α-MG xanthone NO donor compounds, synthesis methods and applications

Abstract

This invention provides a NO donor-based compound, its synthesis method, and its application, belonging to the field of biomedical technology. The synthesis method uses α-MG as a starting material, performing symmetric and asymmetric modifications at the C3 and / or C6 positions of the α-MG oxide structure to introduce a NO donor, thus synthesizing the α-MG oxide-based NO donor compound. This α-MG oxide-based NO donor compound is used against Gram-positive bacteria or as a sensitizer for clinically resistant MRSA strains. The α-MG oxide-based NO donor compound of this invention exhibits strong in vitro and in vivo antibacterial activity, is not prone to drug resistance, and can effectively eliminate biofilm virulence factors of Gram-positive bacteria such as MRSA. It is an active compound for treating Staphylococcus aureus, the main pathogen of bovine mastitis. The synthesis method is simple, has a short route, is clean, and is easily scaled up for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a NO donor compound based on α-MG xanthones, its synthesis method and application. Specifically, it relates to a novel α-dextrin xanthones NO donor compound, its synthesis method and its use against MRSA and other bacteria. Background Technology

[0002] Bovine mastitis seriously threatens the healthy development of the dairy industry. Studies show that about one-third of the world's 220 million dairy cows suffer from various types of mastitis, making it a major cause of economic losses in dairy farming. Globally, mastitis causes economic losses of up to $35 billion annually. Although the causes of this disease are complex, it is mainly caused by bacteria such as Staphylococcus aureus. Currently, the incidence rate is on the rise. Long-term, extensive use of antibiotics in prevention and control not only leads to the emergence of drug-resistant strains but also results in antibiotic residues in dairy products, thus endangering human health. Furthermore, biofilm formation increases the drug resistance of pathogenic bacteria. Statistics show that two-thirds of current microbial infections originate from biofilm formation, which enhances pathogenicity and drug resistance. Therefore, addressing the economic losses and human health problems caused by bovine mastitis, developing novel Gram-positive drugs against Staphylococcus aureus and other bacteria, and developing potentiators to reduce drug resistance caused by biofilm formation are urgently needed.

[0003] Natural antimicrobial agents refer to a class of compounds with antimicrobial activity extracted from certain plants or animals. Examples include animal-derived natural antimicrobial peptides, chitosan derivatives extracted from shrimp and crab shells, and extracts from Andrographis paniculata, Houttuynia cordata, mangosteen peel, garlic, and mint. Natural antimicrobial agents have advantages such as good biocompatibility, low toxicity, and wide availability. However, their application prospects are limited by disadvantages such as weak antimicrobial activity, short duration of antimicrobial activity, and poor drug-like properties. This patent uses α-mangostin (α-MG), the main active ingredient of the traditional Chinese medicine mangosteen, as the research framework. α-mangostin is recognized as a potent pharmacologically active xanthones, including antibacterial, anti-inflammatory, antifungal, antioxidant, and anti-allergic activities. Meanwhile, the xanthone core structure of α-oxanthones possesses natural cell membrane targeting properties, inspiring many scientists to use it as a lead compound for structural modification. Based on the antibacterial activity of gaseous molecule NO and its ability to inhibit biofilm formation and eliminate existing biofilms (Identification of a novel nitric oxide donor peptide with dual biofilm clearance and antibacterial activity for intervention in device-associated infections, Medicinal Chemistry, 2020, 63:9127-9135), and the ability of aromatic nitro groups to control the release of NO, stronger anti-Gram-positive bacterial activity and elimination of drug resistance caused by biofilms can be obtained than lead compounds, including strong antibacterial activity against methicillin-resistant Staphylococcus aureus (Design and synthesis of amphiphilic xanthone membrane-targeting antibacterial agents to improve membrane selectivity, Medicinal Chemistry, 2013, 56, 2359-2373). Reports have indicated that introducing simple sugars such as mannose onto the 6-hydroxyl group of α-oxanthones can address the poor water solubility of lead compounds while simultaneously improving the targeted antibacterial activity of α-oxanthones glycosides (oxanthones esters and sugar derivatives and their preparation and antibacterial uses, CN115260146B). However, no oxanthones NO donor derivatives have been reported for pathogenic infections and antibiotic resistance caused by drug-resistant colonies and biofilm-bearing bacterial communities. This invention uses oxanthones α-MG as a lead compound and, through symmetrical or asymmetrical methods, introduces an aromatic NO donor skeleton, rationally modifying the α-MG chemical space to achieve better antibacterial activity. Simultaneously, α-MG acts as a drug target carrier, synergistically with NO to scavenge biofilms and prevent drug resistance. A series of oxanthones NO donor derivatives with good inhibitory activity against Gram-positive bacteria and reduction of Gram-positive bacterial biofilms are obtained. These derivatives can effectively address MRSA-induced mastitis or biofilm infections in dairy cows. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a novel antibacterial compound based on α-MG xanthones, its synthesis method, and its application. Specifically, α-MG is used as a lead compound, and symmetrical and asymmetric modifications are made to the C3 and / or C6 positions of the xanthone α-MG structure to introduce a NO donor. A series of NO donor compounds are synthesized, and their antibacterial and anti-biofilm activities are screened to obtain novel α-MG xanthones based NO donor compounds with good antibacterial activity and the ability to inhibit and clear biofilms. The NO donor compounds of this invention have a unique design, which is beneficial to improving molecular drugability and targeted controlled release of NO donors. The synthesis conditions are mild, the reaction reagents are readily available, and the reaction route is short. The α-MG xanthones based NO donor compounds can target and disrupt cell wall biosynthesis, damage cell membrane structure, leading to increased permeability, outflow of contents, blocking biofilm formation, and clearing mature biofilms. They can be used as novel broad-spectrum antibacterial drugs and synergists for reducing biofilm resistance.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A NO donor compound based on α-MG xanthones, wherein the general structural formula of the α-MG xanthones NO donor compound is:

[0007]

[0008] R1 and R2 are independent H or NO donors, and there is at least one NO donor among R1 and R2.

