Isoflavone amine copolymers, their preparation methods and antibacterial applications

Abstract

This invention discloses a series of isoflavulol amine copolymers, their preparation methods, and their antibacterial applications. This series of compounds uses fulvulin as the parent compound, converting fulvulin into isoflavulin. Then, isoflavulin, as the lipophilic moiety, is linked to various aliphatic amines and heterocyclic amines via alkyl linkages of different lengths, synthesizing a series of isoflavulol amine copolymers. Further structural optimization can be performed by introducing methyl groups at the nitrogen atom site to form nitrogen cations. The isoflavulol amine copolymers prepared by this invention exhibit good antibacterial effects against Staphylococcus aureus ATCC 29213 and various methicillin-resistant Staphylococcus aureus (MRSA), and possess advantages such as good water solubility, low biotoxicity, and high yield, showing promise for further development into semi-synthetic antibacterial drugs for clinical use.

Description

Technical Field

[0001] This invention belongs to the technical field of isoflavone derivatives, specifically relating to isoflavone amine copolymers, their preparation methods, and antibacterial applications. Background Technology

[0002] Hops (Humulus lupulus L.), also known as snake flower or hops, are perennial climbing herbaceous plants belonging to the genus Humulus in the family Moraceae. They have bitter and aromatic properties and are mainly used in the brewing industry. C. Beer constituents as potential cancer chemopreventive agents[J]. European Journal of Cancer, 2005, 41(13): 1941-1954). It has been reported that hop extracts, such as polyphenols and acyl glucose, also have antioxidant, estrogenic, sedative and potential cancer chemopreventive activities (Gerhauser C, Alt A, Heiss E, et al. Cancer chemopreventive activity of Xanthohumol, a natural product derived from hop[J]. Molecular Cancer Therapeutics, 2002, 1(11): 959-969). Hops contain abundant flavonoids, among which xanthohumol (XN) and isoxanthohumol (IXN) have attracted attention due to their good biological activity (Biendl M, Methner FJ, Stettner G, et al. Brewing trials with axanthohumol-enriched hop product[J]. Brauwelt International, 2004(3):182-185).

[0003] Flavourol is a flavonoid containing an isoprenyl group derived from hops. It belongs to the chalcone family and accounts for only 0.1% to 1% of the dry weight of hops. Its molecular formula is C2. 21 H 22O5, with a molecular weight of 354.4. In 1931, Power et al. first discovered fulvicol in hops and successfully isolated and named it (Power FB, Tutin F, Rogerson H. The constituents of hops[J]. Chemical Society. 1913, 103: 1267-1292). Flavfulvicol has various biological activities, such as effective prevention of cancer, anti-oxidation, antiviral, prevention and treatment of diabetes, and anti-atherosclerosis. During the brewing process of beer, xanthohumol is converted into its isomer, isoflavone, which reduces its biological activity. Therefore, the content of xanthohumol in commercial beers around the world is very low (the highest in traditional pale beer is 0.15 mg / L), while the content of isoflavone is very high (0.04-3.44 mg / L). Studies have shown that isoflavone also has a variety of biological activities such as anti-tumor, anti-inflammatory and lipid metabolism regulation (Wunderlich S, Zürcher A, Back W. Enrichment of xanthohumol in the brewing process[J].Molecular Nutrition&Food Research,2005,49(9):874-881).

[0004] Yang et al. studied the effects of xanthohumol and isoxanthohumol on apoptosis and adipogenesis in 3T3L1 cells. In this study, adipocytes were treated with several concentrations of xanthohumol and isoxanthohumol. Both xanthohumol and isoxanthohumol reduced the survival rate of adipocytes and increased apoptosis and the production of reactive oxygen species. During the differentiation phase, both xanthohumol and isoxanthohumol reduced adipogenesis (Yang JY, Della-Fera MA, Rayalam S, et al. Effect of xanthohumol and isoxanthohumol on 3T3-L1 cell apoptosis and adipogenesis[J].Apoptosis,2007,12(11):1953-1963.). Active species (such as hydroxyl groups, hydrogen peroxide, and hydrogen peroxide) can cause oxidative modification of low-density lipoprotein (LDL), which is usually associated with the development of atherosclerosis. Similar studies by Stevens et al. have shown that flavonol and isoflavone can also inhibit hydrogen peroxide-mediated LDL oxidation at low micromolar concentrations (Stevens JF, Miranda CL, Frei B, et al. Inhibition of peroxynitrite-mediated LDL oxidation by prenylated flavonoids: the α,β-unsaturated keto functionality of 2'-hydroxychalcones as a novel antioxidant pharmacophore[J]. Chemical Research in Toxicology, 2003, 16(10): 1277-1286.). Xanthohumol exhibits broad inhibitory activity in various cancer cell lines, such as breast, colon, ovarian, and prostate cancer cell lines [90-92]. Xanthohumol shows broad inhibitory effects in the initiation stage (reactive molecules, DNA damage), promotion stage (mutation, cell structure changes), and progression stage (uncontrolled cell growth, tumor, metastasis) of carcinogenesis (Colgate EC, Miranda CL, Stevens JF, et al. Xanthohumol, apoptosis and NF-kappaB activation in prostate epithelial cells[J]. Cancer Letters, 2007, 246(1-2): 201-209).Currently, there is limited research on the structural modification and antibacterial activity of isoflavones, and its water solubility is also poor. Summary of the Invention

[0005] Purpose of the invention: To address the above-mentioned technical problems, this invention provides a method for preparing and applying isoflavone amine copolymers. The isoflavone amine copolymers have good antibacterial effects against Staphylococcus aureus ATCC 29213 and various clinically isolated Gram-positive bacteria such as MRSA, and solve the problem of poor water solubility.

[0006] Technical Solution: To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0007] An isoflavone amine copolymer or its quaternary ammonium salt, wherein the structure of the isoflavone amine copolymer is shown below:

[0008]

[0009] Where n = 3, 4, or 5, R 1 R 2 Selected independently Among them, R 3 and R 4 Independently selected from C1-C6 alkyl groups, R 5 R 6 Independently selected from H or C1-C3 alkyl groups, A is selected from atoms C, N, S or O.

[0010] As a preferred embodiment, n = 3, 4, or 5, R 3 R 4 Independently selected from C1-C4 alkyl groups, R 5 R 6 It is independently selected from H or methyl, and A is selected from atoms C, N, S or O.

[0011] As a preferred option, R 1 =R 2 And all selected from When n,R 3 and R 4 They are:

[0012] (1) n = 3, R 3 = R 4 = -(CH2)2CH3(2) n = 3, R 3 = R 4 = -(CH2)3CH3

[0013] (3) n = 3, R 3 = R 4= -CH2CH(CH3)2(4) n = 4, R 3 = R 4 = -(CH2)2CH3

[0014] (5) n = 4, R 3 = R 4 = -(CH2)3CH3 (6) n = 4, R 3 = R 4 = -CH2CH(CH3)2

[0015] (7) n = 5, R 3 = R 4 = -(CH2)2CH3(8) n = 5, R 3 = R 4 = -(CH2)3CH3

[0016] (9) n = 5, R 3 = R 4 = -CH2CH(CH3)2;

[0017] R 1 =R 2 And all selected from When, n, R 1 and R 2 They are:

[0018] (10) n = 3,

[0019] (12) n = 3,

[0020] (14) n = 3,

[0021] (16) n = 3,

[0022] (18) n = 4,

[0023] (20) n = 4,

[0024] (22) n = 4,

[0025] (24) n = 5,

[0026] (26) n = 5,

[0027] (28) n = 5,

[0028] (30) n = 5,

[0029] The above selections represent compounds 1-30 in the following examples.

[0030] As a specific implementation, the quaternary ammonium salt is generated by reacting isoflavone alcohol amine copolymers with haloalkanes and methylating their nitrogen atoms.

[0031] Preferably, the quaternary ammonium salt is selected from the following compounds:

[0032]

[0033] Where n = 4 (compound 31) or 5 (compound 32).

[0034] The aforementioned quaternary ammonium salts are produced by reacting isoflavone amine copolymers 5 and 8 with iodomethane to methylate their nitrogen atoms, thereby generating isoflavone amine copolymers 31-32.

[0035]

[0036] Preferably, the molar ratio of the isoflavone amine copolymers 5 and 8 to the iodomethane base is 1:9-1:10, the reaction temperature is 45-60℃, and the reaction solvent is acetonitrile.

[0037] The present invention also provides a method for preparing the aforementioned isoflavone amine copolymer, comprising the following steps:

[0038] Using fulvicol as a substrate, it was converted to its isomer, isoflavol, under alkaline conditions. Then, the phenolic hydroxyl groups at both ends of isoflavol were introduced into bromoalkane to synthesize intermediate a1-3. Subsequently, intermediate a1-3 was reacted with different aliphatic amines and nitrogen-containing heterocyclic amines under alkaline conditions to generate a series of isoflavol amine copolymers.

[0039]

[0040] Among them, R 1 R 2 R 3 R 4 R 5 R 6 The same applies to n and A as described above.

[0041] As a specific implementation scheme, the preparation method of isoflavol is as follows: using fulvol as a substrate, it is reacted under low-temperature alkaline conditions to generate its isomer, isoflavol:

[0042]

[0043] Preferably, the alkali in the alkaline conditions is selected from NaOH, the reaction temperature is 0±2℃, and the reaction solvent is NaOH solution.

[0044] As a specific implementation scheme, the intermediate a1-3 is prepared as follows: isoflavone alcohol is reacted with dibromoalkane under alkaline conditions to generate intermediate a1-3:

[0045]

[0046] Preferably, the base in the alkaline conditions is K2CO3, the molar ratio of isoflavone to the base is 1:4-1:5, the molar ratio of isoflavone to dibromoalkane is 1:4-1:5, the reaction temperature is 45-60℃, and the reaction solvent is acetone.

[0047] As a specific implementation scheme, the intermediate a1-3 reacts with different aliphatic amines and nitrogen-containing heterocyclic amines under alkaline conditions, wherein the base in the alkaline conditions is K2CO3, the molar ratio of intermediate a1-3 to the base is 1:4-1:5, the molar ratio of intermediate a1-3 to different aliphatic amines and nitrogen-containing heterocyclic amines is 1:4-1:5, the reaction temperature is 45-60℃, and the reaction solvent is acetonitrile.

