A fusidic acid derivative and its preparation method and application

Abstract

The present invention discloses a fusidic acid derivative and a preparation method and application thereof. The present invention derivatizes the 3-OH site of fusidic acid through a linker with a disulfide bond, introduces different terminal functional groups on the one hand, and adjusts the properties of the fusidic acid molecule, such as the introduction of an amino group or a carboxyl group can change the water solubility of the molecule under different pH conditions, and the polyethylene glycol structure can change the systemic circulation characteristics of the molecule, etc. At the same time, active functional groups such as alkynes, amino groups, and carboxyl groups provide sites for further modification, thereby modularly constructing more functional fusidic acid derivatives. The disulfide bond in the linker can effectively maintain the antibacterial activity of fusidic acid, and in the presence of a polypeptide adjuvant, effective killing of Gram-negative bacteria is achieved. The preparation method of the present invention is simple, and the product separation and purification is easy.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry, and in particular relates to a fusidic acid derivative and a preparation method and application thereof. Background Art

[0002] Fusidic acid (FA) is a tetracyclic triterpenoid compound isolated from fungi. It was first isolated from Fusidium coccineum in 1960. FA acts as an inhibitor of protein synthesis and can bind to EF-G-GDP to block peptide synthesis. Since 1962, FA has been clinically used for systemic and localized staphylococcal infections, including methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococcal infections. Although FA is effective against Gram-positive bacteria, it cannot be taken up by Gram-negative bacteria due to the obstruction of the outer membrane of Gram-negative bacteria, so it is ineffective against Gram-negative bacteria.

[0003] FA and its derivatives have been shown to have a wide range of pharmacological activities, including antibacterial, antiparasitic, antituberculosis, anticancer, tumor multidrug resistance (MDR) reversal, anti-inflammatory, antifungal, and antiviral activities in vitro and in vivo. However, the semi-synthesis, bioactivity, and structure-activity relationship (SAR) studies of FA derivatives have remained in the development stage for nearly 20 years. Despite the extensive research and development of FA in recent years, there are still considerable challenges in its effective derivatization due to the limited number of studies on its pharmacological activities and mechanisms of action to date. FA has a short half-life after oral absorption, and therefore, frequent dosing is necessary to maintain adequate blood drug concentrations, which leads to large fluctuations in drug concentrations and increases the risk of adverse clinical outcomes, which limits the application of FA in clinical use. Considering that triterpenes are known to have a wide range of pharmacological activities, other new pharmacological effects of FA remain to be discovered. In view of the above challenges, the following aspects will be of great value to future drug development and clinical application research of FA:

[0004] (1) Prolonging the half-life of FA by adopting appropriate formulations or chemical methods. For example, FA can be administered in a liposome-encapsulated or structurally modified form that blocks the 21-COOH metabolic site.

[0005] (2) From the perspective of pharmacology and mechanism, further research is needed to investigate the potential pharmacological activity of FA and expand its scope of use. At the same time, more research on the mechanism of action will provide a better understanding of how FA works. In addition, highly sensitive in vitro studies do not always produce the same results in vivo, so a large number of in vivo studies must be conducted to verify its effectiveness.

[0006] (3) Synthesize new derivatives by structural modification at the confirmed modification sites or other potentially available sites of FA to explore new drugs with higher activity and better pharmaceutical properties.

[0007] (4) As a clinically applied drug, FA has the potential to be repositioned as an anti-tuberculosis or anti-malarial drug, which requires more research in these areas. The anti-tumor, tumor MDR reversal, anti-inflammatory, and antifungal activities of FA are newly discovered biological activities in recent years and have great research value. Summary of the invention

[0008] The primary purpose of the present invention is to provide a fusidic acid derivative having FA-like activity, wherein the fusidic acid derivative molecule itself is ineffective against Gram-negative bacteria, but can effectively kill bacteria in the presence of a polypeptide adjuvant;

[0009] Another object of the present invention is to provide a method for preparing the above-mentioned fusidic acid derivatives.

[0010] Another object of the present invention is to provide the use of the above-mentioned fusidic acid derivatives in the preparation of antibacterial drugs. The fusidic acid derivatives can be covalently cross-linked with the polypeptide SLAP-S25 to prepare antibacterial composite molecular drugs.

