Multifunctional quinolone anti-biofilm prodrug as well as preparation method and application thereof

Abstract

The invention provides a multifunctional quinolone anti-biofilm prodrug as well as a preparation method and application thereof. The multifunctional quinolone anti-biofilm prodrug has a structure as shown in a formula I, a formula II, a formula III, a formula IV, a formula V or a formula VI. According to the invention, amino ferrocene, a borate group and a quinolone drug are integrated into a prodrug system ABP-QLs, so that the anti-MRSA biofilm effect is realized from multiple dimensions. According to the method, amino ferrocene is utilized to cyclically generate a large amount of ROS to destroy a first defense line, namely an EPS matrix, of a biofilm, and active molecules AF-QLs are promoted to penetrate through the biofilm to remove bacteria in the biofilm while EPS is destroyed. The ABP-QLs disclosed by the invention has a relatively good removal effect on a biofilm, relatively low cytotoxicity and relatively high safety in application to preparation of a prodrug; according to the invention, the quinolone medicines are modified, so that the application of the quinolone medicines in the field of biofilms is widened, and a new thought is provided for removing means of the biofilms.

Description

Technical Field

[0001] The present invention belongs to the technical field of compound medicine, and particularly relates to a multifunctional quinolone antibiofilm prodrug and its preparation method and application. Background Art

[0002] The resistance and tolerance of antibiotics pose a great challenge to the current treatment of bacterial infections. Methicillin-resistant Staphylococcus aureus is a common opportunistic pathogen, often showing high drug resistance due to the formation of biofilms. The complex physical and chemical properties of bacterial biofilms endow them with multiple drug resistance mechanisms. Inhibiting the formation of biofilms and removing mature biofilms have become the focus of preventing and treating related diseases. As a by-product of cellular energy metabolism, reactive oxygen species play important roles in many physiological and pathological processes. Recent studies have found that non-lethal concentrations of antibiotics mediate the accumulation of reactive oxygen species, which can increase the drug resistance and tolerance of bacteria by inducing MarA and SoxS. However, when there is too much reactive oxygen species in the body, it will damage the structure and function of biofilms, and even damage mitochondria, etc., causing cell lysis and thus causing damage to the body.

[0003] Due to the drug resistance and tolerance caused by biofilms, which pose a serious threat to humans and animals and may lead to incurable diseases and death, there is an urgent need to develop new anti-drug-resistant candidate drugs for the treatment of bacterial infections. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a multifunctional quinolone antibiofilm prodrug (ABP-QLs) and its preparation method and application. This prodrug has good biofilm-clearing activity against methicillin-resistant Staphylococcus aureus.

[0005] The present invention provides a multifunctional quinolone antibiofilm prodrug, having the structures of Formula I, Formula II, Formula III, Formula IV, Formula V or Formula VI:

[0006]

[0007]

[0008] In Formulas III to VI, X is selected from -CF or -N; R 1 is independently selected from -CH2CH3 or cyclopropyl; R 2 is independently selected from -C or -H; R 3 is independently selected from -H, -OCH3 or none;

[0009] In Formulas III to V, R 4 is selected from -H or -OCH3.

[0010] The present invention provides a method for preparing the multifunctional quinolone antibiofilm prodrug described in the above technical solution, comprising the following steps:

[0011] Mix ferroceneformic acid, diphenylphosphoryl azide, triethylamine and toluene, and react to obtain 1-azido-1'-ferrocenyl ethanone;

[0012] React 4-(hydroxymethyl)phenylboronic acid pinacol ester and 1-azido-1'-ferrocenyl ethanone in a toluene solution to obtain (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2-ethoxycarbonyl ferrocene cyanamide;

[0013] React a linker, (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2-ethoxycarbonyl ferrocene cyanamide, cesium carbonate, tetrabutylammonium iodide, and potassium iodide in DMF to obtain an intermediate compound; the linker is selected from m-xylene dibromide, 1,3-dibromopropane, ethylene glycol ditosylate or tetraethylene glycol ditosylate;

[0014] React the intermediate compound, a quinolone series compound and N,N-diisopropylethylamine in DMF to obtain a multifunctional quinolone antibiofilm prodrug;

[0015] The quinolone series compounds are selected from norfloxacin, ciprofloxacin, enoxacin, gatifloxacin, sparfloxacin, moxifloxacin or pipemidic acid.

[0016] The present invention provides an application of the multifunctional quinolone antibiofilm prodrug described in the above technical solution in the preparation of a drug for antibiofilm.

[0017] The present invention provides a multifunctional quinolone antibiofilm prodrug having a structure of Formula I, Formula II, Formula III, Formula IV, Formula V or Formula VI. The present invention integrates aminoferrocene, borate group and quinolone drugs into the prodrug system ABP-QLs to achieve the anti-MRSA biofilm effect from multiple dimensions. The present invention utilizes the cyclic generation of a large amount of ROS by aminoferrocene to destroy the first line of defense of the biofilm - the EPS matrix. While the EPS is being destroyed, it promotes the penetration of the active molecule AF-QLs through the biofilm to eliminate the bacteria inside the film. The ABP-QLs of the present invention have a good scavenging effect on the biofilm, with low cytotoxicity and high safety in the application of preparing prodrugs; the modification of quinolone drugs by the present invention broadens the application of quinolone drugs in the field of biofilms and provides a new idea for the means of eliminating biofilms. Description of the Drawings

[0018] Figure 1 1H NMR spectrum of compound 2 in Example 1;

[0019] Figure 2 1H NMR spectrum of Compound 2 in Example 1;

[0020] Figure 3 1H NMR spectrum of Compound 3 in Example 1;

[0021] Figure 4 13C NMR spectrum of Compound 3 in Example 1;

[0022] Figure 5 1H NMR spectrum of Compound 6 in Example 1;

[0023] Figure 6 13C NMR spectrum of Compound 6 in Example 1;

[0024] Figure 7 1H NMR spectrum of ABP-QLs prodrug 7a in Example 1;

[0025] Figure 8 13C NMR spectrum of ABP-QLs prodrug 7a in Example 1;

[0026] Figure 9 1H NMR spectrum of Compound 8 in Example 2;

[0027] Figure 10 13C NMR spectrum of Compound 8 in Example 2;

[0028] Figure 11 1H NMR spectrum of ABP-QLs prodrug 9a in Example 2;

[0029] Figure 12 13C NMR spectrum of ABP-QLs prodrug 9a in Example 2;

[0030] Figure 13 1H NMR spectrum of ABP-QLs prodrug 5e in Example 3;

[0031] Figure 14 13C NMR spectrum of ABP-QLs prodrug 5e in Example 3;

[0032] Figure 15 1H NMR spectrum of ABP-QLs prodrug 9c in Example 4;

[0033] Figure 16 13C NMR spectrum of ABP-QLs prodrug 9c in Example 4;

[0034] Figure 17 1H NMR spectrum of Compound 10 in Example 5;

[0035] Figure 18 13C NMR spectrum of compound 10 in Example 5;

[0036] Figure 19 1H NMR spectrum of ABP-QLs prodrug 11a in Example 5;

[0037] Figure 20 13C NMR spectrum of ABP-QLs prodrug 11a in Example 5;

[0038] Figure 21 Biofilm eradication of ABP-QLs prodrug 9c and ABP-QLs prodrug 5e against MRSA25-1. Detailed implementation mode

[0039] The present invention provides a multifunctional quinolone antibiofilm prodrug having the structure of Formula I, Formula II, Formula III, Formula IV, Formula V or Formula VI:

[0040]

[0041] In Formulas III to VI, X is selected from -CF or -N; R 1 is independently selected from -CH2CH3 or cyclopropyl; R 2 is independently selected from -C or -H; R 3 is independently selected from -H, -OCH3 or blank;

[0042] In Formulas III to V, R 4 is selected from -H or -OCH3.