[0048] Finally, this invention provides the use of the aforementioned isoflavone amine copolymers or their quaternary ammonium salts in the preparation of antibacterial drugs. Preferably, their use is in the preparation of drugs effective against Staphylococcus aureus ATCC 29213 and various methicillin-resistant Staphylococcus aureus (MRSA).

[0049] This invention relates to isoflavone amine copolymers designed by mimicking the structure and function of antimicrobial peptides. Their antimicrobial target is the bacterial cell membrane. Evaluation of the antimicrobial activity of all target compounds revealed that the vast majority of isoflavone amine copolymers exhibited strong antimicrobial activity against Staphylococcus aureus (S. aureus) ATCC 29213 and clinically isolated methicillin-resistant Staphylococcus aureus (MRSA). Notably, isoflavone amine copolymers with nitrogen atom methylation showed even stronger antimicrobial effects against MRSA, with MIC values ​​reaching 0.25 μg / mL against multiple MRSA strains, demonstrating superior antimicrobial efficacy compared to the positive control drug vancomycin. All synthesized isoflavone amine copolymers exhibited low hemolysis rates against sheep erythrocytes and possessed good water solubility and stability; therefore, these compounds have broad clinical application prospects.

[0050] Technical effects: The isoflavone amine copolymer prepared by this invention has good antibacterial effect against Staphylococcus aureus ATCC 29213 and various methicillin-resistant Staphylococcus aureus (MRSA), and has the advantages of good water solubility, low biotoxicity and high yield. It is expected to be further developed into a semi-synthetic antibacterial drug for clinical use. Attached Figure Description

[0051] Figure 1 The dynamic bactericidal curve of compound 32 is shown.

[0052] Figure 2 The in vivo blood routine and blood biochemical indices of compound 32.

[0053] Figure 3 The change in bacterial load on mouse skin of compound 32.

[0054] Figure 4 The image shows the 1H-NMR spectrum of compound 32.

[0055] Figure 5 The image shows the 13C-NMR spectrum of compound 32. Detailed Implementation

[0056] The invention will be further illustrated in detail below through examples.

[0057] Example 1: Preparation of isoflavone

[0058] 100 mg (0.282 mmol) of xanthohumol was weighed and placed in a round-bottom flask. 100 mL of 1% NaOH solution was added, and the mixture was stirred at 0 °C for 4 h. The reaction was monitored by TLC until it was complete. The mixture was then acidified with 50% H2SO4, and a large amount of precipitate was formed. The precipitate was purified by column chromatography to obtain the product isofanthohumol, with a yield of 90%.

[0059] Example 2 Preparation of intermediate a1-3

[0060] Weigh 100 mg (0.282 mmol) of isoflavone into a round-bottom flask, add 5 mL of acetone, stir to dissolve, add 1,3-dibromopropane (285 mg, 1.412 mmol) or 1,4-dibromobutane (487 mg, 1.412 mmol) or 1,5-dibromopentane (325 mg, 1.412 mmol), then add 195 mg (1.412 mmol) of K2CO3, stir the reaction at 50 °C, and monitor the reaction until the end of the reaction by TLC. Filter to remove K2CO3, concentrate the filtrate under reduced pressure, and separate by column chromatography (petroleum ether: acetone = 5:1) to obtain the pale yellow product a1-3, with a yield of 41-55%.

[0061] Example 3 Preparation of Compound 1

[0062] Intermediate a (0.1 mmol) was placed in a round-bottom flask and dissolved in anhydrous acetonitrile. The corresponding amine (0.5 mmol) was then added, followed by K2CO3 (0.5 mmol). The mixture was stirred at 50 °C and monitored by TLC until the reaction was complete. K2CO3 was removed by filtration, and the filtrate was concentrated under reduced pressure and separated by PTLC (dichloromethane:methanol = 8:1) to obtain the pure target compound.

[0063] The physicochemical properties of compound 1 are as follows:

[0064] 1) Brown liquid;

[0065] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (400MHz) characteristics:

[0066] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 37.6%, 1 H NMR(400MHz CDCl3)δ:7.34(d,J=8.8Hz,2H,-Ph),6.89(d,J=8.4Hz,2H,-Ph),6.10(s,1H,-Ph),5.30(dd,J=12.8,2.8Hz,1H,-CH=),5 .13(t,J=6.8Hz,1H,-CH-),4.09(t,J=6.0Hz,2H,-CH2-),4.02(t,J=6.0Hz,2H,-CH2-),3.90(s,3H,-OCH3),3.25(d,J=6. 8Hz,2H,-CH2-),2.94-3.01(m,1H,-CH2-),2.77(dd,J=16.4,2.8Hz,1H,-CH2-),2.65-2.71(m,4H,-CH2-),2.42-2.50(m, 8H,-CH2-),1.97-2.00(m,4H,-CH2-),1.61(d,J=11.6Hz,6H,-CH3),1.45-1.56(m,8H,-CH2-),0.86-0.91(m,12H,-CH3); 13 C NMR(100MHz CDCl3)δ:190.2,162.7,161.1,160.6,158.9,131.0,127.5,122.7,114.4,110.1,105.9,89. 3,78.4,66.5,66.1,56.1,50.6,50.5,45.6,29.6,25.7,21.9,17.9,11.87,11.8; HRMS(ESI)C 39 H 60 N₂O₅[M+H]+ calcd=637.4580; found=637.4579.

[0067] Example 4 Compound 2

[0068] Compound 2 was synthesized using the method described in Example 3. The physicochemical properties of compound 2 are as follows:

[0069] 1) Brown liquid;

[0070] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (400MHz) characteristics

[0071] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 39.4%, 1 H NMR(400MHz CDCl3)δ:7.34(d,J=8.4Hz,2H,-Ph),6.90(d,J=8.8Hz,2H,-Ph),6.10(s,1H,-Ph),5.30(dd,J=12.8,2.8Hz,1H,-CH=),5.13(t, J=7.2Hz,1H,-CH-),4.08(t,J=6.0Hz,2H,-CH2-),4.01(t,J=6.0Hz,2H,-CH2-),3.90(s,3H,-OCH3),3.26(d,J=6.8Hz,2H,-CH2 -),2.94-3.01(m,1H,-CH2-),2.76(dd,J=16.4,3.2Hz,1H,-CH2-),2.62-2.67(m,4H,-CH2-),2.43-2.50(m,8H,-CH2-),1.94-1 .99(m,4H,-CH2-),1.62(d,J=10.4Hz,6H,-CH3),1.40-1.47(m,8H,-CH2-),1.27-1.33(m,8H,-CH2-),0.87-0.91(m,12H,-CH3). 13 C NMR(100MHz CDCl3)δ:190.3,162.7,161.1,160.6,159.0,131.2,131.0,127.4,122.7,114.4,110.1,105.9,89.3, 78.3,66.5,66.1,56.0,53.9,53.7,50.4,45.5,28.9,25.8,21.9,20.69,20.6,17.8,14.0; HRMS(ESI)C 43 H 68 N₂O₅[M+H] +calcd=693.5206; found=693.5207.

[0072] Example 5 Compound 3

[0073] Compound 3 was synthesized using the method described in Example 3. The physicochemical properties of compound 3 are as follows:

[0074] 1) Brown liquid;

[0075] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (400MHz) characteristics:

[0076] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 43.2%. 1 H NMR(400MHz CDCl3)δ:7.34(d,J=8.4Hz,2H,-Ph),6.89(d,J=8.4Hz,2H,-Ph),6.10(s,1H,-Ph),5.30(dd,J=12.8,2.4Hz,1H,-CH=) ,5.13-5.16(m,1H,-CH-),4.10(t,J=6.0Hz,2H,-CH2-),4.03(t,J=6.4Hz,2H,-CH2-),3.90(s,3H,-OCH3),3.25(d,J= 6.8Hz,2H,-CH2-),2.94-3.02(m,1H,-CH2-),2.77(dd,J=16.4,2.8Hz,1H,-CH2-),2.49(d,J=6.4Hz,4H,-CH2-),2.06 (d,J=6.4Hz,8H,-CH2-),1.88(d,J=5.6Hz,4H,-CH2-),1.61-1.68(m,10H,-CH-,-CH3),0.84(d,J=6.4Hz,24H,-CH3); 13 C NMR(100MHz CDCl3)δ:190.4,162.9,161.1,160.6,159.0,131.2,131.0,127.4,122.7,114.5,110. 0,89.1,78.3,66.5,64.0,56.0,51.5,45.5,38.7,25.8,21.9,20.9,17.8; HRMS(ESI)C 43 H 68 N₂O₅[M+H] + calcd=693.5206; found=693.5216.

[0077] Example 6 Compound 4

[0078] Compound 4 was synthesized using the method described in Example 3. The physicochemical properties of compound 4 are as follows:

[0079] 1) Yellow liquid;

[0080] 3) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (400MHz) characteristics:

[0081] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 52.7%. 1 H NMR(400MHz CDC13)δ:7.34(d,J=8.8Hz,2H,-Ph),6.88(d,J=8.8Hz,2H,-Ph),6.09(s,1H,-Ph),5.31(dd,J=12 .4,2.8Hz,1H,-CH=),5.10(t,J=7.2Hz,1H,-CH-),4.08-4.11(m,2H,-CH2-),4.00(t,J=5.6Hz,2H, -CH2-),3.91(s,3H,-OCH3),3.24(d,J=7.2Hz,2H,-CH2-),2.77-2.98(m,14H,-CH2-),1.84-1.91 (m,8H,-CH2-),1.67-1.77(m,8H,-CH2-),1.60(d,J=16.8Hz,6H,-CH3),0.93-0.99(m,12H,-CH3); 13 C NMR(100MHz CDCl3)δ:190.3,162.2,161.1,160.6,158.6,131.5,131.1,127.5,122.5,114.4,109.9,106.1,89 .3,78.3,67.3,66.9,56.0,53.9,52.3,52.1,45.5,25.9,21.9,17.8,17.1,17.0,11.3; HRMS(ESI)C 41 H 64 N₂O₅[M+H] + calcd=665.4893; found=665.4891.