[0009] The NO donor is WR3, where W is X, O, or CO, and R3 is... n = 0, 1, or 2;

[0010] When R4 is NO2, R5, R6, or R7 are each independent of H, CH3, CF3, or X; when R5 is NO2, R4, R6, or R7 are each independent of H, CH3, CF3, or X; when R6 is NO2, R4, R5, or R7 are each independent of H, CH3, CF3, or X; when R7 is NO2, R4, R5, or R6 are each independent of H, CH3, CF3, or X; X is F, Cl, Br, or I.

[0011] Specifically, the structural formula of the α-MG xanthone-based NO donor compound is as follows:

[0012]

[0013] R1 and R2 are independent NO donors.

[0014] Furthermore, R1 and R2 are each any of the following independent structural formulas:

[0015]

[0016] Furthermore, R1 and R2 are each any of the following independent structural formulas:

[0017]

[0018] Furthermore, the structural formula of the α-MG xanthone-based NO donor compound is as follows:

[0019]

[0020] Where R1 is any of the following structural formulas:

[0021]

[0022] Furthermore, R1 can be any of the following structural formulas:

[0023]

[0024] A method for synthesizing the above-mentioned NO donor-based α-MG xanthones, wherein the method uses α-MG as a starting material and performs symmetric and asymmetric modifications at the C3 and / or C6 positions of the α-MG xanthone structure to introduce a NO donor, thereby synthesizing the α-MG xanthones-based NO donor compound; the specific chemical reaction equation is as follows:

[0025]

[0026] Furthermore, the synthetic method involves reacting α-MG with an acyl halide derivative, a haloalkane, or an alcohol derivative under alkaline conditions to undergo a substitution reaction, thereby obtaining the α-MG-based xanthone NO donor compound.

[0027] Furthermore, the synthesis method includes the following steps:

[0028] S1. Mix α-MG, anhydrous solvent and anhydrous alkaline reagent to obtain solution A;

[0029] S2. Take one of the following: an anhydrous solvent containing an acyl halide derivative, an anhydrous solvent containing a haloalkane, and an anhydrous solvent containing an alcohol derivative. Slowly add it dropwise to solution A to carry out a pre-reaction. Then heat it to reflux to carry out the reaction. After the reaction is complete, concentrate it to obtain a semi-solid residue.

[0030] S3. The semi-solid residue is recrystallized with an organic solvent to obtain the α-MG xanthone NO donor-based compound.

[0031] Furthermore, the acyl halide derivative is R3COX, the haloalkane is R3X, and the alcohol derivative is R3OH;

[0032] Among them, R3 is n = 0, 1, or 2;

[0033] When R4 is NO2, R5, R6 or R7 are each independent of H, CH3, CF3 or X;

[0034] When R5 is NO2, R4, R6 or R7 are each independent of H, CH3, CF3 or X;

[0035] When R6 is NO2, R4, R5 or R7 are each independent of H, CH3, CF3 or X;

[0036] When R7 is NO2, R4, R5 or R6 are each independent of H, CH3, CF3 or X;

[0037] X is F, Cl, Br, or I.

[0038] Furthermore, the anhydrous alkaline reagent is potassium carbonate, triethylamine, hydroxylamine, sodium hydroxide, potassium hydroxide, cesium carbonate, N,N-diisopropylethylamine, ammonia or calcium hydroxide;

[0039] The anhydrous solvent is tetrahydrofuran, 1,4-dioxane, acetone, N,N-dimethylformamide, or acetonitrile;

[0040] The pre-reaction temperature was room temperature, and the time was 30–60 min.

[0041] The reflux temperature is 45–65°C, preferably 50°C, and the reflux time is 1–5 hours.

[0042] The organic solvent used for recrystallization is at least one of toluene, xylene, benzene, n-hexane, and cyclohexane, with toluene and n-hexane being preferred.

[0043] The application of the above-mentioned α-MG xanthonone NO donor compound in the preparation of anti-Gram-positive bacteria or as a sensitizer for clinically resistant strains of MRSA.

[0044] Furthermore, the α-MG xanthone-based NO donor compound is used to inhibit or eliminate Gram-positive bacteria and / or Gram-negative biofilms;

[0045] Furthermore, the α-MG xanthones NO donor compounds are used in animal husbandry, including ranches, to reduce or even replace antibiotics; or as treatments for diseases such as mastitis in dairy cows; or as molecular probes to reveal the antibacterial or drug resistance-reducing mechanisms of xanthones; or for the inhibition or removal of Gram-positive bacterial biofilms in medical devices, etc.

[0046] The beneficial effects of the present invention, including an α-MG xanthone-based NO donor compound, its synthesis method, and its application, are as follows:

[0047] The α-MG xanthonone NO donor compounds of this invention have the advantages of strong in vitro and in vivo antibacterial activity and low resistance to drug resistance. They can also effectively eliminate biofilm virulence factors of Gram-positive bacteria such as MRSA, and are active compounds for treating Staphylococcus aureus, the main pathogen of bovine mastitis.