[0043] In the present invention, -Bpin in Formulas I, II, III, IV, V or VI is

[0044] In the present invention, R 3 is selected from none, that is, R does not exist 3 .

[0045] In the present invention, in Formulas III to VI, X = -CF, R 1 = -CH2CH3, R 2 = -C, R 3 = -H, R 4 = -H;

[0046] or X = -CF, R 1 = cyclopropyl, R 2 = -C, R 3 = -OCH3, R 4 = -OCH3;

[0047] or X = -CF, R 1 = cyclopropyl, R 2=-C, R 3 =-H, R 4 =-H;

[0048] or X = -CF, R 1 =-CH2CH3, R 2 =-N, no R 3 , R 4 =-H;

[0049] or X = -N, R 1 =-CH2CH3, R 2 =-N, no R 3 , R 4 =-H.

[0050] In the present invention, in formula VI, X = -CF, R 1 =-CH2CH3, R 2 =-C, R 3 =-H, R 4 =-H;

[0051] or X = -CF, R 1 =cyclopropyl, R 2 =-C, R 3 =-OCH3, R 4 =-OCH3.

[0052] In the present invention, the multifunctional quinolone antibiofilm prodrug is the compound prodrug 5a, prodrug 5b, prodrug 5c, prodrug 5d, prodrug 5e, prodrug 5f, prodrug 5g, prodrug 7a, prodrug 7b, prodrug 7c, prodrug 7d, prodrug 7e, prodrug 9a, prodrug 9b, prodrug 9c, prodrug 9d, prodrug 9e, prodrug 11a or prodrug 11b;

[0053] Among them, prodrug 5a is X = -CF, R in formula III 1 =-CH2CH3, R 2 =-C, R 3 =-H, R 4 =-H; Prodrug 7a is X = -CF, R in formula V 1 =-CH2CH3, R 2 =-C, R 3 =-H, R 4 =-H; Prodrug 9a is X = -CF, R in formula IV 1 =-CH2CH3, R 2 =-C, R 3 =-H, R 4 =-H; Prodrug 11a is X = -CF, R in formula VI 1 =-CH2CH3, R 2 =-C, R3 = -H, R 4 = -H.

[0054] Prodrug 5b has X = -CF in Formula III, R 1 = cyclopropyl, R 2 = -C, R 3 = -OCH3, R 4 = -OCH3, Prodrug 7b has X = -CF in Formula V, R 1 = cyclopropyl, R 2 = -C, R 3 = -OCH3, R 4 = -OCH3, Prodrug 9b has X = -CF in Formula IV, R 1 = cyclopropyl, R 2 = -C, R 3 = -OCH3, R 4 = -OCH3, Prodrug 11b has X = -CF in Formula VI, R 1 = cyclopropyl, R 2 = -C, R 3 = -OCH3, R 4 = -OCH3.

[0055] Prodrug 5c has X = -CF in Formula III, R 1 = cyclopropyl, R 2 = -C, R 3 = -H, R 4 = -H, Prodrug 7c has X = -CF in Formula V, R 1 = cyclopropyl, R 2 = -C, R 3 = -H, R 4 = -H, Prodrug 9c has X = -CF in Formula IV, R 1 = cyclopropyl, R 2 = -C, R 3 = -H, R 4 = -H.

[0056] Prodrug 5d has X = -CF in Formula III, R 1 = -CH2CH3, R 2 = -N, no R 3 , R 4 = -H; Prodrug 7d has X = -CF in Formula V, R 1 = -CH2CH3, R 2 = -N, no R 3 , R 4 = -H; Prodrug 9d has X = -CF in Formula IV, R 1 = -CH2CH3, R 2 = -N, no R 3 , R4 = -H.

[0057] The prodrug 5e is such that in formula III, X = -N, R 1 = -CH2CH3, R 2 = -N, without R 3 , R 4 = -H; the prodrug 7e is such that in formula V, X = -N, R 1 = -CH2CH3, R 2 = -N, without R 3 , R 4 = -H; the prodrug 9e is such that in formula IV, X = -N, R 1 = -CH2CH3, R 2 = -N, without R 3 , R 4 = -H.

[0058] In specific embodiments of the present invention, the multifunctional quinolone antibiofilm prodrugs (ABP-QLs) are specifically prodrug 7a, prodrug 9a, prodrug 5e, prodrug 9c, and prodrug 11a:

[0059]

[0060]

[0061]

[0062] The above-mentioned multifunctional quinolone antibiofilm prodrugs provided by the present invention have a good effect on clearing biofilms, have relatively low cytotoxicity, and are highly safe in the application of preparing prodrugs; by modifying quinolone drugs, the present invention broadens the application of quinolone drugs in the field of biofilms and provides a new idea for the means of clearing biofilms.

[0063] The present invention provides a preparation method for the above-mentioned multifunctional quinolone antibiofilm prodrug according to the above technical solution, comprising the following steps:

[0064] Mix ferroceneformic acid, diphenylphosphoryl azide, triethylamine, and toluene, and react to obtain 1-azido-1'-ferrocenyl ethanone;

[0065] React 4-(hydroxymethyl)phenylboronic acid pinacol ester and 1-azido-1'-ferrocenyl ethanone in a toluene solution to obtain (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2-ethoxycarbonylferrocene cyanamide;

[0066] React a linker, (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2-ethoxycarbonylferrocenecarbamide, cesium carbonate, tetrabutylammonium iodide, and potassium iodide in DMF to obtain an intermediate compound; the linker is selected from m-xylylene dibromide, 1,3-dibromopropane, ethylene glycol ditosylate, and tetraethylene glycol ditosylate;

[0067] React the intermediate compound, a quinolone series compound, and N,N-diisopropylethylamine in DMF to obtain a multifunctional quinolone antibiofilm prodrug;

[0068] The quinolone series compounds are selected from norfloxacin, ciprofloxacin, enoxacin, gatifloxacin, sparfloxacin, moxifloxacin, or pipemidic acid.

[0069] The preparation method of the ABP-QLs prodrug provided by the present invention is simple, the raw materials are cheap, and it is prepared into an antibiofilm prodrug, having a large market space.

[0070] In the present invention, the molar ratio of ferroceneformic acid, diphenylphosphoryl azide, and triethylamine is 1:(1.4 - 1.6):(1.9 - 2.1).