[0082] Example 7 Compound 5

[0083] Compound 5 was synthesized using the method described in Example 3. The physicochemical properties of compound 5 are as follows:

[0084] 1) Yellow liquid;

[0085] 2) The nuclear magnetic resonance spectrum of this compound ( 1H NMR (400MHz) characteristics:

[0086] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 52.4%. 1 H NMR(400MHz CDCl3)δ:7.34(d,J=8.4Hz,2H,-Ph),6.88(d,J=8.8Hz,2H,-Ph),6.09(s,1H,-Ph),5.31(d d,J=12.8,3.2Hz,1H,-CH=),5.12(t,J=7.2Hz,1H,-CH-),4.05(t,J=6.0Hz,2H,-CH2-),3. 99(t,J=5.2Hz,2H,-CH2-),3.91(s,3H,-OCH3),3.25(d,J=7.2Hz,2H,-CH2-),2.94-3.01( m,1H,-CH2-),2.62-2.82(m,13H,-CH2-),1.77-1.88(m,8H,-CH2-),1.52-1.64(m,14H,-CH 3, -CH2-),1.31(q,J=7.2Hz,8H,-CH2-),0.90-0.95(m,12H,-CH3); 13 C NMR(100MHz CDCl3)δ:190.2,162.2,161.1,160.6,158.6,131.6,131.1,127.5,122.5,114.4,110.0,106.2,89.2,78. 3,67.5,66.8,56.1,52.1,52.0,45.5,28.9,25.8,25.6,25.5,21.9,20.2,20.24,17.8,13.6; HRMS(ESI)C 45 H 72 N₂O₅[M+H] + calcd=721.5519; found=721.5517.

[0087] Example 8 Compound 6

[0088] Compound 6 was synthesized using the method described in Example 3. The physicochemical properties of compound 6 are as follows:

[0089] 1) Brown liquid;

[0090] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (400MHz) characteristics:

[0091] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 41.9%.1 H NMR(400MHz CDC13)δ:7.34(d,J=8.4Hz,2H,-Ph),6.89(d,J=8.8Hz,2H,-Ph),6.09(s,1H,-Ph),5.30(dd,J=12.8,3.2Hz,1H,-CH =),5.14(t,J=6.8Hz,1H,-CH-),4.03(t,J=6.0Hz,2H,-CH2-),3.97(t,J=6.0Hz,2H,-CH2-),3.90(s,3H,-OCH3),3.2 6(d,J=7.2Hz,2H,-CH2-),2.94-3.01(m,1H,-CH2-),2.77(dd,J=16.4,2.8Hz,1H,-CH2-),2.39(s,4H,-CH2-),1.98- 2.23(m,8H,-CH2-),1.79-1.87(m,6H,-CH2-),1.62-1.69(m,12H,-CH-,-CH2-,-CH3),0.88(d,J=6.4Hz,24H,-CH3); 13 CNMR(100MHz CDCl3)δ:190.2,162.7,161.1,160.6,159.0,131.4,131.0,127.4,122.7,114.5,110.2,105.0, 89.3,78.4,68.0,64.0,56.1,54.9,45.7,29.4,25.8,22.7,21.9,21.0,20.9,17.8; HRMS(ESI)C 45 H 72 N₂O₅[M+H] + calcd=721.5519; found=721.5522.

[0092] Example 9 Compound 7

[0093] Compound 7 was synthesized using the method described in Example 3. The physicochemical properties of compound 7 are as follows:

[0094] 1) Yellow liquid;

[0095] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (600MHz) characteristics:

[0096] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 46.4%. 1H NMR(600MHz CDCl3)δ:7.34(d,J=9Hz,2H,-Ph),6.88(d,J=8.4Hz,2H,-Ph),6.08(s,1H,-Ph),5.31(dd,J=12.6,2.4Hz,1H,- CH=),5.11-5.14(m,1H,-CH-),4.03(t,J=6.6Hz,2H,-CH2-),3.96(t,J=6.6Hz,2H,-CH2-),3.91(s,3H,-OCH3) ,3.24(d,J=7.2Hz,2H,-CH2-),2.94-2.99(m,1H,-CH2-),2.88-2.90(m,2H,-CH2-),2.77-2.85(m,11H,-CH2-) ,1.71-1.88(m,16H,-CH2-),1.60(d,J=21.0Hz,6H,-CH3),1.51-1.55(m,4H,-CH2-),0.95-0.97(m,12H,-CH3); 13 C NMR(150MHz CDCl3)δ:190.1,162.5,161.1,160.6,158.9,131.3,131.0,127.4,122.7,114.4,110.1,106.0,89.3,78.3,67.7,67. 4,56.1,54.2,54.1,52.7,52.5,45.5,28.8,28.7,25.8,23.7,23.6,21.9,17.8,17.4,17.3,11.43,11.4; HRMS(ESI)C 43 H 68 N₂O₅[M+H] + calcd=639.5206; found=693.5212.

[0097] Example 10 Compound 8

[0098] Compound 8 was synthesized using the method described in Example 3. The physicochemical properties of compound 8 are as follows:

[0099] 1) Yellow liquid;

[0100] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (600MHz) characteristics:

[0101] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 51.3%. 1H NMR(600MHz CDCl3)δ:7.34(d,J=8.4Hz,2H,-Ph), 6.88(d,J=9.0Hz,2H,-Ph), 6.08(s,1H,-Ph), 5.30(dd,J=12.6,3.0Hz,1H,-CH=), 5.12-5.14(m,1H,-CH-),4.02(t,J=6.0Hz,2H,-CH2-),3.96(t,J=6.6Hz,2H,-CH2-),3.90(s,3H,-OCH3),3.24(d,J=6.6 Hz,2H,-CH2-),2.94-2.99(m,1H,-CH2-),2.74-2.80(m,13H,-CH2-),1.81-1.87(m,4H,-CH2-),1.71(d,J=7.2Hz,4H,- CH2-),1.58-1.64(m,14H,-CH2-,-CH3),1.49-1.53(m,4H,-CH2-),1.32-1.36(m,8H,-CH2-),0.92-0.95(m,12H,-CH3); 13 C NMR(150MHz CDCl3)δ:190.2,162.6,161.1,160.6,158.9,131.3,131.0,127.4,122.7,114.4,110.1,105.9,89.3,78.3,67.8,67.5,5 6.1,52.7,52.5,52.3,52.2,45.5,28.9,28.8,25.8,23.88,23.8,23.0,21.9,20.46,20.4,17.8,13.8,13.7; HRMS(ESI)C 47 H 76 N₂O₅[M+H] + calcd=749.5832; found=749.5842.

[0102] Example 11 Compound 9

[0103] Compound 9 was synthesized using the method described in Example 3. The physicochemical properties of compound 9 are as follows:

[0104] 1) Brown solid;

[0105] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (600MHz) characteristics:

[0106] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 36.8%. 1H NMR(600MHz CDCl3) δ: 7.34 (d, J = 9.0 Hz, 2H, - Ph), 6.89 ( d, J = 9.0 Hz, 2 H, - Ph), 6.09 ( s, 1 H, - Ph), 5.31 ( dd, J = 13.2, 3.0 Hz, 1 H, - CH =), 5.13 (t, J = 7. 2Hz,1H,-CH-),4.03(t,J=6.0Hz,2H,-CH2-),3.96(t,J=6.6Hz,2H,-CH2-),3.91(s,3H,-OCH3),3.26(d,J=3.0Hz,2H,-CH2-),3.09- 3.12(m,1H,-CH2-),2.95-3.00(m,2H,-CH2-),2.78(dd,J=16.2,3.0Hz,1H,-CH2-),2.30(s,10H,-CH2-),1.80-1.86(m,8H,-CH-,-C H2-),1.62(d,J=17.4Hz,8H,-CH2-,-CH3),1.50(d,J=6.6Hz,4H,-CH2-),1.39(t,J=7.2Hz,2H,-CH2-),0.96(d,J=7.2Hz,24H,-CH3); 13 CNMR(150MHz CDCl3)δ:190.2,162.7,161.1,160.6,159.0,131.2,131.0,127.4,122.7,114.4,110.2,105.9,89.3,78 .3,68.1,67.8,56.0,55.0,54.8,45.8,45.5,29.0,25.8,23.8,21.9,21.2,21.1,17.8,8.6; HRMS(ESI)C 47 H 76 N₂O₅[M+H] + calcd=749.5832; found=749.5837.

[0107] Example 12 Compound 10

[0108] Compound 10 was synthesized using the method described in Example 3. The physicochemical properties of compound 10 are as follows:

[0109] 1) Orange liquid;

[0110] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (400MHz) characteristics:

[0111] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 51.6%. 1H NMR(400MHz CDCl3)δ:7.34(d,J=8.4Hz,2H,-Ph),6.90(d,J=8.4Hz,2H,-Ph),6.10(s,1H,-Ph),5.31-5.35(m,1H,-CH=),5. 12(t,J=6.8Hz,1H,-CH-),4.09(t,J=6.4Hz,2H,-CH2-),4.03(t,J=6.4Hz,2H,-CH2-),3.91(s,3H,-OCH3),3.7 2-3.75(m,8H,-CH2-),3.25(d,J=6.8Hz,2H,-CH2-),2.94-3.01(m,1H,-CH2-),2.77(dd,J=16.4,3.2Hz,1H,-C H2-),2.54(t,J=7.2Hz,4H,-CH2-),2.49(s,8H,-CH2-),1.96-2.05(m,4H,-CH2-),1.62(d,J=8.8Hz,6H,-CH3); 13 C NMR(100MHzCDCl3)δ:190.2,162.5,161.1,160.6,158.8,131.4,131.1,127.4,122.7,114.4,11 0.1,106.0,89.3,78.3,66.5,66.3,65.8,55.5,53.5,53.4,45.5,25.8,21.9,17.9.HRMS(ESI)C 35 H 48 N₂O₇[M+H] + calcd=609.3540; found=609.3545.

