Honokiol phosphine salt derivative as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of organic synthesis, and provides a honokiol phosphine salt derivative as well as a preparation method and application thereof, and a novel magnolol derivative is designed and synthesized by integrating a cationic head group and a hydrophobic chain. The compound shows potent activity (MIC = 1-4 [mu] g / mL) on a series of gram-positive bacteria, meanwhile, the hemolytic activity is extremely low (HC50 = 106.9 [mu] g / mL), and the cytotoxicity is also low (CC50 = 19.25 [mu] g / mL). In addition, the derivative also has the capabilities of rapid sterilization, low drug resistance, good plasma stability and capability of inhibiting biofilm formation and destroying mature biofilms. And compared with vancomycin, the vancomycin-containing antibacterial peptide has the advantage that the bacterial load can be obviously reduced.

Description

Technical Field

[0001] This invention relates to the field of organic synthesis technology, and in particular to a honokiol phosphine salt derivative, its preparation method, and its application. Background Technology

[0002] The continued escalation of antimicrobial resistance (AMR) has become one of the most serious challenges in global public health. The widespread emergence and spread of multidrug-resistant bacteria (MDRs) has severely weakened the clinical efficacy of traditional antibiotics, directly leading to prolonged courses of infectious diseases, an increasing medical burden, and a significant rise in mortality rates worldwide. According to current statistics, more than 700,000 people die annually from drug-resistant bacterial infections; without effective interventions, this number is projected to exceed 10 million by 2050, posing a profound threat to human health.

[0003] Of particular concern are ESKAPE pathogens, which not only effectively evade the antibacterial effects of commonly used drugs but are also a major source of infection within healthcare facilities, making them extremely difficult to treat and often leading to severe illness or even death. Meanwhile, the global antibiotic development pipeline has been stagnant for a long time, with almost no new antibiotics with entirely novel structures or mechanisms of action entering the market in recent decades, further exacerbating the AMR crisis.

[0004] Currently, the ever-accelerating pace of bacterial evolution coupled with the slow and stalled progress in discovering new antibiotics has created a widening "research and development gap." This precarious disparity highlights humanity's vulnerability in the face of drug resistance and urgently calls for accelerated development of alternative therapies with novel mechanisms of action to address this escalating global health crisis. Summary of the Invention

[0005] The purpose of this invention is to overcome the deficiencies in the prior art and provide a honokiol phosphine salt derivative, its preparation method, and its application.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a honokiol phosphine salt derivative, the structure of which is shown below: ; Among them, R 2 Independent as , , , , , , or .

[0007] The present invention also provides a method for preparing the aforementioned honokiol phosphine salt derivative, comprising the following steps: (1) An amino compound, dichloromethane, 4-bromobutylacetic acid ester and triethylamine were reacted to obtain compound 1; (2) Compound 1, compound 2, organic base, coupling reagent and acetonitrile were mixed and subjected to amide coupling reaction to obtain compound 3; (3) Mix compound 3, base and solvent, carry out hydrolysis reaction, and then acidify to obtain compound 4; (4) Compound 4, carbon tetrabromide, triphenylphosphine and dichloromethane were subjected to a bromination reaction to obtain compound 5; (5) React compound 5, triphenylphosphine and acetonitrile to obtain the above-mentioned honokiol phosphine salt derivative.

[0008] Preferably, the amino compound in step (1) is R. 2 -NH2,R 2 for , , , , , , or ; In step (1), the ratio of amino compound, dichloromethane, 4-bromobutylacetic acid ester and triethylamine is 13~14 mmol: 10~30 mL: 13~14 mmol: 14.5~15.5 mmol; The reaction in step (1) is carried out at a temperature of 20-30°C for 2-4 hours. The structural formula of compound 1 is as follows: ; Among them, R in compound 1 2 for , , , , , , or .

[0009] Preferably, the structural formula of compound 2 in step (2) is as follows: ; In step (2), the organic base is one or more of N,N-diisopropylethylamine, triethylamine, and pyridine; The coupling reagent in step (2) is one or more of HATU, TBTU and BOP; In step (2), the molar ratio of compound 1, compound 2, organic base and coupling reagent is 5~6:2~3:7.5~8.5:5~6; The temperature for the amide coupling reaction in step (2) is 20~30℃; the time is 6~8h. The structural formula of compound 3 mentioned in step (2) is as follows: ; In compound 3, R 2 Independent as , , , , , , or .

[0010] Preferably, the alkali mentioned in step (3) is one or more of sodium hydroxide, potassium hydroxide and lithium hydroxide; The solvent in step (3) comprises methanol and water, wherein the volume ratio of methanol to water is 1~10:1~10; In step (3), the ratio of compound 3, base and solvent is 1~3 mmol: 8~12 mmol: 10~30 mL; The hydrolysis reaction in step (3) is carried out at a temperature of 50~70℃ for 5~10h. The target pH value for acidification in step (3) is ≤3; The structural formula of compound 4 in step (3) is: ; In compound 4, R 2 Independent as , , , , , , or .

[0011] Preferably, the ratio of compound 4, carbon tetrabromide, triphenylphosphine and dichloromethane used in step (4) is 1~2 mmol: 2~4 mmol: 2~4 mmol: 10~30 mL; The bromination reaction in step (4) is carried out at a temperature of 20-30°C for 4-6 hours. The structural formula of compound 5 in step (4) is: ; In compound 5, R 2 Independent as , , , , , , or .

[0012] Preferably, the ratio of compound 5, triphenylphosphine and acetonitrile in step (5) is 0.1~1 mmol: 4~6 mmol: 5~20 mL; The reaction in step (5) is carried out at a temperature of 70-90°C for 12-24 hours.

[0013] The present invention also provides the application of the aforementioned honokiol phosphine salt derivatives in the preparation of antibacterial drugs.

