Vancomycin derivative as well as preparation and antibacterial application thereof

Abstract

The invention discloses a vancomycin derivative as well as preparation and antibacterial application thereof. The structural general formula of the derivative is formula I, vancomycin is taken as an initial raw material, and a natural product coumarin group is introduced through hydrazide substitution or selective esterification, so that the target vancomycin derivative is prepared. A biological activity experiment shows that a natural product coumarin is introduced into a carboxyl position, the limitation of an antibacterial spectrum is broken through, and the derivative has broad-spectrum, high-efficiency and low-toxicity antibacterial activity and can effectively inhibit Gram-negative bacterium virulence factors such as removal of a biofilm of escherichia coli, and the derivative is simple in preparation method, short in route, clean in process and easy to amplify industrialization.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a vancomycin derivative, its preparation and antibacterial use. Background Art

[0002] One of the most severe challenges faced by medicine in the 21st century is the contradiction between the exponential growth of pathogen drug resistance and the continuous shrinkage of the new antibacterial drug R & D pipeline. Especially for the treatment of Gram-negative bacterial infections, no new drugs have been launched in the past fifty years. The essence of this crisis lies in the dual limitations of the traditional antibiotic development paradigm: on the one hand, the mechanism of action based on a single target is prone to cause target mutations leading to drug resistance; on the other hand, the existing molecular structures are difficult to break through the dual defenses of the Gram-negative bacterial outer membrane penetration barrier and the drug resistance efflux pump system.

[0003] As the "last line of defense" of glycopeptide antibiotics, vancomycin blocks transglycosylation and transpeptidation reactions by specifically binding to the terminal D-Ala-D-Ala dipeptide of the Gram-positive bacterial cell wall precursor. It is an antibiotic with definite curative effect and relatively high safety for the treatment of severe infections caused by methicillin-resistant Staphylococcus aureus, methicillin-resistant coagulase-negative Staphylococcus and Enterococcus, including septicemia, pulmonary infection, and skin and soft tissue infection. However, it cannot cross the Gram-negative bacterial outer membrane and has no anti-negative bacterial activity. Existing reports are based on the vancomycin backbone, introducing lipophilic long chains, penetrating peptides, guanidyl groups, tetraalkylammonium, arginine and other functional groups at multiple positions to insert and disrupt the membrane, break through the antibacterial spectrum limitation and show strong anti-negative bacterial activity. (Monoamine or guanidine-modified norvancomycin and vancomycin overcome multidrug resistance by enhancing lipid II binding and increasing membrane activity, Journal of Medicinal Chemistry, 2024, 67, 20639-20663; Research progress of semi-synthetic glycopeptide antibiotics: 2014-2022, ACS Infectious Diseases, 2022, 8, 1381-1407). However, these vancomycin derivatives cause cumulative toxicity or hemolysis risk.

[0004] It is reported that among the 64 antibacterial drugs approved by the FDA from 1981 to 2019, 58% directly originated from natural products or their semi-synthetic derivatives. This is attributed to the structural advantages of natural products, which have a pharmacodynamic backbone optimized by biological evolution and inherent biocompatibility. Through structural modification, the pharmacokinetic limitations of the natural structure can be broken through and the antibacterial spectrum can be expanded. Natural product coumarin compounds have transmembrane anti-negative bacteria, but their strong hemolytic property limits their clinical application (The antibacterial activity and structure-activity relationship of coumarin derivatives, European Journal of Medicinal Chemistry, 2020, 207, 112832). Therefore, using natural products such as coumarin to modify vancomycin is expected to improve its ability to penetrate the outer membrane, expand its antibacterial spectrum, and open up new directions and strategies for the transformation of old drugs. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies of the above-mentioned existing technologies. Using vancomycin as a lead compound, structural modification is carried out on its carboxyl group, and different lengths of hydroxycoumarin are introduced through hydrazide or amide or ester to synthesize a series of vancomycin derivatives, and screening is carried out on antibacterial activity, safety, and anti-biofilm activity, etc., to obtain a novel broad-spectrum, highly efficient, and low-toxic vancomycin derivative with anti-biofilm activity.

[0006] The object of the present invention is achieved through the following technical solutions:

[0007] In the first aspect, the present invention relates to a vancomycin derivative and a pharmaceutically acceptable salt thereof, such as hydrochloride, etc. The structure of the vancomycin derivative of the present invention is as shown in the general formula I in Figure 1 , where X = -NH-, NH-NH- or -O-; n is an integer between 8 and 12, such as 8, 9, 10, 11, 12, and preferably 8, 9 or 10; Preferably (substituted at the 7-position of coumarin).

[0008] The preferred general formula structure of the vancomycin derivative is: in general formula I, X = -NH- or NH-NH-; n is an integer between 8 and 12, and preferably 8, 9 or 10;

[0009] In the vancomycin derivative of the present invention, preferably in general formula I, X = -NH- or NH-NH-; n = 8, 9 or 10;

[0010] Particularly preferred compounds are A4 or B4, and their structural formulas are as follows:

[0011]

[0012]

[0013] In the second aspect, the preparation method of the vancomycin derivative of the present invention uses vancomycin as a starting material, and couples hydroxycoumarin and the carboxyl group of vancomycin through hydrazide or amide or ester. In the case where the target compound is A4 or B4, according to the Figure 5 , Figure 6 shown synthetic route schematic diagram, the compound represented by structural formula I is obtained through different chemical reaction combinations. The specific preparation method is as follows.

[0014] S1 Under nitrogen protection, at 0 - 5 °C, dissolve hydroxycoumarin in an anhydrous solvent, add 1 - 8 times the corresponding carbon-length bromoester or bromo-chain, add a base, and then continue to add a methanol solution of hydrazine hydrate or ammonia, and react at 40 - 75 °C for 4 - 10 hours. After separating and purifying the intermediate, intermediates a1 - a4 or b1 - b4 are obtained.