[0112] Example 13 Compound 11

[0113] Compound 11 was synthesized using the method described in Example 3. The physicochemical properties of compound 11 are as follows:

[0114] 1) Brown liquid;

[0115] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (400MHz) characteristics:

[0116] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 55.4%. 1H NMR(400MHz CDC13)δ:7.34(d,J=8.4Hz,2H,-Ph),6.89(d,J=8.8Hz,2H,-Ph),6.09(s,1H,-Ph),5.31(dd ,J=12.4,2.4Hz,1H,-CH=),5.11(t,J=7.2Hz,1H,-CH-),4.08(t,J=6.0Hz,2H,-CH2-),4.01( t,J=6.4Hz,2H,-CH2-),3.91(s,3H,-OCH3),3.24(d,J=6.8Hz,2H,-CH2-),2.94-3.01(m,1H, -CH2-),2.60-2.82(m,21H,-CH2-),2.00-2.03(m,4H,-CH2-),1.62(d,J=10.8Hz,6H,-CH3); 13 C NMR(100MHz CDCl3)δ:190.1,162.5,161.1,160.6,158.9,131.4,131.1,127.5,122.7,114.5,110.2,106.0, 89.3,78.3,66.3,65.9,56.1,55.8,55.0,54.9,45.6,27.4,27.5,25.8,21.9,17.8; HRMS(ESI)C 35 H 48 N₂O₅S₂[M+H] + calcd=641.3083; found=641.3084.

[0117] Example 14 Compound 12

[0118] Compound 12 was synthesized using the method described in Example 3. The physicochemical properties of compound 12 are as follows:

[0119] 1) Brown liquid;

[0120] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (400MHz) characteristics:

[0121] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 40.7%, 1H NMR(400MHz CDCl3)δ:7.34(d,J=8.8Hz,2H,-Ph),6.90(d,J=8.4Hz,2H,-Ph),6.10(s,1H,-Ph),5.30(dd,J=12.8,2.8Hz,1 H,-CH=),5.13(t,J=6.8Hz,1H,-CH-),4.07(t,J=6.4Hz,2H,-CH2-),4.01(t,J=6.4Hz,2H,-CH2-),3.91(s,3H, -OCH3),3.25(d,J=7.2Hz,2H,-CH2-),2.94-3.01(m,1H,-CH2-),2.76(dd,J=16.4,2.8Hz,1H,-CH2-),2.44-2 .55(m,12H,-CH2-),1.98-2.06(m,4H,-CH2-),1.58-1.64(m,14H,-CH2-,-CH3),1.45(d,J=3.6Hz,4H,-CH2-); 13 CNMR(150MHzCDCl3)δ:190.2,162.7,161.1,160.6,159.0,131.3,131.0,127.4,122.7,114.5,110.2,105.9,89.3, 78.4,66.8,66.4,55.9,54.6,54.5,45.6,29.6,26.6,26.5,25.85,25.8,25.6,24.28,24.2,21.9,17.8; HRMS(ESI)C 37 H 52 N₂O₅[M+H] + calcd=605.3954; found=605.3959.

[0122] Example 15 Compound 13

[0123] Compound 13 was synthesized using the method described in Example 3. The physicochemical properties of compound 13 are as follows:

[0124] 1) Yellow liquid;

[0125] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (400MHz) characteristics:

[0126] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 38.1%. 1H NMR(400MHz CDC13)δ:7.34(d,J=8.4Hz,2H,-Ph),6.89(d,J=8.4Hz,2H,-Ph),6.10(s,1H,-Ph),5.29(d,J=16.4Hz,1H,-CH=) ,5.14(s,1H,-CH-),4.07(t,J=5.6Hz,2H,-CH2-),4.01(d,J=4.0Hz,2H,-CH2-),3.91(s,3H,-OCH3),3.25(d,J=6 .4Hz,2H,-CH2-),2.94-3.01(m,5H,-CH2-),2.77-2.81(m,1H,-CH2-),2.24-2.64(m,6H,-CH-,-CH2-),2.01-2.0 4(m,4H,-CH2-),1.61-1.67(m,14H,-CH2-,-CH3),1.33-1.41(m,4H,-CH2-),1.10(dd,J=18.0,6.0Hz,6H,-CH3); 13 C NMR(100MHzCDCl3)δ:190.2,162.5,161.1,160.6,158.8,131.5,131.1,127.5,122.6,114.5,110.2,106 .0,89.3,78.4,66.6,66.1,56.1,52.0,50.4,45.5,29.4,26.2,25.8,25.2,24.7,21.9,17.8; HRMS(ESI)C 39 H 56 N₂O₅[M+H] + calcd=633.4267; found=633.4265.

[0127] Example 16 Compound 14

[0128] Compound 14 was synthesized using the method described in Example 3. The physicochemical properties of compound 14 are as follows:

[0129] 1) Brown liquid;

[0130] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (600MHz) characteristics:

[0131] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 39.7%, 1H NMR(600MHz CDCl3)δ:7.34(d,J=8.4Hz,2H,-Ph),6.90(d,J=9.0Hz,2H,-Ph),6.10(s,1H,-Ph),5.31(dd,J=12.6,2.4Hz,1H,-CH=) ,5.14-5.16(m,1H,-CH-),4.08(t,J=6.0Hz,2H,-CH2-),4.01(t,J=6.0Hz,2H,-CH2-),3.91(s,3H,-OCH3),3.23-3.29( m,2H,-CH2-),2.84-3.00(m,5H,-CH2-),2.77(dd,J=16.8,3.0Hz,1H,-CH2-),2.51-2.53(m,4H,-CH2-),2.01-2.05(m, 4H,-CH2-),1.86-1.89(m,3H,-CH-,-CH2-),1.57-1.73(m,17H,-CH2-,-CH3,-CH-),0.86(dd,J=6.6,2.4Hz,6H,-CH3); 13 C NMR(100MHz CDCl3)δ:190.2,162.7,161.1,160.6,159.0,131.3,131.0,127.4,122.7,114.5,110.2,105.9,89.3,78.3,66.8,66.4,62.1,61. 9,56.0,55.69,55.6,54.1,54.0,45.5,32.9,32.8,31.0,30.9,26.6,26.5,25.8,25.4,25.3,21.9,19.75,19.7,17.9; HRMS(ESI)C 39 H 56 N₂O₅[M+H] + calcd=633.4267; found=633.4268.

[0132] Example 17 Compound 15

[0133] Compound 15 was synthesized using the method described in Example 3. The physicochemical properties of compound 15 are as follows:

[0134] 1) Brown liquid;

[0135] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (400MHz) characteristics:

[0136] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 37.2%, 1H NMR(400MHz CDCl3)δ:7.34(d,J=8.4Hz,2H,-Ph),6.89(d,J=8.8Hz,2H,-Ph),6.10(s,1H,-Ph),5.30(dd,J=12.4,2.8Hz,1H,- CH=),5.12(t,J=7.2Hz,1H,-CH-),4.08(t,J=6.0Hz,2H,-CH2-),4.02(t,J=6.4Hz,2H,-CH2-),3.91(s,3H,-OCH3) ,3.24(d,J=6.8Hz,2H,-CH2-),2.97-3.05(m,5H,-CH2-),2.76-2.81(m,1H,-CH2-),2.57-2.65(m,4H,-CH2-),2.0 0-2.10(m,8H,-CH2-),1.62-1.67(m,10H,-CH-,-CH2-,-CH3),1.31-1.42(m,6H,-CH2-),0.93-0.95(m,6H,-CH3); 13 C NMR(100MHz CDCl3)δ:190.2,162.6,161.1,160.7,158.9,131.4,131.0,127.4,122.7,114.5,110.0,106.0,89.3,78.3,66 .7,66.1,56.1,55.5,54.0,53.8,45.5,33.7,33.3,30.5,30.3,26.5,26.2,25.8,21.9,21.7,17.9; HRMS(ESI)C 39 H 56 N₂O₅[M+H] + calcd=633.4267; found=633.4272.

[0137] Example 18 Compound 16

[0138] Compound 16 was synthesized using the method described in Example 3. The physicochemical properties of compound 16 are as follows:

[0139] 1) Orange liquid;

[0140] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (400MHz) characteristics:

[0141] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 64.6%. 1H NMR(400MHz CDCl3)δ:7.34(d,J=8.4Hz,2H,-Ph), 6.90(d,J=8.8Hz,2H,-Ph), 6.10(s,1H,-Ph), 5.30(dd,J=12.4,2.8Hz, 1H,-CH=),5.12(t,J=6.8Hz,1H,-CH-),4.08(t,J=6.0Hz,2H,-CH2-),4.01(t,J=6.4Hz,2H,-CH2-),3.91(s, 3H,-OCH3),3.25(d,J=7.2Hz,2H,-CH2-),2.94-3.01(m,1H,-CH2-),2.77(dd,J=16.4,3.2Hz,1H,-CH2-),2. 49-2.57(m,18H,-CH2-),2.30(s,8H,N-CH3,-CH2-),1.96-2.02(m,4H,-CH2-),1.62(d,J=8.8Hz,6H,-CH3); 13 C NMR(100MHz CDCl3)δ:190.3,162.6,161.1,160.6,159.0,131.2,131.1,127.4,122.6,114.4,110.2,105.9,89.3, 78.3,66.6,66.3,58.1,56.1,55.0,53.1,53.0,45.9,45.5,26.7,26.6,25.8,21.9,17.8; HRMS(ESI)C 37 H 54 N4O5[M+H] + calcd=635.4172; found=635.4175.

[0142] Example 19 Compound 17

[0143] Compound 17 was synthesized using the method described in Example 3. The physicochemical properties of compound 17 are as follows:

[0144] 1) Orange liquid;

[0145] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (400MHz) characteristics:

[0146] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 69.8%. 1H NMR(400MHz CDCl3)δ:7.34(d,J=8.4Hz,2H,-Ph),6.89(d,J=8.4Hz,2H,-Ph),6.08(s,1H,-Ph),5.31(dd,J=12.4,2.4Hz,1H,-C H=),5.12(t,J=6.8Hz,1H,-CH-),4.05(t,J=6.0Hz,2H,-CH2-),3.98(t,J=6.0Hz,2H,-CH2-),3.90(s,3H,-OCH3),3 .71-3.74(m,8H,-CH2-),3.25(d,J=7.2Hz,2H,-CH2-),2.94-3.01(m,1H,-CH2-),2.77(dd,J=16.4,2.8Hz,1H,-CH 2-),2.42-2.47(m,12H,-CH2-),1.79-1.88(m,4H,-CH2-),1.70-1.72(m,4H,-CH2-),1.61(d,J=10.8Hz,6H,-CH3); 13 C NMR(100MHz CDCl3)δ:190.2,162.6,161.1,160.6,159.0,131.2,131.0,127.4,122.6,114.4,110.1,105 .9,89.2,78.3,68.0,67.6,66.8,58.4,53.6,45.5,27.1,25.8,22.9,21.9,17.8; HRMS(ESI)C 37 H 52 N₂O₇[M+H] + calcd=637.3853; found=637.3854.