[0048] The present invention relates to α-MG xanthone-based NO donor compounds that focus on their drug-like properties and reduce drug resistance caused by bacterial community biofilm formation, achieved through strategies such as symmetrical or asymmetrical introduction of aromatic NO donor fragments;

[0049] The present invention relates to α-MG xanthones NO donor compounds, which are obtained by using α-MG lead compounds as starting materials and through different chemical reaction combinations. The overall synthesis conditions are mild, the reaction reagents are readily available, the reaction route is short, the selectivity is strong, the yield is high, the reaction process is green and clean, environmentally friendly, and easy to produce on a large scale.

[0050] The mechanism of action of the α-MG xanthones NO donor compounds involved in this invention has a strong ability to depolarize cell membranes, increase cell membrane permeability, disrupt cell membrane integrity, and lead to enhanced permeability and outflow of contents, thereby inhibiting the activity of Gram-positive bacteria. Simultaneously, it controls the release of NO gas, inhibiting the formation of Gram-positive bacterial biofilms and eliminating mature biofilms. Therefore, on the one hand, it can be used against Gram-positive bacteria, including MRSA, or as a sensitizer for clinically resistant MRSA strains. By inhibiting and eliminating Gram-positive or Gram-negative biofilms, it achieves a synergistic effect in treating diseases such as mastitis in dairy cows caused by Staphylococcus aureus, thus providing a new type of input for reducing or even replacing antibiotics in the livestock industry. On the other hand, it can be used as a molecular probe to reveal the antibacterial or resistance-reducing mechanisms of xanthones, as well as to inhibit and eliminate Gram-positive bacterial biofilms in medical devices.

[0051] All the α-MG xanthone-based NO donor compounds involved in this invention have good inhibitory effects on the activity of Gram-positive bacteria, including MRSA, or can eliminate the formation of Gram-positive bacterial biofilms.

[0052] The antibacterial mechanism of all the α-MG xanthonone NO donor compounds involved in this invention is unique. It achieves antibacterial effect and reduces drug resistance caused by biofilm by targeting and inhibiting bacterial cell wall synthesis and destroying cell membrane structure, while controlling the release of NO, inhibiting the formation of biofilms of Gram-positive bacteria and clearing mature biofilms.

[0053] All of the α-MG xanthonone NO donor compounds involved in this invention can be used for the prevention and control of mastitis in dairy cows, and to reduce or delay the resistance of Staphylococcus aureus to veterinary clinical antibiotics. Attached Figure Description

[0054] Figure 1 This is a transmission electron microscope image of Staphylococcus aureus biofilm removal based on α-MG xanthone NO donor compound 3 in Example 6 of the present invention. Detailed Implementation

[0055] The present invention will be described in detail below with reference to examples. The following examples are implemented based on the technical solution of the present invention, using α-MG as a lead compound, and subjecting it to acylation or etherification to prepare NO donor compounds based on α-twiscinoxanthone with different substitution positions. Detailed implementation methods and specific operating procedures are provided, which will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

[0056] Example 1: Synthesis of C3 or C6 position acylation based on α-MG xanthone NO donor compounds

[0057] I. Synthesis of Compound 1 Based on α-MG Xanthone NO Donor

[0058] S1. Preparation of solution A and solution B

[0059] In a dry, flat-bottomed flask, add 1 mmol of α-MG, 30 mL of acetone, and 3 mL of triethylamine (pH 10) to form solution A.

[0060] Solution B was prepared by mixing 1 mmol of 3,5-dinitrobenzoyl chloride with 20 mL of tetrahydrofuran (THF) in a dry separatory funnel.

[0061] S2, Substitution reaction

[0062] Under nitrogen protection, solution B was gradually added dropwise to solution A, and the mixture was stirred at room temperature for 30 minutes to pre-react. The resulting mixture was then refluxed at 50°C with continuous stirring. After the reaction was completed using a TLC monitor, reflux was stopped, and the mixture was concentrated by evaporation under vacuum to obtain a semi-solid residue. The specific chemical reaction equations are as follows:

[0063]

[0064] Wherein, the structural formula of R1 is

[0065] S3, Post-processing

[0066] A saturated sodium bicarbonate solution was added to the semi-solid residue, and the mixture was gently stirred for 10 min. The mixture was filtered, washed with distilled water, and the solid product was collected. The solid product was recrystallized from toluene to give 63.46 mg of a pale yellow solid, which was compound 1 based on the α-MG xanthonone NO donor, with a yield of 41%. The NMR spectrum of compound 1 based on the α-MG xanthonone NO donor is as follows: 1 HNMR(400MHz, CDCl3)δ=13.75(s,1H),9.71(s,3H),7.20(s,1H),6.72(s,1H),6.89(s,1H),5.27-5.12( m,2H),4.15-4.04(m,3H),3.80(s,3H),3.57-3.55(d,2H),1.84(s,3H),1.70(s,3H),1.58-1.56(d,6H). 13 C NMR(100MHz, CDCl3)δ=182.50,161.35,160.71,156.01,155.58,153.83,153.70,149.00,132.88,132.53,129.94, 127.27,125.88,121.46,115.84,114.21,109.36,105.12,99.88,62.65,60.62,26.74,25.65,22.46,17.99,14.30.