[0015] The anhydrous solvent is selected from dimethyl sulfoxide, N,N-dimethylformamide, acetone, acetonitrile, tetrahydrofuran, and 1,4-dioxane, and preferably acetone; the base is selected from potassium carbonate, triethylamine, sodium hydroxide, potassium hydroxide, cesium carbonate, etc., and preferably potassium carbonate; the preferred reaction temperature is 55-75 °C;

[0016] S2 Under nitrogen protection, at 0 °C, vancomycin is dissolved in an anhydrous solvent, and substituents coumarins a1-a4 or b1-b4 with corresponding chain lengths are added respectively, and the reaction is carried out at 40-75 °C for 4-10 hours. After separating and purifying the intermediate, the final products A1-A4 and B1-B4 are obtained.

[0017] The anhydrous solvents are dimethyl sulfoxide and N,N-dimethylformamide, and preferably a mixture of dimethyl sulfoxide and N,N-dimethylformamide in equal proportions; the condensing agents are HBTU and iPr2NEt, iPr2NEt and EDCI, DMAP and DCC, etc., and preferably HBTU and iPr2NEt. The molar ratio of the selected base to vancomycin is 1-4:1, preferably 2:1; the selected reaction temperature is 50-75 °C.

[0018] In a third aspect, the present invention relates to the antibacterial use of the vancomycin derivative, which has a strong ability against Gram-negative bacteria, increases the permeability of the cell membrane, destroys the integrity of the cell wall, and thus exerts a broad-spectrum and highly efficient antibacterial activity. It can be used for broad-spectrum and highly efficient antibacterial and for clearing biofilm formation, and can be used as a sensitizer and synergist for clinically drug-resistant strains.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The vancomycin derivatives synthesized in the present invention focus on their drug-like properties, break through the antibacterial spectrum limitation, and reduce clinical bacterial drug resistance. All the antibacterial activities or reduction of biofilms involved in the present invention increase the antibacterial spectrum of the existing antibiotic vancomycin. Its mechanism is unique. It is a method that inhibits the activities of Gram-positive or negative bacteria and eliminates the formation of their biofilms by doubly destroying the outer membrane and inhibiting the biosynthesis of the bacterial cell wall, as well as forming virulence factors such as biofilms. The vancomycin derivatives involved in the present invention have good activities against Gram-positive bacteria including MRSA and Gram-negative bacteria including Escherichia coli, can clear the formation of their biofilms, and enhance the antibacterial activities of existing antibiotics. All the synthetic routes of the present invention are short, the yields are high, the reaction process is green and clean, friendly to the environment, and easy for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the molecular structure diagram of vancomycin;

[0021] Figure 2 It is the general structural formula I of the vancomycin derivative;

[0022] Figure 3 , Figure 4 are the general structural formulas of vancomycin derivatives A1 - A4 (n = 2, 4, 6, 8) and B1 - B4 (n = 2, 4, 6, 8) respectively;

[0023] Figure 5 , Figure 6 are the schematic diagrams of the synthetic routes of vancomycin derivatives A1 - A4 and B1 - B4 respectively; where:

[0024] Figure 5 : i) CH3CO(CH2) n N2H4, K2CO3, 50 °C, DMF, 8 h; ii) HBTU, iPr2NEt, DMF / DMSO = 1:1, rt.

[0025] Figure 6 : i) NH2(CH2) n Br, K2CO3, 50 °C, DMF, 8 h; ii) HBTU, iPr2NEt, DMF / DMSO = 1:1, rt. Detailed implementation manners

[0026] The present invention will be described in detail below in conjunction with embodiments.

[0027] Example 1 Synthesis of coumarin intermediates a1 - a4 and b1 - b4

[0028] The corresponding bromine chain (1.5 mmol) was added to a solution of 7 - hydroxycoumarin (1.5 mmol) and potassium carbonate (3 mmol) in acetone (50 mL) and stirred. The mixture was heated under reflux, and the reaction was monitored by thin - layer chromatography. After cooling, the mixture was separated and the solvent was evaporated. The crude product was purified by column chromatography to obtain compounds a1 - a4 and b1 - b4.

[0029] Example 2 Synthesis of vancomycin - coumarin derivatives A1 - A4 and B1 - B4

[0030] Vancomycin (1 mmol) was dissolved in a solution of 10 ml of DMF and DMSO in equal proportions, then 1 mmol of diisopropyl ethylamine and HBTC were added, and finally a1 was added and reacted for 4 - 10 hours. After the reaction was completed, the product was concentrated, and the concentrated solution was freeze - dried and purified by a liquid chromatograph to obtain compound A1. The methods for obtaining A2 - A4 and B1 - B4 are the same and will not be elaborated.

[0031] (R)-N-((3S,6R,7R,22R,23S,26S,36R,38aR)-3-(2-Amino-2-oxoethyl)-44-(((2S,3R,4S,5S,6R)-3-(((2S,4S,5S,6S)-4-Amino-5-hydroxy-4,6-dimethyltetrahydro-2H-pyran-2-yl)oxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-19,45-dichloro-7,22,28,30,32-pentahydroxy-2,5,24,38,39-pentaoxo-26-(2-(2-((2-Oxo-2H-chromen-7-yl)oxy)acetyl)hydrazine-1-carbonyl)-2,3,4,5,6,7,23,24,25,26,36,37,38,38a-tetradecahydro-1H,22H-23,36-(iminomethano)-8,11:18,21-divinylene-13,16:31,35-di(methylene)benzo[n][1]oxa[6,9]diazacyclohexadec[4,5-d][1]oxa[7,17]diazacyclotetracos-6-yl)-4-methyl-2-(methylamino)pentanamide (A1)

[0032] (R)-N-((3S,6R,7R,22R,23S,26S,36R,38aR)-3-(2-Amino-2-oxoethyl)-44-(((2S,3R,4S,5S,6R)-3-(((2S,4S,5S,6S)-4-Amino-5-hydroxy-4,6-dimethyltetrahydro-2H-pyran-2-yl)oxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-19,45-dichloro-7,22,28,30,32-pentahydroxy-2,5,24,38,39-pentaoxo-26-(2-(4-((2-Oxo-2H-chromen-7-yl)oxy)butanoyl)hydrazine-1-carbonyl)-2,3,4,5,6,7,23,24,25,26,36,37,38,38a-tetradecahydro-1H,22H-23,36-(iminomethano)-8,11:18,21-divinylene-13,16:31,35-di(methylene)benzo[n][1]oxa[6,9]diazacyclohexadec[4,5-d][1]oxa[7,17]diazacyclotetracos-6-yl)-4-methyl-2-(methylamino)pentanamide (A2)