[0014] This invention provides a honokiol phosphine salt derivative, which is a novel class of honokiol derivatives designed and synthesized by integrating a cationic head group with a hydrophobic chain. This compound exhibits potent activity (MIC = 1~4 μg / mL) against a range of Gram-positive bacteria, while displaying extremely low hemolytic activity (HC). 50 =106.9 μg / mL), and also low cytotoxicity (CC). 50 =19.25 μg / mL). Furthermore, this derivative exhibits rapid bactericidal activity, low resistance, good plasma stability, and the ability to inhibit biofilm formation and disrupt mature biofilms. Mechanistic studies confirm that this derivative exerts its effects by targeting the cell membrane, characterized by loss of membrane potential, increased membrane permeability, accumulation of reactive oxygen species (ROS), and leakage of cell contents, ultimately leading to rapid bacterial death. More importantly, it demonstrates superior antibacterial efficacy in vivo, significantly reducing bacterial load compared to vancomycin. Attached Figure Description

[0015] Figure 1 This is a graph showing the results of hemolytic activity and cytotoxicity tests of derivative 10b in Example 1; Figure 2 The diagram shows the biological fluid stability and bactericidal effect of derivative 10b in Example 1; Figure 3 This is a graph showing the in vivo antibacterial efficacy of derivative 10b in Example 1; Figure 4 This diagram illustrates the morphological differences between various skin lesions. Detailed Implementation

[0016] This invention provides a honokiol phosphine salt derivative, the structure of which is shown below: ; Among them, R 2 Independent as , , , , , , or .

[0017] The present invention also provides a method for preparing the aforementioned honokiol phosphine salt derivative, comprising the following steps: (1) An amino compound, dichloromethane, 4-bromobutylacetic acid ester and triethylamine were reacted to obtain compound 1; (2) Compound 1, compound 2, organic base, coupling reagent and acetonitrile were mixed and subjected to amide coupling reaction to obtain compound 3; (3) Mix compound 3, base and solvent, carry out hydrolysis reaction, and then acidify to obtain compound 4; (4) Compound 4, carbon tetrabromide, triphenylphosphine and dichloromethane were subjected to a bromination reaction to obtain compound 5; (5) React compound 5, triphenylphosphine and acetonitrile to obtain the above-mentioned honokiol phosphine salt derivative.

[0018] In this invention, the amino compound mentioned in step (1) is R. 2 -NH2,R 2 for , , , , , , or .

[0019] In this invention, the preferred ratio of amino compound, dichloromethane, 4-bromobutylacetic acid ester and triethylamine in step (1) is 13-14 mmol: 10-30 mL: 13-14 mmol: 14.5-15.5 mmol, more preferably 13.2-13.8 mmol: 15-25 mL: 13.2-13.8 mmol: 14.6-15.4 mmol, and even more preferably 13.4-13.6 mmol: 18-22 mL: 13.4-13.6 mmol: 14.8-15.2 mmol.

[0020] In this invention, the reaction temperature in step (1) is preferably 20-30°C, more preferably 22-28°C, and even more preferably 24-26°C; the reaction time is preferably 2-4 h, more preferably 2.5-3.5 h, and even more preferably 2.8-3.2 h. After the reaction is complete, the solvent is removed, and the residue is directly purified by rapid column chromatography (CH2Cl2 / MeOH) to obtain the target product compound 1.

[0021] In this invention, the structural formula of compound 1 is as follows: ; Among them, R in compound 1 2 for , , , , , , or .

[0022] In this invention, the structural formula of compound 2 in step (2) is as follows: .

[0023] In this invention, compound 2 is prepared as follows: (I) Magnolol, ethyl bromoacetate, potassium carbonate and acetonitrile were subjected to an alkylation reaction to obtain the reactants; (II) The reactants, sodium hydroxide and solvent were subjected to hydrolysis and acidification to obtain compound 2.

[0024] In this invention, the preferred ratio of magnolol, ethyl bromoacetate, potassium carbonate, and acetonitrile is 11-12 mmol: 30-35 mmol: 25-30 mmol: 5-10 mL, more preferably 11.2-11.8 mmol: 31-34 mmol: 26-29 mmol: 6-9 mL, and even more preferably 11.4-11.6 mmol: 32-33 mmol: 27-28 mmol: 7-8 mL.

[0025] In this invention, the temperature of the alkylation reaction in step (I) is preferably 60~70°C, more preferably 62~68°C, and even more preferably 64~66°C; the time is preferably 5~10h, more preferably 6~9h, and even more preferably 7~8h.

[0026] In this invention, the preferred ratio of solvent, sodium hydroxide and magnolol in step (II) is 60-100 mL: 110-120 mmol: 11-12 mmol, more preferably 65-95 mL: 112-118 mmol: 11.2-11.8 mmol, and even more preferably 70-90 mL: 114-116 mmol: 11.4-11.6 mmol; the solvent is an aqueous methanol solution, and the preferred volume ratio of methanol to water is 1-10: 1-10, more preferably 2-8: 2-8, and even more preferably 4-6: 4-6. The preferred temperature for the hydrolysis reaction is 50-70℃, more preferably 55-65℃, and even more preferably 58-62℃; the preferred time is 4-6h, more preferably 4.5-5.5h, and even more preferably 4.8-5.2h; after the hydrolysis reaction, the concentrated mixture is acidified using hydrochloric acid; the target pH value for acidification is preferably ≤3, more preferably ≤2.8, and even more preferably ≤2.5; after acidification, the mixture is filtered and dried to obtain compound 2.

[0027] In this invention, the organic base in step (2) is one or more of N,N-diisopropylethylamine, triethylamine and pyridine.

[0028] In this invention, the coupling reagent in step (2) is one or more of HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate), TBTU (O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboric acid), and BOP (benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate).

[0029] In this invention, the molar ratio of compound 1, compound 2, organic base and coupling reagent in step (2) is preferably 5~6:2~3:7.5~8.5:5~6, more preferably 5.2~5.8:2.2~2.8:7.6~8.4:5.2~5.8, and even more preferably 5.4~5.6:2.4~2.6:7.8~8.2:5.4~5.6.