[0011] The present invention is achieved by providing a fusidic acid derivative, the chemical structure of which is shown in the following formula (I):

[0012]

[0013] In formula (I), R1 is -(CH2) m -COO-, m=3-4; R2 is selected from -(CH2) n -, n = 1 to 4 or -(CH2CH2O) x -, x=1 to 6, and R3 is selected from any one of alkynyl, amino, azadiphenylcyclooctyneamino, azido, and carboxyl.

[0014] Preferably, it is characterized in that, in formula (I),

[0015] R1 is -(CH2)3-COO-, R2 is -CH2-, and R3 is alkynyl;

[0016] R1 is -(CH2)3-COO-, R2 is -CH2CH2-, and R3 is alkynyl;

[0017] R1 is -(CH2)3-COO-, R2 is -CH2CH2-, and R3 is azaphenylcyclooctyneamido;

[0018] R1 is -(CH2)3-COO-, R2 is -(CH2CH2O)4-, and R3 is alkynyl;

[0019] R1 is -(CH2)3-COO-, R2 is -(CH2CH2O)4-, and R3 is an azido group;

[0020] R1 is -(CH2)4-COO-, R2 is -CH2-, and R3 is alkynyl;

[0021] R1 is -(CH2)4-COO-, R2 is -CH2CH2-, and R3 is alkynyl;

[0022] R1 is -(CH2)4-COO-, R2 is -CH2CH2-, and R3 is azaphenylcyclooctyneamido;

[0023] R1 is -(CH2)4-COO-, R2 is -(CH2CH2O)4-, and R3 is alkynyl;

[0024] R1 is -(CH2)4-COO-, R2 is -(CH2CH2O)4-, and R3 is an azido group

[0025] The present invention further discloses a method for preparing the above-mentioned fusidic acid derivative, which comprises the following steps:

[0026] (1) dissolving an alkynyl compound in ultra-dry tetrahydrofuran (THF), adding a base 1 under a nitrogen atmosphere and an ice bath, stirring and reacting for 0.5 to 1.5 hours, adding triphenylmethyl mercaptan, stirring and reacting for 0.5 to 1.5 hours; slowly heating to room temperature, stirring overnight, extracting, concentrating the organic phase under reduced pressure, and purifying after the reaction to obtain compound 1; wherein the structural formula of the alkynyl compound is: R3-R2-X, X is any one of Cl, Br, and I; the R2 group is selected from -(CH2) n -or-(CH2CH2O) x -, wherein n=1-4, x=1-6; the R3 group is selected from any one of an alkynyl group and an azadibenzocyclooctynylamide group;

[0027] The base 1 is any one of 1,8-diazabicyclo[5.4.0]undec-7-ene, triethylamine and N,N-diisopropylethylamine;

[0028] (2) Compound 1 was dissolved in a mixture of ultra-dry dichloromethane (DCM) and a strong acid solution in a volume ratio of 1:1, stirred in an ice-water bath for reaction, triethylsilane was added, and the temperature was gradually raised to room temperature for reaction for 4 h, washed, dried, and concentrated under reduced pressure to obtain compound 2;

[0029] (3) Compound 2 is dissolved in ultra-dry dichloromethane, base 2 is added, and the reaction is stirred at room temperature for 20 to 40 minutes, 2,2'-dithiodipyridine is slowly added, and the reaction is stirred for 11 to 13 hours. After the reaction is completed, the reaction is filtered, and the collected filtrate is extracted, the organic phase is concentrated under reduced pressure, and purified to obtain compound 3; wherein the base 2 is selected from any one of 1,8-diazabicyclo[5.4.0]undec-7-ene, triethylamine and N,N-diisopropylethylamine;

[0030] (4) Compound 3 is dissolved in methanol, and acid 1 is added. Under a nitrogen atmosphere and an ice-water bath, a solution of 4-mercaptobutyric acid or 5-mercaptovaleric acid dissolved in methanol is added dropwise, and the mixture is heated to room temperature and reacted overnight to obtain compound 4; wherein the acid 1 is selected from any one of glacial acetic acid, formic acid, and carbonic acid;

[0031] (5) Compound 4 was dissolved in ultra-dry dichloromethane until completely dissolved, and then stirred in a nitrogen atmosphere and ice-water bath. A base of 3,4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The reaction was continued with stirring for 25 to 35 minutes under ice-water bath conditions. Fusidic acid was added, and the mixture was gradually returned to room temperature. The reaction was continued with stirring overnight. The mixture was concentrated under reduced pressure and purified to obtain a fusidic acid derivative. The base 3 was selected from any one of 1,8-diazabicyclo[5.4.0]undec-7-ene, triethylamine and N,N-diisopropylethylamine.