[0033] (R)-N-((3S,6R,7R,22R,23S,26S,36R,38aR)-3-(2-Amino-2-oxoethyl)-44-((2S,3R,4S,5S,6R)-3-((2-oxo-2H-chromen-7-yl)oxy)hexanoyl)hydrazine-1-carbonyl)-2,3,4,5,6,7,23,24,25,26,36,37,38,38a-tetrahydro-1H,22H-23,36-(carbamimidoyl)-8,11,18,21-divinyl-13,16:31,35-di(formyl)benzo[n][1]oxa[6,9]diazacyclohexane[4,5-d][1]oxacycloheptan-6-yl)-4-methyl-2-(methylamino)pentanamide (A3)

[0034] ((((2S,4S,5S,6S)-4-Amino-5-hydroxy-4,6-dimethyltetrahydro-2H-pyran-2-yl)oxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-19,45-dichloro-7,22,28,30,32-pentahydroxy-2,5,24,38,39-pentaoxo-26-(2-(8-((2-oxo-2H-chromen-7-yl)oxy)octanoyl)hydrazine-1-carbonyl)-2,3,4,5,6,7,23,24,25,26,36,37,38,38a-tetradecahydro-1H,22H-23,36-(iminomethane)-8,11:18,21-divinyl-13,16:31,35-di(methylene)benzo[n][1]oxa[6,9]diazacyclohexadecane[4,5-d][1]oxa[7,17]diazacyclotetracosane-6-yl)-4-methyl-2-(methylamino)pentanamide (A4)

[0035] (3S,6R,7R,22R,23S,26S,36R,38aR)-3-(2-Amino-2-oxoethyl)-44-(((2S,3R,4S,5S,6R)-3-(((2S,4S,5S,6S)-4-amino-5-hydroxy-4,6-dimethyltetrahydro-2H-pyran-2-yl)oxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-19,45-dichloro-7,22,28,30,32-pentahydroxy-6-((R)-4-methyl-2-(methylamino)pentanamido)-2,5,24,38,39-pentaoxo-N-(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)-2,3,4,5,6,7,23,24,25,26,36,37,38,38a-tetradecahydro-1H,22H-23,36-(iminomethano)-8,11:18,21-divinylene-13,16:31,35-di(methylene)benzo[n][1]oxa[6,9]diazacyclohexadec[4,5-d][1]oxa[7,17]diazacyclotetracos-26-carboxamide (B1)

[0036] (3S,6R,7R,22R,23S,26S,36R,38aR)-3-(2-Amino-2-oxoethyl)-44-(((2S,3R,4S,5S,6R)-3-(((2S,4S,5S,6S)-4-amino-5-hydroxy-4,6-dimethyltetrahydro-2H-pyran-2-yl)oxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-19,45-dichloro-7,22,28,30,32-pentahydroxy-6-((R)-4-methyl-2-(methylamino)pentanamido)-2,5,24,38,39-pentaoxo-N-(6-((2-oxo-2H-chromen-7-yl)oxy)hexyl)-2,3,4,5,6,7,23,24,25,26,36,37,38,38a-tetradecahydro-1H,22H-23,36-(iminomethano)-8,11:18,21-divinylene-13,16:31,35-di(methylene)benzo[n][1]oxa[6,9]diazacyclohexadec[4,5-d][1]oxa[7,17]diazacyclotetracos-26-carboxamide (B2)

[0037] (3S,6R,7R,22R,23S,26S,36R,38aR)-3-(2-Amino-2-oxoethyl)-44-(((2S,3R,4S,5S,6R)-3-(((2S,4S,5S,6S)-4-amino-5-hydroxy-4,6-dimethyltetrahydro-2H-pyran-2-yl)oxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-19,45-dichloro-7,22,28,30,32-pentahydroxy-6-((R)-4-methyl-2-(methylamino)pentanamido)-2,5,24,38,39-pentaoxo-N-(6-((2-oxo-2H-chromen-7-yl)oxy)hexyl)-2,3,4,5,6,7,23,24,25,26,36,37,38,38a-tetradecahydro-1H,22H-23,36-(iminomethanediyl)-8,11:18,21-divinyl-13,16:31,35-bis(methylene)benzo[n][1]oxa[6,9]diazacyclohexadecano[4,5-d][1]oxa[7,17]diazacyclotetracosane-26-carboxamide (B3)

[0038] (3S,6R,7R,22R,23S,26S,36R,38aR)-3-(2-Amino-2-oxoethyl)-44-(((2S,3R,4S,5S,6R)-3-(((2S,4S,5S,6S)-4-amino-5-hydroxy-4,6-dimethyltetrahydro-2H-pyran-2-yl)oxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-19,45-dichloro-7,22,28,30,32-pentahydroxy-6-((R)-4-methyl-2-(methylamino)pentanamido)-2,5,24,38,39-pentaoxo-N-(8-((2-oxo-2H-chromen-7-yl)oxy)octyl)-2,3,4,5,6,7,23,24,25,26,36,37,38,38a-tetradecahydro-1H,22H-23,36-(iminomethanediyl)-8,11:18,21-divinyl-13,16:31,35-bis(methylene)benzo[n][1]oxa[6,9]diazacyclohexadecano[4,5-d][1]oxa[7,17]diazacyclotetracosane-26-carboxamide (B4)

[0039] The structural formulas of vancomycin derivatives A1 - A4 and B1 - B4 are shown in Figure 3 and Figure 4 respectively.

[0040] Minimum inhibitory concentration test of vancomycin derivatives in Example 3

[0041] 1) Experimental principle

[0042] This experiment is based on the growth of bacteria in Mueller-Hinton (MH) medium in 96-well plates. After a period of growth, due to the large quantity, white precipitates will appear, making it easy to distinguish whether there is bacterial growth. The bacteriostatic agent is diluted in a serial dilution method. After adding it to the bacterial suspension and culturing for a period of time, the last well without precipitate is the minimum inhibitory concentration (MIC).