[0147] Example 20 Compound 18

[0148] Compound 18 was synthesized using the method described in Example 3. The physicochemical properties of compound 18 are as follows:

[0149] 1) Brown liquid;

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

[0151] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 59.4%. 1H NMR(400MHz CDC13)δ:7.34(d,J=8.4Hz,2H,-Ph),6.89(d,J=8.8Hz,2H,-Ph),6.08(s,1H,-Ph),5.31(dd,J=12.8,2.8 Hz,1H,-CH=),5.12(t,J=7.2Hz,1H,-CH-),4.04(t,J=6.4Hz,2H,-CH2-),3.97(t,J=6.4Hz,2H,-CH2-),3. 90(s,3H,-OCH3),3.25(d,J=7.2Hz,2H,-CH2-),2.94-3.01(m,1H,-CH2-),2.69-2.82(m,17H,-CH2-),2.4 3-2.47(m,4H,-CH2-),1.78-1.86(m,4H,-CH2-),1.68-1.73(m,4H,-CH2-),1.61(d,J=11.6Hz,6H,-CH3); 13 C NMR(100MHz CDCl3)δ:190.2,162.6,161.1,160.6,159.0,131.3,131.0,127.4,122.7,114.4,110.2,106 .0,89.3,78.3,68.0,67.6,58.7,56.1,54.9,45.5,27.8,27.1,25.8,21.9,17.8; HRMS(ESI)C 37 H 52 N₂O₅S₂[M+H] + calcd=669.3396; found=669.3397.

[0152] Example 21 Compound 19

[0153] Compound 19 was synthesized using the method described in Example 3. The physicochemical properties of compound 19 are as follows:

[0154] 1) Yellow liquid;

[0155] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (400MHz) characteristics:

[0156] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 33.7%. 1H NMR(400MHz CDCl3)δ:7.26(d,J=8.4Hz,2H,-Ph),6.80(d,J=8.4Hz,2H,-Ph),6.02(s,1H,-Ph),5.24 (dd,J=12.4,2.8Hz,1H,-CH=),5.02(t,J=7.2Hz,1H,-CH-),4.00(t,J=6.0Hz,2H,-CH2- ),3.93(t,J=6.0Hz,2H,-CH2-),3.84(s,3H,-OCH3),3.17(d,J=6.8Hz,2H,-CH2-),2.70 -2.93(m,14H,-CH2-),1.77-1.99(m,16H,-CH2-),1.53(d,J=14.4Hz,10H,-CH2-,-CH3); 13 CNMR(100MHz CDCl3)δ:190.0,162.3,161.1,160.6,158.6,131.6,131.1,127.5,122.7,114.5,110.0,106.1,89.3,78.3, 67.5,66.8,57.6,57.5,53.6,53.4,45.5,38.7,28.9,26.8,26.7,25.8,24.4,23.8,22.0,17.8; HRMS(ESI)C 39 H 56 N₂O₅[M+H] + calcd=633.4267; found=633.4272.

[0157] Example 22 Compound 20

[0158] Compound 20 was synthesized using the method described in Example 3. The physicochemical properties of compound 20 are as follows:

[0159] 1) Yellow liquid;

[0160] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (400MHz) characteristics:

[0161] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 39.4%. 1H NMR(400MHz CDCl3)δ:7.34(d,J=8.8Hz,2H,-Ph),6.89(d,J=8.4Hz,2H,-Ph),6.08(s,1H,-Ph),5.30(dd,J=12.4,2.8Hz,1H,-CH=),5 .13(t,J=7.2Hz,1H,-CH-),4.04(t,J=6.4Hz,2H,-CH2-),3.98(t,J=6.4Hz,2H,-CH2-),3.90(s,3H,-OCH3),3.26(d,J=6 .8Hz,2H,-CH2-),2.89-3.01(m,3H,-CH2-),2.77-2.82(m,3H,-CH2-),2.40-2.54(m,4H,-CH-,-CH2-),2.19-2.27(m,2H ,-CH2-),1.76-1.84(m,4H,-CH2-),1.62-1.71(m,16H,-CH2-,-CH3),1.28-1.42(m,6H,-CH2-),1.09-1.13(m,6H,-CH3); 13 C NMR(100MHz CDCl3)δ:190.2,162.6,161.2,160.6,158.9,131.3,131.0,127.4,122.6,114.5,110.2,105.9,89.3, 78.3,68.0,67.6,56.1,53.3,51.7,45.5,29.7,27.3,27.2,25.8,21.9,21.6,21.4,17.8; HRMS(ESI)C 41 H 60 N₂O₅[M+H] + calcd=661.4580; found=661.4578.

[0162] Example 23 Compound 21

[0163] Compound 21 was synthesized using the method described in Example 3. The physicochemical properties of compound 21 are as follows:

[0164] 1) Yellow liquid;

[0165] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (400MHz) characteristics:

[0166] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 32.1%. 1H NMR(400MHz CDCl3)δ:7.34(d,J=8.8Hz,2H,-Ph), 6.89(d,J=8.4Hz,2H,-Ph), 6.08(s,1H,-Ph), 5.30(dd,J=12.8, 2.8Hz,1H,-CH=),5.13(t,J=6.8Hz,1H,-CH-),4.04(t,J=6.4Hz,2H,-CH2-),3.97(t,J=6.4Hz,2H,-CH 2-),3.90(s,3H,-OCH3),3.26(d,J=6.8Hz,2H,-CH2-),2.76-3.01(m,6H,-CH2-),2.39-2.44(m,4H,- CH2-),1.77-1.88(m,6H,-CH2-),1.53-1.72(m,22H,-CH-,-CH2-,-CH3),0.85(q,J=3.2Hz,6H,-CH3); 13 C NMR(100MHz CDCl3)δ:190.3,162.7,161.1,160.6,159.0,131.2,131.0,127.4,122.7,114.4,110.2,105.9,89.3,78.4,68.2,67.8,61. 8,58.6,56.0,53.9,45.5,32.9,32.8,30.9,30.8,27.34,27.3,25.8,25.3,25.2,23.2,23.1,21.9,19.7,17.8; HRMS(ESI)C 41 H 60 N₂O₅[M+H] + calcd=661.4580; found=661.4581.

[0167] Example 24 Compound 22

[0168] Compound 22 was synthesized using the method described in Example 3. The physicochemical properties of compound 22 are as follows:

[0169] 1) Brown liquid;

[0170] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (400MHz) characteristics:

[0171] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 42.8%. 1H NMR(400MHz CDCl3)δ:7.33(d,J=8.8Hz,2H,-Ph),6.88(d,J=8.8Hz,2H,-Ph),6.08(s,1H,-Ph),5.30(dd,J=12.8,2.8Hz,1H,-CH=),5.1 3(t,J=7.2Hz,1H,-CH-),4.04(t,J=6.4Hz,2H,-CH2-),3.97(t,J=6.4Hz,2H,-CH2-),3.90(s,3H,-OCH3),3.25(d,J=6.8Hz, 2H,-CH2-),2.92-3.01(m,5H,-CH2-),2.76(dd,J=16.4,3.2Hz,1H,-CH2-),2.41-2.47(m,4H,-CH2-),1.94-2.04(m,4H,-CH 2-),1.71-1.86(m,8H,-CH2-),1.61-1.66(m,10H,-CH-,-CH2-,-CH3),1.27-1.37(m,6H,-CH2-),0.92-0.94(m,4H,-CH2-); 13 C NMR(100MHz CDCl3)δ:190.3,162.7,161.1,160.6,159.0,131.2,131.0,127.4,122.6,114.4,110.1,105.9,89.3,78.4,68.2, 67.6,58.5,56.1,53.86,53.8,45.5,33.8,33.6,30.6,30.5,27.3,25.8,23.2,23.1,21.9,21.7,17.8; HRMS(ESI)C 41 H 60 N₂O₅[M+H] + calcd=611.4580; found=661.4588.

[0172] Example 25 Compound 23

[0173] Compound 23 was synthesized using the method described in Example 3. The physicochemical properties of compound 23 are as follows:

[0174] 1) Brown liquid;

[0175] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (400MHz) characteristics:

[0176] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 59.3%. 1H NMR(400MHz CDC13)δ:7.34(d,J=8.8Hz,2H,-Ph),6.89(d,J=8.8Hz,2H,-Ph),6.08(s,1H,-Ph),5.30(dd,J=12.8,2.8Hz,1H,-CH =),5.12(t,J=7.2Hz,1H,-CH-),4.04(t,J=6.0Hz,2H,-CH2-),3.97(t,J=6.4Hz,2H,-CH2-),3.90(s,3H,-OCH3),3. 25(d,J=6.8Hz,2H,-CH2-),2.94-3.01(m,1H,-CH2-),2.77(dd,J=16.4,3.2Hz,1H,-CH2-),2.41-2.49(m,20H,-CH2-) -),2.30(d,J=3.2Hz,6H,N-CH3),1.78-1.87(m,4H,-CH2-),1.66-1.72(m,4H,-CH2-),1.61(d,J=10.8Hz,6H,-CH3); 13 CNMR(100MHz CDCl3)δ:190.3,162.7,161.1,160.5,158.9,131.2,131.1,127.4,122.6,114.4,110.1,105.8,89.2,78 .3,68.1,67.7,58.1,58.0,56.0,55.0,54.9,52.9,45.9,45.5,27.2,25.8,23.4,21.9,17.8; HRMS(ESI)C 39 H 58 N4O5[M+H] + calcd=663.4485; found=663.4487.