[0032] Preferably, in step (1), the alkynyl compound is selected from any one of bromopropyne, 4-chloro-1-butyne, 5-chloro-1-pentyne, 6-chloro-1-hexyne, 4-bromo-1-butyne, 5-bromo-1-pentyne, 6-bromo-1-hexyne, 4-iodo-1-butyne, 5-iodo-1-pentyne, 6-iodo-1-hexyne, propyne-polyethylene glycol bromo-bromo-azadibenzocyclooctyne.

[0033] Preferably, in step (1), the molar ratio of the alkynyl compound, the base 1, and triphenylmethyl mercaptan is 1:1-1.5:1-1.5;

[0034] In step (3), the molar ratio of the compound 2, the base 2, and 2,2'-dithiodipyridine is 1:1-3:0.5-1;

[0035] In step (4), the molar ratio of compound 3 to acid 1 is 1:10;

[0036] In step (5), the molar ratio of the compound 4, the base 3, and fusidic acid is 1:1-1.5:0.5-1.

[0037] Preferably, in step (2), the strong acid solution is a trifluoroacetic acid solution with a pH of 1; and in step (4), the methanol is HPLC grade methanol.

[0038] Preferably, in step (4), the preparation process of 4-mercaptobutyric acid is as follows: 4-bromobutyric acid and thiourea are dissolved in an ethanol solution, refluxed for 4 hours, sodium hydroxide solution dissolved in ethanol is added to the reaction flask, refluxed for 3 hours, precipitated by filtration, precipitated by adding water, pH adjusted to acidic, organic phase extracted, concentrated under reduced pressure, and dried to obtain 4-mercaptobutyric acid;

[0039] The preparation process of 5-mercaptovaleric acid is as follows: 5-bromovaleric acid and thiourea are dissolved in an ethanol solution, refluxed for 4 hours, a sodium hydroxide solution dissolved in ethanol is added to a reaction flask, the reflux reaction is continued for 3 hours, a precipitate is filtered, water is added to dissolve the precipitate, the pH is adjusted to be acidic, an organic phase is extracted, and reduced pressure is reduced to concentrate, and 5-mercaptovaleric acid is obtained after drying.

[0040] Preferably, the extraction agent is analytical grade dichloromethane DCM, the purification is separation and purification by silica gel column chromatography, and the drying is drying over anhydrous Na2SO4.

[0041] The invention further discloses the application of the fusidic acid derivatives in the preparation of antibacterial drugs.

[0042] The present invention overcomes the deficiencies of the prior art and provides a fusidic acid derivative and a preparation method and application thereof. The present invention derivatizes the 3-OH site of fusidic acid through a linker having a disulfide bond, introduces different terminal functional groups on the one hand, and adjusts the properties of the fusidic acid molecule, for example, the introduction of an amino group or a carboxyl group can change the water solubility of the molecule under different pH conditions, and the polyethylene glycol structure can change the systemic circulation characteristics of the molecule, etc. At the same time, active functional groups such as alkynes, amino groups, and carboxyl groups provide sites for further modification, thereby modularly constructing more functional fusidic acid derivatives. The disulfide bond in the linker can effectively maintain the antibacterial activity of fusidic acid, and in the presence of a polypeptide adjuvant, achieves effective killing of Gram-negative bacteria.

