSO) δ 171.08, 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 N3O3S[M+H] + =400.1617, and 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%.

[0051] NMR spectrum analysis data of compound 3: 1 H NMR (400MHz, DMSO-d6) 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 (101 MHz, DMSO) δ 171.00, 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 N3O3S[M+H] + =442.2086, and 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%.

[0052] NMR spectrum analysis data of compound 4: 1 H NMR(400MHz, DMSO-d6)δ8.62(t,J=6.4Hz,1H),8.27(t,J=5.7Hz,1H),7.95–7.87 (m,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),4.36–4.21(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 (101 MHz, DMSO) δ 171.02, 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 N4O4S[M+H] + =457.1831, and 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%.

[0053] NMR spectrum analysis data of compound 5: 1H NMR (400MHz, DMSO-d6) 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 (101 MHz, DMSO) δ 171.02, 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 N4O4S[M+H] + =533.2144, and 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%.

[0054] NMR spectrum analysis data of compound 6: 1 H NMR(400MHz,DMSO-d6)δ8.38(t,J=5.7Hz,1H),7.90(d,J=7.5Hz,4H),7.88(s,1H),7.75 (dd,J=7.8,3.7Hz,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); 13C NMR (101 MHz, DMSO-d6) δ 171.04, 155.95, 143.74 (d, J = 4.0 Hz), 140.69, 134.84, 127.63, 127.03 (d, J = 3.4 Hz), 125.31 (d, J = 6.7 Hz), 120.24, 120.08, 65.79, 54.54, 46.59, 38.32, 36.50, 32.99, 29.01, 25.34, 17.47; Molecular weight of the compound: Theoretical molecular weight C 28 H 28 F3N3O3S[M+H] + =544.1803, and 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%.

[0055] NMR spectrum analysis data of compound 7: 1 H NMR (400MHz, DMSO-d6) 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-d6) δ 170.95, 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; Molecular weight of the compound: Theoretical molecular weight C 27 H 29 N3O3S[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%.

[0056] NMR spectrum analysis data of compound 8: 1 H NMR (400MHz, DMSO-d6) 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-d6) δ 170.96, 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; Molecular weight of the compound: Theoretical molecular weight C 28 H 31 N3O3S[M+H] + =490.2086, and 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%.

[0057] NMR spectrum analysis data of compound 9: 1H NMR(400MHz,DMSO-d6)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=6.7H z,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-d6) δ 170.98, 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; Molecular weight of the compound: Theoretical molecular weight C 28 H 31 N3O4S[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%.

[0058] NMR spectrum analysis data of compound 10: 1 H NMR(400MHz, DMSO-d6)δ8.36(t,J=5.8Hz,1H),7.89(t,J=8.0Hz,5H),7.81–7.7 1(m,3H),7.45–7.39(m,6H),7.35–7.28(m,6H),7.26–7.21(m,2H),5.38(s,1H) ,4.42–4.32(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); 13C NMR (101 MHz, DMSO-d6) δ 170.79, 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; Molecular weight of the compound: Theoretical molecular weight C 33 H 33 N3O3S[M+H] + =552.7050, and 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%.

[0059] NMR spectrum analysis data of compound 11: 1 H NMR (400MHz, DMSO-d6) 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 CNMR (101 MHz, DMSO) δ 170.38, 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; Molecular weight of the compound: Theoretical molecular weight C 39 H 37 N3O3S[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%.

[0060] 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 (101 MHz, DMSO-d6) δ 171.58, 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 N7O4S[M+H] + =594.2641, and the molecular weight determined by mass spectrometry was 594.2702. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 98.56%.

[0061] 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 N7O4S[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%.

[0062] NMR spectrum analysis data of compound 14: 1H NMR (400MHz, DMSO-d6) 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=7.9 Hz,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-d6) δ 171.04, 155.95, 143.74 (d, J = 4.0 Hz), 140.69, 134.84, 127.63, 127.03 (d, J = 3.4 Hz), 125.31 (d, J = 6.7 Hz), 120.24, 120.08, 65.79, 54.54, 46.59, 38.32, 36.50, 32.99, 29.01, 25.34, 17.47; Molecular weight of the compound: Theoretical molecular weight C 25 H 31 N3O3S[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%.