[0043] 2) Experimental method

[0044] According to the NCCLS experimental operation requirements, Staphylococcus aureus ATCC 29213, Staphylococcus aureus ATCC 6538, Staphylococcus aureus MRSA2, Escherichia coli ATCC 25922, etc. are used as test evaluation strains. After the strains are revived, they are cultured until the logarithmic growth phase for standby. Vancomycin and its derivatives are dissolved in dimethyl sulfoxide to 2 mg / mL. Take out the 96-well plate, add 25.6 μL to the first column, add MH (B) medium to 200 μL, and dilute it in a serial dilution method to the 10th column. The 11th column is the medium (negative) control, and the 12th column is the bacterial suspension (positive) control. Vancomycin is used as the drug control, and the results are observed after culturing at 37 °C for 18 - 24 h. The minimum inhibitory concentration of the drug control should be within the reference range, and the experimental results of the same batch are considered reliable.

[0045] 3) Experimental results

[0046] The antibacterial activities of vancomycin and its derivatives against standard strains and multi-drug resistant strains of Escherichia coli and Staphylococcus aureus were detected by the microdilution method, and the results are shown in Table 1.

[0047] Table 1 Minimum inhibitory concentration of vancomycin and its derivatives against bacteria

[0048]

[0049] 4) Experimental conclusion

[0050] From the data in Table 1, it can be seen that vancomycin (VAN) has antibacterial effects on Gram-positive bacteria but is ineffective against Escherichia coli. The activities of vancomycin derivatives A1 - A3 and B1 - B3 against Gram-positive bacteria decreased, and no antibacterial activities against Escherichia coli were shown. However, unexpectedly, A4 and B4 showed broad-spectrum antibacterial activities; although the antibacterial activities against Staphylococcus aureus decreased slightly, their activities against Escherichia coli reached 2 μg / mL and 4 μg / mL respectively. This shows that when the carbon chain is extended to 8 carbon atoms, vancomycin derivatives can penetrate the outer membrane of Gram-negative bacteria.

[0051] Example 4 Inhibition of biofilm by vancomycin and its derivatives

[0052] 1) Experimental principle

[0053] Quantitative crystal violet assay of biofilms is a commonly used biological experimental method for measuring the permeability of biological membranes such as cell membranes, mitochondria, and endoplasmic reticulum. This method is based on crystal violet dye, which can bind to biological membranes and diffuse into the cell interior through the cell membrane. After staining, by measuring the absorption degree of the dye in the cells, the permeability of the biological membrane can be indirectly reflected. The absorbance (OD value) of the stained cells can be measured by a spectrophotometer. The absorbance is proportional to the concentration of crystal violet in the cells, and the concentration of crystal violet in the cells is related to the permeability of the biological membrane. Therefore, by measuring the absorbance, the permeability of the biological membrane can be indirectly calculated.

[0054] 2) Experimental method

[0055] Biofilm formation inhibition experiment: Inoculate an overnight bacterial culture into LB (3 mL) and incubate statically at 37 °C until the culture reaches mid-log phase, then dilute 1:100 and set aside. Biofilm inhibition assays were performed by adding bacterial suspension (100 μL) to 96-well plates containing 100 μL of compounds at different concentrations (0 to 128 μg / ml). Then the 96-well plates were placed in an incubator at 37 °C for 12 - 16 hours. After completion, the bacteria were removed from the 96-well plates, washed 3 times with phosphate-buffered saline (1×PBS), then 0.1% crystal violet was added and left at room temperature for 15 - 20 minutes, then the crystal violet was removed and the plates were washed 3 times with PBS. Finally, ethanol was added to the 96-well plates to dissolve the crystal violet adsorbed on the plates, and the plates were placed on a microplate reader to read the absorbance value at 595 nm. The minimum polymer concentration at which the biofilm was reduced by 90% compared to the untreated control was defined as the minimum inhibitory biofilm concentration. Each experiment was repeated at least three times. Biofilm inhibition rate (%) = (ODcontrol - ODsample) / ODcontrol × 100.

[0056] Biofilm eradication experiment: First, incubate in 96-well plates at 37 °C for 12 hours to obtain mature biofilms. After removing the planktonic bacteria, the biofilms were treated with compounds at different concentrations at 37 °C for 12 hours, and the other steps were similar to the inhibition experiment. As described above, biofilm biomass analysis was performed on the samples, and the removal rates of biofilm concentration and biofilm biomass were calculated. Biofilm eradication rate (%) = (ODcontrol - ODsample) / ODcontrol × 100.

[0057] 3) Experimental results

[0058] The inhibitory effects of vancomycin derivatives on Staphylococcus aureus ATCC6538 biofilms and Escherichia coli ATCC25922 were measured by the crystal violet method, and the results are shown in Table 2.

[0059] Table 2 Minimum inhibitory concentration of vancomycin and its derivatives against bacterial biofilms

[0060]

[0061] 4) Experimental conclusions

[0062] As can be seen from the data in Table 2, both vancomycin derivatives A4 and B4 have good inhibitory effects on the biofilms of Gram-positive and Gram-negative bacteria, and A4 has a better effect.

[0063] Example 5 Hemolytic activity detection of vancomycin derivatives

[0064] 1) Experimental principle

[0065] The hemolysis experiment is a common experimental method for detecting the activity of blood antibodies. Its principle is based on the phenomenon of red blood cell lysis under the action of specific antibodies.

[0066] 2) Experimental method

[0067] Add 4% rabbit red blood cells (100 μL) to a 96-well plate containing 100 μL of compounds at different concentrations (0 to 128 μg / ml). The positive control is 1% Triton-X 100, and the negative control is PBS. Incubate the 96-well plate in a thermostat at 37 °C and 60 rpm for 1 hour. After incubation, centrifuge the plate at 1000 g for 3 minutes. Take the supernatant (100 μL) and measure the absorbance of each well at 540 nm. Each experimental data is repeated three times.

[0068] 3) Experimental results

[0069] The experimental results are shown in Table 3. All compounds do not cause hemolysis at a concentration of 256 μg / mL.

[0070] Table 3. Hemolytic activity of vancomycin derivatives

[0071]

[0072] 4) Experimental conclusions

[0073] Vancomycin derivatives A4 and B4 have good biofilm clearance activity and are not easily induced to cause red blood cell hemolysis.