[0071] In the present invention, the molar ratio of 4-hydroxymethylphenylboronic acid pinacol ester and 1-azido-1'-ferrocenylethanone is 1.9 - 2.2:1; specifically, it can be 1.9:1, 2.0:1, 2.1:1, or 2.2:1. The reaction temperature of 4-hydroxymethylphenylboronic acid pinacol ester and 1-azido-1'-ferrocenylethanone is 98 - 103 °C, and the reaction time is 3.5 - 4.5 h; the reaction is carried out under magnetic stirring; in specific examples, the reaction temperature of 4-hydroxymethylphenylboronic acid pinacol ester and 1-azido-1'-ferrocenylethanone is 100 °C, and the time is 4 h. After the reaction is completed, it is monitored by TLC analysis; after the solvent is concentrated under reduced pressure, it is purified by column chromatography (petroleum ether / ethyl acetate) to obtain (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2-ethoxycarbonylferrocenecarbamide.

[0072] In the present invention, the molar ratio of (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2-ethoxycarbonylferrocenecarbamide and the linker is 1:1.9 - 2.2; specifically, it can be 1:1.9, 1:2.0, 1:2.1, or 1:2.2.

[0073] In the present invention, the temperature for preparing the intermediate compound is room temperature of 20 - 35 °C or 55 - 65 °C, and the reaction time is 4 - 6 h; after the reaction is completed, it is monitored by TLC analysis. It is quenched with ultrapure water and extracted with ethyl acetate (3×10). The organic phase is concentrated under reduced pressure and purified by column chromatography (petroleum ether / ethyl acetate) to obtain the intermediate compound.

[0074] In the present invention, different intermediate compounds are obtained through different linkers; the shown linkers are selected from m - dibenzyl bromide, 1,3 - dibromopropane, ethylene glycol ditosylate, tetraethylene glycol ditosylate; in specific embodiments, the shown intermediate compounds are compound 4 (N - ferrocenyl - 2 - (4 - pinacol boronic acid ester phenyl) - 2 - (4 - bromophenyl) acetamide), compound 6 (N - ferrocenyl - 2 - (4 - pinacol boronic acid ester phenyl) - 2 - bromoacetamide), compound 8 (N - ferrocenyl - [2 - (2 - tosyl oxyethoxy) ethyl] - (4 - pinacol borate benzoxy) acetamide) or compound 10 (N - ferrocenyl - 2 - [(4 - pinacol boronic acid ester phenyl) methoxy] ethyl 4 - methylbenzenesulfonic acid carbamate).

[0075] In the present invention, the molar ratio of the intermediate compound to the quinolone series compounds is 1:(1.4 - 1.6); specifically, it can be 1:1.4, 1:1.5 or 1:1.6.

[0076] In the present invention, the quinolone series compounds (QLs) are selected from norfloxacin, ciprofloxacin, enoxacin, gatifloxacin, sparfloxacin, moxifloxacin or pipemidic acid. In the present invention, the temperature for the reaction of the intermediate compound, the quinolone series compound and N,N - diisopropylethylamine in DMF is room temperature, and the room temperature is preferably 15 - 30 °C; the reaction time is 11 - 13 h. After the reaction is completed, it is monitored by TLC analysis; the reaction solution is concentrated under reduced pressure, a small amount of dichloromethane is added, and then purified by column chromatography (dichloromethane / methanol) to obtain the prodrug compound ABP - QLs.

[0077] In a specific embodiment of the present invention, the preparation route 1 of the multifunctional quinolone antibiofilm prodrug with the structure of formula V is as follows:

[0078]

[0079] In a specific embodiment of the present invention, the preparation route 2 of the multifunctional quinolone antibiofilm prodrug with the structures of formula IV and formula VI is as follows:

[0080]

[0081] Route 2;

[0082] In a specific embodiment of the present invention, the preparation route 3 of the multifunctional quinolone antibiofilm prodrug with the structures shown in Formula I, Formula II and Formula III is as follows:

[0083]

[0084] The present invention makes use of the characteristic of high H2O2 content in the biofilm microenvironment, and innovatively integrates a borate ester group (H2O2-responsive site), AF (ROS amplifier, i.e., ferrocenyl group), and quinolone (chemotherapeutic drug) into the prodrug system ABP-QLs to achieve the anti-MRSA biofilm effect in multiple dimensions.

[0085] The present invention realizes the recognition and activation of the MRSA biofilm microenvironment through the H2O2-responsive group, improving the therapeutic effect and safety of the drug; uses AF to cyclically generate a large amount of ROS to destroy the first line of defense of the biofilm - the EPS matrix; while the EPS is being destroyed, promotes the penetration of the active molecule AF-QLs through the biofilm to eliminate the bacteria inside the film.

[0086] The p-QM formed by the activation and self-cleavage of the prodrug provided by the present invention further amplifies the oxidative stress level by alkylating intracellular GSH, achieving a self-sensitizing effect.

[0087] The present invention provides an application of the multifunctional quinolone antibiofilm prodrug described in the above technical solution in the preparation of an antibiofilm drug.

[0088] In the present invention, the antibiofilm drug is an anti-methicillin-resistant Staphylococcus aureus biofilm drug.

[0089] In order to further illustrate the present invention, the following examples are used to describe in detail a multifunctional quinolone antibiofilm prodrug provided by the present invention, its preparation method and application, but they should not be construed as limiting the protection scope of the present invention.

[0090] Example 1

[0091] Preparation of the prodrug with m-xylene dibromide as the linker

[0092] This example prepares the multifunctional quinolone antibiofilm prodrug compound 7a:

[0093]

[0094] The preparation method of the compound with the structure shown in Formula I is as follows;

[0095] (1) Synthesis of 1-azido-1'-ferrocenyl ethanone (Compound 2)

[0096] Add ferrocenecarboxylic acid (10 mmol, 1 equiv.), 10 mL of toluene solvent, diphenylphosphoryl azide (15 mmol, 1.5 equiv.), and triethylamine (20 mmol, 2 equiv.) to a 50 mL round-bottom flask, and stir at room temperature for 6 hours. Monitor by TLC. After the reaction is completed, quench with ultrapure water, extract with ethyl acetate (4×70), collect and combine the organic phases, then extract with 2% HCl, and finally wash with saturated NaHCO3. Collect the organic phase, concentrate under reduced pressure, and separate and purify by column chromatography (petroleum ether / ethyl acetate) to obtain a red solid, which is compound 2 (2.14 g, 84%).

[0097] The 1H-NMR spectrum of the product is as follows Figure 1 : 1 1H-NMR (600 MHz, CDCl3) δ 4.81 (t, J = 2.0 Hz, 2H), 4.51 (t, J = 2.0 Hz, 2H), 4.25 (s, 5H).

[0098] The 13C-NMR spectrum of the product is as follows Figure 2 , 13 13C-NMR (150 MHz, CDCl3) δ 72.79, 70.50, 70.36.

[0099] The high-resolution mass spectrometry results are as follows: HRMS (ESI) calculated for C 11 H9FeN3O [M+Na + + is 277.9993, found 277.9987.

[0100] The synthesis procedure is as follows

[0101]

[0102] (2) Synthesis of (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2-ethoxycarbonylferrocenecarboxamide (Compound 3)

[0103] Add 4-(hydroxymethyl)phenylboronic acid pinacol ester (2.0 mmol, 2 equiv.), compound 2 (1.0 mmol, 1 equiv.), and 5 mL of toluene to a 50 mL sealed tube in sequence, and react in a 100 °C sand bath for 4 hours. Monitor by TLC. After the reaction is completed, directly concentrate under reduced pressure, and separate and purify by column chromatography (PE / EA) to obtain a yellow solid, which is compound 3 (327.3 mg, 71%).