[0063] NMR spectrum analysis data of compound 15: 1 H NMR (400MHz, DMSO-d6) 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); 13C NMR (101 MHz, DMSO-d6) δ 170.96, 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; Molecular weight of the compound: Theoretical molecular weight C 33 H 33 N3O3S[M+H] + =552.2243, and 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%.

[0064] NMR spectrum analysis data of compound 16: 1 H NMR(400MHz,DMSO-d6)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),2.9 2–2.87(m,1H),2.82(dd,J=13.6,5.4Hz,2H),2.62(dd,J=13.7,8.9Hz,1H); 13 C NMR (101 MHz, 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; Molecular weight of the compound: Theoretical molecular weight C 31 H 31 N3O3S[M+H] + =526.2086, and 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%.

[0065] NMR spectrum analysis data of compound 17:1 H NMR (400MHz, DMSO-d6) 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),4.3 7–4.28(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 (101 MHz, DMSO) δ 169.81, 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 N2O4S[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%.

[0066] NMR spectrum analysis data of compound 18: 1 H NMR(400MHz, DMSO-d6)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,1 6H),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 (101 MHz, DMSO) δ 169.72, 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 N3O3S[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%.

[0067] NMR spectrum analysis data of compound 19: 1 H NMR (400MHz, DMSO-d6) 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 (101 MHz, DMSO) δ 171.59, 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; Molecular weight of the compound: Theoretical molecular weight C 45 H 49 N5O4S[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%.

[0068] NMR spectrum analysis data of compound 20: 1H NMR (400MHz, 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 (101 MHz, DMSO) δ 170.97, 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; Molecular weight of the compound: Theoretical molecular weight C 50 H 50 N4O4S[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%.

[0069] NMR spectrum analysis data of compound 21: 1 H NMR(400MHz,DMSO-d6)δ9.06(t,J=5.9Hz,1H),8.55–8.28(m,3H),7.40–7.20(m,21 H),4.49(dd,J=15.2,6.3Hz,1H),4.29(dd,J=15.2,5.3Hz,1H),2.51–2.38(m,3H); 13 C NMR (101 MHz, DMSO) δ 166.75, 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 29H 28 N2OS[M+H] + =453.1922, and 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%.

[0070] NMR spectrum analysis data of compound 22: 1 H NMR(400MHz, DMSO-d6)δ9.00–8.69(m,2H),8.22(d,J=5.4Hz,2H),7.92(q,J=8.2, 5.7Hz,3H),7.40–7.05(m,21H),4.49(p,J=8.0Hz,1H),4.24(dd,J=15.6,5.8Hz,1 H),3.78(q,J=5.8Hz,2H),2.68(tt,J=11.1,5.2Hz,2H),2.37(ddd,J=33.2,11.4, 7.4Hz,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 (101 MHz, DMSO) δ 169.24, 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; Molecular weight of the compound: Theoretical molecular weight C 35 H 40 N4O2S[M+H] + =581.2872, and 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%.

[0071] NMR spectrum analysis data of compound 23: 1 H NMR (400MHz, 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 (101 MHz, DMSO) δ 167.39, 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 N3OS[M+H] + =406.1875, and 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%.

[0072] NMR spectrum analysis data of compound 24: 1 H NMR (400MHz, DMSO-d6) 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=13.7,6.8Hz,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 (101 MHz, DMSO) δ 169.05, 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 N3O3S[M+H] + =540.2243, the molecular weight determined by mass spectrometry was 540.2456. The purity of the compound determined by reverse phase high performance liquid chromatography (RP-HPLC) was 99.02%.

[0073] NMR spectrum analysis data of compound 25: 1H NMR (400MHz, DMSO-d6) 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,20 H),4.33(td,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 (101 MHz, DMSO) δ 172.63, 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; Molecular weight of the compound: Theoretical molecular weight C 38 H 37 N3O2S[M+H] + =600.2606, and 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%.

[0074] NMR spectroscopy 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) δ 172.32, 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 N7O4S[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%.

[0075] NMR spectrum analysis data of compound 27: 1 H NMR (400MHz, DMSO-d6) 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,1H),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 (101 MHz, DMSO) δ 169.24, 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; Molecular weight of the compound: Theoretical molecular weight C 36 H 43 N5O2S[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%.

