Amphipathic osthole-quaternary ammonium salt heterozygote as well as preparation method and antibacterial application thereof

Abstract

The invention discloses a series of amphiphilic cnidium lactone-quaternary ammonium salt heterozygotes as well as a preparation method and antibacterial application thereof. According to the series of compounds, a natural compound cnidium lactone is taken as a lead, a hydrophilic quaternary ammonium salt fragment is introduced to a methoxy group through structure optimization, so that a series of amphiphilic cnidium lactone-quaternary ammonium salt heterozygotes are prepared, and the structural general formula of the amphiphilic cnidium lactone-quaternary ammonium salt heterozygotes is shown as a formula (I). The series of compounds have high in-vivo and in-vitro antibacterial activity on Staphylococcus aureus ATCC 29213 and clinically separated methicillin-resistant Staphylococcus aureus (MRSA), and the antibacterial activity of the compounds is equivalent to that of a clinical first-line drug vancomycin. Through structural optimization, the water solubility and antibacterial activity of the compound are enhanced, and the compound has relatively low in-vivo and in-vitro toxicity and hemolytic activity and is not easy to generate drug resistance. Therefore, the amphiphilic cnidium lactone-quaternary ammonium salt heterozygotes have the potential of being further developed into novel antibacterial drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical chemistry, and specifically relates to an amphiphilic cumin-quat hybrid and a preparation method and antibacterial application thereof. BACKGROUND

[0002] Antibiotic resistance has become a serious threat to global human health. Methicillin-resistant Staphylococcus aureus (MRSA) as a super drug-resistant bacteria can cause pneumonia, necrotizing fasciitis, septicemia, infective endocarditis and osteomyelitis and other serious invasive infections. Due to its strong virulence and strong drug resistance, it poses a great threat to the clinic (M. Liang, X. Ge, H. Xua, et al., Phytochemicals with activity against methicillin-resistant Staphylococcus aureus, Phytomedicine, 2022, 100, 154073.). Therefore, it is urgent to develop new antibacterial agents to combat MRSA infection. There are few related studies on cumin in terms of structural modification and activity against drug-resistant bacteria, and the water solubility of the parent itself is relatively poor, and the activity is relatively poor, which has a great room for improvement.

[0003] In recent years, antimicrobial peptides (AMPs) have become a hot topic in the development of antimicrobial drugs due to their broad-spectrum antimicrobial effects (J. Fu, T. Liu, X. Feng, et al., A perfect pair: Stabilized black phosphorous nanosheets engineering with antimicrobial peptides for robust multidrug resistant bacteria eradication, Adv. Healthcare Mater. 2022, 11, 2101846; Y. Zhu, W. Hao, X. Wang, et al., Antimicrobial peptides, conventional antibiotics, and their synergistic utility for the treatment of drug-resistant infections, Med. Res. Rev. 2022, 42, 1377-1422.). However, the systemic and local toxicity of AMPs, instability in the production process, pro-inflammatory and anti-inflammatory effects of byproducts, poor oral bioavailability and in vivo efficacy, and high preparation costs hinder the therapeutic application of AMPs (N. Chen, C. Jiang, Antimicrobial peptides: Structure, mechanism, and modification, Eur. J. Med. Chem. 2023, 255, 115377; A. R. P. Silva, M. S. J. Rabelo, et al., Recent advances in the design of antimicrobial peptide conjugates, J. Mater. Chem. B. 2022, 10, 3587-3600.). Osthole is a natural coumarin compound, widely exists in Cnidium Cusson, Angelica L., Heracleum L. (Apiaceae) and Citrus (Cruciferae) and other plants, has neuroprotective, anti-inflammatory and anticancer effects (Z. Liu, M. Han, X. Yan, et al., Design, synthesis, and biological evaluation of novel osthole-based isoxazoline derivatives as insecticide candidates, J. Agric. Food Chem. 2022, 70, 7921-7928.). Especially in the field of pesticides, due to its significant inhibitory activity on plant pathogenic fungi, osthole has been successfully developed as a natural antifungal agent (K. Hu, R. Li, F. Mo, et al., Natural product osthole can significantly disrupt cell wall integrity and dynamic balance of Fusarium oxysporum, Pestic. Biochem. Physiol. 2023, 196, 105623.). However, there are few reports on its antibacterial activity against zoonotic pathogens. Previously, we found that osthole had antibacterial activity against some clinical MRSA isolates with minimum inhibitory concentrations (MICs) ≥64 μg / mL, but its solubility and antibacterial activity need to be further improved. Therefore, aiming at the shortcomings of natural products osthole and AMPs, we conceived to combine AMP mimics with osthole to construct osthole-AMP mimic hybrids. This strategy takes osthole as the hydrophobic part, designs AMP mimics and connecting modules to construct osthole-AMP mimic hybrids with similar charge distribution, hydrophobicity and amphiphilicity as AMPs, aiming to obtain amphiphilic osthole-AMP mimic hybrids with high activity and low toxicity, and to improve the water solubility of the parent osthole. SUMMARY

[0004] Invention purposes: In view of the above technical problems, the present application provides an amphiphilic cumin-quaternary ammonium salt hybrid and a preparation method and antibacterial application thereof, which has good in vitro and in vivo bacteriostatic effect on S.aureus ATCC 29213 and various clinical isolated MRSA and other gram-positive bacteria, and solves the problem of poor water solubility.

[0005] Technical solutions: In order to achieve the above invention purposes, the technical solutions adopted by the present application are as follows:

[0006] The amphiphilic cumin-quaternary ammonium salt hybrid has the following structure shown in formula (I):

[0007]

[0008] Among them, n=3, 4 or 5, R is Among them, R 1 and R 2 are independently selected from H or C1-C8 alkyl.

[0009] As a preferred solution, the R 1 and R 2 are independently selected from H or C2-C6 alkyl.

[0010] As a preferred compound, the n, R 1 and R 2 are one of the following combinations:

[0011] (1) n=3, R 1 =H, R 2 =-CH2CH3;(2) n=4, R 1 =H, R 2 =-CH2CH3;

[0012] (3) n=5, R 1 =H, R2=-CH2CH3;(4) n=3, R 1 =R 2 =-CH2CH3;

[0013] (5) n=4, R 1 =R 2 =-CH2CH3;(6) n=5, R 1 =R 2 =-CH2CH3;

[0014] (7) n=3, R 1 =H, R2 = -(CH2)2CH3; (8) n = 4, R 1 = H, R 2 = -(CH2)2CH3;

[0015] (9) n = 5, R 1 = H, R 2 = -(CH2)2CH3; (10) n = 3, R 1 = R 2 = -(CH2)2CH3;

[0016] (11) n = 4, R 1 = R 2 = -(CH2)2CH3; (12) n = 5, R 1 = R 2 = -(CH2)2CH3;

[0017] (13) n = 3, R 1 = H, R 2 = -(CH2)3CH3; (14) n = 4, R 1 = H, R 2 = -(CH2)3CH3;

[0018] (15) n = 5, R 1 = H, R 2 = -(CH2)3CH3; (16) n = 3, R 1 = R 2 = -CH(CH3)2;

[0019] (17) n = 4, R 1 = R 2 = -CH(CH3)2; (18) n = 5, R 1 = R 2 = -CH(CH3)2;

[0020] (19) n = 3, R 1 = H, R2= -(CH2)4CH3; (20) n = 4, R 1 = H, R2= -(CH2)4CH3;

[0021] (21) n = 5, R 1 = H, R2= -(CH2)4CH3; (22) n = 3, R1 = R 2 = -(CH2)3CH3;

[0022] (23) n = 4, R 1 = R 2 = -(CH2)3CH3; 1 = R 2 = -(CH2)3CH3;

[0023] (25) n = 3, R 1 = H, R2 = -(CH2)5CH3; 1 = H, R2 = -(CH2)5CH3;

[0024] (27) n = 5, R 1 = H, R2 = -(CH2)5CH3; 1 = R 2 = -CH2CH(CH3)2;

[0025] (29) n = 4, R 1 = R 2 = -CH2CH(CH3)2; 1 = R 2 = -CH2CH(CH3)2.

[0026] The above specific selection represents compounds 1-30 in the following examples (each compound corresponds to the same combination of numbers as described above).

[0027] The present application also provides a preparation method of the amphiphilic cymarin-quaternary ammonium salt hybrid, comprising the following steps:

[0028] (1) taking cymarin as a substrate, converting the methoxyl group thereof into a phenolic hydroxyl group under the action of boron tribromide to obtain an intermediate a;

[0029] (2) reacting the intermediate a with different dibromoalkanes under alkaline conditions to synthesize an intermediate b;

[0030] (3) reacting the intermediate b with a small molecule peptide mimic c again to generate the amphiphilic cymarin-quaternary ammonium salt hybrid, and the reaction formula is shown as follows:

[0031]

[0032] wherein, R and n are the same as described above.

[0033] Preferably, in step (1), the molar ratio of the reaction of osthole and boron tribromide is 1:1-1:5, the reaction temperature is -40-0℃, and the reaction solvent is anhydrous dichloromethane.

[0034] Preferably, in step (2), the base in the alkaline condition is K2CO3, the molar ratio of the reaction of intermediate a and the base is 1:1-1:3, the molar ratio of the reaction of intermediate a and dibromoalkane is 1:1.5-1:3, the reaction temperature is 45-60℃, and the reaction solvent is anhydrous acetone.

[0035] Preferably, in step (3), the preparation method of intermediate c comprises the following steps: substitution reaction of amine RH and bromoacetyl bromide under alkaline condition to generate bromoacetamide, and then substitution reaction of bromoacetamide and dimethylamine under alkaline condition to generate small molecular peptidomimetic c:

[0036]

[0037] wherein R is as defined above.

[0038] Further preferably, the preparation method of intermediate c is as follows:

[0039]

[0040] wherein R 1 and R 2 are as defined above.

[0041] Further preferably, the molar ratio of the reaction of various amine RH with different carbon chain length and bromoacetyl bromide is 1:1.5-1:2.5, the selected base is K2CO3, the reaction temperature is 0℃ to room temperature, the reaction time is 2-8h, and the reaction solvent is anhydrous dichloromethane; the molar ratio of the reaction of bromoamide and dimethylamine is 1:1.5-1:3, the reaction temperature is room temperature, the reaction time is 12-18h, and the reaction solvent is anhydrous acetone.

[0042] Preferably, in step (3), the molar ratio of the reaction of intermediate b and intermediate c is 1:2-1:3, the reaction temperature is 70-80℃, and the reaction solvent is anhydrous ethanol.

[0043] The application finally provides the use of the amphiphilic osthole-quaternary ammonium salt hybrid in the preparation of antibacterial drugs. Preferably, the use in the preparation of drugs for inhibiting Staphylococcus aureus (Staphylococcus aureus ATCC 29213) and various clinical methicillin-resistant Staphylococcus aureus (MRSA).