[0074] Example 6 Cytotoxicity detection of vancomycin derivatives

[0075] 1) Experimental principle of CCK-8 method

[0076] The principle of the CCK-8 method is based on the fact that this reagent contains a water-soluble tetrazolium salt, chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt. Under the action of the electron carrier 1-methoxy-5-methylphenazinium methyl sulfate (1-Methoxy PMS), it is reduced by dehydrogenases in cells to a highly water-soluble yellow formazan dye. The amount of formazan produced is proportional to the number of living cells. Therefore, this property can be used to directly measure cell proliferation and analyze its toxicity.

[0077] 2) Experimental method

[0078] Add 100 μL of cell suspension into 96 wells, with each well containing approximately 5000 cells. After shaking the cells evenly, place the 96-well plate in an incubator at 37 °C for 24 hours. After discarding the supernatant, add 100 μL of compounds with different concentrations. Place the 96-well plate in an incubator at 37 °C for 72 hours. Finally, under dark conditions, add 5 μL of CCK-8 solution to each well. Incubate the 96-well plate in an incubator at 37 °C for 4 hours, and measure the OD value at 450 nm using a microplate reader. The inhibition rate of the cells was obtained under the action of compounds with different concentrations, and the experiment was repeated three times.

[0079] 3) Experimental results

[0080] The experimental results are shown in Table 3. All compound concentrations of IC 50 showed no toxicity at 256 μg / mL.

[0081] 4) Experimental conclusion

[0082] Both Compound A4 and B4 have good safety.

[0083] Table 3. Toxicity of vancomycin derivatives

[0084]

[0085] 1H NMR of vancomycin derivatives A1 - A4 and B1 - B4 (A1)

[0086] 11H NMR (400 MHz, DMSO-d6) δ 9.85 (s, 1H), 8.98 (d, J = 3.2 Hz, 2H), 8.65 (s, 1H), 8.23 (s, 1H), 7.92 (s, 2H), 7.86 (dd, J = 2.1, 1.0 Hz, 1H), 7.74 (dd, J = 11.0, 1.0 Hz, 1H), 7.64–7.56 (m, 2H), 7.43 (ddd, J = 7.5, 2.0, 1.0 Hz, 1H), 7.35 (dd, J = 1.9, 1.0 Hz, 1H), 7.33–7.25 (m, 2H), 7.21 (s, 1H), 7.07 (d, J = 2.1 Hz, 1H), 7.05–6.98 (m, 2H), 6.80 (ddd, J = 4.3, 2.1, 1.0 Hz, 2H), 6.53 (ddd, J = 7.5, 1.9, 1.0 Hz, 1H), 6.43 (d, J = 7.4 Hz, 1H), 6.34 (d, J = 1.9 Hz, 1H), 6.26–6.15 (m, 2H), 6.11–6.00 (m, 2H), 5.89–5.74 (m, 3H), 5.67 (d, J = 1.1 Hz, 1H), 5.16–5.06 (m, 3H), 5.00–4.78 (m, 4H), 4.75 (d, J = 12.5 Hz, 1H), 4.60 (t, J = 7.0 Hz, 1H), 4.27 (t, J = 6.9 Hz, 1H), 3.95–3.57 (m, 7H), 3.55–3.41 (m, 2H), 2.79 (dd, J = 12.4, 7.0 Hz, 1H), 2.13 (dd, J = 12.4, 7.0 Hz, 1H), 1.91 (dd, J = 13.2, 7.0 Hz, 1H), 1.82–1.64 (m, 4H), 1.59 (dd, J = 13.2, 7.0 Hz, 1H), 1.11–0.81 (m, 12H). 1313C NMR (100 MHz, DMSO-d6) δ 172.14, 171.96, 171.03, 170.76, 168.98, 168.66, 168.53, 167.64, 166.64, 160.59, 157.28, 155.92, 155.52, 154.17, 152.31, 149.50, 142.92, 138.90, 138.06, 137.99, 135.52, 134.44, 128.39, 128.15, 126.62, 126.22, 125.76, 125.64, 124.12, 121.49, 117.42, 116.64, 112.23, 112.05, 110.82, 108.35, 104.42, 102.73, 101.51, 101.49, 97.69, 79.24, 76.87, 71.25, 70.98, 70.64, 69.50, 65.97, 64.09, 61.09, 60.78, 58.73, 57.21, 55.54, 55.01, 54.29, 54.00, 51.64, 39.52, 37.04, 33.19, 32.36, 26.39, 23.83, 21.97, 16.29.

[0087] (A2)

[0088] 11H NMR (400 MHz, DMSO-d6) δ 10.08 (s, 1H), 9.96 (s, 1H), 9.46 (s, 1H), 8.98 (s, 1H), 8.23 (s, 0H), 7.84–7.66 (m, 2H), 7.50 (s, 2H), 7.12–6.90 (m, 4H), 6.70 (d, J = 1.0 Hz, 1H), 6.54 (dd, J = 2.0, 0.9 Hz, 0H), 6.30 (d, J = 2.1 Hz, 0H), 6.24–6.13 (m, 1H), 5.90–5.74 (m, 3H), 5.33 (t, J = 5.0 Hz, 1H), 5.10 (dd, J = 5.0, 4.0 Hz, 2H), 4.99–4.81 (m, 2H), 4.63 (t, J = 7.0 Hz, 1H), 4.08 (t, J = 6.9 Hz, 1H), 3.97–3.55 (m, 4H), 3.53–3.37 (m, 1H), 2.81 (dd, J = 12.4, 7.0 Hz, 1H), 2.56 (dd, J = 12.4, 7.0 Hz, 1H), 2.37 (s, 2H), 2.17–1.94 (m, 2H), 1.87–1.68 (m, 2H), 1.62–1.48 (m, 1H), 1.32 (t, J = 7.0 Hz, 1H), 1.20–1.05 (m, 3H), 0.98–0.82 (m, 5H). 13 13C NMR (100 MHz, DMSO-d6) δ 172.96, 172.77, 172.41, 171.84, 171.57, 169.79, 169.48, 169.35, 168.45, 162.70, 161.40, 158.09, 156.73, 156.09, 154.98, 153.12, 150.31, 143.73, 139.71, 138.87, 138.81, 136.33, 135.25, 128.96, 128.77, 127.44, 127.03, 126.58, 126.45, 124.94, 122.30, 118.23, 117.45, 113.25, 113.04, 112.87, 109.17, 105.23, 103.54, 102.30, 101.64, 98.50, 80.05, 77.68, 72.06, 71.80, 71.45, 70.32, 67.51, 64.90, 61.90, 61.60, 59.54, 58.03, 56.35, 55.82, 55.10, 54.81, 52.46, 40.33, 37.85, 34.00, 33.18, 31.33, 27.20, 25.07, 24.64, 22.78, 17.10, -10.21.