[0030] In this invention, acetonitrile is used in excess in step (2) to provide a reaction environment.

[0031] In this invention, the mixing in step (2) involves initially mixing compound 2, organic base, coupling reagent and acetonitrile for 10 min, and then adding compound 1 to carry out amide coupling reaction.

[0032] In this invention, the temperature of the amide coupling reaction in step (2) is preferably 20~30℃, more preferably 22~28℃, and even more preferably 24~26℃; the time is preferably 6~8h, more preferably 6.5~7.5h, and even more preferably 6.8~7.2h.

[0033] In this invention, after the amide coupling reaction in step (2) is completed, standard aqueous phase post-treatment is performed, including acidification and ethyl acetate extraction, and finally compound 3 is purified by silica gel column chromatography.

[0034] In this invention, the structural formula of compound 3 in step (2) is as follows: ; In this invention, R in compound 3 2 Independent as , , , , , , or .

[0035] In this invention, the alkali mentioned in step (3) is one or more of sodium hydroxide, potassium hydroxide and lithium hydroxide.

[0036] In this invention, the solvent in step (3) comprises methanol and water, and the volume ratio of methanol to water is preferably 1~10:1~10, more preferably 2~8:2~8, and even more preferably 4~6:4~6; In this invention, the preferred ratio of compound 3, base and solvent in step (3) is 1~3 mmol: 8~12 mmol: 10~30 mL, more preferably 1.5~2.5 mmol: 9~11 mmol: 15~25 mL, and even more preferably 1.8~2.3 mmol: 10~10.5 mmol: 16~24 mL.

[0037] In this invention, the temperature of the hydrolysis reaction in step (3) is preferably 50~70℃, more preferably 55~65℃, and even more preferably 58~62℃; the time is preferably 5~10h, more preferably 6~9h, and even more preferably 7~8h.

[0038] In this invention, after hydrolysis in step (3) is completed, methanol is evaporated for acidification.

[0039] In this invention, the target pH value for acidification in step (3) is preferably ≤3, more preferably ≤2.8, and even more preferably ≤2.5. After acidification, the product is extracted with ethyl acetate, and the organic phase is dried with Na2SO4 and concentrated to obtain a crude product, which is directly used in subsequent reactions.

[0040] In this invention, the structural formula of compound 4 in step (3) is as follows: .

[0041] In this invention, R in compound 4 2 Independent as , , , , , , or .

[0042] In this invention, the preferred ratio of compound 4, carbon tetrabromide, triphenylphosphine and dichloromethane in step (4) is 1~2 mmol: 2~4 mmol: 2~4 mmol: 10~30 mL, more preferably 1.2~1.8 mmol: 2.5~3.5 mmol: 2.5~3.5 mmol: 15~25 mL, and even more preferably 1.4~1.6 mmol: 2.6~3.4 mmol: 2.6~3.4 mmol: 18~22 mL.

[0043] In this invention, compound 4, carbon tetrabromide, triphenylphosphine and dichloromethane are mixed. The mixing temperature is preferably -5~5°C, more preferably -4~4°C, and even more preferably -2~2°C. The mixing time is preferably ≥20 min, more preferably ≥25 min, and even more preferably ≥30 min. Stirring is maintained during the mixing process. After the mixing is completed, a bromination reaction is carried out.

[0044] In this invention, the temperature of the bromination reaction in step (4) is preferably 20~30℃, more preferably 22~28℃, and even more preferably 24~26℃; the time is preferably 4~6h, more preferably 4.5~5.5h, and even more preferably 4.8~5.2h; stirring is maintained during the reaction; after the reaction is completed, the solvent is removed, the residue is extracted with ethyl acetate, the organic phase is separated, dried and concentrated, and purified by silica gel rapid column chromatography to obtain the bromination product compound 5.

[0045] In this invention, the structural formula of compound 5 in step (4) is as follows: .

[0046] In this invention, R in compound 5 2 Independent as , , , , , , or .

[0047] In this invention, the preferred ratio of compound 5, triphenylphosphine and acetonitrile in step (5) is 0.1~1mmol:4~6mmol:5~20mL, more preferably 0.2~0.8mmol:4.5~5.5mmol:10~15mL, and even more preferably 0.4~0.6mmol:4.8~5.2mmol:12~13mL.

[0048] In this invention, the reaction temperature in step (5) is preferably 70-90°C, more preferably 75-85°C, and even more preferably 78-82°C; the reaction time is preferably 12-24 h, more preferably 14-20 h, and even more preferably 16-18 h. After the reaction is complete, the solvent is evaporated, and the crude product is ground with anhydrous diethyl ether. The obtained solid is collected by filtration, which is the honokiol phosphine salt derivative.

[0049] The present invention also provides the application of the aforementioned honokiol phosphine salt derivatives in the preparation of antibacterial drugs.

[0050] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0051] Example 1

[0052] The amino compound is R 2 -NH2, when R 2 Select as When the corresponding compound is a, then the choice is... When the corresponding compound is b, then choose... When the corresponding compound is c, then the choice is... When the corresponding compound is d, choose... When the corresponding compound is e, then the choice is... When the corresponding compound is f, then the choice is... When the corresponding compound is g, choose When the time is right, the corresponding compound is h.

[0053] 13.67 mmol of an amino compound, 20 mL of dichloromethane, 13.67 mmol of 4-bromobutylacetic acid, and 15.04 mmol of triethylamine were mixed and reacted. After the reaction was completed, the solvent was removed, and the residue was directly purified by rapid column chromatography (CH2Cl2 / MeOH) to obtain the corresponding compounds 1a to 1h. The reaction conditions and yields are recorded in Table 1.

[0054] Table 1. Reaction conditions and yields of compounds 1a-1h

[0055] 11.26 mmol of magnolol, 33.78 mmol of ethyl bromoacetate, and 28.16 mmol of K₂CO₃ were alkylated in 8 mL of acetonitrile at 65 °C for 8 hours. After solvent removal, the residue was hydrolyzed in 80 mL of MeOH / H₂O (1:1 v / v) with 111.26 mmol of NaOH at 60 °C for 5 hours. After concentrating the mixture, the aqueous phase was acidified to pH 3 by colorimetry. The precipitated solid was filtered and dried to give compound 2 as a white solid in 65% yield.