[0044] The compounds of the present application are designed and synthesized by referring to the structure and function of antibacterial peptides, and a series of amphiphilic osthole-quaternary ammonium salt hybrids are obtained. The parent structure in the derivative is beneficial to the insertion of the compound into the bacterial phospholipid bilayer membrane as a hydrophobic part, and the hydrophilic cationic part is beneficial to the interaction with the negatively charged bacterial cell membrane, thereby leading to the death of bacteria. The antibacterial activity evaluation of all target compounds shows that all target compounds exhibit good in vitro antibacterial activity against Staphylococcus aureus ATCC29213 and clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), and the minimum inhibitory concentration (MIC≤128 μg / mL). In particular, the in vitro and in vivo anti-MRSA activity of the preferred compound 27 is comparable to that of the positive control drug vancomycin. In addition, the target compounds have low hemolytic activity, in vivo toxicity, good water solubility and stability. Therefore, such compounds have broad clinical application prospects.

[0045] Technical effects: The amphiphilic osthole-quaternary ammonium salt hybrid prepared by the present application has good in vivo and in vitro bacteriostatic effect on Staphylococcus aureus ATCC29213 and various methicillin-resistant Staphylococcus aureus (MRSA) and other gram-positive bacteria, and improves the water solubility, reduces the biological toxicity, and has a high yield, and is expected to be further developed as a potential antibacterial drug in clinical application. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 is the dynamic bactericidal curve of compound 27.

[0047] Figure 2 is the in vivo blood routine and blood biochemical index of compound 27.

[0048] Figure 3 is the change of the bacterial load of the mouse skin of compound 27.

[0049] Figure 4 is the 1H-NMR spectrum of compound 27.

[0050] Figure 5 is the 13C-NMR spectrum of compound 27. DETAILED DESCRIPTION

[0051] The present application is further described in detail by the following examples.

[0052] Preparation of intermediate a in example 1

[0053] Weigh appropriate amounts of substrate osthol (1 mmol) and boron tribromide (BBr3) (1.3 mmol) into a 25 mL round-bottom flask, add 3 mL of anhydrous toluene to dissolve them, heat under reflux at 110 °C with stirring, and detect the reaction by thin-layer chromatography (TLC) until the reaction is complete. Filter the reaction solution, wash thoroughly with dichloromethane, concentrate the filtrate under reduced pressure, and separate the intermediate a by column chromatography.

[0054] Example 2 Preparation of intermediate b1-3

[0055] Weigh appropriate amounts of intermediate a (1 mmol) and potassium carbonate (3 mmol) into a 25 mL round-bottom flask, add 3 mL of acetone to dissolve them, then add 1,3-dibromoethane / 1,4-dibromopropane / 1,5-dibromobutane (3 mmol), heat and stir at 50 °C, detect the reaction by thin-layer chromatography (TLC) until the reaction is complete, extract with ethyl acetate (3 × 30 mL), combine the organic layers, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and separate by column chromatography to obtain intermediate b1-3.

[0056] The physicochemical properties of intermediate b1 are as follows:

[0057] 1) White solid;

[0058] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (400MHz) characteristics:

[0059] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 68%, 1 H NMR (400MHz CDCl3) δ: 7.81 (d, J = 8.4Hz, 1H, -Ph), 7.68 (d, J = 8.4Hz, 1H, -Ph), 7.01 (d, J = 8.4Hz, 1H, -Ph), 6.45 (d, J = 8.4Hz, 1H, -Ph), 5.75 (m, 1H, -C H =CH2), 4.08 (t, J = 7.2Hz, 2H, -OCH2-), 3.50 (t, J = 7.2Hz, 2H, -CH2Br), 3.30 (t, J = 6. 0Hz,2H,-CH2-),2.13(m,2H,-CH2-),1.83(s,3H,-CH3),1.67(s,3H,CH3); MS(ESI)C 17 H 20 BrO3[M+H] + calcd=351.05; found=351.11.

[0060] The physicochemical properties of intermediate b2 are as follows:

[0061] 1) White solid;

[0062] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (400MHz) characteristics:

[0063] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 78%, 1 H NMR (400MHz CDCl3) δ: 7.80 (d, J = 8.4Hz, 1H, -Ph), 7.69 (d, J = 8.4Hz, 1H, -Ph), 7.02 (d, J = 8.4Hz, 1H, -Ph), 6.47 (d, J = 8.4Hz, 1H, -Ph), 5.72 (m, 1H, -C H =CH2),4.07(t,J=7.2Hz,2H,-OCH2-),3.52(t,J=7.2Hz,2H,-CH2Br),3.33(t,J=6.0Hz,2H,-C H2-),2.01(m,2H,-CH2-),1.85(m,2H,-CH2-),1.83(s,3H,-CH3),1.67(s,3H,CH3); MS(ESI)C 18 H 22 BrO3[M+H] + calcd=365.07; found=365.09.

[0064] The physicochemical properties of intermediate b3 are as follows:

[0065] 1) White solid;

[0066] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (400MHz) characteristics:

[0067] Using CDCl3 as solvent, the peaks were assigned as follows: Yield: 80%. 1 H NMR (400MHz CDCl3) δ: 7.82 (d, J = 8.4Hz, 1H, -Ph), 7.66 (d, J = 8.4Hz, 1H, -Ph), 7.00 (d, J = 8.4Hz, 1H, -Ph), 6.44 (d, J = 8.4Hz, 1H, -Ph), 5.73 (m, 1H, -C H=CH2), 4.06 (t, J = 7.2 Hz, 2H, -OCH2-), 3.51 (t, J = 7.2 Hz, 2H, -CH2Br), 3.32 (t, J = 6.0 Hz, 2H, -CH2-), 2.10 (m, 2H, -CH2-), 1.92 (m, 2H, -CH2-), 1.83 (s, 3H, -CH3), 1.67 (s, 3H, CH3), 1.29 (m, 2H, -CH2-); MS (ESI) C 19 H 24 BrO3[M+H] + calcd = 379.08; found = 379.13.

[0068] Preparation of Example 3 Intermediate c

[0069] The corresponding amine (1 mmol) was weighed into a 50 mL round-bottom flask, 2 mL of anhydrous dichloromethane was added to dissolve it, and then potassium carbonate (1.5 mmol) was added. At 0°C, bromoacetyl bromide (1.5 mmol) was slowly added dropwise to the reaction solution, and the reaction was continued for half an hour, after which it was transferred to room temperature. TLC detection was performed to determine the end of the reaction, and ethyl acetate extraction, anhydrous sodium sulfate drying, and reduced-pressure concentration were performed. Column chromatography was used to isolate the corresponding bromoacetamide.

[0070] Dimethylamine (1.5 mmol) and the corresponding bromoacetamide (1 mmol) were weighed into a 25 mL round-bottom flask, 3 mL of acetone was added to dissolve them, and an appropriate amount of potassium carbonate (1.5 mmol) was added. The reaction was carried out at room temperature, and TLC detection was performed to determine the end of the reaction. Ethyl acetate extraction, anhydrous sodium sulfate drying, and reduced-pressure concentration were performed. Column chromatography was used to isolate intermediate c.

[0071] Example 4 Compound 1

[0072] Intermediate b1-3 (1 mmol) and intermediate c (3 mmol) were weighed into a 25 mL round-bottom flask, 2 mL of anhydrous acetonitrile was added to dissolve them, and the reaction was stirred at 78°C. TLC detection was performed to determine the end of the reaction, and preparative thin-layer chromatography (dichloromethane:methanol = 10:1) was used to isolate the pure target compound.

[0073] The physical and chemical properties of compound 1 are as follows:

[0074] 1) light yellow liquid;

[0075] 2) the nuclear magnetic resonance spectrum (1H NMR, 400 MHz) of the compound is characterized by: 1 H / 13 C NMR, 400 MHz) is as follows:

[0076] In CDCl3 as the solvent, each peak is assigned as follows:1 H NMR (400 MHz CDCl3) δ: 8.80 (s, 1H, -NH-), 7.60 (d, J = 9.6 Hz, 1H, -CH=CH-), 7.29 (d, J = 8.4 Hz, 1H, -Ph), 6.79 (d, J = 8.8 Hz, 1H, -Ph), 6.25 (d, J = 9.6 Hz, 1H, -CH=CH-), 5.12 (t, J = 6.0 Hz, 1H, -CH=C(CH3)2), 4.73 (s, 2H, -CH2-), 4.22 (s, 2H, -CH2-), 3.91 (s, 2H, -CH2-), 3.50 (d, J = 7.2 Hz, 2H, -CH2-), 3.47 (s, 6H, N-CH3), 3.30 (t, J = 6.0 Hz, 2H, -CH2-), 2.48 (s, 2H, -CH2-), 1.83 (s, 3H, -CH3), 1.67 (s, 3H, CH3), 1.19 (t, J = 7.2 Hz, 3H, -CH3); 13 C NMR (100 MHz CDCl3) δ: 162.3, 161.0, 158.3, 152.8, 143.6, 132.8, 126.5, 121.2, 117.9, 113.6, 108.1, 64.9, 64.0, 62.9, 52.5, 34.8, 25.8, 23.5, 22.0, 18.2, 14.1. HRMS (ESI) C 23 H 33 N2O4[M-Br] + calcd = 401.2435; found = 401.2451.

[0077] Example 5 Compound 2

[0078] Compound 2 was synthesized by the method described in Example 4, and the physical and chemical properties of Compound 2 were as follows:

[0079] 1), light yellow liquid;

[0080] 2), the nuclear magnetic resonance spectrum of the compound (H / 1 H / 13 C NMR, 400 MHz) characteristics:

[0081] In CDCl3as solvent, each peak is attributed to: 1H NMR (400 MHz CDCl3) δ: 8.83 (s, 1H, -NH-), 7.60 (d, J = 9.6 Hz, 1H, -CH=CH-), 7.28 (d, J = 8.8 Hz, 1H, -Ph), 6.82 (d, J = 8.8 Hz, 1H, -Ph), 6.23 (d, J = 9.6 Hz, 1H, -CH=CH-), 5.14 (t, J = 6.4 Hz, 1H, -CH=C(CH3)2), 4.68 (s, 2H, -CH2-), 4.15 (s, 2H, -CH2-), 3.79 (s, 2H, -CH2-), 3.50 (d, J = 7.2 Hz, 2H, -CH2-), 3.42 (s, 6H, N-CH3), 3.32 (s, 2H, -CH2-), 2.10 (s, 2H, -CH2-), 1.97 (s, 2H, -CH2-), 1.83 (s, 3H, -CH3), 1.65 (s, 3H, CH3), 1.19 (t, J = 6.8 Hz, 3H, -CH3); 13 C NMR (100 MHz CDCl3) δ: 162.3, 161.1, 158.8, 152.8, 143.6, 132.7, 126.4, 121.2, 117.8, 113.3, 108.2, 67.3, 65.8, 63.3, 51.9, 34.7, 26.1, 25.8, 22.0, 19.9, 18.1, 14.1; HRMS (ESI) C 24 H 35 N2O4[M-Br] + calcd = 415.2591; found = 415.2610.