[0104] The 1H-NMR spectrum of the product is as follows Figure 3 , and the results are as follows 11H NMR (600 MHz, CDCl3) δ 7.81 (d, J = 7.5 Hz, 2H), 7.37 (d, J = 7.5 Hz, 2H), 5.16 (s, 2H), 4.48 (s, 2H), 4.13 (d, J = 7.8 Hz, 5H), 3.96 (s, 2H), 1.33 (s, 13H).

[0105] The 1H NMR spectrum of the product is as follows Figure 4 , and the results are as follows: 13 13C-NMR (150 MHz, CDCl3) δ 171.28, 153.74, 139.36, 135.13, 127.26, 95.56, 83.97, 69.25, 66.82, 64.52, 60.73 (d, J = 68.6 Hz), 24.96, 21.15, 14.29.

[0106] The results of high-resolution mass spectrometry are as follows: HRMS (ESI) calculated for C 24 H 28 BFeNO4 [M + H + + molecular weight is 462.1534, found 462.1501.

[0107] The synthesis steps are as follows:

[0108]

[0109] (3) Synthesis of N-ferrocenyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2-bromoacetamide (Compound 6)

[0110] Compound 3 (0.1 mmol, 1 equiv.), 1,3-dibromopropane (0.2 mmol, 2 equiv.), Cs2CO3 (0.3 mmol, 3 equiv.), TBAI (50%), KI (20%), and 1 mL of DMF were added to a 10 mL reaction tube and stirred at room temperature for 5 hours. Monitored by TLC. After completion of the reaction, it was extracted with EA (5 × 3), the combined organic layers were collected, washed with brine, dried over Na2SO4, the solvent was removed by rotary evaporation under reduced pressure, and then purified by column chromatography (PE:EA = 7:1) to obtain a pale yellow solid (48.8 mg, 84%).

[0111] The 1H NMR spectrum of the product is as follows Figure 5 , and the results are as follows: 1H-NMR(600MHz, Acetone-d6) δ 7.74 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 5.21 (s, 2H), 4.54 (s, 2H), 4.12 (s, 5H), 3.99 (t, J = 2.0 Hz, 2H), 3.89–3.84 (m, 2H), 3.57 (t, J = 6.5 Hz, 2H), 2.31–2.25 (m, 2H), 1.31 (s, 12H).

[0112] The 1H-NMR spectrum of the product is as Figure 6 , and the results are as follows: 13 C-NMR(150MHz, Acetone-d6) δ 205.36, 140.12, 134.83, 130.08, 127.23, 83.76, 70.90, 68.92, 66.90, 64.34, 62.04, 37.21, 31.73, 31.26, 24.37.

[0113] The results of high-resolution mass spectrometry are as follows: HRMS(ESI) calculated for C 27 H 33 BBrFeNO4 [M+Na + molecular weight is 604.0935, found to be 604.0960.

[0114] The synthesis procedure is as follows:

[0115]

[0116] (4) Synthesis of ABP-QLs prodrug 7a

[0117] To a 10 mL reaction tube, add compound 6 (0.1 mmol, 1 equiv.), norfloxacin (0.2 mmol, 2 equiv.), N,N-diisopropylethylamine (0.3 mmol, 3 equiv.), 1 mL of DMF, and stir in a 60 °C sand bath for 8 h. Monitor by TLC. After the reaction is complete, extract with EA (5×3), collect and combine the organic layers, wash with brine, dry over Na2SO4, remove the solvent under reduced pressure, and then separate and purify by column chromatography (DCM / MeOH) to obtain a pale yellow solid, which is ABP-QLs prodrug 7a (45.9 mg, 56%).

[0118] The 1H-NMR spectrum of the product is as Figure 7 , and the results are as follows: 1H-NMR (400 MHz, CDCl3) δ 8.60 (s, 1H), 7.94 (d, J = 12.8 Hz, 1H), 7.75 (d, J = 7.6 Hz, 2H), 7.33 (d, J = 7.6 Hz, 2H), 7.20 (s, 1H), 5.14 (s, 2H), 4.29 (s, 4H), 4.06 (s, 5H), 3.93 (s, 2H), 3.69 (s, 2H), 3.28 (s, 4H), 2.62 (s, 4H), 2.45 (s, 2H), 1.88 (s, 2H), 1.52 (s, 3H), 1.25 (s, 12H).

[0119] 1H-NMR of the product is as follows Figure 8 , and the results are as follows: 13 13C-NMR (150 MHz, DMSO-d6) δ 176.59, 166.63, 154.15, 152.49, 148.97, 140.29, 137.62, 135.05, 127.84, 119.68, 111.65, 107.49, 106.26, 84.14, 69.26, 67.05, 64.67, 56.50, 55.27, 52.79, 49.55, 30.21, 25.41, 25.05, 19.03, 14.84.

[0120] The results of high-resolution mass spectrometry are as follows: HRMS (ESI) calculated for C 43 H 50 BFFeN4O7 [M + H + + is 821.3184. Found 821.3176.

[0121] The synthetic procedure is as follows:

[0122]

[0123] Example 2

[0124] Preparation of Diethylene Glycol Di-p-toluenesulfonate as a Linker Prodrug

[0125] In this example, compound 9a was prepared:

[0126]

[0127] (1) Synthesis of 1-Azido-1'-ferrocenyl ethanone (Compound 2)

[0128] As shown in step (1) of Example 1.

[0129] (2) Synthesis of Fc-NH-Bpin (Compound 3)

[0130] As shown in step (2) of Example 1.

[0131] (3) Synthesis of N-ferrocenyl-[2-(2-(p-toluenesulfonyloxy)ethoxy)ethyl]-(4-pinacolborate benzyloxy)acetamide (Compound 8)

[0132] Compound 3 (0.1 mmol, 1 equiv.), diethylene glycol ditosylate (0.5 mmol, 5 equiv.), Cs2CO3 (0.6 mmol, 6 equiv.), TBAI (50%), KI (20%), and 1 mL of DMF were added to a 10 mL reaction tube and stirred at 80 °C for 6 hours. Monitored by TLC. After completion of the reaction, it was extracted with EA (5×3), the organic layers were combined, washed with brine, dried over Na2SO4, and the solvent was removed by distillation under reduced pressure. The crude product was separated and purified by column chromatography (PE / EA) to obtain a yellow liquid, which is Compound 8 (25.3 mg, 36%).

[0133] The 1H NMR spectrum of the product is as Figure 9 , and the results are as follows: 1 1H-NMR (400 MHz, Acetone-d6) δ 7.82 (s, 1H), 7.80 (s, 1H), 7.77 (s, 1H), 7.75 (s, 1H), 7.48 (s, 1H), 7.46 (s, 1H), 7.44 (s, 1H), 7.42 (s, 1H), 5.20 (s, 2H), 4.19–4.16 (m, 2H), 4.15 (s, 5H), 4.00 (t, J = 2.0 Hz, 2H), 3.88 (t, J = 6.0 Hz, 2H), 3.68 (t, J = 6.1 Hz, 4H), 2.86 (s, 2H), 2.45 (s, 3H), 1.34 (s, 12H).