[0067] II. Synthesis of NO donor compounds 2–14 based on α-MG xanthones

[0068] Following the method used in step one for the synthesis of NO donor compound 1 based on α-MG xanthones, NO donor compounds 2–14 were synthesized. The process steps, parameters, and raw materials were basically the same, the only difference being the type of acyl chloride derivative. In the synthesis of NO donor compound 1 based on α-MG xanthones, the acyl chloride derivative was 3,5-dinitrobenzoyl chloride, while in the synthesis of NO donor compounds 2–14 based on α-MG xanthones, the acyl chloride derivatives were, in order, 4-nitrobenzyl bromide, 2-chloro-4-nitrobenzoyl chloride, 4-nitrobenzoyl chloride, 4-methyl-3-nitrobenzoyl chloride, 3-nitrobenzoyl chloride, 2- Trifluoromethyl-4-nitrobenzoyl chloride, 2-methyl-4-nitrobenzoyl chloride, 4-chloro-3-nitrobenzoyl chloride, 2-nitrobenzoyl chloride, 2-chlorobenzoyl chloride, 2,4-dichlorobenzoyl chloride, 2-chloro-4-dimethylaminebenzoyl chloride, and benzoyl chloride were used to obtain the corresponding crude products (i.e., semi-solid residues). All were then subjected to the same post-treatment method, recrystallization from toluene, to sequentially obtain pale yellow solids 2–14 (i.e., compounds 2–14 based on α-MG xanthones (NO donors)), with yields of 46%, 46%, 46%, 43%, 57%, 51%, 47%, 41%, 38%, 61%, 63%, 55%, and 70%, respectively. It can be seen that the method of this invention for synthesizing compounds based on α-MG xanthones (NO donors) has a yield of 30–70%.

[0069] The chemical reaction equations for the synthesis of compounds 2–14 based on α-MG xanthone NO donors are as follows:

[0070]

[0071] Among them, the structural formulas corresponding to R1 in α-MG xanthone NO donor compounds 2–14 are as follows:

[0072]

[0073] The yield of compound 2, based on the α-MG xanthone NO donor, was 46%, and it was a pale yellow solid. The NMR spectrum is as follows: 1H NMR (400MHz, CDCl3) δ6.68(s,1H),6.64(s,1H),6.14-6.07(m,2H),5.47(t,J=12.0Hz,2H),5.37(t,12.0Hz,2H),5.24(t,J=8.0,1H),5.13 (t,J=8.0,1H),4.65-4.61(m,4H),4.22-3.98(m,2H),3.52(s,3H),3.33-3.22(m,2H),2.47(s,3H),1.84(s,3H),1.76(s,3H),1.68(s,6H). 13 C NMR (100MHz, CDCl3) δ175.23,168.32,161.23,157.11,155.28,155.20,147.20,146.16,139.77,133.54,132.17,130.11,127 .23,125.12,121.12,119.21,118.15,115.21,108.11,99.23,97.05,69.36,26.04,25.85,25.71,23.17,20.78,18.31,17.55.

[0074] The yield of compound 3, based on the α-MG xanthone NO donor, was 46%, and it was a pale yellow solid. The NMR spectrum is as follows: 1 H NMR(400MHz, CDCl3)δ6.70(s,1H),6.65(s,1H),6.32-6.21(m,2H),5.63(t,J= 16.0Hz,2H),5.12(t,J=16.0Hz,2H),5.06(t,J=4.0Hz,1H),5.02(t,J=4.0Hz, 1H),4.76-4.70(m,4H),4.20(d,2H),3.82(d,J=4.0Hz,3H),3.38(d,J=44.0Hz ,2H),3.08(m,1H),1.82(s,3H),1.74(s,3H),1.65(s,6H),1.43(s,3H),1.41(s 3H). 13C NMR (100MHz, CDCl3) δ177.70,175.40,161.52,155.96,155.72,154.25,150.38,144.05,138.13,132.20,131.88,130.90,123.80,122.36,1 18.24,118.11,114.28,112.68,98.95,98.87,96.81,96.72,69.68,6 0.76,34.30,34.12,25.98,23.20,19.16,18.47,18.16,17.99,17.96.

[0075] The yield of compound 4, based on the α-MG xanthone NO donor, was 46%, and it was a pale yellow solid. The NMR spectrum is as follows: 1 H NMR (400MHz, CDCl3) δ13.70(s,1H),6.74(s,1H),6.60(s,1H),6.17 -5.98(m,1H),5.77(d,J=12.0Hz,1H),5.56(d,J=12.0Hz,1H),5.20(t,J=8.0Hz,1H),5.18(t,J=8.0Hz,1H),4.60(d,J= 4.0Hz,2H),4.13-4.09(m,2H),3.90(s,3H),3.32(d,J=8.0Hz,2H),2.34(s,3H),1.85(s,3H),1.78(s,3H),1.68(s,6H). 13 C NMR (100MHz, CDCl3) δ182.88,168.66,161.24,157.66,157.64,154.70,144.52,136.72,132.26,132.10,123.16,122. 86,121.90,121.50,116.10,112.00,107.22,100.26,100.08,99.56,98.43,69.72,60.92,26.40,26.05,25.85,22.45.

[0076] The yield of compound 5, based on the α-MG xanthone NO donor, was 43%, and it was a pale yellow solid. The NMR spectrum is as follows: 1H NMR(400MHz, CDCl3)δ13.70(s,1H),6.74(s,1H),6.60(s,1H),6.24-6.16(m,1H) ,5.52(d,J=12.0Hz,1H),5.38(d,J=12.0Hz,1H),5.24(t,J=8.0Hz,1H),5.12(t,J =8.0Hz,1H),4.70(d,J=4.0Hz,2H),4.16-4.10(m,2H),3.62(s,3H),3.20(d,J=8. 0Hz,2H),2.86-2.82(m,1H),1.86(s,3H),1.74(s,3H),1.68(s,6H),1.36(s,3H). 13 C NMR (100MHz, CDCl3) δ183.68,172.68,162.80,156.32,154.20,152.20,148.20,136.20,132.22,131.90,131.82,123.01,1 22.90,121.60,121.46,118.56,116.22,112.18,106.94,100.92,70.52,60.92,34.32,25.90,25.70,22.14,18.19,17.86.