[0082] Example 6 Compound 3

[0083] Compound 3 was synthesized by the method described in Example 4, and the physical and chemical properties of compound 3 are as follows:

[0084] 1) light yellow liquid;

[0085] 2) the nuclear magnetic resonance spectrum (H / 1 H / 13 C NMR, 400 MHz) characteristics of the compound are:

[0086] In CDCl3as solvent, each peak is assigned as: 1H NMR (400 MHz CDCl3) δ: 8.87 (s, 1H, -NH-), 7.60 (d, J = 9.2 Hz, 1H, -CH=CH-), 7.27 (d, J = 9.6 Hz, 1H, -Ph), 6.82 (d, J = 8.8 Hz, 1H, -Ph), 6.22 (d, J = 9.6 Hz, 1H, -CH=CH-), 5.17 (t, J = 7.2 Hz, 1H, -CH=C(CH3)2), 4.67 (s, 2H, -CH2-), 4.08 (t, J = 6.0 Hz, 2H, -CH2-), 3.67 (s, 2H, -CH2-), 3.50 (d, J = 6.8 Hz, 2H, -CH2-), 3.41 (s, 6H, N-CH3), 3.30 (t, J = 6.4 Hz, 2H, -CH2-), 1.95 (s, 4H, -CH2-), 1.83 (s, 3H, -CH3), 1.66 (s, 3H, CH3), 1.63 (s, 2H, -CH2-), 1.19 (t, J = 7.2 Hz, 3H, -CH3); 13 C NMR (100 MHz CDCl3) δ: 162.4, 161.3, 159.2, 152.8, 143.8, 132.5, 126.4, 121.2, 117.8, 113.0, 108.1, 67.9, 66.2, 63.2, 52.1, 34.7, 28.6, 25.9, 22.9, 22.7, 22.0, 18.1, 14.1; HRMS (ESI) C 25 H 37 N2O4[M-Br] + calcd = 429.2748; found = 429.2764.

[0087] Example 7 Compound 4

[0088] Compound 4 was synthesized by the method described in Example 4, and the physical and chemical properties of compound 4 were as follows:

[0089] 1) light yellow liquid;

[0090] 2) the nuclear magnetic resonance spectrum (H / C NMR, 400 MHz) characteristics of the compound: 1 H / 13 C NMR, 400 MHz) characteristics:

[0091] In CDCl3 solvent, each peak is assigned as follows: 1H NMR (400 MHz CDCl3) δ: 7.60 (d, J = 9.6 Hz, 1H, -CH=CH-), 7.27 (d, J = 9.2 Hz, 1H, -Ph), 6.80 (d, J = 8.8 Hz, 1H, -Ph), 6.24 (d, J = 9.6 Hz, 1H, -CH=CH-), 5.15 (t, J = 6.8 Hz, 1H, -CH=C(CH3)2), 5.00 (s, 2H, -CH2-), 4.20 (t, J = 5.6 Hz, 2H, -CH2-), 4.14-4.18 (m, 2H, -CH2-), 3.65 (s, 6H, N-CH3), 3.50 (d, J = 7.2 Hz, 4H, -CH2-), 3.36-3.39 (m, 2H, -CH2-), 2.40-2.44 (m, 2H, -CH2-), 1.83 (s, 3H, -CH3), 1.66 (s, 3H, CH3), 1.25-1.29 (m, 3H, -CH3), 1.11-1.15 (m, 3H, -CH3); HRMS (ESI) C 25 H 37 N2O4[M-Br] + calcd = 429.2748; found = 429.2764.

[0092] Example 8 Compound 5

[0093] Compound 5 was synthesized using the method described in Example 4. The physical and chemical properties of compound 5 are as follows:

[0094] 1) light yellow liquid;

[0095] 2) the nuclear magnetic resonance spectrum (H / 1 H / 13 C NMR, 400 MHz) characteristics of the compound are as follows:

[0096] In CDCl3as solvent, wherein each peak is assigned as: 1H NMR (400 MHz CDCl3) δ: 7.60 (d, J = 9.6 Hz, 1H, -CH=CH-), 7.27 (d, J = 10.8 Hz, 1H, -Ph), 6.84 (d, J = 8.4 Hz, 1H, -Ph), 6.22 (d, J = 9.2 Hz, 1H, -CH=CH-), 5.15 (t, J = 6.8 Hz, 1H, -CH=C(CH3)2), 4.93 (s, 2H, -CH3), 4.13 (t, J = 5.2 Hz, 2H, -CH2-), 4.02 (t, J = 7.2 Hz, 2H, -CH2-), 3.59 (s, 6H, N-CH3), 3.48 (t, J = 6.8 Hz, 4H, -CH2-), 3.34-3.37 (m, 2H, -CH2-), 1.96-2.04 (m, 4H, -CH2-), 1.82 (s, 3H, -CH3), 1.65 (s, 3H, CH3), 1.24 (t, J = 7.2 Hz, 3H, -CH3), 1.11 (t, J = 7.2 Hz, 3H, -CH3); 13 CNMR (100 MHz CDCl3) δ: 162.2, 161.2, 159.0, 152.8, 143.7, 132.6, 126.4, 121.2, 117.7, 113.2, 108.3, 67.4, 64.8, 61.5, 52.2, 42.2, 40.9, 26.1, 25.9, 22.1, 19.9, 18.1, 14.4, 12.8. HRMS (ESI) C 26 H 39 N2O4[M-Br] + calcd = 443.2904; found = 443.2922.

[0097] Example 9 Compound 6

[0098] Compound 6 was synthesized by the method described in Example 4, and the physical and chemical properties of compound 6 are as follows:

[0099] 1) light yellow liquid;

[0100] 2) the nuclear magnetic resonance spectrum of the compound (H 1 H / 13 C NMR, 400 MHz) characteristics:

[0101] In CDCl3 as solvent, each peak is assigned as: 1H NMR (400 MHz CDCl3) δ: 7.60 (d, J = 9.6 Hz, 1H, -CH=CH-), 7.27 (d, J = 6.8 Hz, 1H, -Ph), 6.81 (d, J = 8.8 Hz, 1H, -Ph), 6.22 (d, J = 9.2 Hz, 1H, -CH=CH-), 5.18 (t, J = 7.2 Hz, 1H, -CH=C(CH3)2), 4.95 (s, 2H, -CH2-), 4.06 (t, J = 6.0 Hz, 2H, -CH2-), 3.89-3.94 (m, 2H, -CH2-), 3.60 (s, 6H, N-CH3), 3.50-3.52 (m, 4H, -CH2-), 3.33-3.39 (m, 2H, -CH2-), 1.92-1.95 (m, 2H, -CH2-), 1.85-1.88 (m, 2H, -CH2-), 1.83 (s, 3H, -CH3), 1.66 (s, 3H, CH3), 1.59-1.63 (m, 2H, -CH2-), 1.25 (t, J = 6.8 Hz, 3H, -CH3), 1.11 (t, J = 6.8 Hz, 3H, -CH3); 13 C NMR (100 MHz CDCl3) δ: 162.3, 161.3, 159.3, 152.8, 143.7, 132.4, 126.3, 121.2, 117.8, 113.0, 108.2, 67.9, 64.8, 61.3, 52.5, 42.2, 41.0, 28.6, 25.8, 22.9, 22.8, 22.0, 18.0, 14.5, 12.8; HRMS (ESI) C 27 H 41 N2O4[M-Br] + calcd = 457.3061; found = 457.3080.

[0102] Example 10 Compound 7

[0103] Compound 7 was synthesized by the method described in Example 4, and the physical and chemical properties of compound 7 are as follows:

[0104] 1) light yellow liquid;

[0105] 2) the nuclear magnetic resonance spectrum (H / 1 H / 13 C NMR, 400 MHz) characteristics of the compound are as follows:

[0106] In CDCl3 as solvent, each peak is assigned as follows: 1H NMR (400 MHz CDCl3) δ: 8.79 (s, 1H, -NH-), 7.60 (d, J = 9.6 Hz, 1H, -CH=CH-), 7.28 (d, J = 8.8 Hz, 1H, -Ph), 6.80 (d, J = 8.8 Hz, 1H, -Ph), 6.24 (d, J = 9.6 Hz, 1H, -CH=CH-), 5.12 (t, J = 6.8 Hz, 1H, -CH=C(CH3)2), 4.73 (s, 2H, -CH2-), 4.21 (t, J = 5.2 Hz, 2H, -CH2-), 3.90-3.94 (m, 2H, -CH2-), 3.50 (d, J = 6.8 Hz, 2H, -CH2-), 3.47 (s, 6H, N-CH3), 3.22-3.25 (m, 2H, -CH2-), 2.49 (s, 2H, -CH2-), 1.83 (s, 3H, -CH3), 1.67 (s, 3H, CH3), 1.59-1.62 (m, 2H, -CH2-), 0.92 (t, J = 7.6 Hz, 3H, -CH3); 13 CNMR (100 MHz CDCl3) δ: 162.4, 161.0, 158.3, 152.8, 143.5, 132.7, 126.5, 121.2, 117.9, 113.6, 108.1, 64.9, 64.0, 62.9, 52.5, 41.5, 25.7, 23.5, 22.2, 22.0, 18.2, 11.5; HRMS (ESI) C 24 H 35 N2O4[M-Br] + calcd = 415.2591; found = 415.2608.

[0107] Example 11 Compound 8

[0108] Compound 8 was synthesized using the method described in Example 4, and the physical and chemical properties of compound 8 are as follows:

[0109] 1) light yellow liquid;

[0110] 2) the nuclear magnetic resonance spectrum of this compound (H 1 H / 13 C NMR, 400 MHz) characteristics:

[0111] In CDCl3as solvent, each peak is attributed to: 1H NMR (400 MHz CDCl3) δ: 8.83 (s, 1H, -NH-), 7.60 (d, J = 9.2 Hz, 1H, -CH=CH-), 7.28 (d, J = 8.8 Hz, 1H, -Ph), 6.82 (d, J = 8.4 Hz, 1H, -Ph), 6.23 (d, J = 9.2 Hz, 1H, -CH=CH-), 5.14 (t, J = 6.0 Hz, 1H, -CH=C(CH3)2), 4.69 (s, 2H, -CH2-), 4.15 (s, 2H, -CH2-), 3.79 (s, 2H, -CH2-), 3.50 (d, J = 6.8 Hz, 2H, -CH2-), 3.41 (s, 6H, N-CH3), 3.24 (s, 2H, -CH2-), 2.10 (s, 2H, -CH2-), 1.97 (s, 2H, -CH2-), 1.83 (s, 3H, -CH3), 1.65 (s, 3H, CH3), 1.58-1.61 (m, 2H, -CH2-), 0.93 (t, J = 7.2 Hz, 3H, -CH3); HRMS (ESI) C 25 H 37 N2O4[M-Br] + calcd = 429.2748; found = 429.2766.