[0056] 2.61 mmol of compound 2, 7.83 mmol of DIPEA, and 5.22 mmol of HATU were mixed in acetonitrile for 10 min. Then, 5.22 mmol of compounds 1a to 1h were added separately to carry out amide coupling reaction. After the reaction was completed, the mixture was post-treated with standard aqueous phase, including acidification and ethyl acetate extraction. Finally, the mixture was purified by silica gel column chromatography to obtain compounds 3a to 3h. The reaction conditions and yields are recorded in Table 2.

[0057] Table 2 Reaction conditions and yields of compounds 3a-3h (Table 2)

[0058] 2 mmol of compounds 3a-3h were hydrolyzed in 20 mL of methanol / water solution (volume ratio 5:8) at 60 °C for 8 h with 10 mmol of sodium hydroxide. After hydrolysis, the methanol was evaporated and the aqueous solution was acidified to pH 3 with hydrochloric acid. After acidification, the solution was extracted with ethyl acetate. The organic phase was dried over Na2SO4 and concentrated to obtain crude products 4a-4h, which were directly used in subsequent reactions.

[0059] 1.5 mmol of 4a-4h, 3 mmol of carbon tetrabromide, 3 mmol of triphenylphosphine, and 20 mL of dichloromethane were mixed and stirred at 0 °C for 20 min, and then bromination was carried out. After the reaction was completed, the solvent was removed, the residue was extracted with ethyl acetate, the organic phase was separated, dried and concentrated, and purified by silica gel column chromatography to obtain the brominated product compounds 5a-5h. The reaction conditions and yields are recorded in Table 3.

[0060] Table 3 Reaction conditions and yields of compounds 5a-5h

[0061] 0.5 mmol of compounds 5a–5h, 5 mmol of triphenylphosphine, and 10 mL of acetonitrile were mixed and reacted separately. After the reaction was completed, the solvent was evaporated, and the crude product was ground with anhydrous diethyl ether. The obtained solid was collected by filtration, which is the honokiol phosphine salt derivative 10a–10h. The reaction conditions and yields are recorded in Table 4.

[0062] Table 4 shows the reaction conditions and yields of magnolol phosphine salt derivatives over 10 years to 10 hours.

[0063] The 1H NMR data of magnolol phosphine salt derivative 10a are as follows: 1 ¹H NMR (500 MHz, CDCl₃) δ 7.85–7.63 (m, 30H), 7.31–7.19 (m, 2H), 7.09–7.00 (m, 2H), 6.77 (t, J = 8.9 Hz, 2H), 6.02–5.85 (m, 2H), 5.13–4.94 (m, 4H), 4.75–4.49 (m, 4H), 4.00–3.70 (m, 4H), 3.49–3.14 (m, 12H), 2.10–1.88 (m, 4H), 1.69–1.41 (m, 8H), 1.36–1.16 (m, 6H), 0.93–0.81 (m, 6H). The carbon spectral data are as follows: 13 C NMR(125 MHz, CDCl3)δ167.87,167.82,155.11,153.55,137.53,136.79,135.00,134.96,133.73,133.70,133.65,133.62,131.38 ,131.18,131.07,130.57,130.48,130.40,128.55,128.26,128.07,118.50,118.47,118.42,117.82,117.79,117.74,11 5.65, 115.60, 113.10, 111.16, 68.19, 67.49, 67.21, 46.58, 46.34, 43.77, 43.47, 39.35, 34.28, 30.98, 30.79, 29.63, 27.77, 27.72, 27.63, 22.23, 22.15, 21.83, 21.75, 20.12, 20.08, 20.05, 19.90, 19.69, 13.89, 13.84, 13.77. Mass spectrometry data are from HRMS (ESI) C. 74 H 84 N2O4P2Br2[M-2Br] / 2 + calcd=563.2948;found=563.2957

[0064] The 1H NMR data of magnolol phosphine salt derivative 10b are as follows: 1H NMR (500 MHz, CDCl3) δ7.90–7.61(m,30H),7.47(td,J=7.6,2.9 Hz,1H),7.32(ddt,J=14.7,8.9,4.1 Hz,3H),7.12–6.98(m,2H),6.76(dt,J=8.6,4.1 Hz,1H),5.95(dtt,J=16.5,9.9,6.5 Hz,2H),5.16–4.88(m,4H),4.60(d,J=51.3 Hz,4H),4.03–3.67(m,4H),3.57–3.11(m,12H),2.09(d,J=60.2 Hz, 6H), 1.56 (d, J = 105.0 Hz, 6H), 1.33–1.16 (m, 12H), 0.85 (dt, J = 13.5, 6.7 Hz, 6H). Carbon spectral data are as follows: 13 C NMR(125 MHz, CDCl3)δ167.89,155.17,153.61,137.59,136.86,134.98,133.86,133.83,133.78,133.76,133.70,133.6 5,132.13,132.05,131.96,131.22,131.12,130.60,130.51,130.42,130.40,128.73,128.60,128.55,128.52, 128.46, 128.30, 128.07, 118.58, 117.92, 115.66, 113.64, 113.04, 111.20, 67.44, 67.27, 46.90, 46.62, 43.87, 39.40, 34.35, 31.56, 31.46, 28.96, 28.78, 26.56, 26.45, 22.58, 22.55, 19.85, 14.06, 14.00. Mass spectrometry data are from HRMS (ESI) C. 78 H 92 N2O4P2Br2[M-2Br] / 2 + calcd=591.3261;found=591.3264.