[0077] The yield of compound 6, based on the α-MG xanthone NO donor, was 57%, and it was a pale yellow solid. The NMR spectrum is as follows: 1 H NMR (400MHz, CDCl3) δ7.20(s,1H),6.52(s,2H),6.21(s,1H),5.28 -5.10(m,2H),4.22(s,2H),3.76(s,3H),3.28(d,2H),2.50(s,3H),1.80(s,3H),1.76(s,3H),1.68(s,3H). 13 CNMR (100MHz, CDCl3) δ176.22,171.11,159.84,159.82,154.80,153.22,148.14,142.80,137.23,131.59,123.65,12 1.24,118.63,114.24,109.15,101.48,100.71,61.90,61.87,25.82,22.88,21.52,18.32,17.92.TOF-MS,m / z:[M+Na + ], calcd for C 26 H 28 NaO7 +,475.1727,found:475.1738.

[0078] The yield of compound 7, based on the α-MG xanthone NO donor, was 51%, and it was a pale yellow solid. The NMR spectrum is as follows: 1 H NMR (400MHz, CDCl3) δ7.16(s,1H),6.52(s,1H),6.42(s,2H),5.22(t,J=4.0Hz,1H),5.14(t,J=4.0Hz,1H),4.12(d,J=8.0Hz,2H) ,3.80(s,3H),3.60(d,J=8.0Hz,2H),3.10(m,1H),1.80(s,3H),1.76(s,3H),1.70(s,3H),1.66(s,3H),1.45(s,3H),1.43(s,3H). 13 CNMR (100MHz, CDCl3) δ176.76,171.28,158.82,154.65,153.86,147.36,141.76,136.16,132.96,122.86,120.36 ,117.56,113.32,108.32,100.42,100.44,99.54,67.16,60.86,33.55,25.46,24.83,21.76,17.80,17.30,17.06.

[0079] The yield of compound 8, based on the α-MG xanthone NO donor, was 47%, and it was a pale yellow solid. The NMR spectrum is as follows: 1 H NMR (400MHz, CDCl3) δ13.62(s,1H),6.78(s,1H),6.59(s,1H),5.26-5.14(m,2H),4.05(d,J=4.0 Hz,2H),3.78(s,3H),3.33(d,J=4.0Hz,2H),2.34(s,3H),1.83(s,3H),1.78(s,3H),1.68(s,6H). 13 C NMR (100MHz, CDCl3) δ182.62,168.93,161.14,156.05,155.30,154.47,153.76,143.01,137.40,132.36,132.22,1 23.09,121.66,116.07,112.27,107.02,101.80,100.30,62.09,26.73,25.93,25.86,22.44,21.22,18.32,17.96.

[0080] The yield of compound 9, based on the α-MG xanthone NO donor, was 41%, and it was a pale yellow solid. The NMR spectrum is as follows: 1 H NMR (400MHz, CDCl3) δ13.62(s,1H),6.73(s,1H),6.61(s,1H),6.55(s,1H),5.22(t,J=4.0Hz,1H),5.15(d,J=4.0Hz,1H),4.03 (d,J=4.0Hz,1H),3.76(s,3H),3.30(d,J=4.0Hz,2H),2.88-2.86(m,1H),1.84(s,3H),1.74(s,3H),1.68(s,6H),1.36(s,3H). 13 C NMR (100MHz, CDCl3) δ182.42,175.11,160.95,155.80,155.74,155.14,152.10,148.20,142.82,137.24,132. 22,122.30,121.55,116.66,112.22,106.56,101.20,99.80,61.80,34.20,26.26,25.22,23.22,18.82,17.88.

[0081] The yield of compound 10, based on the α-MG xanthone NO donor, was 38%, and it was a pale yellow solid. The NMR spectrum is as follows: 1 H NMR (400MHz, CDCl3) δ13.70(s,1H),6.66(s,1H),6.24(s,1H),5.50(t,1H),5.44(t,J=8.0Hz,1H),4.24( d,J=4.0Hz,2H),3.96(s,3H),3.62(d,J=4.0Hz,2H),2.62(s,3H),2.04(s,6H),1.86(s,3H),1.80(s,3H). 13 C NMR (100MHz, CDCl3) δ183.06,168.26,162.16,160.20,154.60,148.80,146.52,139.14,135.02,132.20,1 23.10,121.64,117.08,110.49,109.28,103.84,93.35,61.82,26.24,25.96,21.55,21.06,18.31,18.02.

[0082] The yield of compound 11, based on the α-MG xanthone NO donor, was 61%, and it was a pale yellow solid. The NMR spectrum is as follows: 1H NMR (400MHz, CDCl3) δ13.62(s,1H),6.80(s,1H),6.58(s,1H),5.30(t,J=8.0Hz,1H),5.20(t,J=8.0Hz,1H),4.06(d,J=4.0Hz,2H),3 .80(s,3H),3.50(d,J=4.0Hz,2H),2.90-2.84(m,1H),1.82(s,3H),1.72(s,3H),1.70(s,3H),1.62(s,3H),1.48(s,3H),1.32(s,3H). 13 C NMR (100MHz, CDCl3) δ182.88,175.22,161.32,156.16,155.28,154.22,152.32,141.38,136.40,132.38,12 2.86,121.80,121.76,116.74,112.22,106.10,101.78,100.26,62.23,34.60,26.74,25.95,25.87,18.03.