[0112] Example 12 Compound 9

[0113] Compound 9 was synthesized using the method described in Example 4. The physicochemical properties of compound 9 are as follows:

[0114] 1) light yellow liquid;

[0115] 2) the nuclear magnetic resonance spectrum (H 1 H / 13 C NMR, 400 MHz) characteristics of the compound are:

[0116] in CDCl3, where each peak is assigned: 1H NMR (400 MHz CDCl3) δ: 8.84 (s, 1H, -NH-), 7.60 (d, J = 9.2 Hz, 1H, -CH=CH-), 7.27 (d, J = 9.2 Hz, 1H, -Ph), 6.82 (d, J = 8.8 Hz, 1H, -Ph), 6.22 (d, J = 9.2 Hz, 1H, -CH=CH-), 5.17 (t, J = 7.2 Hz, 1H, -CH=C(CH3)2), 4.68 (s, 2H, -CH2-), 4.08 (t, J = 5.6 Hz, 2H, -CH2-), 3.67 (s, 2H, -CH2-), 3.50 (d, J = 7.2 Hz, 2H, -CH2-), 3.41 (s, 6H, N-CH3), 3.23 (s, 2H, -CH2-), 1.95 (s, 4H, -CH2-), 1.83 (s, 3H, -CH3), 1.66 (s, 3H, CH3), 1.57-1.63 (m, 4H, -CH2-), 0.92 (t, J = 7.2 Hz, 3H, -CH3); 13 C NMR (100 MHz CDCl3) δ: 162.5, 161.3, 159.2, 152.9, 143.7, 132.5, 126.3, 121.2, 117.8, 113.1, 108.3, 67.8, 66.1, 63.2, 52.2, 41.5, 28.7, 25.9, 22.9, 22.7, 22.2, 22.0, 18.0, 11.6; HRMS (ESI) C 26 H 39 N2O4[M-Br] + calcd = 443.2904; found = 443.2921.

[0117] Example 13 Compound 10

[0118] Compound 10 was synthesized using the method described in Example 4, and the physical and chemical properties of compound 10 are as follows:

[0119] 1) light yellow liquid;

[0120] 2) the nuclear magnetic resonance spectrum of this compound (H / 1 H / 13 C NMR, 400 MHz) characteristics:

[0121] In CDCl3as solvent, each peak is attributed to: 1H NMR (400 MHz CDCl3) δ: 7.60 (d, J = 9.6 Hz, 1H, -CH=CH-), 7.27 (d, J = 9.2 Hz, 1H, -Ph), 6.80 (d, J = 8.8 Hz, 1H, -Ph), 6.24 (d, J = 9.6 Hz, 1H, -CH=CH-), 5.14 (t, J = 6.8 Hz, 1H, -CH=C(CH3)2), 4.92 (s, 2H, -CH2-), 4.21 (t, J = 5.2 Hz, 2H, -CH2-), 4.14-4.18 (m, 2H, -CH2-), 3.63 (s, 6H, N-CH3), 3.50 (d, J = 7.2 Hz, 2H, -CH2-), 3.38 (t, J = 7.6 Hz, 2H, -CH2-), 3.24 (t, J = 8.0 Hz, 2H, -CH2-), 2.41 (s, 2H, -CH2-), 1.83 (s, 3H, CH3), 1.67 (s, 3H, CH3), 1.50-1.61 (m, 4H, -CH2-), 0.97 (t, J = 7.2 Hz, 3H, -CH3), 0.87 (t, J = 7.2 Hz, 3H, -CH3); 13 C NMR (100 MHz CDCl3) δ: 162.5, 161.1, 158.5, 152.8, 143.6, 132.6, 126.4, 121.2, 117.9, 113.4, 108.2, 65.0, 62.9, 61.3, 52.9, 49.3, 48.1, 43.3, 25.7, 23.6, 22.1, 22.0, 20.7, 18.1, 11.3, 11.2; HRMS (ESI) C 27 H 41 N2O4[M-Br] + calcd = 457.3061; found = 457.3077.

[0122] Example 14 Compound 11

[0123] Compound 11 was synthesized by the method described in Example 4, and the physical and chemical properties of compound 11 were as follows:

[0124] 1) light yellow liquid;

[0125] 2) the nuclear magnetic resonance spectrum of the compound (H / 1 H / 13 C NMR, 400 MHz) characteristics:

[0126] In CDCl3 as solvent, each peak is assigned: 1H NMR (400 MHz CDCl3) δ: 7.60 (d, J = 9.6 Hz, 1 H, -CH=CH-), 7.28 (d, J = 8.8 Hz, 1 H, -Ph), 6.84 (d, J = 8.8 Hz, 1 H, -Ph), 6.23 (d, J = 9.2 Hz, 1 H, -CH=CH-), 5.15 (t, J = 6.8 Hz, 1 H, -CH=C(CH3)2), 4.87 (s, 2 H, -CH2-), 4.15 (s, 2 H, -CH2-), 4.03 (s, 2 H, -CH2-), 3.59 (s, 6 H, N-CH3), 3.49 (d, J = 7.2 Hz, 2 H, -CH2-), 3.39 (s, 2 H, -CH2-), 3.22 (t, J = 8.4 Hz, 2 H, -CH2-), 1.98 (s, 4 H, -CH2-), 1.82 (s, 3 H, -CH3), 1.65 (s, 3 H, CH3), 1.49-1.59 (m, 4 H, -CH2-), 0.95 (t, J = 7.2 Hz, 3 H, -CH3), 0.86 (t, J = 7.6 Hz, 3 H, -CH3); HRMS (ESI) C 28 H 43 N2O4[M-Br] + calcd = 471.3217; found = 471.3237.

[0127] Example 15 Compound 12

[0128] Compound 12 was synthesized using the method described in Example 4. The physical and chemical properties of compound 12 are as follows:

[0129] 1) light yellow liquid;

[0130] 2) the nuclear magnetic resonance spectrum (H 1 H / 13 C NMR, 400 MHz) characteristics of the compound are as follows:

[0131] In CDCl3 solvent, the assignment of each peak is as follows: 1H NMR (400 MHz CDCl3) δ: 7.60 (d, J = 9.6 Hz, 1H, -CH=CH-), 7.27 (d, J = 7.2 Hz, 1H, -Ph), 6.81 (d, J = 8.4 Hz, 1H, -Ph), 6.22 (d, J = 9.2 Hz, 1H, -CH=CH-), 5.18 (t, J = 7.2 Hz, 1H, -CH=C(CH3)2), 4.87 (s, 2H, -CH2-), 4.06 (t, J = 6.0 Hz, 2H, -CH2-), 3.88-3.92 (m, 2H, -CH2-), 3.59 (s, 6H, N-CH3), 3.50 (d, J = 7.2 Hz, 2H, -CH2-), 3.38 (t, J = 7.2 Hz, 2H, -CH2-), 3.23 (t, J = 7.6 Hz, 2H, -CH2-), 1.91-1.95 (m, 2H, -CH2-), 1.85-1.89 (m, 2H, -CH2-), 1.83 (s, 3H, -CH3), 1.66 (s, 3H, CH3), 1.59-1.64 (m, 4H, -CH2-), 1.52-1.56 (m, 2H, -CH2-), 0.96 (t, J = 7.2 Hz, 3H, -CH3), 0.87 (t, J = 7.2 Hz, 3H, -CH3); 13 C NMR (100 MHz CDCl3) δ: 162.7, 161.3, 159.3, 152.8, 143.7, 132.4, 126.3, 121.2, 117.8, 113.0, 108.2, 67.9, 64.8, 61.3, 52.7, 49.3, 48.1, 28.6, 25.8, 22.8, 22.8, 22.1, 22.0, 20.7, 18.0, 11.3; HRMS (ESI) C 29 H 45 N2O4[M-Br] + calcd = 485.3374; found = 485.3391.

[0132] Example 16 Compound 13

[0133] Compound 13 was synthesized using the method described in Example 4, and the physical and chemical properties of compound 13 are as follows:

[0134] 1) light yellow liquid;

[0135] 2) the nuclear magnetic resonance spectrum (H / 1 H / 13 C NMR, 400 MHz) characteristics of this compound are as follows:

[0136] In CDCl3 as solvent, each peak is assigned:1 H NMR (400 MHz CDCl3) δ: 8.81 (s, 1H, -NH-), 7.60 (d, J = 9.6 Hz, 1H, -CH=CH-), 7.29 (d, J = 8.8 Hz, 1H, -Ph), 6.80 (d, J = 8.4 Hz, 1H, -Ph), 6.25 (d, J = 9.2 Hz, 1H, -CH=CH-), 5.12 (t, J = 6.8 Hz, 1H, -CH=C(CH3)2), 4.73 (s, 2H, -CH2-), 4.21 (t, J = 5.2 Hz, 2H, -CH2-), 3.90-3.94 (m, 2H, -CH2-), 3.50 (d, J = 7.2 Hz, 2H, -CH2-), 3.46 (s, 6H, N-CH3), 3.26-3.29 (m, 2H, -CH2-), 2.46-2.50 (m, 2H, -CH2-), 1.83 (s, 3H, -CH3), 1.67 (s, 3H, CH3), 1.54-1.58 (m, 2H, -CH2-), 1.35-1.39 (m, 2H, -CH2-), 0.88 (t, J = 7.2 Hz, 3H, -CH3); 13 C NMR (100 MHz CDCl3) δ: 162.4, 161.0, 158.3, 152.8, 143.6, 132.8, 126.5, 121.2, 117.9, 113.6, 108.1, 64.9, 63.9, 63.0, 53.4, 52.5, 39.6, 30.9, 25.8, 23.6, 22.0, 20.2, 18.2, 13.6; HRMS (ESI) C 25 H 37 N2O4[M-Br] + calcd = 429.2748; found = 429.2752.

[0137] Example 17 Compound 14

[0138] Compound 14 was synthesized by the method described in Example 4, and the physical and chemical properties of compound 14 are as follows:

[0139] 1) light yellow liquid;

[0140] 2) the nuclear magnetic resonance spectrum of the compound (H / 1 H / 13 C NMR, 400 MHz) characteristics:

[0141] In CDCl3 as solvent, each peak is assigned: 1H NMR (400 MHz CDCl3) δ: 8.83 (s, 1H, -NH-), 7.60 (d, J = 9.6 Hz, 1H, -CH=CH-), 7.28 (d, J = 8.8 Hz, 1H, -Ph), 6.82 (d, J = 8.4 Hz, 1H, -Ph), 6.23 (d, J = 9.6 Hz, 1H, -CH=CH-), 5.14 (t, J = 6.8 Hz, 1H, -CH=C(CH3)2), 4.69 (s, 2H, -CH2-), 4.15 (s, 2H, -CH2-), 3.79 (s, 2H, -CH2-), 3.48-3.52 (m, 2H, -CH2-), 3.41 (s, 6H, N-CH3), 3.26 (s, 2H, -CH2-), 2.10 (s, 2H, -CH2-), 1.97 (s, 2H, -CH2-), 1.83 (s, 3H, -CH3), 1.65 (s, 3H, CH3), 1.55-1.58 (m, 2H, -CH2-), 1.36-1.39 (m, 2H, -CH2-), 0.89 (t, J = 7.2 Hz, 3H, -CH3); 13 C NMR (100 MHz CDCl3) δ: 162.5, 161.2, 159.0, 152.9, 143.7, 132.7, 126.5, 121.3, 117.8, 113.4, 108.3, 67.4, 65.9, 63.5, 52.0, 39.7, 30.9, 26.1, 25.8, 22.1, 20.2, 20.0, 18.1, 13.6. HRMS (ESI) C 26 H 39 N2O4[M-Br] + calcd = 443.2904; found = 443.2911.