[0076] 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 (101 MHz, 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. Molecular weight of the compound: Theoretical molecular weight C 27 H 31 N7O4S[M+H] +=568.2556, and 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%.

[0077] NMR spectroscopy 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 N7O4S[M+H] + =612.2818, and 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%.

[0078] NMR spectrum analysis data of compound 30: 1 H NMR(400MHz, DMSO-d6)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); 13C NMR (101 MHz, DMSO) δ 169.73, 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 N5O2S[M+H] + =442.2198, and 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%.

[0079] NMR spectrum analysis data of compound 31: 1 H NMR (400MHz, DMSO-d6) 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); 13 C NMR (101 MHz, DMSO) δ 170.18, 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; Molecular weight of the compound: Theoretical molecular weight C 27 H 31 N7O4S[M+H] + =550.2158, and 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%.

[0080] NMR spectroscopy data of compound 32: 1H 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=4.8Hz,1H),4.99(s,1H),4.52(dt,J=8.1,4.6Hz,1H),4.08(t,J=6.3Hz,2H),3.28(qdt,J=14.8,4 .9,4.0Hz,2H),3.06–2.96(m,4H).13C NMR (101MHz,DMSO-d6)δ172.14,166.98,144.41,141.66,139.46,134.60,129.08,128.74,128.66,128.01,127.91,127.90,127.89,127.71,61.65,55.16,42.27,41.66,33.13. Molecular weight data of compound: theoretical molecular weight C 27 H 31 N7O4S[M+H] + =510.2137, and 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%.

[0081] NMR spectrum analysis data of compound 33: 1 H NMR (400MHz, DMSO-d6) 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 C NMR (101 MHz, DMSO) δ 171.08, 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 33N3O2S[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%.

[0082] NMR spectrum analysis data of compound 34: 1 H NMR(400MHz,DMSO-d6)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); 13 C NMR (101 MHz, DMSO) δ 171.14, 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 FeN3O2S[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%.

[0083] NMR spectroscopy data of compound 35: 1H 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 (101 MHz, DMSO-d6) δ 172.32, 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 N7O4S[M+H] + =566.2511, and 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%.

[0084] Example 5. Determination of the antibacterial activity of the compounds in vitro

[0085] The in vitro antibacterial activity of compounds 1-28 and control antibiotics was determined using the following bacterial strains: Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 29213), Bacillus subtilis (ATCC 23857), Staphylococcus epidermidis (ATCC 12228), Candida albicans (ATCC 14053), Enterococcus faecalis (ATCC 19433), and Enterococcus gallinarum (ATCC 51299). These strains are standard strains obtained from the American Type Culture Collection. In addition, the clinically isolated drug-resistant strains MSRA S2, MSRA S3, and MSRA S4 were provided by the First Hospital of Lanzhou University.

[0086] The minimum inhibitory concentration (MIC) of each compound against the tested strains was determined using the standard two-fold dilution method recommended by the National Center for Clinical and Laboratory Standards (NCCLS). Briefly, an appropriate amount of frozen bacterial or fungal culture was transferred to fresh MH / SD medium and incubated overnight at 37°C on a shaker at 180 rpm. The culture was transferred a second time and incubated on a shaker for another 4-5 hours to obtain bacteria in the logarithmic growth phase. 1×105 CFU / mL of the culture was then added to a 96-well plate, 100 μL per well. Following this, 100 μL of peptide at twice the final concentration (1-256 μM) was added to each well in a two-fold dilution. Fresh culture medium was used as a negative control, and triplicates were performed for each concentration. After drug addition, the 96-well plate was incubated in a 37°C constant temperature and humidity incubator for 12 hours before observation. The minimum inhibitory concentration (MIC) of the compound was determined as the concentration of the first clear well after the well that became visibly turbid, i.e., the MIC for the specific strain. The results are shown in Table 1.