[0134] The 13C NMR spectrum of the product is as Figure 10 , and the results are as follows: 13 13C-NMR (100 MHz, Acetone-d6) δ 144.93, 140.09, 134.78, 133.41, 130.00, 127.84, 127.10, 101.44, 83.70, 69.69, 68.80, 68.39, 66.77, 64.25, 62.99, 62.83, 62.78, 62.72, 39.35, 24.34, 20.71.

[0135] The high-resolution mass spectrometry results are as follows: HRMS (ESI) calculated for C 35 H 42 BFeNO8S [M+H +The molecular weight is 704.2152, and the measured value is 704.2113.

[0136] The synthesis steps are as follows:

[0137]

[0138] (4) Synthesis of ABP-QLs prodrug 9a

[0139] Add compound 8 (0.1 mmol, 1 equiv.), norfloxacin (0.2 mmol, 2 equiv.), N,N-diisopropylethylamine (0.6 mmol, 6 equiv.), and 1 mL of DMF to a 10 mL reaction tube, and stir in a 60 °C sand bath for 8 h. Monitor by TLC. After the reaction is complete, extract with EA (5×3), collect and combine the organic layers, wash with brine, dry over Na2SO4, remove the solvent by distillation under reduced pressure, and then separate and purify by column chromatography (DCM / MeOH) to obtain a dark yellow solid (45.1 mg, 53%).

[0140] The 1H-NMR spectrum of the product is as Figure 11 , and the results are as follows: 1 1H-NMR (400 MHz, CDCl3) δ 15.07 (s, 1H), 7.98 (s, 1H), 7.74 (d, J = 6.8 Hz, 3H), 7.31 (d, J = 7.1 Hz, 3H), 5.13 (s, 2H), 4.39 (s, 4H), 4.08 (s, 5H), 3.93 (s, 6H), 3.65 (s, 6H), 3.33 (s, 2H), 2.68 (s, 4H), 1.26 (s, 12H), 1.18 (s, 3H).

[0141] The 13C-NMR spectrum of the product is as Figure 12 , and the results are as follows: 13 13C-NMR (100 MHz, DMSO-d6) δ 176.61, 166.59, 154.56, 152.08, 148.99, 140.31, 137.63, 135.01, 134.71, 128.50, 127.56, 125.96, 111.75, 111.53, 107.54, 84.14, 69.23, 68.50, 67.03, 64.67, 57.45, 53.15, 49.92, 49.57, 29.51, 25.43, 25.09, 21.25, 19.04, 14.81.

[0142] The high-resolution mass spectrometry results are as follows: HRMS (ESI) calculated for C 44 H 52 BFFeN4O8 [M+H+ The molecular weight is 851.3290, and the measured value is 851.3276.

[0143] The synthesis steps are as follows:

[0144]

[0145] Example 3

[0146] Preparation of Prodrug with m-Xylylene Dibromide as Linker

[0147] In this example, the multifunctional quinolone antibiofilm prodrug compound 5e was prepared:

[0148]

[0149] (1) Synthesis of 1-Azido-1'-ferrocenylacetone (Compound 2)

[0150] As shown in step (1) of Example 1.

[0151] (2) Synthesis of Fc-NH-Bpin (Compound 3)

[0152] As shown in step (2) of Example 1.

[0153] (3) Synthesis of N-Ferrocenyl-2-(4-(4,4,5,5-Tetramethyl-1,3,2-Dioxaborolan-2-Yl)phenyl)-2-(4-Bromophenyl)Acetamide (4)

[0154] Add m-xylylene dibromide (0.2 mmol, 2 equiv.), compound 3 (0.1 mmol, 1 equiv.), Cs2CO3 (0.3 mmol, 3 equiv.), TBAI (50%), KI (20%) to a 10 mL reaction tube, and 1 mL of DMF. Stir at room temperature for 4 hours. Monitor by TLC. After the reaction is completed, quench with ultrapure water. Extract with EA (3×20), collect and combine the organic phases, concentrate under reduced pressure, and purify by column chromatography (PE:EA = 7:1) to obtain a yellow oil, which can be used directly in the next step without further purification.

[0155] The synthesis steps are as follows:

[0156]

[0157] (4) Synthesis of ABP-QLs Prodrug 5e

[0158] Compound 4 (0.1 mmol, 1 equiv.), pipemidic acid (0.2 mmol, 2 equiv.), DIPEA (0.3 mmol, 3 equiv.), and 1 mL of DMF were added to a 10 mL reaction tube and stirred at room temperature for 8 h. Monitored by TLC. After the reaction was completed, it was extracted with EA (5×3), the organic layers were collected and combined, washed with brine, dried over Na2SO4, the solvent was removed by distillation under reduced pressure, and then separated and purified by column chromatography (DCM:MeOH = 25:1) to obtain a yellow solid (59.3 mg, 67%).

[0159] The 1H NMR spectrum of the product is as Figure 13 , and the results are as follows: 1 1H-NMR (600 MHz, DMSO-d6) δ 8.91 (s, 1H), 7.98 (d, J = 13.6 Hz, 1H), 7.57 (s, 2H), 7.29 (q, J = 7.1, 6.5 Hz, 2H), 7.17 (d, J = 16.9 Hz, 4H), 5.15 (s, 2H), 4.88 (s, 2H), 4.47 (d, J = 8.9 Hz, 2H), 4.41 - 4.33 (m, 3H), 4.05 (s, 5H), 4.02 (s, 1H), 3.96 (t, J = 2.0 Hz, 2H), 3.75 (s, 1H), 3.71 (s, 3H), 3.44 (s, 2H), 2.44 (s, 3H), 1.30 (d, J = 7.1 Hz, 3H), 1.21 (s, 12H).

[0160] The 1H NMR spectrum of the product is as Figure 14 , and the results are as follows: 13 13C-NMR (150 MHz, DMSO-d6) δ 176.85, 166.37, 150.37, 148.21, 146.50, 145.32, 134.99, 128.94, 128.06, 127.12, 125.72, 119.92 (d, J = 22.1 Hz), 113.13, 108.61, 84.18, 71.92, 69.35 (d, J = 4.0 Hz), 64.70, 62.74, 62.20, 52.75, 47.69, 47.21 (d, J = 7.5 Hz), 25.14, 15.17.

[0161] The high-resolution mass spectrometry results are as follows: HRMS (ESI) calculated for C 46 H 51 BFeN6O7 [M + H + the molecular weight is 867.3334, found 867.3344.

[0162] The synthetic procedure is shown below:

[0163]

[0164] Example 4

[0165] Preparation of Prodrug with Diethylene Glycol Di-p-toluenesulfonate as Linker

[0166] In this example, the multifunctional quinolone antibiofilm prodrug compound 9c was prepared as follows:

[0167]

[0168] (1) Synthesis of 1-azido-1'-ferrocenyl ethanone (Compound 2)

[0169] As shown in step (1) of Example 1.

[0170] (2) Synthesis of Fc-NH-Bpin (Compound 3)

[0171] As shown in step (2) of Example 1.