[0142] Example 18 Compound 15

[0143] Compound 15 was synthesized using the method described in Example 4, and the physical and chemical properties of compound 15 are as follows:

[0144] 1) light yellow liquid;

[0145] 2) the nuclear magnetic resonance spectrum (H / 1 H / 13 C NMR, 400 MHz) characteristics of the compound:

[0146] In CDCl3 as solvent, each peak is assigned: 1H NMR(600MHz CDC13)δ:8.82(s,1H,-NH-),7.60(d,J=9.0Hz,1H,-CH=CH-),7.27(d,J=8.4Hz,1H,-Ph),6.82(d,J=8.4Hz,1H,-Ph),6.22(d,J= 9.6Hz,1H,-CH=CH-),5.18(t,J=7.2Hz,1H,-CH=C(CH3)2),4.65(s,2H,-CH2-),4.08(t,J=6.0Hz,2H,-CH2-),3.66-3.69(m,2H,- CH2-),3.51(d,J=7.2Hz,2H,-CH2-),3.40(s,6H,N-CH3),3.24-3.28(m,2H,-CH2-),1.94-1.96(m,4H,-CH2-),1.83(s,3H,-CH3 ),1.66(s,3H,CH3),1.62-1.64(m,2H,-CH2-),1.55-1.57(m,2H,-CH2-),1.34-1.38(m,2H,-CH2-),0.89(t,J=7.2Hz,3H,-CH3); 13 C NMR(150MHz CDCl3)δ:162.5,161.3,159.2,152.8,143.7,132.5,126.3,121.2,117.8,113.0,108.2,67. 9,66.0,63.2,52.2,39.6,30.9,28.6,25.8,22.9,22.7,22.0,20.2,18.0,13.6; HRMS(ESI)C 27 H 41 N₂O₄[M-Br] + calcd=457.3061; found=457.3069.

[0147] Example 19 Compound 16

[0148] Compound 16 was synthesized using the method described in Example 4. The physicochemical properties of compound 16 are as follows:

[0149] 1) A light yellow liquid;

[0150] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H / 13 C NMR (400MHz) characteristics:

[0151] Using CDCl3 as solvent, the peak assignments are as follows: 1H NMR (400 MHz, CDC13) δ: 7.60 (d, J = 9.6 Hz, 1H, -CH=CH-), 7.27 (d, J = 8.8 Hz, 1H, -Ph), 6.81 (d, J = 8.8 Hz, 1H, -Ph), 6.24 (d, J = 9.2 Hz, 1H, -CH=CH-), 5.15 (t, J = 6.8 Hz, 1H, -CH=C(CH3)2), 4.98 (s, 2H, -CH2-), 4.22 (s, 2H, -CH2-), 4.09-4.14 (m, 2H, -CH2-), 3.62 (s, 6H, N-CH3), 3.48-3.52 (m, 2H, -CH2-), 2.41 (s, 2H, -CH2-), 1.89 (s, 2H, -CH2-), 1.83 (s, 3H, -CH3), 1.67 (s, 3H, CH3), 1.37 (d, J = 6.8 Hz, 6H, -CH3), 1.26 (d, J = 6.0 Hz, 6H, -CH3).

[0152] Example 20 Compound 17

[0153] Compound 17 was synthesized using the method described in Example 4. The physical and chemical properties of compound 17 are as follows:

[0154] 1) light yellow liquid;

[0155] 2) the nuclear magnetic resonance spectrum of the compound 1 H / 13 C NMR, 400 MHz) characteristics:

[0156] in CDC13 solvent, wherein each peak is assigned: 1 H NMR (400 MHz, CDC13) δ: 7.60 (d, J = 9.6 Hz, 1H, -CH=CH-), 7.27 (d, J = 8.8 Hz, 1H, -Ph), 6.85 (d, J = 8.8 Hz, 1H, -Ph), 6.22 (d, J = 8.8 Hz, 1H, -CH=CH-), 5.15 (t, J = 6.4 Hz, 1H, -CH=C(CH3)2), 4.91 (s, 2H, -CH2-), 4.14 (t, J = 6.0 Hz, 2H, -CH2-), 4.03 (s, 2H, -CH2-), 3.55 (s, 6H, N-CH3), 3.46-3.51 (m, 4H, -CH2-), 1.97-2.02 (m, 4H, -CH2-), 1.83 (s, 3H, -CH3), 1.65 (s, 3H, CH3), 1.36 (d, J = 6.8 Hz, 6H, -CH3), 1.25 (d, J = 6.4 Hz, 6H, -CH3); HRMS (ESI) C 28H 43 N2O4[M-Br] + calcd = 471.3217; found = 471.3234.

[0157] Example 21 Compound 18

[0158] Compound 18 was synthesized using the method described in Example 4, and the physical and chemical properties of compound 18 are as follows:

[0159] 1) light yellow liquid;

[0160] 2) the nuclear magnetic resonance spectrum of the compound (400MHz) is characterized by: 1 H / 13 C NMR, 400MHz) is characterized by:

[0161] In CDCl3, each peak is assigned as follows: 1 H NMR (400MHz CDCl3) δ: 7.60 (d, J = 9.6 Hz, 1H, -CH=CH-), 7.27 (d, J = 7.2 Hz, 1H, -Ph), 6.81 (d, J = 8.4 Hz, 1H, -Ph), 6.22 (d, J = 9.6 Hz, 1H, -CH=CH-), 5.18 (t, J = 7.2 Hz, 1H, -CH=C(CH3)2), 4.92 (s, 2H, -CH2-), 4.08 (s, 2H, -CH2-), 3.89 (s, 2H, -CH2-), 3.56 (s, 6H, N-CH3), 3.50 (d, J = 7.2 Hz, 2H, -CH2-), 3.43-3.48 (m, 2H, -CH2-), 1.93 (s, 2H, -CH2-), 1.83 (s, 3H, -CH3), 1.66 (s, 3H, CH3), 1.62 (s, 2H, -CH2-), 1.40 (s, 2H, -CH2-), 1.36 (d, J = 6.8 Hz, 6H, -CH3), 1.27 (s, 6H, -CH3); 13 CNMR (100MHz CDCl3) δ: 161.7, 161.3, 159.3, 152.8, 143.7, 132.4, 126.3, 121.2, 117.8, 113.0, 108.2, 68.0, 64.7, 62.4, 52.6, 48.8, 46.7, 28.7, 25.9, 22.9, 22.0, 21.0, 20.3, 18.1; HRMS (ESI) C 29 H 45 N2O4[M-Br] + calcd = 485.3374; found = 485.3392.

[0162] Example 22 Compound 19

[0163] Compound 19 was synthesized by the method described in Example 4, and the physicochemical properties of compound 19 are as follows:

[0164] 1) light yellow liquid;

[0165] 2) the nuclear magnetic resonance spectrum of the compound (H NMR, 400MHz) is characterized by: 1 H / 13 C NMR, 400MHz) is characterized by:

[0166] In CDCl3, each peak is assigned as follows: 1 H NMR (400MHz CDCl3) δ: 8.83 (s, 1H, -NH-), 7.61 (d, J = 9.6 Hz, 1H, -CH=CH-), 7.29 (d, J = 8.4 Hz, 1H, -Ph), 6.79 (d, J = 8.4 Hz, 1H, -Ph), 6.25 (d, J = 9.6 Hz, 1H, -CH=CH-), 5.12 (t, J = 6.4 Hz, 1H, -CH=C(CH3)2), 4.73 (s, 2H, -CH2-), 4.22 (s, 2H, -CH2-), 3.90 (s, 2H, -CH2-), 3.50 (d, J = 6.8 Hz, 2H, -CH2-), 3.46 (s, 6H, N-CH3), 3.25 (s, 2H, -CH2-), 2.48 (s, 2H, -CH2-), 1.83 (s, 3H, -CH3), 1.67 (s, 3H, CH3), 1.56-1.60 (m, 2H, -CH2-), 1.30 (s, 4H, -CH2-), 0.86 (t, J = 6.4 Hz, 3H, -CH3); 13 C NMR (100MHz CDCl3) δ: 162.3, 161.0, 158.2, 152.8, 143.6, 132.8, 126.5, 121.3, 117.8, 113.7, 108.0, 64.9, 64.2, 63.0, 52.5, 39.9, 29.2, 28.6, 25.9, 23.7, 22.1, 22.0, 18.2, 13.9; HRMS (ESI) C 26 H 39 N2O4[M-Br] + calcd = 443.2904; found = 443.2911.

[0167] Example 23 Compound 20

[0168] Compound 20 was synthesized by the method described in Example 4, and the physicochemical properties of compound 20 are as follows:

[0169] 1) pale yellow liquid;

[0170] 2) the nuclear magnetic resonance spectrum of the compound 1 H / 13 C NMR, 400MHz) characteristics:

[0171] In CDCl3as solvent, where each peak is assigned: 1 H NMR (400MHz CDCl3) δ: 8.82 (s, 1H, -NH-), 7.60 (d, J = 9.2 Hz, 1H, -CH=CH-), 7.28 (d, J = 8.8 Hz, 1H, -Ph), 6.82 (d, J = 8.4 Hz, 1H, -Ph), 6.23 (d, J = 9.6 Hz, 1H, -CH=CH-), 5.14 (t, J = 6.8 Hz, 1H, -CH=C(CH3)2), 4.70 (s, 2H, -CH2-), 4.15 (s, 2H, -CH2-), 3.79 (s, 2H, -CH2-), 3.50 (d, J = 6.8 Hz, 2H, -CH2-), 3.41 (s, 6H, N-CH3), 3.26 (s, 2H, -CH2-), 2.10 (s, 2H, -CH2-), 1.97 (s, 2H, -CH2-), 1.83 (s, 3H, -CH3), 1.65 (s, 3H, CH3), 1.58 (s, 2H, -CH2-), 1.31 (s, 4H, -CH2-), 0.86 (t, J = 6.4 Hz, 3H, -CH3); 13 C NMR (100MHz CDCl3) δ: 162.4, 161.1, 158.9, 152.8, 143.6, 132.7, 126.4, 121.2, 117.8, 113.3, 108.2, 67.3, 65.8, 63.4, 51.9, 39.9, 29.1, 28.6, 26.1, 25.8, 22.2, 22.1, 20.0, 18.1, 14.0; HRMS (ESI) C 27 H 41 N2O4[M-Br] + calcd = 457.3061; found = 457.3069.