[0087] Table 1: Minimum inhibitory concentration (MIC) of compounds 1-35 against test bacteria (μg / mL)

[0088] As shown in Table 1, compounds 1-35 all exhibited good antibacterial activity against the test bacteria, with minimum inhibitory concentrations ranging from 1.56 μg / mL to 125 μg / mL. In particular, compounds 10, 25, 28, 29, and 32 exhibited strong 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 exhibited strong inhibitory effects against a variety of test bacteria, but showed poor inhibitory effects against a few test bacteria, suggesting potential for development as inhibitors against a variety of test bacteria. Compounds 16, 6, 7, 8, 9, 15, 18, 27, 33, and 34 also exhibited strong inhibitory effects against a variety of test bacteria, but showed poor inhibitory effects against a few test bacteria, suggesting potential for development as inhibitors against a variety of test bacteria.

[0089] Example 6. Compounds induce bacterial cell membrane depolarization

[0090] 6.1 Effects of Compound 1-35 on the Membrane Potential of Staphylococcus aureus Cells

[0091] The membrane potential was detected using the fluorescent dye DiOC2(3). When the membrane potential was high, the dye aggregated to form polymers and produced red fluorescence when excited by light of a certain wavelength; when the membrane potential was low, the dye could not aggregate and produced green fluorescence when excited by light of a certain wavelength. The relative ratio of red and green fluorescence was used to measure the depolarization ratio. Before the test, Staphylococcus aureus was cultured in a 37°C constant temperature oscillator to the logarithmic phase. The logarithmic phase bacteria were centrifuged at 1500 rpm 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 485 nm, emission wavelengths were 530 nm and 630 nm). The negative control was PBS and the positive control was 1% Triton X-100.

[0092] Figure 1 shows that compounds 1-35 can produce membrane depolarization on bacterial cell membranes, destroying the cell membrane potential and thus exerting a bactericidal effect. In detail, 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, compound 1-35 and the positive control 1% Triton X-100 could both cause a decrease (Figure 1), indicating that compound 1-32 caused cell membrane depolarization and destroyed its membrane potential after interacting with bacteria.

[0093] 6.2 Effects of Compounds on Bacterial Uptake of the Fluorescent Dye Propidium Iodide (PI)

[0094] PI is a DNA-binding dye that can penetrate damaged cell membranes, intercalate into nucleic acids, and emit fluorescence. This assay was used to detect bacterial cell death. Compound 1-35 at a concentration of 4× the MIC was incubated with logarithmic-phase Staphylococcus aureus for 30 minutes. Then, a 1 mg / mL PI solution (final concentration 100 μg / mL) was added. After incubation at 37°C in the dark for 15 minutes, the cells were washed with PBS, and the PI-positive rate was determined by flow cytometry. As shown in Figure 2, compound 1-35, like the positive control 1% Triton X-100, also caused PI to enter the cells, indicating that compound 1-35 can disrupt cell integrity, leading to leakage of cellular contents and subsequent bacterial death.

[0095] Example 7. Bactericidal kinetics of compounds 1-35

[0096] The bactericidal kinetics of compounds 1-35 against Staphylococcus aureus ATCC 29213 were determined by colony forming unit (CFU) counting method.6 Staphylococcus aureus (S. aureus) at 100 CFU / mL was added to a 96-well plate. Compounds 1-35 (final concentrations of 2×MIC and 4×MIC) were then added and incubated at 37°C. Appropriate amounts of the bacterial solution were removed at intervals of 0, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and 12 hours, and diluted to the appropriate multiple. 100 μL of the diluted solution was evenly spread onto MH solid medium using a spreading rod. After overnight incubation, colonies were counted. Figure 3 shows that compound 1-35, at a concentration of 2×MIC, was able to kill all bacteria within 4 hours, demonstrating its rapid bacterial killing ability. The positive control, vancomycin, killed the test bacteria in 6 hours.

[0097] Example 8. Anti-biofilm effect of compound 1-35

[0098] The crystal violet method was used to determine the inhibitory effect of compound 1-35 on the biofilm formation of Staphylococcus aureus. 6 Logarithmic phase Staphylococcus aureus (CFU / mL) was added to a 96-well plate, and then 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 surface planktonic bacteria, washed with PBS, fixed with methanol, and stained with crystal violet solution for 15 minutes. After that, 95% ethanol was added to dissolve the crystal violet in the biofilm and measure the OD. 570 According to the formula, biofilm formation inhibition rate (%) = [1-(OD 570 - OD of negative control group 570 ) / (positive control group OD 570 - OD of negative control group 570 As shown in FIG4 , the inhibition rate of compounds 1-35 on biofilm formation was >50%, and some compounds could completely inhibit the formation of biofilm.