[0065] The 1H NMR data of magnolol phosphine salt derivative 10c are as follows 1¹H NMR (500 MHz, DMSO-d⁶) δ 7.97–7.61 (m, 30H), 7.33–7.26 (m, 2H), 7.06–6.93 (m, 2H), 6.83–6.74 (m, 2H), 6.04–5.87 (m, 2H), 5.12–4.90 (m, 4H), 4.83–4.70 (m, 4H), 3.61 (d, J = 11.9 Hz, 4H), 3.34–3.13 (m, 12H), 1.80–1.38 (m, 12H), 1.23 (d, J = 17.3 Hz, 20H), 0.84 (t, J = 6.8 Hz, 6H). The carbon spectrum data are as follows: 13 C NMR(125 MHz, DMSO-d6)δ167.63,167.33,167.23,155.25,153.67,138.39,137.38,137.33,135.38, 134.01,133.92,130.80,130.76,130.70,130.66,128.48,119.19,118.50,116.03,116.01, 63.19, 55.31, 46.65, 45.77, 44.31, 39.08, 34.46, 31.65, 29.24, 29.12, 28.88, 28.74, 28.28, 28.16, 27.61, 26.78, 26.70, 26.63, 22.52, 20.66, 20.26, 19.67, 14.39. Mass spectrometry data are from HRMS (ESI) C. 82 H 100 N2O4P2Br2[M-2Br] / 2 + calcd=619.3574;found=619.3579.

[0066] The 10-day proton NMR data of magnolol phosphine salt derivatives are as follows: 1H NMR(500 MHz, CDCl3)δ7.81–7.52(m,30H),7.39(td,J=7.7,2.9 Hz,1H),7.23(s,2H),7.03–6.92(m,2H),6.70–6.65(m,1H),5.88(dtt,J=16.5,9.8,6.6 Hz,2H),5.08–4.80(m,4H),4.53(d,J=48.7 Hz,4H),3.95–3.60(m,4H),3.42–3.07(m,12H),1.93(dp,J=23.6,6.3 Hz, 4H), 1.64–1.36 (m, 8H), 1.24–1.10 (m, 28H), 0.79 (td, J=7.0, 2.0 Hz, 6H). Carbon spectral data are as follows: 13 C NMR(125 MHz, CDCl3)δ167.85,153.61,137.59,136.86,134.93,133.84,133.81,133.76,133.73,133.23,132.13, 132.05,131.96,131.12,130.57,130.50,130.47,130.40,130.37,128.55,128.46,128.30,128.06,118.6 2,117.93,115.65,115.62,111.18,67.32,46.85,46.54,39.40,34.36,31.88,31.85,29.63,29.58,29.55,29.50,29.43,29.33,29.30,29.27,29.01,28.81,27.79,26.92,26.79,22.65,14.10. Mass spectrometry data are from HRMS (ESI) C. 86 H 108 N2O4P2Br2[M-2Br] / 2 + calcd=647.3887;found=647.3889.

[0067] The 1H NMR data of magnolol phosphine salt derivative 10e are as follows: 1¹H NMR (500 MHz, CDCl₃) δ 7.87–7.61 (m, 30H), 7.58–7.44 (m, 2H), 7.10–6.99 (m, 2H), 5.95 (dddd, J=21.1, 14.3, 10.4, 5.6 Hz, 2H), 5.14–4.88 (m, 4H), 4.82–4.44 (m, 4H), 3.99–3.66 (m, 4H), 3.53–3.12 (m, 12H), 2.00 (dt, J=24.0, 6.8 Hz, 4H), 1.71–1.43 (m, 8H), 1.23 (d, J=10.6 Hz, 36H), 0.87 (td, J=6.9, 2.2 Hz, 6H). The carbon spectrum data are as follows: 13 C NMR(125 MHz, CDCl3)δ167.86,155.16,153.59,137.57,136.85,135.00,134.94,133.84,133.81,133.78,133.73,133.70,133.23,132.88,13 2.12,132.04,131.97,131.41,131.24,131.12,130.57,130.50,130.47,130.40,130.37,128.59,128.55,128.46,128.28,118.55,1 17.90, 115.65, 115.62, 112.94, 111.17, 67.30, 67.21, 46.83, 46.51, 43.81, 43.45, 39.39, 34.34, 31.89, 29.62, 29.59, 29.55, 29.50, 29.44, 29.35, 29.31, 28.99, 28.80, 27.79, 26.99, 26.92, 26.79, 22.66, 22.40, 22.30, 22.00, 21.89, 19.80, 14.11. Mass spectrometry data are from HRMS (ESI) C. 90 H 116 N2O4P2Br2[M-2Br] / 2 + calcd=675.4200;found=675.4205.

[0068] The 10f proton NMR data of magnolol phosphine salt derivatives are as follows: 1¹H NMR (500 MHz, CDCl₃) δ 7.84–7.54 (m, 28H), 7.29–7.23 (m, 4H), 7.01–6.91 (m, 2H), 6.78 (dd, J=18.0, 8.5 Hz, 2H), 5.83 (dddt, J=19.4, 16.6, 9.9, 6.7 Hz, 2H), 5.04–4.79 (m, 4H), 4.57 (d, J=92.7 Hz, 4H), 3.92–3.76 (m, 2H), 3.55 (dq, J=14.3, 7.9, 6.8 Hz, 2H), 3.42–3.11 (m, 8H), 2.12–1.91 (m, 20H), 1.75–1.36 (m, 18H). The carbon spectrum data are as follows: 13 C NMR(125 MHz, CDCl3)δ168.23,168.16,155.30,153.70,137.69,137.18,137.10,137.00,135.18,135.15,135.12,133.80,133.72,133.70,133 .64,133.62,132.96,132.12,132.04,131.95,130.94,130.78,130.63,130.61,130.59,130.53,130.51,130.23,128.71,128.56,128 .52,128.46,128.16,128.06,118.36,117.68,115.60,113.71,111.85,70.71,69.56,60.40,59.03,58.93,49.32,43.05,42.66,39.82,39.65,39.45,36.36,36.33,35.51,34.37,33.27,33.15,30.12,30.08,22.83,22.50,22.10,21.07,20.22,20.05. Mass spectrometry data are from HRMS (ESI) C. 86 H 96 N2O4P2Br2[M-2Br] / 2 + calcd=641.3417;found=641.3419.