[0172] Compound 21 of Example 24

[0173] Compound 21 was synthesized by the method described in Example 4, and the physical and chemical properties of compound 21 are as follows:

[0174] 1) pale yellow liquid;

[0175] 2), the nuclear magnetic resonance spectrum of the compound 1 H / 13 C NMR, 400 MHz) characteristics:

[0176] In CDCl3as solvent, each peak is assigned as follows: 1 H NMR (400 MHz CDCl3) δ: 8.87 (s, 1H, -NH-), 7.60 (d, J = 9.6 Hz, 1H, -CH=CH-), 7.26 (d, J = 9.2 Hz, 1H, -Ph), 6.82 (d, J = 8.4 Hz, 1H, -Ph), 6.22 (d, J = 9.6 Hz, 1H, -CH=CH-), 5.17 (t, J = 6.8 Hz, 1H, -CH=C(CH3)2), 4.68 (s, 2H, -CH2-), 4.08 (t, J = 6.0 Hz, 2H, -CH2-), 3.64-3.69 (m, 2H, -CH2-), 3.50 (d, J = 6.8 Hz, 2H, -CH2-), 3.40 (s, 6H, N-CH3), 3.23-3.28 (m, 2H, -CH2-), 1.93-1.97 (m, 4H, -CH2-), 1.83 (s, 3H, -CH3), 1.66 (s, 3H, CH3), 1.56-1.63 (m, 4H, -CH2-), 1.30-1.32 (m, 4H, -CH2-), 0.86 (t, J = 6.4 Hz, 3H, -CH3); 13 C NMR (100 MHz CDCl3) δ: 162.4, 161.2, 159.2, 152.8, 143.7, 132.4, 126.3, 121.2, 117.8, 113.0, 108.2, 67.8, 66.1, 63.2, 52.1, 39.9, 29.1, 28.6, 28.5, 25.8, 22.9, 22.7, 22.2, 22.0, 18.0, 13.9. HRMS (ESI) C 28 H 43 N2O4[M-Br] + calcd = 471.3217; found = 471.3220.

[0177] Example 25 Compound 22

[0178] Compound 22 was synthesized by the method described in Example 4, and the physical and chemical properties of compound 22 are as follows:

[0179] 1), light yellow liquid;

[0180] 2), the nuclear magnetic resonance spectrum of the compound 1 H / 13C NMR, 400 MHz) characteristics:

[0181] In CDCl3as solvent, where each peak is assigned: 1 H NMR (400 MHz, CDCl3) δ: 7.60 (d, J = 9.6 Hz, 1H, -CH=CH-), 7.28 (d, J = 8.4 Hz, 1H, -Ph), 6.80 (d, J = 8.8 Hz, 1H, -Ph), 6.24 (d, J = 9.6 Hz, 1H, -CH=CH-), 5.15 (t, J = 7.2 Hz, 1H, -CH=C(CH3)2), 4.93 (s, 2H, -CH2-), 4.15-4.22 (m, 4H, -CH2-), 3.63 (s, 6H, N-CH3), 3.50 (d, J = 7.2 Hz, 2H, -CH2-), 3.42 (t, J = 7.6H, 2H, -CH2-), 3.27 (t, J = 7.6H, 2H, -CH2-), 2.40 (s, 2H, -CH2-), 1.83 (s, 3H, -CH3), 1.67 (s, 3H, CH3), 1.56-1.60 (m, 2H, -CH2-), 1.48-1.52 (m, 2H, -CH2-), 1.37-1.43 (m, 2H, -CH2-), 1.29-1.33 (m, 2H, -CH2-), 0.94 (t, J = 7.6 Hz, 3H, -CH3), 0.90 (t, J = 7.6 Hz, 3H, -CH3); HRMS (ESI) C 29 H 45 N2O4[M-Br] + calcd = 485.3374; found = 485.3385.

[0182] Compound 23 of Example 26

[0183] Compound 23 was synthesized using the method described in Example 4, and the physical and chemical properties of compound 23 are as follows:

[0184] 1) light yellow liquid;

[0185] 2) the nuclear magnetic resonance spectrum of the compound 1 H / 13 C NMR, 400 MHz) characteristics:

[0186] In CDCl3as solvent, where each peak is assigned: 1H NMR (400 MHz CDCl3) δ: 7.60 (d, J = 9.6 Hz, 1H, -CH=CH-), 7.27 (d, J = 11.2 Hz, 1H, -Ph), 6.85 (d, J = 8.4 Hz, 1H, -Ph), 6.22 (d, J = 9.6 Hz, 1H, -CH=CH-), 5.16 (t, J = 6.8 Hz, 1H, -CH=C(CH3)2), 4.86 (s, 2H, -CH2-), 4.13-4.16 (m, 2H, -CH2-), 4.03 (s, 2H, -CH2-), 3.58 (s, 6H, N-CH3), 3.49 (d, J = 7.2 Hz, 2H, -CH2-), 3.39 (t, J = 8.0 Hz, 2H, -CH2-), 3.25 (t, J = 7.6 Hz, 2H, -CH2-), 1.96-2.04 (s, 4H, -CH2-), 1.82 (s, 3H, -CH3), 1.65 (s, 3H, CH3), 1.57-1.60 (m, 2H, -CH2-), 1.48-1.51 (m, 2H, -CH2-), 1.35-1.39 (m, 2H, -CH2-), 1.28-1.32 (m, 2H, -CH2-), 0.93 (t, J = 7.2 Hz, 3H, -CH3), 0.90 (t, J = 4.4 Hz, 3H, -CH3); HRMS (ESI) C 30 H 47 N2O4[M-Br] + calcd = 499.3530; found = 499.3542.

[0187] Example 27 Compound 24

[0188] Compound 24 was synthesized by the method described in Example 4, and the physical and chemical properties of compound 24 were as follows:

[0189] 1) light yellow liquid;

[0190] 2) the nuclear magnetic resonance spectrum of the compound (H 1 H / 13 C NMR, 400 MHz) characteristics:

[0191] In CDCl3solvent, each peak is assigned: 1H NMR (400 MHz CDCh) δ: 7.60 (d, J = 9.6 Hz, 1H, -CH=CH-), 7.27 (d, J = 6.8 Hz, 1H, -Ph), 6.82 (d, J = 8.8 Hz, 1H, -Ph), 6.22 (d, J = 9.2 Hz, 1H, -CH=CH-), 5.18 (t, J = 6.8 Hz, 1H, -CH=C(CH3)2), 4.84 (s, 2H, -CH2-), 4.08 (s, 2H, -CH2-), 3.90 (s, 2H, -CH2-), 3.58 (s, 6H, N-CH3), 3.50 (d, J = 7.2 Hz, 2H, -CH2-), 3.42 (s, 2H, -CH3), 3.26 (t, J = 7.2 Hz, 2H, -CH2-), 1.93 (s, 2H, -CH2-), 1.88 (s, 2H, -CH2-), 1.83 (s, 3H, -CH3), 1.66 (s, 3H, CH3), 1.60 (s, 4H, -CH2-), 1.48-1.52 (m, 2H, -CH2-), 1.40 (s, 2H, -CH2-), 1.29-1.33 (m, 2H, -CH2-), 0.90-0.96 (m, 6H, -CH3); 13 C NMR (100 MHz CDCh) δ: 162.6, 161.3, 159.3, 152.8, 143.8, 132.4, 126.3, 121.3, 117.8, 113.0, 108.2, 68.0, 64.9, 61.5, 52.8, 47.8, 46.4, 31.0, 29.5, 28.6, 25.8, 22.9, 22.0, 20.2, 20.1, 18.0, 13.9, 13.7; HRMS (ESI) C 31 H 49 N2O4[M-Br] + calcd = 513.3687; found = 513.3693.

[0192] Compound 25 of Example 28

[0193] Compound 25 was synthesized using the method described in Example 4, and the physical and chemical properties of compound 25 are as follows:

[0194] 1) light yellow liquid;

[0195] 2) the nuclear magnetic resonance spectrum (H / 1 H / 13 C NMR, 400 MHz) characteristics of this compound are:

[0196] In CDCh solvent, each peak is assigned as follows: 1H NMR (600 MHz CDCl3) δ: 8.77 (s, 1H, -NH-), 7.61 (d, J = 9.6 Hz, 1H, -CH=CH-), 7.29 (d, J = 8.4 Hz, 1H, -Ph), 6.80 (d, J = 8.4 Hz, 1H, -Ph), 6.25 (d, J = 9.6 Hz, 1H, -CH=CH-), 5.13 (t, J = 4.8 Hz, 1H, -CH=C(CH3)2), 4.69 (s, 2H, -CH2-), 4.22 (t, J = 3.2 Hz, 2H, -CH2-), 3.90-3.93 (m, 2H, -CH2-), 3.50 (d, J = 6.8 Hz, 2H, -CH2-), 3.45 (s, 6H, N-CH3), 3.25 (t, J = 4.8 Hz, 2H, -CH2-), 2.48 (s, 2H, -CH2-), 1.83 (s, 3H, -CH3), 1.67 (s, 3H, CH3), 1.55-1.58 (m, 2H, -CH2-), 1.31-1.33 (m, 2H, -CH2-), 1.26-1.28 (m, 4H, -CH2-), 0.85 (t, J = 4.8 Hz, 3H, -CH3); 13 C NMR (150 MHz CDCl3) δ: 162.4, 161.0, 158.3, 152.8, 143.6, 132.8, 126.5, 121.2, 117.9, 113.6, 108.1, 64.8, 64.0, 63.0, 52.6, 39.9, 31.3, 28.8, 26.6, 25.8, 23.5, 22.5, 22.0, 18.2, 14.0; HRMS (ESI) C 27 H 41 N2O4[M-Br] + calcd = 457.3061; found = 457.3068.

[0197] Compound 26 of Example 29

[0198] Compound 26 was synthesized by the method described in Example 4, and the physical and chemical properties of compound 26 were as follows:

[0199] 1) light yellow liquid;

[0200] 2) the nuclear magnetic resonance spectrum (H / 1 H / 13 C NMR, 400 MHz) characteristics of the compound:

[0201] In CDCl3as solvent, each peak is assigned as follows: 1H NMR(400MHz CDCl3)δ:8.84(s,1H,-NH-),7.60(d,J=9.6Hz,1H,-CH=CH-),7.28(d,J=8.8Hz,1H,-Ph),6.82(d,J=8.8Hz,1H,-Ph),6. 23(d,J=9.2Hz,1H,-CH=CH-),5.14(t,J=6.4Hz,1H,-CH=C(CH3)2),4.69(s,2H,-CH2-),4.15(s,2H,-CH2-),3.78(s,2H, -CH2-),3.50(d,J=6.8Hz,2H,-CH2-),3.41(s,6H,N-CH3),3.26(s,2H,-CH2-),2.10(s,2H,-CH2-),1.97(s,2H,-CH2-) ,1.83(s,3H,-CH3),1.65(s,3H,CH3),1.56-1.59(m,2H,-CH2-),1.26-1.31(m,6H,-CH2-),0.85(t,J=6.4Hz,3H,-CH3); 13 C NMR(100MHz CDCl3)δ:162.4,161.2,158.9,152.9,143.7,132.7,126.5,121.2,117.8,113.3,108.2,67.4, 66.0,63.5,51.9,40.1,31.3,28.9,26.7,26.1,25.8,22.5,22.1,20.0,18.1,14.0; HRMS(ESI)C 28 H 43 N₂O₄[M-Br] + calcd=471.3217; found=471.3230.