[0177] Example 26 Compound 24

[0178] Compound 24 was synthesized using the method described in Example 3. The physicochemical properties of compound 24 are as follows:

[0179] 1) Orange liquid;

[0180] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (600MHz) characteristics:

[0181] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 62.1%. 1H NMR(600MHz CDC13)δ:7.34(d,J=9.0Hz,2H,-Ph),6.89(d,J=9.0Hz,2H,-Ph),6.07(s,1H,-Ph),5.31(dd,J=12.6,3.0Hz,1H,-CH= ),5.13(t,J=7.2Hz,1H,-CH-),4.02(t,J=6.6Hz,2H,-CH2-),3.96(t,J=6.6Hz,2H,-CH2-),3.90(s,3H,-OCH3),3.75 (d,J=3.6Hz,8H,-CH2-),3.25-3.26(m,2H,-CH2-),2.95-3.00(m,1H,-CH2-),2.77(dd,J=16.8,3.0Hz,1H,-CH2-),2 .41-2.50(m,12H,-CH2-),1.80-1.86(m,4H,-CH2-),1.61(d,J=17.4Hz,10H,-CH2-,-CH3),1.49-1.53(m,4H,-CH2-); 13 C NMR(150MHz CDCl3)δ:190.2,162.7,161.1,160.6,159.0,131.2,131.0,127.4,122.7,114.4,110.2,105.9,89.3,78. 3,68.0,67.7,66.6,58.87,58.8,56.0,53.6,53.5,45.5,29.1,29.0,25.8,23.9,21.9,17.8; HRMS(ESI)C 39 H 56 N₂O₇[M+H] + calcd=665.4166; found=665.4170.

[0182] Example 27 Compound 25

[0183] Compound 25 was synthesized using the method described in Example 3. The physicochemical properties of compound 25 are as follows:

[0184] 1) Brown liquid;

[0185] 2) The nuclear magnetic resonance spectrum of this compound ( 1 HNMR (600MHz) characteristics:

[0186] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 59.7%. 1H NMR(600MHz CDCl3)δ:7.34(d,J=8.4Hz,2H,-Ph),6.88(d,J=9.0Hz,2H,-Ph),6.07(s,1H,-Ph),5.31(dd,J=12.6,3.0H z,1H,-CH=),5.13(t,J=7.2Hz,1H,-CH-),4.02(t,J=6.0Hz,2H,-CH2-),3.95(t,J=6.6Hz,2H,-CH2-),3.9 0(s,3H,-OCH3),3.25(d,J=4.2Hz,2H,-CH2-),2.95-3.00(m,1H,-CH2-),2.73-2.81(m,17H,-CH2-),2.45 (s,4H,-CH2-),1.79-1.85(m,4H,-CH2-),1.61(d,J=18.0Hz,10H,-CH2-,-CH3),1.49-1.51(m,4H,-CH2-); 13 C NMR(150MHz CDCl3)δ:190.2,162.7,161.1,160.6,159.0,131.2,131.0,127.4,122.7,114.4,110.1,105.9,89.3, 78.3,68.0,67.7,59.1,56.0,54.97,54.9,45.5,29.0,27.6,27.5,25.8,24.0,21.9,17.8; HRMS(ESI)C 39 H 56 N₂O₅S₂[M+H] + calcd=697.3709; found=697.3711.

[0187] Example 28 Compound 26

[0188] Compound 26 was synthesized using the method described in Example 3. The physicochemical properties of compound 26 are as follows:

[0189] 1) Brown liquid;

[0190] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (600MHz) characteristics:

[0191] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 39.6%, 1H NMR(600MHz CDCl3)δ:7.34(d,J=8.4Hz,2H,-Ph),6.89(d,J=9.0Hz,2H,-Ph),6.08(s,1H,-Ph),5.31(dd,J=12.6,3.0Hz,1H, -CH=),5.13-5.16(m,1H,-CH-),4.02(t,J=6.0Hz,2H,-CH2-),3.95(t,J=6.6Hz,2H,-CH2-),3.90(s,3H,-OCH3) ,3.25-3.27(m,2H,-CH2-),2.95-3.00(m,1H,-CH2-),2.77(dd,J=16.2,3.0Hz,1H,-CH2-),2.45(s,8H,-CH2-), 2.37-2.39(m,4H,-CH2-),1.79-1.85(m,4H,-CH2-),1.60-1.64(m,18H,-CH2-,-CH3),1.45-1.50(m,8H,-CH2-); 13 C NMR(150MHz CDCl3)δ:190.3,162.7,161.1,160.6,159.0,131.2,131.0,127.4,122.7,114.4,110.2,105.9,89.3,78.3,68.1,67.8,59.0 7,59.0,56.0,54.4,54.3,45.5,29.14,29.1,26.2,26.1,25.8,25.5,25.4,24.24,24.2,24.17,24.1,21.9,17.8; HRMS(ESI)C 41 H 60 N₂O₅[M+H] + calcd=661.4580; found=661.4581.

[0192] Example 29 Compound 27

[0193] Compound 27 was synthesized using the method described in Example 3. The physicochemical properties of compound 27 are as follows:

[0194] 1) Brown liquid;

[0195] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (600MHz) characteristics:

[0196] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 36.7%. 1H NMR(600MHz CDCl3)δ:7.34(d,J=8.4Hz,2H,-Ph),6.89(d,J=9.0Hz,2H,-Ph),6.08(s,1H,-Ph),5.31(dd,J=12.6,2.4Hz,1H,-CH=),5.14-5.16(m,1H,-CH-) ,4.02(t,J=6.6Hz,2H,-CH2-),3.96(t,J=6.6Hz,2H,-CH2-),3.91(s,3H,-OCH3),3.23-3.30(m,2H,-CH2-),2.95-3.00(m,1H,-CH2-),2.89(d, J=4.2Hz,2H,-CH2-),2.78(dd,J=16.2,3.0Hz,1H,-CH2-),2.69-2.75(m,2H,-CH2-),2.33-2.43(m,4H,-CH-,-CH2-),2.18-2.23(m,2H,-CH2-) ,1.80-1.85(m,4H,-CH2-),1.54-1.68(m,18H,-CH2-,-CH3),1.44-1.49(m,4H,-CH2-);1.30-1.37(m,4H,-CH2-),1.08(t,J=6.6Hz,6H,-CH3); 13 C NMR(150MHz CDCl3)δ:190.2,162.7,161.1,160.6,159.1,131.2,131.0,127.4,122.7,114.4,110.2,105.9,89.3,78.3,68. 1,67.8,56.0,53.9,52.1,45.5,34.37,34.3,29.2,29.1,25.8,25.1,24.9,24.2,23.8,21.9,17.8; HRMS(ESI)C 43 H 64 N₂O₅[M+H] + calcd=689.4893; found=689.4902.

[0197] Example 30 Compound 28

[0198] Compound 28 was synthesized using the method described in Example 3. The physicochemical properties of compound 28 are as follows:

[0199] 1) Brown liquid;

[0200] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (600MHz) characteristics:

[0201] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 41.2%. 1 H NMR(600MHz CDCl3)δ:7.34(d,J=8.4Hz,2H,-Ph),6.89(d,J=8.4Hz,2H,-Ph),6.08(s,1H,-Ph),5.31(dd,J=13.2,3.0Hz,1H,-C H=),5.13-5.16(m,1H,-CH-),4.02(t,J=6.6Hz,2H,-CH2-),3.96(t,J=6Hz,2H,-CH2-),3.90(s,3H,-OCH3),3.25- 3.27(m,2H,-CH2-),2.86-3.00(m,5H,-CH2-),2.77(dd,J=16.2,3.0Hz,1H,-CH2-),2.35-2.40(m,4H,-CH2-),1.8 0-1.87(m,6H,-CH2-),1.56-1.72(m,22H,-CH-,-CH2-,-CH3),1.46-1.50(m,4H,-CH2-),0.86-0.87(m,6H,-CH3); 13 C NMR(150MHzCDCl3)δ:190.3,162.7,161.1,160.6,159.0,131.2,131.0,127.4,122.7,114.4,110.2,105.9,89.3,78.3,68.1,67.8,61.8,61.7 ,58.9,56.0,53.9,53.8,45.5,32.9,32.8,30.89,30.8,29.15,29.1,26 .3,26.2,25.8,25.29,25.2,24.17,24.1,21.9,19.7,17.8; HRMS(ESI)C 43 H 64 N₂O₅[M+H] + calcd=689.4893; found=689.4896.

[0202] Example 31 Compound 29

[0203] Compound 29 was synthesized using the method described in Example 3. The physicochemical properties of compound 29 are as follows:

[0204] 1) Brown liquid;

[0205] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (600MHz) characteristics:

[0206] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 43.9%. 1 H NMR(600MHz CDCl3)δ:7.34(d,J=8.4Hz,2H,-Ph),6.89(d,J=9.0Hz,2H,-Ph),6.08(s,1H,-Ph),5.31(dd,J=12.6,3.0Hz,1H,-CH=),5.13-5.16 (m,1H,-CH-),4.02(t,J=6.6Hz,2H,-CH2-),3.96(t,J=6.0Hz,2H,-CH2-),3.90(s,3H,-OCH3),3.25-3.27(m,2H,-CH2-),2.94-3.0 0(m,5H,-CH2-),2.77(dd,J=16.8,3.0Hz,1H,-CH2-),2.36-2.41(m,4H,-CH2-),1.95-1.98(m,4H,-CH2-),1.80-1.86(m,4H,-CH2 -),1.61-1.64(m,14H,-CH-,-CH2-,-CH3),1.46-1.51(m,4H,-CH2-),1.32-1.41(m,6H,-CH2-),0.92(dd,J=6.0,2.4Hz,6H,-CH3); 13 C NMR(150MHz CDCl3)δ:190.3,162.7,161.1,160.6,159.0,131.2,131.0,127.4,122.7,114.4,110.2,105.9,89.3,78.3,68.1,67.8,58.8 ,56.0,53.99,53.9,45.5,33.9,33.8,30.6,29.1,29.0,26.5,26.4,25.8,24.17,24.1,21.9,21.79,21.7,17.8; HRMS(ESI)C 43 H 64 N₂O₅[M+H] + calcd=689.4893; found=689.4897.