[0069] The 1H NMR data of 10g of magnolol phosphine salt derivative are as follows: 1¹H NMR (500 MHz, CDCl₃) δ 7.91–7.53 (m, 30H), 7.35–7.27 (m, 4H), 7.27–6.94 (m, 10H), 6.73–6.68 (m, 1H), 6.01–5.81 (m, 2H), 5.15–4.83 (m, 5H), 4.75–4.62 (m, 3H), 4.59–4.40 (m, 4H), 3.80 (d, J = 60.0 Hz, 4H), 3.52–3.22 (m, 8H), 2.14–1.93 (m, 4H), 1.63 (d, J = 32.4 Hz, 4H), 1.34–1.21 (m, 18H). The carbon spectrum data are as follows: 13 C NMR(125 MHz, CDCl3)δ168.66,155.00,153.50,150.60,150.56,137.59,136.82,134.96,133.82,133.80,133.74,1 33.72,133.66,133.16,133.10,131.33,131.17,131.09,130.66,130.52,130.49,130.42,130.39,128.54 ,128.29,128.03,127.81,126.28,125.76,125.39,118.57,117.89,115.66,115.60,113.05,111.09,67.70,67.38,49.29,44.20,43.89,39.40,34.49,34.29,31.36,31.34,31.32,21.98,19.84. Mass spectrometry data are from HRMS (ESI) C 88 H 96 N2O4P2Br2[M-2Br] / 2 + calcd=653.3417;found=653.3421.

[0070] The 10-hour proton NMR data of magnolol phosphine salt derivatives are as follows: 1 ¹H NMR (500 MHz, CDCl₃) δ 7.82–7.59 (m, 28H), 7.54–7.44 (m, 8H), 7.39 (dq, J=7.2, 4.0 Hz, 4H), 7.32 (tt, J=5.5, 2.5 Hz, 9H), 7.24–7.07 (m, 4H), 7.06–6.96 (m, 2H), 6.03–5.78 (m, 2H), 5.13–4.48 (m, 12H), 4.00–3.66 (m, 4H), 3.56–3.16 (m, 8H), 2.14–1.99 (m, 4H). The carbon spectral data are as follows:13 C NMR(125 MHz, CDCl3)δ168.69,155.02,153.49,140.71,140.45,137.56,137.21,137.12,136.77,135.00,133.80,13 3.77,133.71,133.65,132.95,132.12,132.05,131.97,131.94,131.31,131.14,130.53,130.50,130.44,1 30.40, 128.81, 128.77, 128.71, 128.65, 128.56, 128.51, 128.46, 127.51, 127.46, 127.40, 127.20, 126.98, 118.50, 117.83, 115.68, 115.64, 67.50, 49.53, 39.39, 34.41, 34.29, 27.68, 22.51, 19.85. Mass spectrometry data are from HRMS (ESI) C. 92 H 88 N2O4P2Br2[M-2Br] / 2 + calcd=673.3104;found=673.3106.

[0071] In vitro antibacterial test

[0072] Preparation of bacterial suspension: The preserved strain was passaged twice on a suitable solid culture medium to ensure purity and activity. Several morphologically uniform single colonies were picked and suspended in sterile physiological saline or broth, and the turbidity was adjusted to 0.5 McFarland standard (approximately 1–2 × 10⁻⁶). 8 (CFU / mL). The suspension was then diluted with MH broth to prepare a working bacterial suspension with a final concentration of approximately 5 × 10⁻⁶ CFU / mL. 5 CFU / mL.

[0073] ii. Drug dilution and plating: In microplates, the test compound is serially diluted twofold using MH, typically ranging from 0.125 to 128 µg / mL, or adjusted according to preliminary experiments. The final volume of liquid in all wells is 100 µL. Positive growth control wells (containing only MH broth and bacterial culture) and negative sterile control wells (containing only MH broth, without bacterial culture) should be included in the microplate.

[0074] iii. Inoculation and Incubation: Add 100 µL of the prepared working bacterial suspension to all test wells and positive control wells, so that the final inoculation amount per well is approximately 5 × 10⁻⁶. 5CFU / mL. Cover with a sterile cap, gently shake to mix, and then incubate the microplate at 35±2°C under aerobic conditions for 16–20 hours according to CLSI guidelines.

[0075] IV. Result Interpretation: After incubation, the microplate was placed under a white background and contrasting light for visual interpretation. The MIC endpoint was defined as the lowest drug concentration that could completely inhibit the growth of visible bacteria. All assays were repeated at least three times in independent experiments. Results are recorded in Table 5.