[0083] The yield of compound 12, based on the α-MG xanthone NO donor, was 63%, and it was a pale yellow solid. The NMR spectrum is as follows: 1 H NMR(400MHz, CDCl3)δ=13.52(s,1H),8.32(d,1H),7.60(s,1H),7.22(d,1H),7.18(s,1H),6.70(s,1H),5.26(d,J =12.5Hz,2H),4.36–4.08(m,3H),3.78(s,3H),3.74(d,2H),2.06(s,1H),1.86(s,6H),1.72(s,3H),1.70(s,3H). 13 C NMR (100MHz, CDCl3)δ=181.18,162.06,160.62,154.82,153.36,148.30,142.42,139.82,138.20,136.09,134.64,132.16,131.76, 127.38,126.24,122.18,121.52,117.20,110.54,109.22,103.70,93.20,62.22,60.42,26.42,25.78,21.42,18.16,17.84,14.14.

[0084] The yield of compound 13, based on the α-MG xanthone NO donor, was 55%, and it was a pale yellow solid. The NMR spectrum is as follows:1 H NMR (400MHz, CDCl3) δ13.78(s,1H),6.66(s,1H),6.22(s,1H),5.32-5.20(m,2H),4.12(d,J=4.0Hz,2H) ,4.08(t,J=6.4Hz,2H),3.80(s,3H),3.72(t,J=6.4Hz,2H),3.44(d,J=4.0Hz,2H),1.94-1.40(m,20H). 13 C NMR (100MHz, CDCl3) δ182.16,161.66,158.74,156.66,154.05,144.12,137.36,135.22,123.24,122 .76,111.76,108.86,103.77,98.90,93.16,62.97,61.00,32.61,28.96,27.21,26.86,25.54,18.56.

[0085] The yield of compound 14, based on the α-MG xanthone NO donor, was 70%, and it was a pale yellow solid. The NMR spectrum is as follows: 1 H NMR (400MHz, CDCl3) δ13.50(s,1H),6.86(s,1H),6.32(s,1H),5.30-5.20(m,2H),4.20(d,J=6.8Hz,2H),4.18(d,J =8.0Hz,2H),4.02(d,J=8.0Hz,2H),3.68(s,3H),3.62(t,J=6.8Hz,4H),3.32(d,J=6.8Hz,2H),1.78-1.52(m,30H). 13 C NMR (100MHz, CDCl3) δ182.82,162.30,160.86,158.52,155.78,155.22,144.24,138.18,134.76,124.52,123.36,122.38,111.96, 103.92,98.86,89.42,68.79,64.11,63.28,62.66,61.22,60.84,32.76,29.20,28.00,26.04,25.68,22.28,18.32,18.26,17.96.

[0086] Example 2: Minimum inhibitory concentration test of α-MG and NO donor compounds based on α-MG xanthones.

[0087] 1) Experimental Principle

[0088] This experiment is based on the growth of bacteria in MH (Mueller-Hinton Broth) medium in 96-well plates. After a period of time, due to the large amount of bacteria, a white precipitate will be produced, which makes it easy to distinguish whether there is bacterial growth. The antibacterial agent is serially diluted, added to the bacterial suspension, and cultured for a period of time. The last well without precipitate is the minimum inhibitory concentration (MIC).

[0089] 2) Experimental methods

[0090] Following NCCLS experimental procedures, strains including *Staphylococcus aureus* ATCC 29213, *Staphylococcus aureus* ATCC6538, MRSA2, and *Escherichia coli* ATCC 25922 were used. After resuscitation, each strain was cultured to the logarithmic growth phase for later use. Using sterile pipettes, bacterial cultures were taken and added dropwise to 96-well plates. The amount of bacterial culture added depended on experimental needs and bacterial concentration. α-MG and α-MG-based xanthone NO donor compounds 1–14 were dissolved in dimethyl sulfoxide to a concentration of 2 mg / mL, and 25.6 μL of each was added to the first well. Then, MH(B) medium was added to a final volume of 200 μL, followed by serial dilutions up to the 10th well. Vancomycin (the positive control) was treated using the same dissolution, addition, and serial dilution method, and incubated at 37°C for 18–24 h, with results observed. The minimum inhibitory concentration (MIC) of the control drug should be within the reference range; results from the same batch are considered reliable.

[0091] 3) Experimental Results

[0092] The antibacterial activity of α-MG and its derivatives against standard strains and multidrug-resistant strains of Escherichia coli and Staphylococcus aureus was tested by microdilution method. The results are shown in Table 1.

[0093] Table 1. Minimum inhibitory concentrations (MICs) of α-MG and α-MG-based xanthone NO donor compounds 1–14 against bacteria.

[0094]

[0095]

[0096] Note: In Table 1, 1 to 14 represent compounds 1 to 14 based on α-MG xanthone NO donors, respectively.

[0097] 4) Experimental Conclusions

[0098] As shown in Table 1, the lead compound α-MG and α-MG-based xanthones NO donor compounds 1-14 all exhibited antibacterial activity against Gram-positive bacteria and the clinical isolate MRSA2. Among them, α-MG-based xanthones NO donor compounds 1, 3-10 showed stronger antibacterial activity. Example 3: NO release based on α-MG xanthones NO donor compounds.

[0099] 1) Experimental Principle

[0100] The basic principle of NO detection is to indirectly determine the NO content by measuring the concentration of NO in the body or through substances produced by chemical reactions. NO is a highly reactive gas molecule, difficult to detect directly; therefore, it is usually detected by measuring its metabolites or using specific chemical reactions. Chemical colorimetry detects NO by measuring its metabolic products in the body. For example, NO in human serum, in the presence of oxygen and water, can generate nitrite (NO2) at a concentration equal to the molar concentration of NO. - ) and nitrates (NO3) - The concentration of NO was indirectly determined by measuring the rate of change in absorbance value after a chemical reaction to generate a purple-red complex.