[0089] (A3)

[0090] 1 1H NMR (400 MHz, DMSO-d6) δ 10.50 (s, 1H), 10.08 (s, 2H), 9.18 (d, J = 8.1 Hz, 2H), 8.98 (s, 1H), 8.76 (s, 1H), 8.23 (s, 1H), 8.12 (s, 2H), 7.97 (dd, J = 10.8, 1.1 Hz, 1H), 7.88–7.82 (m, 2H), 7.62 (dd, J = 7.4, 1.0 Hz, 1H), 7.55–7.47 (m, 2H), 7.32 (ddd, J = 7.5, 2.0, 1.0 Hz, 1H), 7.28–7.15 (m, 3H), 7.05 (dd, J = 7.5, 2.0 Hz, 1H), 7.01–6.88 (m, 4H), 6.39 (d, J = 1.9 Hz, 1H), 6.28 (d, J = 10.9 Hz, 1H), 6.12 (dd, J = 2.0, 1.0 Hz, 1H), 6.03–5.93 (m, 2H), 5.84–5.76 (m, 3H), 5.14–5.00 (m, 4H), 4.83–4.71 (m, 2H), 4.32–4.06 (m, 5H), 3.84–3.55 (m, 5H), 3.46–3.37 (m, 2H), 3.01 (s, 1H), 2.89–2.77 (m, 2H), 2.57 (dd, J = 12.4, 7.0 Hz, 1H), 2.45–2.32 (m, 2H), 2.31 (s, 3H), 1.88–1.68 (m, 6H), 1.71–1.58 (m, 2H), 1.55–1.31 (m, 4H), 1.18–0.99 (m, 4H), 0.90 (dd, J = 20.0, 6.7 Hz, 6H), 0.79 (s, 3H). 1313C NMR (100 MHz, DMSO-d6) δ 172.96, 172.77, 172.41, 171.84, 171.57, 169.79, 169.48, 169.35, 168.45, 162.70, 161.40, 158.09, 156.73, 156.09, 154.98, 153.12, 150.31, 143.73, 139.71, 138.87, 138.81, 136.33, 135.25, 128.96, 128.77, 127.44, 127.03, 126.58, 126.45, 124.94, 122.30, 118.23, 117.45, 113.25, 113.04, 112.87, 109.17, 105.23, 103.54, 102.30, 101.64, 98.50, 80.05, 77.68, 72.06, 71.80, 71.45, 70.32, 67.70, 64.90, 61.90, 61.60, 59.54, 58.03, 56.35, 55.82, 55.10, 54.81, 52.46, 40.33, 37.85, 35.18, 34.00, 33.18, 29.32, 27.20, 25.78, 25.65, 24.64, 22.78, 17.10.

[0091] (A4)

[0092] 11H NMR (400 MHz, DMSO-d6) δ 10.97 (s, 1H), 10.08 (s, 2H), 9.43 (s, 1H), 9.28 (s, 1H), 8.98 (s, 1H), 8.69 (s, 1H), 8.23 (s, 1H), 7.97 (dd, J = 10.8, 1.1 Hz, 1H), 7.86 (d, J = 2.3 Hz, 3H), 7.68–7.58 (m, 2H), 7.58–7.50 (m, 3H), 7.32 (ddd, J = 7.5, 2.0, 1.0 Hz, 1H), 7.31 (s, 1H), 7.29–7.19 (m, 2H), 7.10 (d, J = 7.5 Hz, 1H), 7.04–6.91 (m, 4H), 6.85 (ddd, J = 6.1, 2.1, 1.0 Hz, 2H), 6.45 (d, J = 2.1 Hz, 1H), 6.32–6.20 (m, 2H), 5.84–5.73 (m, 5H), 5.10 (dd, J = 5.0, 4.0 Hz, 3H), 4.91 (d, J = 7.1 Hz, 1H), 4.79 (d, J = 7.1 Hz, 1H), 4.59 (dt, J = 24.8, 7.0 Hz, 2H), 4.29 (t, J = 7.0 Hz, 1H), 3.96–3.57 (m, 9H), 3.44 (dd, J = 7.0, 4.9 Hz, 1H), 2.85 (dd, J = 3.8, 2.5 Hz, 1H), 2.72 (dd, J = 12.4, 7.0 Hz, 1H), 2.53 (s, 1H), 2.51–2.33 (m, 2H), 2.34 (s, 3H), 2.22 (dd, J = 12.4, 7.0 Hz, 1H), 1.94–1.72 (m, 3H), 1.71 (s, 2H), 1.71–1.52 (m, 2H), 1.56–1.34 (m, 5H), 1.29 (tdd, J = 10.2, 6.1, 3.0 Hz, 1H), 1.20 (d, J = 6.8 Hz, 3H), 0.97–0.84 (m, 8H). 1313C NMR(100MHz, DMSO-d6) δ 172.96, 172.77, 172.41, 171.84, 171.57, 169.79, 169.48, 169.35, 168.45, 162.70, 161.40, 158.09, 156.73, 156.09, 154.98, 153.12, 150.31, 143.73, 139.71, 138.87, 138.81, 136.33, 135.25, 128.96, 128.77, 127.44, 127.03, 126.58, 126.45, 124.94, 122.30, 118.23, 117.45, 113.25, 113.04, 112.87, 109.17, 105.23, 103.54, 102.30, 101.64, 98.50, 80.05, 77.68, 72.06, 71.80, 71.45, 70.32, 68.01, 64.90, 61.90, 61.60, 59.54, 58.03, 56.35, 55.82, 55.10, 54.81, 52.46, 40.33, 37.85, 35.18, 34.00, 33.18, 30.35, 30.14, 29.29, 27.38, 27.20, 26.31, 24.64, 22.78, 17.10.