[0207] Example 32 Compound 30

[0208] Compound 30 was synthesized using the method described in Example 3. The physicochemical properties of compound 30 are as follows:

[0209] 1) Brown liquid;

[0210] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (600MHz) characteristics:

[0211] Using CDCl3 as solvent, the peak assignments are as follows: Yield: 61.3%. 1 H NMR(600MHz CDCl3)δ:7.34(d,J=8.4Hz,2H,-Ph),6.88(d,J=8.4Hz,2H,-Ph),6.07(s,1H,-Ph),5.30(dd,J=13.2,3.0Hz,1H,-CH=),5.13-5 .15(m,1H,-CH-),4.01(t,J=6.0Hz,2H,-CH2-),3.95(t,J=6.6Hz,2H,-CH2-),3.90(s,3H,-OCH3),3.25-3.26(m,2H,-CH2-),2. 95-3.00(m,1H,-CH2-),2.77(dd,J=16.2,3.0Hz,1H,-CH2-),2.49-2.70(m,14H,-CH2-),2.38-2.41(m,6H,-CH2-),2.31(d,J=3 .6Hz,6H,N-CH3),1.79-1.85(m,4H,-CH2-),1.61(d,J=17.4Hz,6H,-CH3),1.56-1.59(m,4H,-CH2-),1.47-1.51(m,4H,-CH2-); 13 C NMR(150MHz CDCl3)δ:190.2,162.7,161.1,160.6,159.0,131.2,131.0,127.4,122.7,114.4,110.2,105.9,89.2,78.3,68.0,67.8,58 .4,58.3,56.0,54.85,54.8,52.9,52.8,45.82,45.8,45.5,29.14,29.1,26.4,25.8,24.06,24.0,21.9,17.8; HRMS(ESI)C 41 H 62 N4O5[M+H] + calcd=691.4798; found=691.4808.

[0212] Example 33 Preparation of compound 31

[0213] Compound 5 (0.1 mmol) was placed in a round-bottom flask, and an appropriate amount of anhydrous acetonitrile was added to dissolve it. Iodomethane solution (1 mmol) was added, and the reaction was stirred in an oil bath at 50 °C. The reaction was monitored by TLC until it was completed. The solvent was removed by concentration, and the target compound was obtained by recrystallization.

[0214] The physicochemical properties of compound 31 are as follows:

[0215] 1) Yellow solid;

[0216] 2) The nuclear magnetic resonance spectrum of this compound ( 1 H NMR (400MHz) characteristics:

[0217] Using DMSO as the solvent, the peaks were assigned as follows: Yield: 33.2%. 1 H NMR(400MHz DMSO)δ:7.40(d,J=8.4Hz,2H,-Ph),6.97(d,J=8.4Hz,2H,-Ph),6.31(s,1H,-Ph),5.40-5.44(m,1H,- CH=),5.08(t,J=6.8Hz,1H,-CH-),4.17(s,2H,-CH2-),4.03-4.06(m,2H,-CH2-),3.81(s,3H,-OCH3), 3.16-3.32(m,14H,-CH2-),2.94-3.01(m,7H,N-CH3,-CH2-),2.63(d,J=14.0Hz,1H,-CH2-),1.79(s, 8H,-CH2-),1.56-1.61(m,14H,-CH2-,-CH3),1.27-1.32(m,8H,-CH2-),0.90(t,J=7.2Hz,12H,-CH3); 13 C NMR(100MHz DMSO)δ:188.4,161.7,160.3,160.1,158.3,131.3,130.2,127.7,122.5,114.2,108.5,105.3,77.7,75. 0,66.5,60.6,60.0,47.5,44.8,25.6,25.5,25.3,23.39,23.3,21.5,19.2,18.3,17.5,13.4; HRMS(ESI)C 47 H 78 I₂N₂O₅[M⁻²I] / 2 + calcd=375.2950; found=375.2958.

[0218] Example 34 Preparation of Compound 32

[0219] Compound 32 was synthesized using the method described in Example 33. The physicochemical properties of compound 32 are as follows:

[0220] 1) Yellow solid;

[0221] 2) The nuclear magnetic resonance spectrum of this compound (1 H NMR (400MHz) characteristics:

[0222] Using DMSO as solvent, the peak assignments are as follows: Yield: 31.3%, 1 H NMR(400MHz DMSO)δ:7.39(d,J=8.4Hz,2H,-Ph),6.95(d,J=8.4Hz,2H,-Ph),6.30(s,1H,-Ph),5.40(dd,J=12.0,2.4Hz,1H,-CH= ),5.06(t,J=6.8Hz,1H,-CH-),4.11(t,J=5.6Hz,2H,-CH2-),3.99(t,J=6.0Hz,2H,-CH2-),3.81(s,3H,-OCH3),3.1 5-3.23(m,14H,-CH2-),2.94-3.01(m,7H,N-CH3,-CH2-),2.62-2.66(m,1H,-CH2-),1.71-1.80(m,8H,-CH2-),1.57 -1.60(m,14H,-CH2-,-CH3),1.44(d,J=5.6Hz,4H,-CH2-),1.25-1.34(m,8H,-CH2-),0.91(t,J=7.2Hz,12H,-CH3); 13 CNMR(100MHz DMSO)δ:188.3,161.8,160.3,160.1,158.5,131.2,130.2,127.7,122.6,114.3,108.6,105.3,77.7,75. 6,67.1,60.4,59.2,47.5,44.8,28.1,28.0,25.5,23.3,22.5,22.4,21.1,19.2,17.5,13.4; HRMS(ESI)C 49 H 82 I₂N₂O₅[M⁻²I] / 2 + calcd=389.3106; found=389.3112.

[0223] Application Example 1: In vitro antibacterial activity assay

[0224] 1. Test bacteria:

[0225] Staphylococcus aureus (ATCC 29213); methicillin-resistant Staphylococcus aureus (MRSA).

[0226] 2. Samples and reagents:

[0227] The samples were: isoflavol, vancomycin, meropenem, and compounds 1-32 prepared in the examples.

[0228] 3. Testing Method:

[0229] According to the Clinical Laboratory Standards Institute (CLSI) standards, the in vitro antibacterial activity of isoflavone, compounds 1-32 of this invention, and the clinical antibacterial drug vancomycin was tested using a 96-well plate and serial dilution method. The minimum drug concentration observed with the naked eye in the smallest completely clear well was defined as the MIC value.

[0230] Table 1. In vitro antibacterial activity (μg / mL) of isoflavone amine compounds 1-32 of the present invention

[0231]

[0232] Note: IXN: Isoflavol; a Ec: Escherichia coli ATCC25922; b Sa: Staphylococcus aureus (ATCC29213); c M-1-10: 10 clinically isolated MRSA strains; d SI: Selectivity Index (HC) 50 / MICs of S.aureus); e Van: Vancomycin; f MEM: Meropenem; g ND: Not determined; experiment repeated 3 times.

[0233] As shown in Table 1, the parent compound isoflavone exhibited poor antibacterial activity against both Gram-positive and Gram-negative bacteria, with MIC values ​​all exceeding 64 μg / mL. After structural modification of isoflavone, except for compounds 3, 6, 9-12, 18, and 25, the antibacterial activity against Staphylococcus aureus and MRSA was improved for the remaining 24 compounds. Among them, compounds 2, 5, 8, 26, and 29 showed MIC values ​​of 4-8 μg / mL against Staphylococcus aureus and MRSA, demonstrating superior antibacterial activity compared to the parent compound isoflavone (>64 μg / mL). Notably, compounds 31-32, further modified to introduce nitrogen ions, not only exhibited strong antibacterial activity against Gram-positive bacteria (MIC values ​​reaching 0.25-1 μg / mL) but also showed significantly improved antibacterial activity against the Gram-negative bacterium Escherichia coli (MIC values ​​reaching 16-32 μg / mL). The selectivity index (SI) can determine the safe range of a drug's efficacy. A higher SI indicates better membrane selectivity and a wider safe range. Table 1 shows that compounds 31-32 have SI values ​​higher than 1280 μg / mL, suggesting good safety and membrane selectivity. Surprisingly, among all the synthesized target compounds, compound 32 exhibits the strongest antibacterial effect. Its MIC values ​​against the five clinically isolated MRSA strains reached 0.25 μg / mL, and its MIC value against Escherichia coli reached 16 μg / mL, demonstrating antibacterial efficacy comparable to the marketed drug vancomycin, while also exhibiting good safety and membrane selectivity. Therefore, it holds promise as a clinical antibacterial drug against methicillin-resistant Staphylococcus aureus.

[0234] Application Example 2: Time-based sterilization kinetics experiment:

[0235] 1. Test bacteria:

[0236] Staphylococcus aureus ATCC 29213; MRSA-3 (clinical isolate).

[0237] 2. Samples and reagents:

[0238] The samples were: vancomycin and compound 32 prepared in the examples.

[0239] 3. Testing Method:

[0240] The dynamic bactericidal curves of target compound 32 against Staphylococcus aureus and MRSA-3 were determined using the plate count method. Single colonies of Staphylococcus aureus and MRSA-3 were picked and added to 1 mL of LB broth in a biosafety cabinet and incubated for 16–18 h in a shaker at 37 °C and 200 rpm. The cultured solution was then diluted 10,000 times with LB broth and incubated for another 2.5 h (early logarithmic growth stage) in a shaker (200 rpm, 37 °C) to determine the final bacterial concentration as 1 × 10⁻⁶. 5 CFU / mL. Then, different concentrations of compound 32 (4×MIC, 8×MIC) were added to the bacterial culture, followed by incubation at 37℃ and 200 rpm on a shaker. Samples were taken at 0, 0.5, 1, 2, 4, 6, and 8 h, with a sample volume of 100 μL each time. The samples were centrifuged (3500 rpm, 4℃), the culture medium was discarded, and the samples were washed 2-3 times with sterile PBS buffer. The final resuspending was in 100 μL of PBS buffer. The treated samples were serially diluted in 96-well plates, with 10 μL of each dilution added to MHB solid medium. Three parallel control groups were set up for each group. The samples were air-dried and incubated at 37℃ for 16-24 h. The colony count was recorded at each time point. Vancomycin (8×MIC) was used as a positive control, and the untreated group served as a blank control. The colony count was recorded the following day, in log10 CFU / mL, and plotted. The results are shown below. Figure 1 As shown.