[0076] Table 5. In vitro antibacterial activity

[0077] In Table 5, bacteria 1: *Staphylococcus aureus* ATCC25923, erythromycin-sensitive strain; bacteria 2: *Staphylococcus aureus* ATCC43300, methicillin-resistant strain; bacteria 3: *Enterococcus faecalis* ATCC29212, vancomycin-sensitive strain; bacteria 4: *Enterococcus faecalis* ATCC51299, vancomycin-resistant strain; bacteria 5: *Bacillus subtilis* ATCC9372, penicillin-sensitive strain; bacteria 6: *Acinetobacter baumannii* ATCC19606, standard strain; bacteria 7: *Escherichia coli* ATCC25922, penicillin-sensitive strain; bacteria 8: *Pseudomonas aeruginosa* ATCC27853, penicillin-sensitive strain. Van represents vancomycin, and Cos represents colistin. Gram-positive bacteria included Staphylococcus aureus ATCC25923, ATCC43300; Enterococcus faecalis ATCC29212, ATCC51299; Bacillus subtilis ATCC9372, and Gram-negative bacteria included Escherichia coli ATCC25922, Acinetobacter baumannii ATCC19606, and Pseudomonas aeruginosa ATCC27853. Vancomycin and colistin were used as baseline control drugs for Gram-positive and Gram-negative bacterial infections, respectively. Table 5 shows that the activity of the derivatives against Gram-positive pathogens exhibited a bell-shaped curve. Activity increased from the C4 chain (10a, MIC = 2-8 µg / mL) to the optimal C6 chain length (10b, MIC = 1-4 µg / mL), defining a critical hydrophobic window. However, further extension to the C8 (10c) and C12 (10d) chains led to a sharp decrease in activity (MIC = 4 to ≥128 µg / mL), indicating that excessive lipophilicity is detrimental. Secondly, replacing the flexible alkyl chain with bulky, conformationally restricted hydrophobic domains (e.g., adamantyl (10f), tert-butylbenzyl (10g), and biphenylmethyl (10h)) significantly improved the compound activity (MIC = 2–64 µg / mL). The compound library exhibited significant Gram-positive bacterial selectivity, likely due to the permeability barrier of the outer membrane of Gram-negative bacteria. Among all derivatives, derivative 10b (C6 alkyl tail) was the most promising candidate. It showed potent activity against Gram-positive strains (MIC = 1–4 µg / mL) and moderate activity against Gram-negative pathogens Acinetobacter baumannii (MIC = 32 µg / mL) and Escherichia coli (MIC = 16 µg / mL).

[0078] Tests of hemolytic activity and cytotoxicity of derivative 10b

[0079] Hemolysis assays were performed using fresh sheep erythrocytes to assess compound-induced erythrocyte membrane damage. A 5% erythrocyte suspension (dissolved in PBS) was treated with a compound dilution ranging from 1 to 512 × MIC. After incubation at 37°C for 1 hour, the culture plates were centrifuged, and the absorbance of the supernatant at 540 nm was measured to determine the released hemoglobin. The percentage of hemolysis was standardized using the following formula: Hemolysis (%) = ((OD_compound - OD_negative control) / (OD_positive control - OD_negative control) × 100%, with PBS as a negative control and 1% Triton X-100 as a positive control. All measurements were performed in triplicate.

[0080] The antiproliferative effect of compound 10b on human hepatocyte LO2 cells was evaluated in a concentration-dependent manner using the CCK-8 assay. The CCK-8 assay quantifies viable cells based on mitochondrial dehydrogenase activity. After seeding and adhesion, cells were exposed to different concentrations of 10b for 24 hours. After incubation, cells were washed and incubated with CCK-8 solution. The formazan dye produced by metabolically active cells was quantified by measuring absorbance at 450 nm. The cell inhibition rate was calculated as follows: Inhibition rate (%) = 1 - ((OD_compound - OD_blank) / (OD_control - OD_blank) × 100%, where blank represents wells without cells and the sample compound, and control represents wells with only cells and no sample compound. To ensure statistical reliability, the entire experiment was performed in triplicate.

[0081] The results of the hemolytic activity and cytotoxicity tests are as follows: Figure 1 As shown, Figure 1 In the table, A represents the hemolytic potential assessment result of derivative 10b, and B represents the cytotoxicity test result; from Figure 1 As can be seen, in the hemolysis model using erythrocytes, 10b exhibits excellent blood compatibility, and the hemolytic effect is negligible. 50 The concentration was 106.9 μg / mL, and even at a concentration of 32 μg / mL, only 6.16% hemolysis was observed, confirming its minimal effect on mammalian erythrocytes. To further assess its biocompatibility, the cytotoxicity of 10b to LO2 hepatocytes was evaluated using the CCK-8 assay. As shown in the figure, the cytotoxicity to LO2 cells was low, and the CCK-8 assay showed low cytotoxicity. 50 The concentration was 19.25 μg / mL. Notably, cell viability remained high within its effective bacterial concentration range (89.24% at 8 μg / mL). Therefore, derivative 10b exhibited good safety and a promising therapeutic window.

[0082] The bactericidal activity of derivative 10b in plasma and complex mammalian body fluids was tested. Its stability in biorelevant environments was investigated by monitoring the change in the minimum bactericidal concentration (MBC) of compound 10b in 50% (v / v) plasma over time. MBC was determined after pre-incubation at 37°C for 0, 3, and 6 hours. Furthermore, to assess how the compound's potency was affected by various blood components, parallel MBC determinations were performed in 50% plasma, 50% serum, and 50% whole blood. MBC was strictly defined as the lowest concentration at which the initial bacterial load decreased by ≥99.9% after 18 hours of incubation; bacterial load was quantified using standard plate counting. Three independent replicate experiments were performed to ensure data reliability, and the results are as follows: Figure 2 As shown, Figure 2 In Figure A, the stability of derivative 10b in plasma is shown, and in Figure B, its bactericidal activity in body fluids is shown. It can be seen that its MBC value against Staphylococcus aureus ATCC25923 is close to the initial value, with only an increase of less than two times, confirming its good plasma stability. Furthermore, derivative 10b maintained potent bactericidal activity when tested in 50% serum and whole blood, with the MBC value increasing only two-fold compared to standard conditions. These results indicate that the potency loss of 10b in biorelevant environments is negligible, which is a key advantage for drug development.