[0093] (B1)

[0094] 11H NMR (400 MHz, DMSO-d6) δ 10.90 (s, 1H), 8.98 (s, 1H), 8.86 (d, J = 4.0 Hz, 2H), 8.69 (d, J = 24.4 Hz, 2H), 8.19 (d, J = 31.9 Hz, 2H), 7.84–7.74 (m, 3H), 7.67–7.42 (m, 4H), 7.36–7.22 (m, 3H), 7.16–6.86 (m, 7H), 6.47–6.32 (m, 3H), 6.19 (d, J = 10.8 Hz, 1H), 5.85–5.61 (m, 6H), 5.14–4.93 (m, 5H), 4.81 (d, J = 7.1 Hz, 1H), 4.49 (t, J = 7.0 Hz, 1H), 4.36–4.22 (m, 1H), 4.15 (ddd, J = 14.6, 7.3, 5.0 Hz, 2H), 4.00 (dt, J = 14.0, 7.0 Hz, 2H), 3.86–3.62 (m, 6H), 3.53–3.39 (m, 2H), 3.06–2.67 (m, 3H), 2.36 (d, J = 38.6 Hz, 4H), 2.14 (dd, J = 12.4, 7.0 Hz, 1H), 1.95 (dd, J = 13.2, 7.0 Hz, 1H), 1.83 (s, 2H), 1.73–1.51 (m, 3H), 1.43–1.22 (m, 3H), 1.18–1.06 (m, 6H), 0.90 (dd, J = 20.0, 6.7 Hz, 6H). 1313C NMR (100 MHz, DMSO-d6) δ 172.96, 171.84, 171.57, 171.15, 169.79, 169.48, 169.35, 167.60, 162.70, 161.40, 158.09, 156.73, 156.09, 154.98, 153.12, 150.31, 143.73, 139.71, 138.87, 138.81, 136.33, 135.25, 128.96, 128.77, 127.44, 127.03, 126.58, 126.45, 124.94, 122.30, 118.23, 117.45, 113.53, 113.25, 113.04, 112.87, 105.23, 103.54, 102.30, 101.64, 98.50, 80.05, 77.68, 72.06, 71.80, 71.45, 70.32, 66.52, 64.90, 61.90, 61.60, 59.54, 58.03, 57.96, 56.35, 55.10, 54.81, 52.46, 40.33, 38.70, 37.85, 34.00, 33.18, 29.56, 27.20, 24.64, 22.78, 17.10.

[0095] (B2)

[0096] 11H NMR (400 MHz, DMSO-d6) δ 10.48 (s, 1H), 9.70 (s, 1H), 9.48 (s, 1H), 9.37 (s, 1H), 8.98 (s, 1H), 8.77 (s, 1H), 8.19 (d, J = 31.9 Hz, 2H), 7.87–7.58 (m, 6H), 7.56–7.47 (m, 1H), 7.40–7.12 (m, 7H), 7.06–6.83 (m, 4H), 6.50–6.33 (m, 2H), 6.27–6.09 (m, 2H), 5.90–5.77 (m, 3H), 5.72 (d, J = 1.0 Hz, 1H), 5.10 (dd, J = 5.0, 4.0 Hz, 3H), 4.87–4.61 (m, 3H), 4.18–4.00 (m, 4H), 3.88–3.55 (m, 7H), 3.39 (dd, J = 7.0, 4.9 Hz, 1H), 2.93 (dd, J = 11.8, 2.1 Hz, 1H), 2.80–2.52 (m, 3H), 2.28 (s, 3H), 2.06 (dd, J = 12.5, 7.1 Hz, 1H), 1.88 (dd, J = 13.3, 7.1 Hz, 1H), 1.81–1.19 (m, 14H), 1.09 (d, J = 6.8 Hz, 3H), 0.90 (dd, J = 20.0, 6.7 Hz, 6H), 0.76 (s, 3H). 13CNMR(100MHz, DMSO-d6) δ 172.96, 171.84, 171.57, 171.15, 169.79, 169.48, 169.35, 168.45, 162.70, 161.40, 158.09, 156.73, 156.09, 154.98, 154.07, 153.42, 151.94, 150.31, 143.73, 139.71, 139.48, 138.81, 136.33, 134.22, 133.41, 128.96, 128.77, 127.84, 127.44, 127.03, 126.58, 126.45, 124.94, 122.30, 120.26, 118.23, 117.45, 113.53, 113.25, 113.04, 112.87, 107.19, 105.23, 103.54, 102.30, 101.64, 98.50, 80.05, 77.68, 72.67, 72.06, 71.80, 70.32, 68.01, 64.90, 61.90, 61.60, 61.38, 59.54, 57.96, 56.35, 55.10, 54.81, 52.46, 40.33, 39.92, 37.85, 34.00, 33.18, 29.44, 29.41, 27.20, 26.42, 25.63, 24.64, 22.78, 17.10.

[0097] (B3)