[0172] (3) Synthesis of N-ferrocenyl-[2-(2-p-toluenesulfonyloxyethoxy)ethyl]-(4-pinacol borate benzoxy)acetamide (Compound 8)

[0173] As shown in step (3) of Example II.

[0174] (4) Synthesis of ABP-QLs Prodrug 9c

[0175] Add compound 8 (0.1 mmol, 1 equiv.), ciprofloxacin (0.2 mmol, 2 equiv.), DIPEA (0.6 mmol, 6 equiv.), and 1 mL of DMF to a 10 mL reaction tube, and stir in a 60 °C sand bath for 18 h. Monitor by TLC. After the reaction is complete, extract with EA (5×3), collect and combine the organic layers, wash with brine, dry over Na2SO4, remove the solvent under reduced pressure, and purify by column chromatography (DCM:MeOH = 25:1) to obtain a dark yellow solid (33.6 mg, 39%)

[0176] The 1H NMR spectrum of the product is as follows Figure 15 , and the results are as follows: 1H-NMR (400 MHz, CDCl3) δ 14.91 (s, 1H), 8.68 (s, 1H), 7.93 (s, 1H), 7.76–7.61 (m, 3H), 7.31 (d, J = 7.5 Hz, 2H), 5.13 (s, 2H), 4.37 (s, 3H), 4.07 (s, 5H), 3.93 (s, 2H), 3.86 (s, 2H), 3.77–3.52 (m, 8H), 3.05 (s, 6H), 1.26 (s, 12H), 1.18 (s, 4H).

[0177] 1H-NMR of the product is as follows Figure 16 , and the results are as follows: 13 13C-NMR (100 MHz, DMSO-d6) δ 176.81, 166.32, 148.23, 145.26, 140.32, 135.00, 127.55, 108.54, 84.14, 69.22, 67.01, 64.65, 56.49, 53.24, 47.70, 25.43, 25.10, 19.04, 15.16.

[0178] The results of high-resolution mass spectrometry are as follows: HRMS (ESI) calculated for C 45 H 52 BFFeN4O8 [M + H + + m / z 863.3290, found 863.3281.

[0179] Example 5

[0180] Preparation of prodrug with tetraethylene glycol ditosylate as linker

[0181] In this example, the multifunctional quinolone antibiofilm prodrug compound 11a was prepared:

[0182]

[0183] (1) Synthesis of 1-azido-1'-ferrocenyl ethanone (Compound 2)

[0184] As shown in step (1) of Example 1.

[0185] (2) Synthesis of Fc-NH-Bpin (Compound 3)

[0186] As shown in step (2) of Example 1.

[0187] (3) Synthesis of N-ferrocenyl-2-[(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methoxy]ethyl 4-methylbenzenesulfonamide (10)

[0188] Compound 3 (0.1 mmol, 1 equiv.), tetraethylene glycol ditosylate (0.5 mmol, 5 equiv.), Cs2CO3 (0.6 mmol, 6 equiv.), TBAI (50%), KI (20%), and 1 mL of DMF were added to a 10 mL reaction tube and stirred at 60 °C for 8 h. Monitored by TLC. After the reaction was completed, it was extracted with EA (5×3), the organic layers were collected and combined, washed with brine, dried over Na2SO4, and the solvent was removed by distillation under reduced pressure. The crude product was separated and purified by a silica gel column (PE:EA = 2:1) to obtain a pale yellow liquid (60.1 mg, 76%).

[0189] The 1H NMR spectrum of the product is as Figure 17 , and the results are as follows: 1 1H-NMR (400 MHz, CDCl3) δ 7.80 (s, 1H), 7.78 (s, 2H), 7.77 (s, 1H), 7.35 (d, J = 7.7 Hz, 2H), 7.31 (d, J = 7.9 Hz, 2H), 5.16 (s, 2H), 4.15 (s, 3H), 4.13 (t, J = 4.9 Hz, 5H), 4.00 (s, 2H), 3.90 (s, 2H), 3.65 (t, J = 4.9 Hz, 6H), 3.58 (s, 4H), 3.55 (s, 4H), 2.42 (s, 3H), 1.33 (s, 12H).

[0190] The 1H NMR spectrum of the product is as Figure 18 , and the results are as follows: 13 13C-NMR (150 MHz, CDCl3) δ 144.88, 139.42, 135.08, 133.08, 129.92, 129.52, 128.07 (d, J = 4.9 Hz), 127.26, 114.68, 83.97, 70.84, 70.78, 70.68, 70.66–70.64 (m), 69.34, 68.81 (d, J = 7.9 Hz), 67.48, 64.90–64.58 (m), 38.04, 24.97, 21.75, 21.16, 14.29

[0191] The high-resolution mass spectrometry results are as follows: HRMS (ESI) calculated for C 39 H 50 BFeNO 10 S [M + H+] molecular weight is 792.2676. Found 792.2649.

[0192] The synthesis procedure is shown as follows:

[0193]

[0194] Synthesis of (4)N-ferrocenyl-2-{2-[2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)ethoxy]ethoxy}-N-[4-fluoro-2-(ethylamino-6-oxo-1,6-dihydropyridin-3-yl]acetamide

[0195] Add compound 10 (0.1 mmol, 1 equiv.), norfloxacin (0.2 mmol, 2 equiv.), DIPEA (0.6 mmol, 6 equiv.), and 1 mL of DMF to a 10 mL reaction tube, and stir in a sand bath at 60 °C for 8 h. Monitor by TLC. After the reaction is complete, extract with EA (5×3), combine the organic layers, wash with brine, dry over Na2SO4, remove the solvent by distillation under reduced pressure, and then separate and purify by column chromatography (DCM:MeOH = 25:1) to obtain a yellow solid (37.5 mg, 40%).

[0196] The 1H NMR spectrum of the product is as Figure 19 follows: 1 1H-NMR (600 MHz, CDCl3) δ 8.65 (s, 1H), 7.98 (s, 1H), 7.77 (d, J = 7.5 Hz, 2H), 7.33 (d, J = 7.5 Hz, 2H), 7.16–6.76 (m, 1H), 5.15 (s, 2H), 4.36 (s, 4H), 4.11 (s, 5H), 3.95 (s, 2H), 3.89 (s, 2H), 3.62 (s, 14H), 3.01 (s, 8H), 1.32 (s, 12H), 1.23 (s, 3H).

[0197] The 1H NMR spectrum of the product is as Figure 20 follows: 13 13C-NMR (150 MHz, DMSO-d6) δ 166.62, 140.33, 137.68, 135.07, 134.75, 127.58, 127.21, 126.03, 125.44, 111.65, 109.37, 107.67, 106.30, 84.22, 79.89-79.18 (m), 71.09, 70.60-70.17 (m), 70.21, 69.28, 64.86–64.59 (m), 58.07, 52.06, 46.85, 25.49, 25.19, 14.97.

[0198] The high-resolution mass spectrometry results are as follows: HRMS (ESI) calculated for C 48 H 60 BFFeN4O 10 + of+ Molecular weight is 939.3814. Actual measurement: 939.3819.

[0199] The synthesis steps are as follows:

[0200]

[0201] Effect test:

[0202] I. Antibacterial activity test

[0203] Determine the minimum inhibitory concentration MIC (μg / mL) value of each test compound by referring to the broth microdilution method described in the guidelines of the Clinical and Laboratory Standards Institute (CLSI).