[0101] 2) Experimental methods

[0102] According to the manufacturer's instructions, the nitrite colorimetric assay kit (Beyotime, China) was used to determine the NO levels in bacteria based on α-MG xanthones NO donor compounds 1–14 by colorimetry. Simply put, the bacterial density of Staphylococcus aureus S.aureus ATCC 6538 or MASA2 was approximately 1 × 10⁻⁶. 8 CFU / mL, treated with NO donor compounds 1–14 based on α-MG xanthones, incubated at 37°C for 20 min, then lysed by ultrasonication in an ice bath at a ratio of 1:5 (cell mass (g) to extract (mL)) for 5 min. The bacterial lysate was then harvested, mixed with Griess reagent at 37°C for 10 min, and measured at 540 nm using a microplate reader. Bacteria treated with 0.4% DMSO in the culture medium served as a negative control (solvent control) for nitrite production background levels, and different concentrations of sodium nitrite were prepared as positive controls. Each experiment was repeated at least three times. The results are shown in Table 2.

[0103] Table 2. NO production in Staphylococcus aureus ATCC6538 and MASA2 based on α-MG xanthones NO donor compounds 1–14.

[0104]

[0105] Note: In Table 2, 1 to 14 represent compounds 1 to 14 based on α-MG xanthone NO donors, respectively.

[0106] 3) Experimental Results

[0107] As can be seen from the data in Table 2, the amount of NO produced in Staphylococcus aureus ATCC 6538 and MASA2 based on α-MG xanthones NO donor compounds 1, 3-10 was higher than that in the solvent control (without added compounds) to varying degrees, with the release amount based on α-MG xanthones NO donor compound 3 being the highest.

[0108] Example 4: α-MG and α-MG-based xanthone NO donor compounds inhibiting biofilm formation

[0109] 1) Experimental Principle

[0110] Quantitative analysis of crystal violet in biological membranes is a commonly used biological experimental method for determining the permeability of biological membranes such as cell membranes, mitochondria, and endoplasmic reticulum. This method is based on the crystal violet dye, which can bind to biological membranes and diffuse into the cell interior through the cell membrane. After staining, the permeability of the biological membrane can be indirectly reflected by measuring the degree of dye absorption in the cell. The absorbance (OD value) of the stained cells can be measured using a spectrophotometer. The absorbance is directly proportional to the concentration of crystal violet in the cell, and the concentration of crystal violet in the cell is related to the permeability of the biological membrane. Therefore, the permeability of biological membranes can be indirectly calculated by measuring absorbance.

[0111] 2) Experimental methods

[0112] LB medium (3 mL) was inoculated with an overnight culture of Staphylococcus aureus ATCC 6538 and incubated statically at 37°C until the culture reached mid-log phase. The culture was then diluted 1:100 to obtain a bacterial suspension and set aside. Biofilm inhibition assays were performed by adding 100 μL of the bacterial suspension to 96-well plates containing 100 μL of α-MG at different concentrations (0–128 μg / mL) and α-MG-based xanthone NO donor compounds 1–14.

[0113] Incubate the 96-well plates at 37°C for 12–16 h. After incubation, remove bacteria from the 96-well plates, wash three times with phosphate-buffered saline (1×PBS), then add 0.1% crystal violet and incubate at room temperature for 15–20 min. Remove the crystal violet and wash three times with PBS buffer. Finally, add ethanol to the 96-well plates to dissolve the adsorbed crystal violet, and read the absorbance at 595 nm using an ELISA reader. The minimum polymer concentration that results in a 90% reduction in biofilm compared to the untreated control (no drug added) will be reported as the minimum inhibitory biofilm concentration of the biomass. Each experiment should be repeated at least three times. Biofilm inhibition rate (%) = (OD control - OD sample) / OD control × 100%.

[0114] The removal experiment began by incubating 96-well plates at 37°C for 12 hours to obtain mature biofilms. After removing planktonic cells, the biofilms were treated with different concentrations of α-MG xanthone-based NO donor compounds 1–14 at 37°C for 12 hours. Other steps were similar to the inhibition experiment. As described above, biofilm biomass was analyzed, and the biofilm concentration and biomass removal rates were calculated. Biofilm eradication rate (%) = (OD control - OD sample) / OD control × 100%.

[0115] 3) Experimental Results

[0116] The inhibitory effects of compounds 1–14 based on α-MG xanthones NO donors on the biofilm of Staphylococcus aureus ATCC 6538 were measured using the crystal violet method, and the results are shown in Table 3.

[0117] Table 3. Minimum inhibitory concentrations of α-MG xanthonone NO donor compounds 1–14 on bacterial biofilm.

[0118]

[0119] Note: In Table 3, 1 to 14 represent compounds 1 to 14 based on α-MG xanthone NO donors, respectively.

[0120] 4) Experimental Conclusions

[0121] As can be seen from the data in Table 3, based on the inhibitory effect of α-MG xanthones NO donor compounds 1, 3-10 on the biofilm of the listed bacterial communities, compound 3 based on α-MG xanthones NO donor showed the best effect.

[0122] Example 5: Detection of hemolytic activity of α-MG and α-MG-based xanthone NO donor compounds

[0123] 1) Experimental Principle

[0124] The hemolysis test is a commonly used experimental method for detecting the activity of antibodies in blood. Its principle is based on the phenomenon that red blood cells dissolve when exposed to specific antibodies.