[0043] Compared with the shortcomings and deficiencies of the prior art, the present invention has the following beneficial effects:

[0044] (1) The preparation method of the present invention is simple and easy to separate and purify;

[0045] (2) The fusidic acid derivatives of the present invention have the advantages of having a disulfide bond and containing an alkyne at the end;

[0046] (3) The present invention utilizes reducing substances in bacteria, and it is expected that the disulfide bonds in fusidic acid derivatives can be opened, thereby releasing active FA and achieving an antibacterial effect;

[0047] (4) The fusidic acid derivatives described in the present invention can effectively kill bacteria in the presence of the polypeptide SLAP-S25. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 is a synthetic route diagram of the fusidic acid derivative C1 in Example 1 of the present invention;

[0049] Figure 2 The H NMR spectrum of the fusidic acid derivative C1 in Example 1 of the present invention;

[0050] Figure 3 is the carbon NMR spectrum of the fusidic acid derivative C1 in Example 1 of the present invention;

[0051] Figure 4 is a synthetic route of the fusidic acid derivative C2 in Example 2 of the present invention;

[0052] Figure 5 is the H NMR spectrum of the fusidic acid derivative C2 in Example 2 of the present invention;

[0053] Figure 6 is a synthetic route diagram of the fusidic acid derivative C3 in Example 3 of the present invention;

[0054] Figure 7 This is the hydrogen nuclear magnetic resonance spectrum of the fusidic acid derivative C3 in Example 3 of the present invention. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0056] Example 1

[0057] The synthetic route of fusidic acid derivative molecule C1 is as follows Figure 1 As shown, the specific steps include:

[0058] (1) Synthesis of Compound 1

[0059] Dissolve triphenylmethanethiol (0.83 g, 3 mmol) in 10 mL of ultra-dry tetrahydrofuran (THF), stir under ice-water bath, add triethylamine (Et3N, 0.84 mL, 6 mmol), continue stirring under ice-water bath for 30 min; add 3-bromopropyne (0.174 mL, 2 mmol) and warm to room temperature for overnight reaction.

[0060] After the reaction was completed by TLC silica gel plate detection, the mixed solution was diluted with 100 mL of dichloromethane and washed three times with deionized water, 20 mL each time, washed once with saturated brine, dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography to obtain compound 1 (0.4662 g, yield 74.14%).

[0061] (2) Synthesis of Compound 2

[0062] Compound 1 (0.9856 g, 3 mmol) was dissolved in a mixed solution of ultra-dry dichloromethane (7 mL) and trifluoroacetic acid (TFA, 5 mL). Under ice bath stirring, triethylsilane (Et3SiH, 0.96 mL, 6 mmol) was slowly added and the temperature was gradually raised to room temperature for 4 h. After the reaction was completed, it was diluted with 200 mL of dichloromethane, washed three times with 20 mL of deionized water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound 2.

[0063] (3) Synthesis of Compound 3

[0064] Compound 2 (0.1296 g, 1.8 mmol) was dissolved in ultra-dry dichloromethane (10 mL), and the reaction was continued under stirring under ice-bath water conditions, and triethylamine (Et3N, 1.295 mL, 9 mmol) was added; 2,2'-disulfide dipyridine (0.6609 g, 3 mmol) solution dissolved in ultra-dry DCM (10 mL) was added to the reaction system and reacted overnight. The crude product was separated and purified by silica gel column chromatography (10:1, PE / EA) to obtain compound 3 (0.1485 g, yield 45.6%).

[0065] (4) Synthesis of Compound 4

[0066] Dissolve 4-bromobutyric acid and thiourea in an ethanol solution, reflux for 4 hours, add a sodium hydroxide solution dissolved in ethanol to the reaction flask, continue to reflux for 3 hours, filter the precipitate, add water to dissolve the precipitate, adjust the pH to acidic, extract, concentrate the organic phase under reduced pressure, and dry to obtain 4-mercaptobutyric acid.

[0067] Compound 3 (0.195 g, 1 mmol) was dissolved in HPLC grade methanol (5 mL), stirred in an ice-water bath, and glacial acetic acid (AcOH, 0.59 mL, 10 mmol) was added. Under a nitrogen atmosphere and with stirring in an ice-water bath, a solution of 4-mercaptobutyric acid (0.103 mL, 1 mmol) dissolved in HPLC grade methanol (2 mL) was added dropwise, and the mixture was warmed to room temperature and reacted overnight.

[0068] After the reaction was completed by silica gel plate detection, the reaction solution was concentrated under reduced pressure to remove the solvent HPLC grade methanol, and then the reaction solution was diluted with 100 mL of dichloromethane and washed three times with 30 mL of deionized water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (10:1, PE / EA) to obtain compound 4 (0.1562 g, yield 76.5%).