[0083] Based on the potent antibacterial activity, good plasma stability, and safety exhibited by compound 10b in vitro, we established a mouse skin abscess model infected with Staphylococcus aureus ATCC25923 to systematically evaluate its in vivo therapeutic effect. The in vivo efficacy of derivative 10b was assessed using the mouse skin abscess model infected with Staphylococcus aureus ATCC 25923. All animal experiments were conducted according to protocols approved by the Animal Ethics Committee of Liaocheng University. Male BALB / c mice (6-8 weeks old, license number: SCXK(LU)20220006) were randomly divided into four groups (n=5 per group): a solvent control group (infection, treated with saline), two treatment groups (infection, administered 5 or 10 mg / kg of 10b, respectively), and a positive control group (infection, treated with 10 mg / kg of vancomycin). Staphylococcus aureus suspension (5 × 10⁻⁶ mg / kg) was injected subcutaneously into the back of the mice. 8 Skin infection was induced using CFU / mL (100 μL). Two hours after infection, mice were given appropriate subcutaneous treatment at the infection site. All animals were sacrificed 24 hours later. Infected skin tissue was collected, fixed with 4% paraformaldehyde, and stained with hematoxylin and eosin (H&E). In vivo antibacterial efficacy was assessed as follows: Figure 3 As shown (where Van is vancomycin), Figure 3In the table, A represents the bacterial load in the skin tissue of infected mice, and B represents the bacterial survival rate. It can be seen that the quantitative bacteriological results indicate that the bacterial load at the infection site was higher in the saline control group, approximately 9.3 log [missing value]. 10 CFU / g. Treatment with 10b at a dose of 5 mg / kg reduced the bacterial load by 1.28 log. 10 CFU / g reduced bacterial survival by 93.38%. In comparison, the 10 mg / kg dose of 10b was more effective, significantly reducing bacterial load by 2.99 log. 10 CFU / g, bacterial survival rate decreased by 99.89%. Notably, at the same dose (10 mg / kg), 10b showed superior antibacterial efficacy compared to vancomycin (vancomycin reduced bacterial load by 1.83 log). 10 (CFU / g, bacterial survival rate decreased by 98.28%), indicating that 10b has stronger in vivo antibacterial ability.

[0084] Morphological differences in different skin lesions, such as Figure 4 As shown in the figure (where Van is vancomycin), skin sections from the saline control group exhibited extensive pustular formation, accompanied by significant vasodilation and dense inflammatory cell infiltration in both the epidermis and dermis. In the low-dose 10b (5 mg / kg) treatment group, both pustular formation and inflammatory cell infiltration were significantly reduced, indicating effective control of the inflammatory response. The high-dose 10b (10 mg / kg) treatment group showed the best histological recovery, with no obvious inflammatory cells observed in either the epidermis or dermis, and the tissue structure was very similar to that of the uninfected control group. This treatment was superior to the vancomycin treatment group under similar conditions. In conclusion, compound 10b demonstrated potent in vivo anti-infective efficacy in a Staphylococcus aureus-induced mouse skin abscess model, warranting further investigation and potentially becoming a candidate drug for antibacterial drug development.

[0085] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A phosphine salt derivative of magnolol, characterized in that, The structures of the honokiol phosphine salt derivatives are shown below: ； Among them, R 2 Independent as , , , , , , or .

2. The method for preparing the magnolol phosphine salt derivative according to claim 1, characterized in that, Includes the following steps: (1) An amino compound, dichloromethane, 4-bromobutylacetic acid ester and triethylamine were reacted to obtain compound 1; (2) Compound 1, compound 2, organic base, coupling reagent and acetonitrile were mixed and subjected to amide coupling reaction to obtain compound 3; (3) Mix compound 3, base and solvent, carry out hydrolysis reaction, and then acidify to obtain compound 4; (4) Compound 4, carbon tetrabromide, triphenylphosphine and dichloromethane were subjected to a bromination reaction to obtain compound 5; (5) React compound 5, triphenylphosphine and acetonitrile to obtain the above-mentioned honokiol phosphine salt derivative.

3. The method for preparing the magnolol phosphine salt derivative as described in claim 2, characterized in that, The amino compound mentioned in step (1) is R 2 -NH2,R 2 for , , , , , , or ; In step (1), the ratio of amino compound, dichloromethane, 4-bromobutylacetic acid ester and triethylamine is 13~14 mmol: 10~30 mL: 13~14 mmol: 14.5~15.5 mmol; The reaction in step (1) is carried out at a temperature of 20-30°C for 2-4 hours. The structural formula of compound 1 is as follows: ; Among them, R in compound 1 2 for , , , , , , or .

4. The method for preparing the magnolol phosphine salt derivative as described in claim 3, characterized in that, The structural formula of compound 2 in step (2) is: ; In step (2), the organic base is one or more of N,N-diisopropylethylamine, triethylamine, and pyridine; The coupling reagent in step (2) is one or more of HATU, TBTU and BOP; In step (2), the molar ratio of compound 1, compound 2, organic base and coupling reagent is 5~6:2~3:7.5~8.5:5~6; The temperature for the amide coupling reaction in step (2) is 20~30℃; the time is 6~8h. The structural formula of compound 3 mentioned in step (2) is as follows: ; R in compound 3 2 Independent as , , , , , , or .

5. The method for preparing the magnolol phosphine salt derivative as described in claim 4, characterized in that, The alkali mentioned in step (3) is one or more of sodium hydroxide, potassium hydroxide and lithium hydroxide; The solvent in step (3) comprises methanol and water, wherein the volume ratio of methanol to water is 1~10:1~10; In step (3), the ratio of compound 3, base and solvent is 1~3 mmol: 8~12 mmol: 10~30 mL; The hydrolysis reaction in step (3) is carried out at a temperature of 50~70℃ for 5~10h. The target pH value for acidification in step (3) is ≤3; The structural formula of compound 4 in step (3) is: ; In compound 4, R 2 Independent as , , , , , , or .

6. The method for preparing the magnolol phosphine salt derivative as described in claim 5, characterized in that, The ratio of compound 4, carbon tetrabromide, triphenylphosphine and dichloromethane used in step (4) is 1~2 mmol: 2~4 mmol: 2~4 mmol: 10~30 mL; The bromination reaction in step (4) is carried out at a temperature of 20-30°C for 4-6 hours. The structural formula of compound 5 in step (4) is: ; In compound 5, R 2 Independent as , , , , , , or .

7. The method for preparing the honokiol phosphine salt derivative as described in claim 6, characterized in that, In step (5), the ratio of compound 5, triphenylphosphine, and acetonitrile is 0.1-1 mmol: 4-6 mmol: 5-20 mL. The reaction in step (5) is carried out at a temperature of 70~90℃ for 12~24h.

8. The use of the magnolol phosphine salt derivative as described in claim 1 in the preparation of antibacterial drugs.

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