[0098] 11H NMR (400 MHz, DMSO-d6) δ 10.31 (s, 1H), 9.41 (s, 1H), 9.13 (s, 1H), 8.98 (s, 1H), 8.74 (s, 1H), 8.23 (s, 1H), 8.14 (d, J = 4.3 Hz, 3H), 7.97 (dd, J = 10.8, 1.1 Hz, 1H), 7.84 (dd, J = 1.9, 1.0 Hz, 1H), 7.66–7.52 (m, 4H), 7.44–7.34 (m, 2H), 7.33–7.18 (m, 5H), 7.16–7.08 (m, 2H), 6.66 (dd, J = 2.0, 1.0 Hz, 1H), 6.42 (d, J = 2.1 Hz, 1H), 6.28 (d, J = 10.9 Hz, 1H), 6.13 (dd, J = 2.0, 0.9 Hz, 1H), 5.95 (d, J = 1.1 Hz, 1H), 5.84–5.75 (m, 2H), 5.67–5.58 (m, 2H), 5.37–5.29 (m, 1H), 5.10 (dd, J = 5.0, 4.0 Hz, 3H), 4.87–4.76 (m, 2H), 4.72 (d, J = 7.0 Hz, 1H), 4.31 (t, J = 7.0 Hz, 1H), 4.08–3.87 (m, 4H), 3.85–3.63 (m, 7H), 3.52–3.42 (m, 2H), 3.16 (s, 2H), 3.05 (s, 1H), 2.98 (dd, J = 5.0, 1.8 Hz, 1H), 2.74 (td, J = 12.2, 4.0 Hz, 1H), 2.60 (dd, J = 12.4, 7.0 Hz, 1H), 2.37 (s, 3H), 2.30 (dd, J = 12.4, 7.0 Hz, 1H), 1.93 (dd, J = 13.2, 7.0 Hz, 1H), 1.85–1.69 (m, 3H), 1.66–1.45 (m, 2H), 1.29–1.07 (m, 7H), 1.04–0.84 (m, 7H), 0.49–0.33 (m, 2H). 1313C NMR (100 MHz, DMSO-d6) δ 172.96, 171.84, 171.57, 171.15, 169.79, 169.48, 169.35, 167.60, 162.70, 161.40, 158.09, 156.73, 156.09, 154.98, 153.12, 150.31, 143.73, 139.71, 138.87, 138.81, 136.33, 135.25, 128.96, 128.77, 127.44, 127.03, 126.58, 126.45, 124.94, 122.30, 118.23, 117.45, 113.53, 113.25, 113.04, 112.87, 105.23, 103.54, 102.30, 101.64, 98.50, 80.05, 77.68, 72.06, 71.80, 71.45, 70.32, 68.01, 64.90, 61.90, 61.60, 59.54, 58.03, 57.96, 56.35, 55.10, 54.81, 52.46, 40.33, 39.92, 37.85, 34.00, 33.18, 29.44, 29.41, 27.20, 26.42, 25.63, 24.64, 22.78, 17.10.

[0099] (B4)

[0100] 11H NMR (400 MHz, DMSO-d6) δ 10.72 (s, 1H), 9.13 (s, 1H), 8.97 (d, J = 8.4 Hz, 2H), 8.54 (s, 1H), 8.23 (d, J = 2.3 Hz, 3H), 8.15 (s, 1H), 7.97 (dd, J = 10.8, 1.0 Hz, 1H), 7.83 (dd, J = 2.0, 1.0 Hz, 1H), 7.62 (dt, J = 7.6, 1.1 Hz, 2H), 7.53 (ddd, J = 17.0, 1.9, 1.0 Hz, 2H), 7.42 (s, 1H), 7.34–7.26 (m, 3H), 7.21 (dd, J = 7.5, 1.9 Hz, 1H), 7.14 (s, 1H), 7.07–6.92 (m, 5H), 6.62 (dd, J = 2.0, 1.0 Hz, 1H), 6.42 (d, J = 1.9 Hz, 1H), 6.36 (dd, J = 2.0, 0.9 Hz, 1H), 6.28 (d, J = 10.9 Hz, 1H), 5.88–5.76 (m, 3H), 5.57–5.46 (m, 1H), 5.38–5.29 (m, 1H), 5.10 (dd, J = 5.0, 4.0 Hz, 3H), 5.01–4.93 (m, 2H), 4.76 (d, J = 7.0 Hz, 1H), 4.39 (t, J = 7.0 Hz, 1H), 4.24–4.05 (m, 3H), 4.01–3.59 (m, 7H), 3.54–3.44 (m, 2H), 3.06–2.96 (m, 2H), 2.59 (dd, J = 12.4, 7.0 Hz, 1H), 2.38 (s, 3H), 2.42–2.27 (m, 2H), 2.23 (s, 1H), 1.77 (s, 2H), 1.99–1.32 (m, 9H), 1.35–1.07 (m, 10H), 0.90 (dd, J = 20.0, 6.8 Hz, 6H). 1313C NMR (100 MHz, DMSO-d6) δ 172.96, 171.84, 171.57, 171.15, 169.79, 169.48, 169.35, 167.60, 162.70, 161.40, 158.09, 156.73, 156.09, 154.98, 153.12, 150.31, 143.73, 139.71, 138.87, 138.81, 136.33, 135.25, 128.96, 128.77, 127.44, 127.03, 126.58, 126.45, 124.94, 122.30, 118.23, 117.45, 113.53, 113.25, 113.04, 112.87, 105.23, 103.54, 102.30, 101.64, 98.50, 80.05, 77.68, 72.06, 71.80, 71.45, 70.32, 68.01, 64.90, 61.90, 61.60, 59.54, 58.03, 57.96, 56.35, 55.10, 54.81, 52.46, 40.33, 39.92, 37.85, 34.00, 33.18, 30.35, 29.41, 29.28, 28.68, 27.20, 26.65, 26.31, 24.64, 22.78, 17.10。

Claims

1. A vancomycin derivative and a pharmaceutically acceptable salt thereof, wherein the structure of the vancomycin derivative is shown in General Formula I: Wherein: X = -NH-, NH-NH- or -O-; n is an integer between 8 and 12; 2. The pharmaceutically acceptable salt of the vancomycin derivative according to Claim 1, wherein the salt is hydrochloride.

3. The vancomycin derivative according to claim 1 and its pharmaceutically acceptable salts, wherein in the general formula I 4. The vancomycin derivative and a pharmaceutically acceptable salt thereof according to Claim 1, wherein in General Formula I, X = -NH- or NH-NH.

5. The vancomycin derivative and a pharmaceutically acceptable salt thereof according to Claim 1, wherein in General Formula I, n = 8, 9 or 10.

6. The vancomycin derivative according to claim 1 and its pharmaceutically acceptable salt, wherein in the general formula I, X = -NH- or NH-NH-; n = 8, 9 or 10; 7. A vancomycin derivative and a pharmaceutically acceptable salt thereof, wherein the structure of the vancomycin derivative is shown as follows: Among them, n＝8。 8. A vancomycin derivative and a pharmaceutically acceptable salt thereof, wherein the structure of the vancomycin derivative is shown as follows: Among them, n＝8。 9. A method for preparing any one of the vancomycin derivatives according to claims 1-8, characterized in that, Using vancomycin as the starting material, hydroxycoumarin and the carboxyl group of vancomycin are coupled through hydrazide, amide or ester.

10. The antibacterial use of any one of the vancomycin derivatives or a pharmaceutically acceptable salt thereof according to Claims 1-8.

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