[0202] Example 30 Compound 27

[0203] Compound 27 was synthesized using the method described in Example 4. The physicochemical properties of compound 27 are as follows:

[0204] 1) A light yellow liquid;

[0205] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H / 13 C NMR (400MHz) characteristics:

[0206] Using CDCl3 as solvent, the peak assignments are as follows: 1H NMR (400 MHz CDCl3) δ: 8.84 (s, 1H, -NH-), 7.60 (d, J = 9.6 Hz, 1H, -CH=CH-), 7.27 (d, J = 9.6 Hz, 1H, -Ph), 6.82 (d, J = 8.4 Hz, 1H, -Ph), 6.22 (d, J = 9.6 Hz, 1H, -CH=CH-), 5.17 (t, J = 6.8 Hz, 1H, -CH=C(CH3)2), 4.67 (s, 2H, -CH2-), 4.08 (t, J = 5.6 Hz, 2H, -CH2-), 3.67 (s, 2H, -CH2-), 3.50 (d, J = 6.8 Hz, 2H, -CH2-), 3.41 (s, 6H, N-CH3), 3.25 (s, 2H, -CH2-), 1.95 (s, 4H, -CH2-), 1.83 (s, 3H, -CH3), 1.66 (s, 3H, CH3), 1.55-1.63 (m, 4H, -CH2-), 1.27 (s, 6H, -CH2-), 0.84 (t, J = 6.0 Hz, 3H, -CH3); 13 C NMR (100 MHz CDCl3) δ: 162.5, 161.3, 159.2, 152.9, 143.7, 132.5, 126.3, 121.2, 118.0, 113.1, 108.3, 67.8, 66.2, 63.3, 52.2, 39.9, 31.5, 29.1, 28.7, 26.8, 26.0, 23.0, 22.8, 22.6, 22.1, 18.1, 14.0; HRMS (ESI) C 29 H 45 N2O4[M-Br] + calcd = 485.3374; found = 485.3374.

[0207] Example 31 Compound 28

[0208] Compound 28 was synthesized by the method described in Example 4, and the physical and chemical properties of compound 28 are as follows:

[0209] 1) light yellow liquid;

[0210] 2) the nuclear magnetic resonance spectrum (H / 1 H / 13 C NMR, 400 MHz) characteristics of the compound:

[0211] In CDCl3as solvent, each peak is assigned: 1H NMR (400 MHz CDCl3) δ: 7.60 (d, J = 9.2 Hz, 1H, -CH=CH-), 7.27 (d, J = 10.0 Hz, 1H, -Ph), 6.80 (d, J = 8.8 Hz, 1H, -Ph), 6.24 (d, J = 9.6 Hz, 1H, -CH=CH-), 5.14 (t, J = 6.4 Hz, 1H, -CH=C(CH3)2), 4.92 (s, 2H, -CH2-), 4.21 (t, J = 5.2 Hz, 2H, -CH2-), 4.14-4.19 (m, 2H, -CH2-), 3.63 (s, 6H, N-CH3), 3.50 (d, J = 6.8 Hz, 2H, -CH2-), 3.35 (d, J = 7.6 Hz, 2H, -CH2-), 3.19 (d, J = 7.6 Hz, 2H, -CH2-), 2.41 (s, 2H, -CH2-), 1.95-2.00 (m, 2H, -CH2-), 1.83 (s, 3H, -CH3), 1.66 (s, 3H, CH3), 0.98 (d, J = 6.4 Hz, 6H, -CH3), 0.88 (d, J = 6.8 Hz, 6H, -CH3); HRMS (ESI) C 29 H 45 N2O4[M-Br] + calcd = 485.3374; found = 485.3386.

[0212] Example 32 Compound 29

[0213] Compound 29 was synthesized using the method described in Example 4. The physical and chemical properties of compound 29 are as follows:

[0214] 1) light yellow liquid;

[0215] 2) the nuclear magnetic resonance spectrum (H / 1 H / 13 C NMR, 400 MHz) characteristics of the compound:

[0216] in CDCl3, where each peak is assigned: 1H NMR (400 MHz CDCl3) δ: 7.60 (d, J = 9.2 Hz, 1 H, -CH=CH-), 7.28 (d, J = 8.4 Hz, 1 H, -Ph), 6.84 (d, J = 8.8 Hz, 1 H, -Ph), 6.23 (d, J = 9.2 Hz, 1 H, -CH=CH-), 5.15 (t, J = 6.8 Hz, 1 H, -CH=C(CH3)2), 4.87 (s, 2 H, -CH2-), 4.14 (s, 2 H, -CH2-), 4.03 (s, 2 H, -CH2-), 3.59 (s, 6 H, N-CH3), 3.49 (d, J = 7.2 Hz, 2 H, -CH2-), 3.34 (d, J = 7.6 Hz, 2 H, -CH2-), 3.18 (d, J = 7.6 Hz, 2 H, -CH2-), 1.95-2.00 (m, 6 H, -CH2-), 1.82 (s, 3 H, -CH3), 1.65 (s, 3 H, CH3), 0.96 (d, J = 6.4 Hz, 6 H, -CH3), 0.88 (d, J = 6.4 Hz, 6 H, -CH3); HRMS (ESI) C 30 H 47 N2O4[M-Br] + calcd = 499.3530; found = 499.3537.

[0217] Example 33 Compound 30

[0218] Compound 30 was synthesized using the method described in Example 4. The physical and chemical properties of compound 30 are as follows:

[0219] 1) light yellow liquid;

[0220] 2) the nuclear magnetic resonance spectrum (H 1 H / 13 C NMR, 400 MHz) characteristics of the compound:

[0221] in CDCl3, where each peak is assigned: 1H NMR (400 MHz, CDC13) δ: 7.60 (d, J = 9.2 Hz, 1H, -CH=CH-), 7.27 (d, J = 7.2 Hz, 1H, -Ph), 6.81 (d, J = 8.4 Hz, 1H, -Ph), 6.22 (d, J = 9.6 Hz, 1H, -CH=CH-), 5.18 (t, J = 6.8 Hz, 1H, -CH=C(CH3)2), 4.88 (s, 2H, -CH2-), 4.07 (s, 2H, -CH2-), 3.90 (s, 2H, -CH2-), 3.60 (s, 6H, N-CH3), 3.50 (d, J = 7.2 Hz, 2H, -CH2-), 3.35 (s, 2H, -CH3), 3.19-3.23 (m, 2H, -CH2-), 1.93-2.00 (m, 4H, -CH2-), 1.88 (s, 2H, -CH2-), 1.83 (s, 3H, -CH3), 1.66 (s, 3H, CH3), 1.61 (s, 2H, -CH2-), 0.97 (d, J = 5.6 Hz, 6H, -CH3), 0.88 (d, J = 6.4 Hz, 6H, -CH3); 13 C NMR (100 MHz, CDC13) δ: 163.8, 161.5, 159.6, 152.9, 143.9, 132.5, 126.4, 121.3, 117.8, 113.2, 108.3, 68.1, 65.0, 61.4, 54.8, 53.4, 52.8, 28.6, 27.8, 26.4, 25.8, 22.9, 22.0, 20.1, 19.9, 18.0; HRMS (ESI) C 31 H 49 N2O4[M-Br] + calcd = 513.3687; found = 513.3701.

[0222] Application Example 1: In vitro antibacterial activity determination

[0223] 1. Test bacteria:

[0224] Staphylococcus aureus ATCC 29213; Escherichia coli ATCC 25922; Methicillin-resistant Staphylococcus aureus (MRSA).

[0225] 2. Samples and reagents:

[0226] The samples are: osthole, vancomycin, meropenem and compounds 1-30 prepared in the examples.

[0227] 3. Test method:

[0228] According to the Clinical and Laboratory Standards Institute (CLSI) guidelines, the in vitro antibacterial activities of the compounds 1-30 and the clinical antibacterial drug vancomycin were tested by the broth microdilution method using 96-well microtiter plates. The MIC was defined as the lowest concentration of the drug that produced complete growth inhibition of the test organism.

[0229] Table 1. MICs of the compounds 1-30 against 10 clinical isolates of MRSA

[0230]

[0231]

[0232] a S.a.: Staphylococcus aureus ATCC 29213; b M11-23: 10 clinical isolates of MRSA; c E.c.: Escherichia coli ATCC 25922; d SI: Selectivity index (HC 50 / MICs of S.aureus); e ND: not determined. f Van: vancomycin; k MEM: meropenem. The experiments were repeated at least 3 times.

[0233] As shown in Table 1, the compounds 1-30 have good antibacterial activities against MRSA. The compounds 2, 5-8, 13-16, 18, 21, 23 and 24 have better antibacterial activities than the parent compound, and the MICs of the compounds 6 and 13 are 1-2 μg / mL, which are close to the MIC of the positive drug vancomycin. The compounds 6 and 13 have stable antibacterial activities against 10 clinical isolates of MRSA, and thus can be used as clinical antibacterial drugs against MRSA.

[0234] Example 2: Time-kill kinetics experiment of the compound 27

[0235] 1. Test bacteria:

[0236] Staphylococcus aureus (ATCC 29213); MRSA-16 (clinical isolate).

[0237] 2. Samples and reagents:

[0238] The sample is: vancomycin and the compound 27 prepared in the example.

[0239] 3. Test method:

[0240] A single colony of Staphylococcus aureus drug-resistant strain MRSA-16 was picked into 1 mL of LB liquid medium and cultured in a shaker (200 rpm, 37°C) for 16-18 h. Then the bacterial solution was diluted 10000 times with LB liquid medium and cultured for 2.5 h, and the bacterial solution was diluted to 1x10 5 CFU / mL. Then different concentrations of compound 27 (4 MIC, 8xMIC) were added to the bacterial solution, and vancomycin (8xMIC) was used as a positive control, and a blank group without drug was set. The shaker was placed in a shaker (200 rpm, 37°C) and continued to be cultured, and 100 μL of each group was taken at 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h after drug addition, centrifuged at 3500 rpm, 4°C for 3 min, the supernatant was removed, washed three times, resuspended with 100 μL of 1xPBS buffer solution, diluted by 2 times, and plate counting was performed, three parallel controls were set for each group, and overnight culture was performed in a 37°C constant temperature incubator, and the next day the number of colonies was counted, and the unit log 10 CFU / mL was plotted, and the results are shown in Figure 1 A and B.