[0204] The steps are as follows:

[0205] (1) Preparation of LB medium: Weigh 10 g of commercially available LB medium into a 500 mL conical flask, add ultrapure water, dissolve it and then transfer it to a volumetric flask. Add water to make the volume up to 500 mL, and then transfer it to a conical flask. After autoclaving for 30 min, cool it for standby.

[0206] (2) Preparation of compound concentration: After dissolving the test compound in 3% DMSO solution, prepare a stock solution with a concentration of 258 μg / mL using LB medium for standby.

[0207] (3) Preparation of bacterial suspension: Take clinical strains, dilute them with medium and culture them in an incubator for 24 h to activate the strains. Prepare the activated bacterial suspension into a bacterial suspension with a concentration equivalent to 1 McFarland turbidity standard, and then dilute it 1000-fold with LB medium, with a concentration equivalent to 1×10 5 CFu / mL of bacterial suspension for standby.

[0208] (4) Method

[0209] Determination was carried out by the two-fold dilution method. First, add 100 μL of LB medium to each well in rows B, C, and D of the 96-well plate. Then, add 100 μL of the compound to be tested to B2, C2, and D2 respectively. After thorough mixing, take 100 μL of the mixed solution and transfer it to B3, C3, and D3. After remixing, take 100 μL of the mixed solution and transfer it to B4, C4, and D4. Repeat the above operation until B11, C11, and D11. Finally, discard 100 μL of the mixed solution from the last three wells, so that the drug concentrations in the second row to the ninth row are 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 respectively, that is, the drugs are added by the two-fold dilution method. Then, use E2 - E4 with 1% DMSO as the solvent control group, F2 - F4 as the drug-free blank control, and G2 - G4 as the ciprofloxacin (CIP) positive control. Add 200 μL of normal saline to the remaining side wells to prevent edge effects. Place the 96-well plate in an incubator at 37 °C for 24 h, and take the minimum inhibitory concentration (MIC) as the concentration at which bacterial growth is completely inhibited in the small wells.

[0210] (5) Results

[0211] The results are shown in Table 1. Most of the compounds showed a certain degree of antibacterial activity. Among them, the prodrug 9b showed significant antibacterial activity against a variety of clinically significant drug-resistant bacteria and sensitive bacteria, including MRSA21 - 5 (MIC: 0.27 μM), S. aureus 25293 (MIC: 0.27 μM), E. faecium 51299 (MIC: 4.41 μM), and E. coli 25922 (MIC: 0.27 μM), showing comparable or even better efficacy than first-line antibiotics such as vancomycin, ampicillin, and ciprofloxacin. The prodrug 9c is a derivative of CIP, which showed better antibacterial activity against MRSA21 - 5 (MIC: 2.31 μM) than ciprofloxacin (MIC: 6.04 μM). The prodrugs 5b and 5e were selective for S. aureus 29213 (MICs were 4.26 μM and 4.18 μM respectively).

[0212] Table 1: MIC values (μg / mL) of some Pro-AF-Qs prodrugs

[0213]

[0214] II. Determination of biofilm clearance

[0215] (1) Experimental principle: Crystal violet is an alkaline dye commonly used in the Gram staining method in microbiology. Its staining principle is mainly based on the chemical binding and physical adsorption of the dye to cell components, and is mainly used to detect and quantify the formation of biofilms. A biofilm is a structured community formed by microorganisms on a solid surface, enclosed in extracellular polymers secreted by the bacteria themselves. The basic principle of the crystal violet staining method is to use the cations in the crystal violet dye to bind to the anions in the polysaccharides, proteins and other structures in the biofilm through electrostatic interactions, forming a dense network structure that can firmly bind crystal violet and is not easily eluted by the decolorizing agent. Finally, after dissolving with a certain reagent, the absorbance is measured by an enzyme-linked immunosorbent assay (ELISA) reader to quantify the biofilm.

[0216] (2) Preparation of bacterial suspensions and compounds

[0217] Preparation of compound concentration: Dissolve the compound to be tested in a 3% DMSO solution and prepare a stock solution with a concentration of 258 μg / mL using LB medium for later use.

[0218] Preparation of LB medium: Weigh 20 g of LB medium into a 1000 mL conical flask, add ultrapure water, dissolve and transfer to a volumetric flask, add water to a volume of 1000 mL, and finally transfer to a conical flask and sterilize by autoclaving for 30 min. Cool and set aside.

[0219] Preparation of bacterial suspension: Take a clinical strain and culture it in a medium for 24 h for activation. Then, prepare the activated bacterial suspension into a bacterial suspension with a concentration equivalent to 1 McFarland standard, and dilute it tenfold with LB medium for later use.

[0220] (3) Experimental procedures

[0221] Based on the MICs determined in the above experiments, the effects of compounds at subinhibitory concentrations on MRSA25-1 biofilms were determined using a two-fold dilution method. First, 100 μL of LB medium was added to each well of a 96-well plate (excluding the edge wells). Then, 100 μL of the test compound at a concentration of 258 μg / mL was added to the sixth column. After thorough mixing, 100 μL of the mixture was transferred to the seventh column. Mixing was repeated again, and 100 μL of the mixture was transferred to the eighth column. Repeating this process for the eleventh column, 100 μL of the mixture was discarded. The drug was then added using a two-fold dilution method, resulting in drug concentrations of 4×MIC, 2×MIC, 1×MIC, 1 / 2×MIC, 1 / 4×MIC, and 1 / 8×MIC in columns 6 to 9, respectively. Columns 2 to 5 served as blank controls without drug addition. Subsequently, 100 μL of the test bacterial solution was added to columns 2 to 11, and the solution was pipetted three times to ensure uniform mixing of the bacterial solution and drug. Add physiological saline to the remaining side wells to prevent edge effects. Place the 96-well plate in a 37°C constant temperature incubator and incubate for 72 hours (take it out every four hours and shake it on a decolorizing shaker for 3 minutes). Take out the 96-well plate, carefully aspirate the floating bacterial solution, and then gently wash it three times with sterile physiological saline to completely remove the residual floating bacteria. Then add 250μL of 2% crystal violet solution and let it stand for 30 minutes. After staining is completed, rinse slowly with physiological saline to wash away excess crystal violet. Finally, add 250μL of anhydrous ethanol to dissolve and fix the biofilm, and use a microplate reader to measure the OD value of each well of the 96-well plate at 570nm to observe the growth of the biofilm.

[0222] (4) Results and Discussion

[0223] Table 2 Inhibitory activity of ABP-QLs prodrugs on biofilm

[0224]

[0225] Results from the biofilm inhibition assay showed that the parent ciprofloxacin exhibited relatively weak antibiofilm activity (only approximately 14.8% inhibition at 1×MIC). In contrast, the synthetic ABP-QLs prodrugs displayed superior biofilm inhibition. Prodrug compounds 5b, 7d, 7e, 9a, and 11a all inhibited biofilm growth by more than 80% at 1×MIC compared to the untreated control. Notably, 9c inhibited biofilm biomass by 70.3% at a lower 1×MIC (2.31 μM) compared to ciprofloxacin (6.04 μM). Furthermore, prodrug 5e, derived from the now-obsolete second-generation quinolone antibiotic pipemidic acid due to its numerous side effects and resistance, exhibited significant antibiofilm activity, achieving a 67.0% biofilm inhibition at 1 / 4×MIC. However, prodrug 9b failed to inhibit biofilm formation and even appeared to promote it, highlighting the complexity of biofilm-bacterial interactions. Overall, the introduction of aminoferrocene significantly enhanced the antibiofilm activity of these compounds, confirming the potential of ABP-QLs as dual-acting agents with both antibacterial and antibiofilm efficacy.