[0069] (5) Synthesis of Compound C1

[0070] Compound 4 (0.140 g, 1.37 mmol) was dissolved in ultra-dry dichloromethane. After complete dissolution, triethylamine (Et3N, 0.192 mL, 2.74 mmol), 4-dimethylaminopyridine (DMAP, 0.017 g, 0.137 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 0.2712 g, 2.74 mmol) were added dropwise under nitrogen atmosphere and stirring in an ice-water bath. The reaction was continued with stirring in an ice-water bath for 30 minutes, and fusidic acid (0.4470 g, 0.91 mmol) was added. The temperature was gradually restored to room temperature and the reaction was continued with stirring overnight.

[0071] After the reaction was completed by silica gel plate detection, the reaction solution was diluted with 100 mL DCM and washed three times with 20 mL deionized water, the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The fusidic acid derivative compound C1 (0.063 g, yield 9.66%) was obtained by silica gel column chromatography for separation and purification.

[0072] Compound C1 was obtained and characterized. Figure 2 , 3 As shown, the chemical structure of the chemical C1 is:

[0073]

[0074] Example 2

[0075] The synthetic route of fusidic acid derivative C2 is as follows Figure 4 As shown, the specific steps include:

[0076] (1) Synthesis of compound TEG-alkyne

[0077] Dissolve tetraethylene glycol (17.3 mL, 100 mmol, 2 eq) in ultra-dry THF (50 mL), place in an ice bath, add sodium hydride (2.6 g, 65 mmol, 0.65 eq, 60% dispersed in mineral oil), react for 1 h in an ice bath, and then add 3-bromopropyne (7.4 mL, 50 mmol, 1 eq, 80% toluene solution) to the reaction solution.

[0078] The mixture was returned to room temperature and stirred overnight. After the reaction was completed as detected by TLC silica gel plate, water (40 mL) was added to quench the reaction, and dichloromethane (100 mL*3) was used for extraction. The organic phases were combined and concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (1:5, PE / EA) and dried to obtain TEG-alkyne (8.1503 g, yield: 70.18%).

[0079] (2) Synthesis of brominated TEG-alkyne

[0080] Compound TEG-alkyne (11.6605 g, 50.2 mmol) was dissolved in 100 mL of ultra-dry DCM and cooled to 0° C. Triphenylphosphine (19.67 g, 75 mmol, 1.5 eq) was added in small amounts several times and stirred for 15 min, and carbon tetrabromide (24.87 g, 75 mmol, 1.5 eq) was added in small amounts several times, and the reaction solution was returned to room temperature and stirred overnight.

[0081] After the reaction was completed, the reaction was detected on a silica gel plate and concentrated under reduced pressure to remove the solvent DCM. The crude product was separated and purified by silica gel column chromatography (1:2, PE / EA) and dried to obtain brominated TEG-alkyne (8.8901 g, yield 60%).

[0082] (3) Synthesis of Compound 1

[0083] Sodium hydride (NaH, content of 60%, 0.7920g, 19.8mmol) was dissolved in ultra-dry N, N-dimethylformamide (20mL) under ice bath, triphenylmethanethiol (Ph3CSH, 4.5606g, 16.5mmol) was added, and the reaction was stirred for 30min under ice bath; brominated TEG-alkyne (3.2432g, 11mmol) was added to the reaction system, and the reaction was transferred to room temperature overnight. After the reaction was completed by silica gel plate detection, the reaction solution was diluted with 200mL of dichloromethane, washed three times with 50mL of deionized water, and concentrated under reduced pressure after drying. The crude product was separated and purified by silica gel column chromatography (2:1, PE / EA), and compound 1 (4.8087g, yield 89.17%) was obtained after drying.

[0084] (4) Synthesis of Compound 2

[0085] Compound 1 (0.9804 g, 2 mmol) was dissolved in a mixture of trifluoroacetic acid (TFA, 5 mL) and dichloromethane (7 mL), ice-bathed, triethylsilane (Et3SiH, 0.320 mL, 2 mmol) was added, and the mixture was reacted at room temperature overnight. After the reaction was completed by silica gel plate detection, the reaction solution was diluted with 100 mL DCM and washed three times with 20 mL deionized water, the organic phase was dried and concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (2: 1, PE / EA), and compound 2 (0.3851 g, 61.24% yield) was obtained after drying.