[0099] Example 9. Determination of drug resistance of Staphylococcus aureus to compounds 10, 28 and 33

[0100] The resistance of S. aureus to representative compounds 10, 28, and 33 after continuous exposure to sub-MIC concentrations was determined by serially measuring the MICs of the compounds against bacteria treated with ampicillin and ciprofloxacin. Ampicillin and ciprofloxacin were used as controls. S. aureus was incubated with sub-MIC concentrations of compounds 10, 28, and 33, along with ampicillin and ciprofloxacin, for 16 hours, followed by MIC determination. This was repeated for 21 passages. Figure 5 shows that over the 21-day bacterial resistance experiment, the MICs of compounds 10, 28, and 33 fluctuated within a range of 1-2 times the MIC, indicating that the bacteria had not developed resistance to these compounds. In contrast, the ciprofloxacin and ampicillin control groups developed resistance to compounds 640 times their original MICs by day 18 and day 17, respectively, demonstrating significant resistance.

[0101] Example 10. In vivo antibacterial activity assay of compounds 10, 28 and 33

[0102] The in vivo antibacterial activity of compounds 10, 28, and 33 was tested in a MRSA-infected mouse pneumonia model. Kunming mice weighing 20 g ± 1 g (purchased from the Animal Center of Lanzhou University) were selected and acclimated to the laboratory for 1 week. They were then randomly divided into groups and anesthetized and administered with bacteria (25 μL at a concentration of 6.0 × 10 9 CFU / mL of logarithmic phase MRSA) to establish a MRSA-infected mouse pneumonia model. The drugs were administered 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 lungs were grinded and smeared aseptically, and then the plates were smeared and cultured at 37°C for 24h for colony counting. As shown in Figure 6, compared with the uninfected group, the lungs of the mice in the infected control group showed obvious congestion and swelling, while the vancomycin-treated group showed local congestion but no obvious swelling. The compound 10, 28 and 33 treatment groups showed only very slight congestion and no obvious swelling. As shown in Figure 7, 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.

[0103] In summary, the present invention changes the amphiphilicity of the backbone by linking different hydrophobic groups and cationic groups on the amino terminus, carboxyl terminus and side chain thiol groups of cysteine ​​to obtain small molecule antimicrobial peptide mimetics that can treat infectious diseases caused by sensitive bacteria and drug-resistant bacteria.

[0104] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions that do not depart from the concept of the present invention are within the scope of protection of the present invention.

Claims

1. A small molecule antimicrobial peptide mimetic, the small molecule antimicrobial peptide mimetic is based on a cysteine backbone structure and is obtained by chemical modification at the amino terminus, carboxyl terminus, or side chain thiol group, and is characterized in that, The structural general formula of the small molecule antibacterial peptide mimetic is as follows: Wherein: R1 is selected from one of; R2 is selected from one of; R3 is selected from one of.

2. The small molecule antimicrobial peptide mimetic according to claim 1, wherein R1 is selected from one of; R2 is selected from one of; R3 is selected from 3. The small molecule antimicrobial peptide mimic according to claim 1, wherein, Its structural formula is one of Formula 1, 5-12, 15-18, 20, 23, 25, 27-30, 32-35:

4. A composition, characterized in that, Its active ingredient comprises at least one of the small molecule antimicrobial peptide mimetics described in any one of claims 1 to 3.

5. The composition according to claim 4, characterized in that, It also comprises an acceptable carrier.

6. The composition according to claim 4, wherein Its dosage form is an oral preparation, an injection, a mucosal administration preparation or a topical preparation.

7. Use of the small molecule antimicrobial peptide mimetic described in any one of claims 1 to 3 in the preparation of an anti-infective drug.

8. The application according to claim 7, characterized in that, The infection is caused by bacteria or fungi.

9. The application according to claim 8, characterized in that, The bacteria or fungi are drug-resistant bacteria or fungi.

10. The application according to claim 9, wherein The drug-resistant bacteria or fungi are selected from methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus.

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