[0226] 3. Test of biofilm removal activity

[0227] (1) Preparation of bacterial solution

[0228] The clinical strain was cultured in a culture medium for 24 hours for activation, and the activated bacterial suspension was prepared into a bacterial suspension with a concentration equivalent to 1 McFarland turbidimetric standard, and then diluted twenty times with LB culture medium for use.

[0229] (2) Experimental steps

[0230] First, add 200 μL of bacterial suspension to each well of a 96-well plate and incubate in a 37°C incubator for 72 hours. Once the biofilm has grown, carefully aspirate the bacterial suspension and wash twice with sterile saline to remove floating bacteria. Next, add 100 μL of the prepared compound at different concentrations (4×MIC, 2×MIC, 1×MIC, 1 / 2×MIC, 1 / 4×MIC, 1 / 8×MIC) and 100 μL of the prepared LB medium to columns 6-9, mix thoroughly, and use columns 2 to 5 as blank controls without drug. Incubate the 96-well plate in a 37°C incubator for 72 hours. Use a decolorizing shaker for 3 minutes every four hours. After 72 hours, remove the 96-well plate, aspirate the floating bacterial suspension, and carefully wash twice with sterile saline to remove floating bacteria. Add 250 μL of 2% crystal violet and stain for 30 minutes. Then slowly wash away the excess crystal violet with normal saline. Finally, add 250 μL of anhydrous ethanol to dissolve the biofilm. Use a microplate reader to measure the OD value of each well of the 96-well plate at 570 nm to observe the removal of the biofilm.

[0231] (3) Results and Discussion

[0232] As Figure 21 shown, the biofilm eradication efficacy of the H2O2-responsive prodrug 9c (biofilm eradication rate of 66.5% at 1×MIC) was 12-fold higher than that of ciprofloxacin (showing only an eradication rate of 5.4% under the same conditions). In addition, the eradication rate of compound 5e against mature biofilms reached 84.2% at 1×MIC, which was 15-fold higher than that of ciprofloxacin. In summary, compounds 9c and 5e have good biofilm-clearing activity and have the potential for further development.

[0233] As can be seen from the above examples, the present invention provides a multifunctional quinolone anti-biofilm prodrug having the structures of Formula I, Formula II, Formula III, Formula IV, Formula V or Formula VI. The present invention integrates aminoferrocene, borate ester groups and quinolone drugs into the prodrug system ABP-QLs to achieve anti-MRSA biofilm effects from multiple dimensions. The present invention utilizes the cyclic generation of a large amount of ROS by aminoferrocene to destroy the first line of defense of the biofilm - the EPS matrix. While the EPS is being destroyed, it promotes the penetration of the active molecule AF-QLs through the biofilm to clear the bacteria inside the film. The ABP-QLs of the present invention have good biofilm-clearing effects, low cytotoxicity, and high safety in the application of preparing prodrugs; the modification of quinolone drugs in the present invention broadens the application of quinolone drugs in the field of biofilms and provides new ideas for the means of clearing biofilms.

[0234] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A multifunctional quinolone antibiofilm prodrug, characterized in that, Having the structures of Formula Ⅰ, Formula Ⅱ, Formula Ⅲ, Formula Ⅳ, Formula Ⅴ or Formula Ⅵ: In Formulae III to VI, X is selected from -CF or -N; R 1 is independently selected from -CH2CH3 or cyclopropyl; R 2 is independently selected from -C or -H; R 3 is independently selected from -H, -OCH3 or none; R in Formulae III to V 4 is selected from -H or -OCH3.

2. The multifunctional quinolone antibiofilm prodrug according to claim 1, wherein In Formulae III to VI, X = -CF, R 1 = -CH2CH3, R 2 = -C, R 3 = -H, R 4 = -H; or X = -CF, R 1 = cyclopropyl, R 2 = -C, R 3 = -OCH3, R 4 = -OCH3; or X = -CF, R 1 = cyclopropyl, R 2 = -C, R 3 = -H, R 4 = -H; or X = -CF, R 1 = -CH2CH3, R 2 = -N, without R 3 , R 4 = -H; or X = -N, R 1 = -CH2CH3, R 2 = -N, without R 3 , R 4 = -H.

3. The multifunctional quinolone antibiofilm prodrug according to claim 1, wherein In Formula VI, X = -CF, R 1 = -CH2CH3, R 2 = -C, R 3 = -H, R 4 = -H; or X = -CF, R 1 = cyclopropyl, R 2 = -C, R 3 = -OCH3, R 4 = -OCH3.

4. A method for preparing the multifunctional quinolone antibiofilm prodrug according to claim 1, comprising the following steps: Mix ferroceneformic acid, diphenylphosphoryl azide, triethylamine and toluene, and react to obtain 1-azido-1'-ferrocenyl ethanone; React 4-hydroxymethylphenylboronic acid pinacol ester and 1-azido-1'-ferrocenyl ethanone in a toluene solution to obtain (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2-ethoxycarbonylferrocene cyanamide; React a linker, (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2-ethoxycarbonylferrocene cyanamide, cesium carbonate, tetrabutylammonium iodide and potassium iodide in DMF to obtain an intermediate compound; the linker is selected from m-xylene dibromide, 1,3-dibromopropane, diethylene glycol ditosylate, tetraethylene glycol ditosylate; React the intermediate compound, a quinolone series compound and N,N-diisopropylethylamine in DMF to obtain a multifunctional quinolone antibiofilm prodrug; The quinolone series compound is selected from norfloxacin, ciprofloxacin, enoxacin, gatifloxacin, sparfloxacin, moxifloxacin or pipemidic acid.

5. The preparation method according to claim 4, characterized in that, The molar ratio of ferroceneformic acid, diphenylphosphoryl azide and triethylamine is 1:(1.4-1.6):(1.9-2.1).

6. The preparation method according to claim 4, characterized in that, The molar ratio of 4-hydroxymethylphenylboronic acid pinacol ester and 1-azido-1'-ferrocenyl ethanone is 1.9-2.2:

1.

7. The preparation method according to claim 4, characterized in that, The molar ratio of (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2-ethoxycarbonylferrocene cyanamide and the linker is 1:1.9-2.

2.

8. The preparation method according to claim 4, characterized in that, The molar ratio of the intermediate compound and the quinolone series compound is 1:(1.4-1.6).

9. Use of the multifunctional quinolone antibiofilm prodrug according to any one of claims 1-3 in the preparation of a drug for antibiofilm.

10. The application according to claim 9, wherein The drug for antibiofilm is a drug for anti-methicillin-resistant Staphylococcus aureus biofilm.

Images