[0086] (5) Synthesis of Compound 3

[0087] Compound 2 (0.3038 g, 1.2235 mmol) was dissolved in ultra-dry DCM (10 mL), triethylamine (Et3N, 0.51 mL, 3.671 mmol) was added, and magnetic stirring was performed under ice-water bath; a solution of 2,2'-disulfide dipyridine (0.4043 g, 1.835 mmol) dissolved in DCM (10 mL) was added dropwise to the reaction system, and the temperature was gradually raised to room temperature for reaction overnight.

[0088] After the reaction was completed by TLC silica gel plate detection, it was diluted with 100 mL of dichloromethane, washed three times with 20 mL of deionized water, dried and concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (1:1, PE / EA), and compound 3 (0.3719 g, yield 84.91%) was obtained after drying.

[0089] (6) Synthesis of Compound 4

[0090] Dissolve 4-bromobutyric acid and thiourea in an ethanol solution, reflux for 4 hours, add a sodium hydroxide solution dissolved in ethanol to the reaction flask, continue to reflux for 3 hours, filter the precipitate, add water to dissolve the precipitate, adjust the pH to acidic, extract, concentrate the organic phase under reduced pressure, and dry to obtain 4-mercaptobutyric acid.

[0091] Compound 3 (0.1071 g, 0.3 mmol) was dissolved in HPLC grade methanol (5 mL), and glacial acetic acid (AcOH, 0.177 mL, 3 mmol) was added under nitrogen atmosphere and stirring in an ice-water bath; 4-mercaptobutyric acid (0.031 mL, 0.3 mmol) was dissolved in HPLC grade methanol (2 mL), added dropwise to the reaction system, and the temperature was raised to room temperature for reaction overnight.

[0092] After the reaction was completed by silica gel plate detection, the reaction solution was diluted with 50 mL of dichloromethane and washed three times with 20 mL of deionized water, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (5:1, DCM / MeOH) and dried to obtain compound 4 (0.074 g, yield 61.34%).

[0093] (7) Synthesis of Compound C2

[0094] Compound 4 (0.109 g, 0.3 mmol) was dissolved in ultra-dry dichloromethane until completely dissolved, and triethylamine (0.084 ml, 0.6 mmol) was added dropwise under nitrogen atmosphere and ice-water bath stirring conditions, and 4-dimethylaminopyridine (DMAP, 0.004 g, 0.03 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 0.115 g, 0.6 mmol) were added, and the reaction was continued under ice-water bath stirring conditions for 30 minutes. Fusidic acid (0.129 g, 0.6 mmol) was added, and the temperature was gradually restored to room temperature, and the reaction was continued to stir overnight.

[0095] After the reaction was completed by silica gel plate detection, the reaction solution was diluted with dichloromethane and washed three times with deionized water, the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and separated and purified by silica gel column chromatography to obtain fusidic acid derivative C2 (0.053 g, yield 20.34%).

[0096] Compound C2 was obtained and characterized. Figure 5 As shown, the chemical structure of the chemical C2 is:

[0097]

[0098] Example 3

[0099] The synthetic route of fusidic acid derivative C3 is as follows Figure 6 As shown, the specific steps include:

[0100] (1) Synthesis of brominated azadibenzocyclooctyne

[0101] 2-Bromoethylamine hydrobromide (1g, 4.88mmol) was dissolved in THF (5mL), ice bath, N, N-diisopropylethylamine (DIPEA, 2.4mL, 13.78mmol), azadibenzocyclooctyne-N-hydroxysuccinimide ester (1.86g, 4.63mmol), and the reaction was continued overnight at room temperature. After the reaction was completed, the reaction solution was diluted with 100mL of dichloromethane and washed three times with 50mL of deionized water, dried and concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (100:1, PE / EA) to obtain bromo-azadibenzocyclooctyne (1.62g, 85.4% yield).