[0241] Figure 1 The results showed that compound 32, at a concentration of 8×MIC, could completely kill Staphylococcus aureus ATCC 29213 and MRSA-3 within 0.5-1 h, while vancomycin at the same concentration only showed a good inhibitory effect. Furthermore, at a concentration of 4×MIC, compound 32 could completely kill Staphylococcus aureus ATCC 29213 and MRSA-3 within 4-8 h, still showing good bactericidal efficacy at this dosage. Therefore, compound 32 can be seen to kill Staphylococcus aureus ATCC 29213 and MRSA-3 efficiently and rapidly in a concentration-dependent manner, effectively avoiding the development of bacterial resistance, and holds promise for development into a rapid-acting antibacterial drug for clinical use.

[0242] Application Example 3: In vivo safety evaluation experiment

[0243] 1. Reagents:

[0244] Compound 32 prepared in the example, 0.9% NaCl.

[0245] 2. Test animals

[0246] SPF grade KM mice (purchased from Beijing Spaford Biotechnology Co., Ltd., weighing 19-22g, 4-6 weeks old).

[0247] 3. Testing Methods

[0248] Animal experiments were conducted according to the "Guidelines for the Care and Use of Laboratory Animals" approved by the Animal Protection and Utilization Committee. Thirty mice were divided into groups of five: Control (0.9% NaCl), 32 (5 mg / kg), 32 (10 mg / kg), 32 (20 mg / kg), 32 (40 mg / kg), and 32 (80 mg / kg). Hair was removed from the backs of the mice using a depilatory cream. After 24 hours of feeding, the mice were anesthetized with a small amount of ether, followed by an injection of 60 μL of 0.9% NaCl and different doses of compound 32. After 24 hours, the mice were observed for mortality and skin abnormalities (e.g., ulceration, redness, etc.). Blood was collected from the eyes of mice at the maximum dose that did not cause adverse reactions for routine blood tests and blood biochemical markers to evaluate the in vivo toxicity of compound 32. The results are as follows: Figure 2 As shown.

[0249] By subcutaneously injecting mice with different concentrations of compound 32, it was found that when the dosage of compound 32 was 80 and 40 mg / kg, some mice died, with survival rates of 40% and 80%, respectively. This indicates that compound 32 has certain in vivo toxicity at concentrations exceeding 40 mg / kg. When the dosage of compound 32 was 20 mg / kg, no mice died, but slight redness and swelling appeared on their backs. When the dosage of compound 32 was 10 mg / kg and 5 mg / kg, no mice died, and no adverse reactions such as redness and swelling occurred at the administration site. Therefore, it can be preliminarily determined that compound 32 has certain in vivo safety and no skin irritation at dosage concentrations ≤10 mg / kg. Therefore, blood samples were taken from mice treated with a dosage of 10 mg / kg for routine blood tests, including platelet count (PLT), hemoglobin (HGB), red blood cell count (RBC), mean corpuscular volume (MCV), hematocrit (HCT), and white blood cell count (WBC). Serum was simultaneously collected for blood biochemistry tests, including urea (UREA), albumin (ALB), and creatinine (CREA). The test results are as follows: Figure 2 As shown, when the drug concentration is 10 mg / kg, the various test indicators of mice are almost the same as those of the Control group. Therefore, it can be seen that compound 32 has certain in vivo drug safety when the drug concentration is ≤10 mg / kg.

[0250] Application Example 4: In vivo anti-MRSA infection activity experiment of compound 32

[0251] 1. Test bacteria:

[0252] MRSA-3 (clinical isolate)

[0253] 2. Samples and reagents:

[0254] The samples were: vancomycin and compound 32 prepared in the examples, and 0.9% NaCl.

[0255] 3. Test animals:

[0256] SPF grade KM mice (purchased from Beijing Spaford Biotechnology Co., Ltd., weighing 19-22g, 4-6 weeks old).

[0257] 4. Testing Method:

[0258] Thirty KM mice were divided into groups of six: a blank control group (injected with only 0.9% NaCl), a model group (injected with MRSA-3 bacterial suspension and 0.9% NaCl), a positive control vancomycin group (injected with MRSA-3 bacterial suspension and vancomycin 4 mg / kg), a high-dose treatment group (injected with MRSA-3 bacterial suspension and 32 mg / kg), and a low-dose treatment group (injected with MRSA-3 bacterial suspension and 32 mg / kg). All mice were shaved from the back and, after 24 hours of feeding, were anesthetized with a small amount of ether. Each group (except the blank control group) was then injected with 60 μL of MRSA-3 bacterial suspension (6 × 10⁻⁶ mg / kg). 8 Subcutaneous abscesses were induced by CFU / mL. Two hours later, 60 μL of different concentrations of the compound, the positive control drug vancomycin, or physiological saline were injected into the abscess sites of mice in each group. After 24 hours, the survival status of the mice in each group was observed. Then, the mice were euthanized by dislocation, and the infected skin was dissected in a biosafety cabinet and added to 1 mL of sterile physiological saline. The tissue fluid was then ground to a fragmented state using a tissue homogenizer (4℃, 70Hz). The tissue fluid from each group was then serially diluted in 96-well plates. 10 μL of each diluted sample was aspirated and dropped onto LB agar plates. Three parallel controls were set for each group. The plates were air-dried and incubated at 37℃ for 18-24 hours. The colony counts were then recorded and plotted. The results are shown below. Figure 3 As shown.

[0259] Figure 3The results showed that the bacterial load in the skin tissue of mice in the Control group was as high as approximately 8.8 log10 CFU / g. After treatment with compound 32 and vancomycin, the bacterial load in the skin tissue of mice in all groups decreased, with the most significant reduction observed after treatment with compound 32 (8 mg / kg), which reduced the bacterial load at the infection site by approximately 5 log10 CFU / g compared to the Control group. Treatment with compound 32 (4 mg / kg) and vancomycin (4 mg / kg) resulted in reductions of approximately 3.2 log10 CFU / g and 1.6 log10 CFU / g, respectively, demonstrating moderate antibacterial effects. Compound 32 showed slightly better antibacterial efficacy than vancomycin. Therefore, this confirms that compound 32 also exhibits good anti-MRSA effects in vivo and holds promise for development into a clinical antibacterial agent.

Claims

1. An isoflavone amine copolymer or a quaternary ammonium salt thereof, wherein the structure of the isoflavone amine copolymer is shown below: ； in, R 1 =R 2 And all selected from When n, R 3 and R 4 They are: (1) n = 3, R 3 = R 4 = -(CH2)2CH3(2) n = 3, R 3 = R 4 = -(CH2)3CH3 (3) n = 3, R 3 = R 4 = -CH2CH(CH3)2(4) n = 4, R 3 = R 4 = -(CH2)2CH3 (5) n = 4, R 3 = R 4 = -(CH2)3CH3 (7) n = 5, R 3 = R 4 = -(CH2)2CH3(8) n = 5, R 3 = R 4 = -(CH2)3CH3; R 1 =R 2 And all selected from When, n, R 1 and R 2 They are: (13) n = 3,R 1 =R 2 = ; (14) n = 3, R 1 =R 2 = ; (15) n = 3, R 1 =R 2 = ; (16) n = 3, R 1 =R 2 = ; (17) n = 4, R 1 =R 2 = ; (19) n = 4, R 1 =R 2 = ; (20) n = 4,R 1 =R 2 = ; (21) n = 4, R 1 =R 2 = ; (22) n = 4, R 1 =R 2 = ;(23) n = 4,R 1 =R 2 = ; (24) n = 5, R 1 =R 2 = ; (26) n = 5, R 1 =R 2 = ; (27) n = 5,R 1 =R 2 = ; (28) n = 5, R 1 =R 2 = ;(29) n = 5, R 1 =R 2 = ; (30) n = 5, R 1 =R 2 = 。 2. The isoflavone alcohol-amine copolymer or its quaternary ammonium salt according to claim 1, characterized in that, The quaternary ammonium salt is generated by reacting isoflavone alcohol amine copolymers with haloalkanes and methylating their nitrogen atoms.

3. The isoflavone alcohol-amine copolymer or its quaternary ammonium salt according to claim 1, characterized in that, The quaternary ammonium salt is selected from the following compounds: ； Where n = 4 or 5.

4. The method for preparing the isoflavone-amine copolymer according to claim 1, characterized in that, Includes the following steps: Using fulvicol as a substrate, it was converted to its isomer, isoflavol, under alkaline conditions. Then, the phenolic hydroxyl groups at both ends of isoflavol were introduced into bromoalkane to synthesize intermediate a1-3. Subsequently, intermediate a1-3 was reacted with different aliphatic amines and nitrogen-containing heterocyclic amines under alkaline conditions to generate a series of isoflavol amine copolymers. ； Among them, R 1 R 2 R 3 R 4 R 5 R 6 And n is as described in claim 1.

5. The method for preparing isoflavone-amine copolymers according to claim 4, characterized in that, The preparation method of the isoflavol is as follows: using fulvol as a substrate, it is reacted under low-temperature alkaline conditions to generate its isomer, isoflavol: 。 6. The method for preparing isoflavone-amine copolymers according to claim 5, characterized in that, The alkaline conditions are based on NaOH, the reaction temperature is 0±2℃, and the reaction solvent is NaOH solution.

7. The method for preparing isoflavone-amine copolymers according to claim 4, characterized in that, The intermediate a1-3 is prepared as follows: isoflavone alcohol is reacted with dibromoalkane under alkaline conditions to generate intermediate a1-3: 。 8. The method for preparing isoflavone-amine copolymers according to claim 7, characterized in that, The alkaline condition is K2CO3, the molar ratio of isoflavone to the alkaline is 1:4-1:5, the molar ratio of isoflavone to dibromoalkane is 1:4-1:5, the reaction temperature is 45-60℃, and the reaction solvent is acetone.

9. The method for preparing isoflavone-amine copolymers according to claim 4, characterized in that, The intermediate a1-3 reacts with different aliphatic amines and nitrogen-containing heterocyclic amines under alkaline conditions, wherein the base in the alkaline conditions is K2CO3, the molar ratio of intermediate a1-3 to the base is 1:4-1:5, the molar ratio of intermediate a1-3 to different aliphatic amines and nitrogen-containing heterocyclic amines is 1:4-1:5, the reaction temperature is 45-60℃, and the reaction solvent is acetonitrile.

10. The use of the isoflavone amine copolymer or its quaternary ammonium salt as described in any one of claims 1-3 in the preparation of antibacterial drugs.

Images