[0241] Figure 1 A and B show that compound 27 completely kills Staphylococcus aureus ATCC29213 and MRSA-3 within 1-2 h under the action of 8xMIC, and has a stronger bactericidal effect than the positive control drug vancomycin under the same concentration. In addition, under the condition of 4xMIC, the number of colonies of Staphylococcus aureus ATCC29213 and MRSA-16 also gradually decreases with the extension of time, showing a good inhibitory effect. The results show that the amphiphilic osthole-quaternary ammonium salt hybrid 27 has a strong bactericidal effect on Staphylococcus aureus ATCC29213 and MRSA-3, and has a dose-dependent effect, and is expected to develop into a clinical rapid antibacterial drug.

[0242] Example 3: Drug resistance induction experiment of compound 27:

[0243] First, the MIC values of compound 27 and the positive control norfloxacin were determined, and then single S. aureus colonies were placed in MHB broth (1.0 mL) containing sub-inhibitory concentrations (1 / 2 MIC) of compound 27 and norfloxacin, and incubated on a shaker (200 rpm, 37 °C) for 12 hours. Then, the bacteria were inoculated onto fresh MHA plates containing sub-inhibitory concentrations (1 / 2 MIC) of compound 27 and norfloxacin, and incubated again at 37 °C for 24 hours. When S. aureus formed single colonies, the above steps were repeated for more than 20 generations, and the MIC value of compound 27 for each generation was recorded using the CLSI broth dilution method, and the results are shown in Table 1. Figure 1 C.

[0244] From Figure 1 The results show that compound 27 did not show any resistance when S. aureus was grown for 20 generations, while the positive control drug norfloxacin showed obvious resistance when the MIC value increased from 1 to 128 μg / mL after S. aureus was grown for 17 generations.

[0245] Example 4: In vivo safety evaluation experiment

[0246] 1. Reagents:

[0247] Compound 27 prepared in the examples, 0.9% NaCl.

[0248] 2. Test animals

[0249] SPF KM mice (purchased from Beijing Sbielfu Biotechnology Co., Ltd., body weight 19-22 g, 4-6 weeks old).

[0250] 3. Test method

[0251] Thirty mice were divided into groups of five each, namely Control group (0.9% NaCl), 13 (5 mg / kg), 27 (10 mg / kg), 27 (20 mg / kg), 27 (40 mg / kg), and 27 (80 mg / kg). The mice were shaved on the back, and then 60 μL of 0.9% NaCl and different concentrations of compound 27 were injected, respectively. After 24 hours, the mice were observed for any abnormalities (such as redness, hardness, ulceration, etc.) on the skin. The mice in the group with the maximum dose that did not cause adverse reactions were sacrificed, and their blood was taken for routine blood tests and blood biochemical index tests to evaluate whether compound 27 had in vivo toxicity to mice, and the results are shown in Table 2. Figure 2

[0252] ​The mice were injected subcutaneously with different concentrations of the compound, and it was found that when the concentration of compound 27 was ≤20 mg / kg, the mice did not show any adverse reactions (such as redness, hardness, ulceration, etc.) on the skin, so routine blood tests and blood biochemical index tests were performed on the mice at this dosage. Figure 2 The blood samples were subjected to routine blood tests and blood biochemical analysis, and the test items included white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), and platelet count (PLT). The statistical results showed that there was no significant difference in the results compared with the Control group (0.9% NaCl) after subcutaneous injection of compound 27 (20 mg / kg) in KM mice. At the same time, by collecting serum for blood biochemical tests, including albumin (ALB), urea (UREA) and creatinine (CREA), there was no significant difference in the corresponding indicators of liver and kidney function in mice compared with the Control group (0.9% NaCl) after subcutaneous injection of compound 27 (20 mg / kg) in KM mice, indicating that compound 27 has certain in vivo safety.

[0253] Example 5: In vivo anti-MRSA infection activity experiment of compound 27

[0254] 1. Test bacteria:

[0255] MRSA-16 (clinical isolate)

[0256] 2. Samples and reagents:

[0257] The samples were: vancomycin and compound 27 prepared in the examples, 0.9% NaCl.

[0258] 3. Test animals:

[0259] SPF KM mice (purchased from Beijing Sbielfu Biotechnology Co., Ltd., body weight 19-22 g, 4-6 weeks old).

[0260] 4. Test method:

[0261] Thirty KM mice were taken, and their backs were depilated. The mice were divided into groups of 6, namely the blank group (no injection of MRSA-16 bacterial solution, no injection of drug solution), the model group (only injection of MRSA-16 bacterial solution), compound 27 (10 mg / kg), compound 27 (5 mg / kg), and positive control vancomycin (5 mg / kg). The mice were anesthetized, and MRSA-3 bacterial solution (60 μL, 6×10 8CFU / mL), 2h after infection, 60μL of different concentrations of compounds, positive drug vancomycin and 0.9% NaCl were injected into the skin infection site respectively, 24h after administration, the mice were sacrificed by dislocation, then the infected skin of mice was aseptically separated, grinded, and counted by a counting plate, the bacterial load of the skin of mice was read at 24h, and the results are shown in Figure 3

[0262] The results show that compared with the model group, the number of MRSA cells in the skin of mice treated with compound 27 and vancomycin is significantly reduced (P<0.0001), and the treatment effect of compound 27 (10mg / kg) is the most significant (P<0.0001), and the bacterial load of the tissue is reduced by about 4.458log 10 CFU / g; while the bacterial load of the tissue of compound 27 (5mg / kg) and vancomycin is reduced by about 2.962log 10 CFU / g and 1.922log 10 CFU / g, showing a moderate antibacterial effect, and the antibacterial effect of compound 27 is slightly better than that of vancomycin (P<0.05). Therefore, it is confirmed that the amphiphilic cuminum-quaternary ammonium salt hybrid 27 has a good treatment effect on the skin abscess of MRSA infected mice, which is better than the control drug vancomycin, and is expected to be developed into an anti-MRSA drug.​

Claims

1. An amphiphilic osthol-quaternary ammonium salt hybrid, the structure of which is shown in formula (I): in, n = 3, 4, or 5, R is Among them, R 1 and R 2 It is independently selected from H or C1-C8 alkyl groups.

2. The amphiphilic osthol-quaternary ammonium salt hybrid according to claim 1, characterized in that, The R 1 and R 2 It is independently selected from H or C2-C6 alkyl groups.

3. The amphiphilic osthol-quaternary ammonium salt hybrid according to claim 1, characterized in that, The n,R 1 and R 2 It is one of the following combinations: (1) n =3, R 1 = H, R 2 = -CH2CH3;(2) n = 4, R 1 = H, R 2 = -CH2CH3; (3) n = 5, R 1 = H, R2= -CH2CH3;(4) n = 3, R 1 = R 2 = -CH2CH3; (5) n = 4, R 1 = R 2 = -CH2CH3;(6) n = 5, R 1 = R 2 = -CH2CH3; (7) n = 3, R 1 = H, R 2 = -(CH2)2CH3;(8) n = 4,R 1 = H, R 2 = -(CH2)2CH3; (9) n = 5, R 1 = H, R 2 = -(CH2)2CH3;(10) n = 3,R 1 = R 2 = -(CH2)2CH3; (11) n = 4, R 1 =R 2 = -(CH2)2CH3;(12) n = 5, R 1 =R 2 = -(CH2)2CH3; (13) n = 3, R 1 = H, R 2 = -(CH2)3CH3;(14) n = 4, R 1 = H, R 2 = -(CH2)3CH3; (15) n = 5, R 1 = H, R 2 = -(CH2)3CH3;(16) n = 3, R 1 = R 2 = -CH(CH3)2; (17) n = 4, R 1 =R 2 = -CH(CH3)2;(18) n = 5, R 1 =R 2 = -CH(CH3)2; (19) n = 3, R 1 = H, R2= -(CH2)4CH3; (20) n = 4, R 1 = H, R2= -(CH2)4CH3; (21) n = 5, R 1 = H, R2= -(CH2)4CH3;(22) n = 3, R 1 = R 2 = -(CH2)3CH3; (23) n = 4, R 1 =R 2 = -(CH2)3CH3;(24) n = 5, R 1 =R 2 = -(CH2)3CH3; (25)n=3,R 1 =H,R2=-(CH2)5CH3;(26)n=4,R 1 =H,R2=-(CH2)5CH3; (27) n = 5, R 1 = H, R2= -(CH2)5CH3;(28) n = 3, R 1 = R 2 = -CH2CH(CH3)2; (29) n = 4, R 1 =R 2 = -CH2CH(CH3)2;(30) n = 5, R 1 =R 2 = -CH2CH(CH3)2。 4. The method for preparing the amphiphilic osthol-quaternary ammonium salt hybrid according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Using osthol as a substrate, its methoxy group is converted into a phenolic hydroxyl group under the action of boron tribromide to obtain intermediate a; (2) Intermediate a reacts with different dibromoalkanes under alkaline conditions to synthesize intermediate b; (3) Intermediate b then reacts with small molecule peptide mimic c to generate the amphiphilic osthol-quaternary ammonium salt hybrid, as shown in the following reaction formula: Wherein, R and n are as described in any one of claims 1-3.

5. The method for preparing the amphiphilic osthol-quaternary ammonium salt hybrid according to claim 4, characterized in that, In step (1), the molar ratio of osthol to boron tribromide is 1:1 to 1:5, the reaction temperature is -40 to 0℃, and the reaction solvent is anhydrous dichloromethane.

6. The method for preparing the amphiphilic osthol-quaternary ammonium salt hybrid according to claim 4, characterized in that, In step (2), the base in the alkaline conditions is K2CO3, the molar ratio of intermediate a to the base is 1:1-1:3, the molar ratio of intermediate a to dibromoalkanes is 1:1.5-1:3, the reaction temperature is 45-60℃, and the reaction solvent is anhydrous acetone.

7. The method for preparing the amphiphilic osthol-quaternary ammonium salt hybrid according to claim 4, characterized in that, In step (3), the preparation method of intermediate c includes the following steps: amine RH reacts with bromoacetyl bromide under alkaline conditions to generate bromoacetamide, and then bromoacetamide reacts with dimethylamine under alkaline conditions to generate small molecule peptide mimic c: Wherein, R is as described in any one of claims 1-3.

8. The method for preparing the amphiphilic osthol-quaternary ammonium salt hybrid according to claim 4, characterized in that, In step (3), the molar ratio of intermediate b to intermediate c is 1:1.5-1:3, the reaction temperature is 80-90℃, and the reaction solvent is anhydrous ethanol.

9. The use of the amphiphilic osthol-quaternary ammonium salt hybrid according to any one of claims 1-3 in the preparation of antibacterial drugs.

Images