[0102] (2) Synthesis of Compound 1

[0103] Sodium hydride (NaH, content of 60%, 0.1584 g, 3.96 mmol) was dissolved in ultra-dry N, N-dimethylformamide (5 mL) under ice bath, triphenylmethanethiol (Ph3CSH, 0.9121 g, 3.3 mmol) was added, and the reaction was stirred for 30 min under ice bath condition; brominated azadibenzocyclooctyne (0.902 g, 2.2 mmol) was added to the reaction system, and the reaction was transferred to room temperature overnight.

[0104] After the reaction was completed by silica gel plate detection, the reaction solution was diluted with 100 mL of dichloromethane, washed three times with 50 mL of deionized water, dried and concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (20:1, PE / EA) and dried to obtain compound 1 (0.9882 g, yield 74.12%).

[0105] (3) Synthesis of Compound 2

[0106] Compound 1 (0.909 g, 1.5 mmol) was dissolved in a mixture of trifluoroacetic acid (TFA, 5 mL) and dichloromethane (7 mL). Triethylsilane (Et3SiH, 0.320 mL, 2 mmol) was added under ice bath conditions and the mixture was reacted at room temperature overnight.

[0107] After the reaction was completed by silica gel plate detection, the reaction solution was diluted with 100 mL DCM and washed three times with 20 mL deionized water, and the organic phase was dried and concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (2:1, PE / EA) to obtain compound 2 (0.4355 g, yield 79.76%) after drying.

[0108] (5) Synthesis of Compound 3

[0109] Compound 2 (0.4355 g, 1.196 mmol) was dissolved in ultra-dry DCM (10 mL), and triethylamine (Et3N, 0.51 mL, 3.671 mmol) was added. Under nitrogen atmosphere and stirring in an ice-water bath, a solution of 2,2'-disulfide dipyridine (0.4043 g, 1.835 mmol) dissolved in DCM (10 mL) was added dropwise to the reaction system, and the temperature was gradually raised to room temperature for reaction overnight.

[0110] After the reaction was completed by TLC silica gel plate detection, it was diluted with 100 mL of dichloromethane, washed three times with 20 mL of deionized water, dried and concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (1:1, PE / EA) and dried to obtain compound 3 (0.409 g, yield 72.3%).

[0111] (6) Synthesis of Compound 4

[0112] Dissolve 4-bromobutyric acid and thiourea in an ethanol solution, reflux for 4 hours, add a sodium hydroxide solution dissolved in ethanol to the reaction flask, continue to reflux for 3 hours, filter the precipitate, add water to dissolve the precipitate, adjust the pH to acidic, extract, concentrate the organic phase under reduced pressure, and dry to obtain 4-mercaptobutyric acid.

[0113] Compound 3 (0.2838 g, 0.6 mmol) was dissolved in HPLC grade methanol (5 mL), and glacial acetic acid (AcOH, 0.177 mL, 3 mmol) was added under nitrogen atmosphere and stirring in an ice-water bath; 4-mercaptobutyric acid (0.062 mL, 0.6 mmol) was dissolved in HPLC grade methanol (2 mL), added dropwise to the reaction system, and the temperature was raised to room temperature for reaction overnight.

[0114] After the reaction was completed by silica gel plate detection, the reaction solution was diluted with 50 mL of dichloromethane and washed three times with 20 mL of deionized water, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (5:1, DCM / MeOH) and dried to obtain compound 4 (0.1340 g, yield 46.33%).

[0115] (6) Synthesis of Compound C3

[0116] Compound 4 (0.2 g, 0.415 mmol) was dissolved in ultra-dry dichloromethane (10 mL) until completely dissolved, stirred under ice-water bath, triethylamine (0.122 mL, 0.8 mmol) was added dropwise, 4-dimethylaminopyridine (DMAP, 0.005 g, 0.04 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 0.115 g, 0.6 mmol) was added, and the reaction was continued under ice-water bath with stirring for 30 minutes. Fusidic acid (0.153 g, 0.8 mmol) was added, and the temperature was gradually restored to room temperature, and the reaction was continued with stirring overnight.

[0117] After the reaction was completed by silica gel plate detection, the reaction solution was diluted with dichloromethane and washed three times with deionized water. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The fusidic acid derivative C3 (0.064 g, yield 15.93%) was obtained by separation and purification by silica gel column chromatography. Figure 6 The compounds shown.