[0125] 2) Experimental methods

[0126] 100 μL of 4% rabbit erythrocytes were added to 96-well plates containing 100 μL of different concentrations (0–128 μg / ml) of α-MG and NO donor compounds 1–14 based on α-MG xanthones. 1% Triton-X 100 was used as a positive control, and PBS buffer as a negative control. The 96-well plates were incubated at 37°C and 60 rpm for 1 h. After incubation, the 96-well plates were centrifuged at 1000 g for 3 min. 100 μL of the supernatant was collected, and the absorbance of each well was measured at 540 nm. Each experiment was repeated three times.

[0127] 3) Experimental Results

[0128] The experimental results are shown in Table 4. Based on the α-MG xanthone NO donor compounds 1-14, none of them were hemolytic at a concentration of 128 μg / mL.

[0129] Table 4. Hemolytic activity of α-MG and NO donor compounds 1–14 based on α-MG xanthones.

[0130]

[0131]

[0132] 4) Experimental Conclusions

[0133] Based on the fact that α-MG xanthone NO donor compounds 1, 3-10 have good biofilm clearance activity and are less likely to induce erythrocyte hemolysis.

[0134] Example 6: Observation of the effects of α-MG xanthonone NO donor compounds on bacterial morphology under transmission electron microscopy.

[0135] 1) Experimental Principle

[0136] Transmission electron microscopy allows observation of the morphology of sample tissues. Ultrathin sections with a thickness of 10–100 nm are prepared using ultrathin sectioning techniques. The changes in bacterial morphology after incubation with α-MG xanthones NO donor compound 3 are observed under a transmission electron microscope.

[0137] 2) Experimental methods

[0138] Staphylococcus aureus ATCC6538, revived to the logarithmic growth phase, was incubated with different concentrations of α-MG xanthones (NO donor compounds) for 2 hours. After centrifugation and discarding the culture medium, the cells were washed 2-3 times with PBS buffer, fixed with 2.5% glutaraldehyde, dehydrated, sputter-coated with gold, and observed under a transmission electron microscope. The cell wall thickness was measured. This group was designated as the drug-treated group. A control group (no drugs were added) was used.

[0139] 3) Experimental Results

[0140] Staphylococcus aureus, after incubation with α-MG xanthones NO donor compound 3, was compared with a control group, and the results are as follows: Figure 1 As shown in the figure, the biofilm in the blank group (without any drug) was clearly visible, while the biofilm in the drug-treated group was significantly cleared. Among them, Figure 1 The left image is a transmission electron microscope (TEM) image of the blank control group, and the right image is a TEM image of Staphylococcus aureus ATCC6538 after incubation with α-MG xanthonone NO donor compound 3 at a concentration of 32 μg / mL and a series of treatments (i.e., the drug-treated group).

[0141] 4) Experimental Conclusions

[0142] The experimental results show that, based on the α-MG xanthone NO donor compound 3, after interacting with bacteria for a certain period of time, it can effectively remove the biofilm of Gram-positive bacteria.

[0143] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A NO donor compound based on α-MG xanthone, characterized in that, The structural formula of the α-MG xanthone NO donor compound is as follows: Where R1 is any of the following structural formulas:

2. A method for synthesizing a NO donor compound based on an α-MG xanthone as described in claim 1, characterized in that, The synthetic method uses α-MG as the starting material, modifies the C6 position of the α-MG structure of xanthones to introduce a NO donor, and synthesizes the α-MG xanthones-based NO donor compound.

3. The method for synthesizing NO donor compounds based on α-MG xanthones according to claim 2, characterized in that, The synthesis method involves reacting α-MG with an acyl halide derivative under alkaline conditions to undergo a substitution reaction, thereby obtaining the α-MG-based xanthone NO donor compound.

4. The method for synthesizing NO donor compounds based on α-MG xanthones according to claim 3, characterized in that, The synthesis method includes the following steps: S1. Mix α-MG, anhydrous solvent and anhydrous alkaline reagent to obtain solution A; S2. Take an anhydrous solvent containing an acyl halide derivative and slowly add it dropwise into solution A to carry out a pre-reaction. Then heat it to reflux to carry out the reaction. After the reaction is complete, concentrate it to obtain a semi-solid residue. S3. The semi-solid residue is recrystallized with an organic solvent to obtain the α-MG xanthone NO donor-based compound.

5. The method for synthesizing α-MG xanthone-based NO donor compounds according to claim 3 or 4, characterized in that, The halogen in acyl halide derivatives is F, Cl, Br or I.

6. The method for synthesizing NO donor compounds based on α-MG xanthones according to claim 4, characterized in that, Anhydrous alkaline reagents include potassium carbonate, triethylamine, hydroxylamine, sodium hydroxide, potassium hydroxide, cesium carbonate, N,N-diisopropylethylamine, ammonia, or calcium hydroxide. The anhydrous solvent is tetrahydrofuran, 1,4-dioxane, acetone, N,N-dimethylformamide, or acetonitrile; The pre-reaction temperature was room temperature, and the time was 30–60 min. The reflux temperature is 45–65℃ and the time is 1–5 hours; The organic solvent used for recrystallization is at least one of toluene, xylene, benzene, n-hexane, and cyclohexane.

7. The use of the α-MG xanthonone-based NO donor compound of claim 1 in the preparation of anti-Staphylococcus aureus or as a sensitizer for clinically resistant MRSA strains.

8. The application of the α-MG xanthone-based NO donor compound according to claim 7, characterized in that, The α-MG xanthonone-based NO donor compounds are used to inhibit or remove Staphylococcus aureus biofilms.

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