[0118] Compound C3 was obtained and characterized. Figure 7 As shown, the chemical structure of the chemical C3 is:

[0119]

[0120] Embodiments 4 to 10

[0121] Examples 4 to 10 of the present invention are substantially the same as the above-mentioned Example 1, except that the reaction raw materials are different, as shown in Table 1 below:

[0122] Table 1 Comparison of the differences between Examples 4 to 10 and Example 1

[0123]

[0124] In Table 1, the preparation process of the above 5-mercaptovaleric acid is as follows: 5-bromovaleric acid and thiourea are dissolved in an ethanol solution, refluxed for 4 hours, a sodium hydroxide solution dissolved in ethanol is added to a reaction flask, the reflux reaction is continued for 3 hours, the precipitate is filtered, water is added to dissolve the precipitate, the pH is adjusted to acidic, the organic phase is extracted, and reduced pressure is concentrated, and 5-mercaptovaleric acid is obtained after drying.

[0125] Application Example In vitro antibacterial test of fusidic acid derivatives in Escherichia coli

[0126] Take E. coli that has been cultured in a constant temperature incubator (37°C) for 18 to 24 hours, take 0.5 turbidity standard bacterial suspension to 0.5 turbidity standard solution, and dilute the above bacterial suspension to 1:100 with LB broth for later use. Add LB broth to the first column of the 96-well plate, add 100 μL of LB broth to the remaining wells, use a spray gun to draw 100 μL of the solution in the first column of the well plate to the second column, mix well and draw 100 μL to the third column, and dilute to the 9th column by analogy, and draw 100 μL from the 9th column of the well plate and discard it. The 10th column is a growth control without antibiotics. Add 100 μL of diluted bacterial solution to each well of the 96-well plate (the final concentrations of antibiotics in the first column to the 9th column are 256, 128, 64, 32, 16, 8, 4, 2, and 1 μg / mL, respectively). The inoculated 96-well plate was placed in a 37°C incubator for incubation for 16 to 20 hours, and the growth of bacteria was observed to obtain the minimum inhibitory concentration (MIC).

[0127] A similar method was used to study the inhibitory effect of fusidic acid derivative compounds and fusidic acid derivative compounds in combination with peptide SLAP-S25 on Escherichia coli: LB broth (100 μL, containing 8 μg / mL SLAP-S25) was added to all wells of a 96-well plate, and 512 μg / mL of antibiotics (100 μL, 512 μg / mL) was added to the first column of the wells and mixed thoroughly. From the first column of the wells, multiple dilutions were performed to the 9th column, and 100 μL was drawn from the 9th column of the wells and discarded. The 10th column of the wells was a growth control without antibiotics. 100 μL of the diluted bacterial suspension was added to each well (the concentrations of antibiotics in the 1st to 9th columns of the wells were 256, 128, 64, 32, 16, 8, 4, 2, and 1 μg / mL, respectively, and the concentration of SLAP-S25 was 4 μg / mL). The inoculated 96-well plate was placed in a 37°C incubator for 16 to 20 hours to observe the growth of bacteria, and the minimum inhibitory concentration (MIC) of the drug for the drug-resistant bacteria to be tested when SLAP-S25 was used in combination with antibiotics was obtained. The results are shown in Table 1 below:

[0128] Table 2 Growth of Escherichia coli (+) after different medications

[0129]

[0130]

[0131] Table 2 shows the growth of Escherichia coli (+) when fusidic acid derivatives and fusidic acid derivatives are used in combination with the peptide SLAP-S25. The anti-Gram-negative bacteria ability of the FA derivatives was tested using Escherichia coli as a model. The results in Table 2 show that the FA derivatives can effectively kill bacteria in the presence of a polypeptide adjuvant. At the same time, the different bioorthogonal functional groups carried by the prepared FA derivatives can be used for covalent cross-linking with polypeptides to prepare antibacterial composite molecules, improve delivery efficiency and drug synergy, and obtain better antibacterial molecules.

[0132] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A fusidic acid derivative, characterized in that The chemical structural formula of the fusidic acid derivative is shown in the following formula (I):

2. The method for preparing the fusidic acid derivative according to claim 1, characterized in that: The method comprises the following steps:

3. Use of the fusidic acid derivative according to claim 1 in the preparation of antibacterial drugs.

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