Cryptotanshinone derivative and fosfomycin combined antibacterial drug as well as preparation method and application thereof

Abstract

The invention discloses a cryptotanshinone derivative and fosfomycin combined antibacterial drug and a preparation method and application thereof.The combined antibacterial drug is composed of a cryptotanshinone derivative and fosfomycin, and the cryptotanshinone derivative is obtained by introducing a specific functional group to a specific position of cryptotanshinone; therefore, the derivative has stronger antibacterial activity and better stability. Functional groups can be hydroxyl, carboxyl, amino and the like, and specific introduction positions and functional group types can be optimized and determined according to experiments. The cryptotanshinone derivative has higher antibacterial activity and better stability, and the treatment effect can be remarkably improved when the cryptotanshinone derivative is combined with fosfomycin for use.

Description

Technical Field

[0001] The present invention relates to the pharmaceutical field, and in particular to a novel cryptotanshinone derivative and fosfomycin combined antibacterial drug, a preparation method thereof, and applications thereof. Background Art

[0002] With the widespread use of antimicrobial drugs, bacterial resistance is becoming an increasingly serious problem. In particular, infections caused by fosfomycin-resistant Staphylococcus aureus pose a significant challenge to clinical treatment. Existing treatments often have limited effectiveness and are prone to developing resistance. Therefore, the development of new antimicrobial drugs has become a hot topic in current research.

[0003] Cryptotanshinone, as a natural product, has multiple biological activities such as antibacterial and anti-inflammatory properties. However, the antibacterial effect of cryptotanshinone when used directly is not ideal and may have certain side effects. Fosfomycin is a broad-spectrum antibiotic that has antibacterial effects on a variety of bacteria, but it is not very effective against fosfomycin-resistant strains. Therefore, the combined use of cryptotanshinone and fosfomycin has become a potential way to solve the problem of drug-resistant bacterial infections by enhancing antibacterial activity and reducing the development of drug resistance.

[0004] However, existing cryptotanshinone and fosfomycin combination regimens have some problems, such as irrational drug ratios and unstable efficacy. Therefore, the development of a novel cryptotanshinone derivative combined with fosfomycin as an antibacterial drug to improve therapeutic efficacy and reduce the development of drug resistance is of great clinical significance. Summary of the Invention

[0005] The object of the present invention is to provide an antibacterial drug combining a cryptotanshinone derivative and fosfomycin, which has stronger antibacterial activity, lower drug resistance and better stability.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The combined antibacterial drug of the present invention comprises a cryptotanshinone derivative and fosfomycin. Cryptotanshinone derivatives are obtained by introducing specific functional groups at specific positions in cryptotanshinone, which imparts them with enhanced antibacterial activity and improved stability. The functional groups can be hydroxyl, carboxyl, amino, etc. The specific introduction position and type of functional groups can be determined through experimental optimization.

[0008] The weight ratio of cryptotanshinone derivatives to fosfomycin is 1:(10 ～ 150), preferably 1:40. This ratio range has been optimized through experiments to ensure the stability and therapeutic effect of the drug in the body.

[0009] The combined antibacterial drug of the present invention can be used to treat Gram-positive bacterial infections, particularly fosfomycin-resistant Staphylococcus aureus infections. The administration method can be oral, injection, or topical, and the specific administration method can be determined according to the condition and patient condition.

[0010] The preparation method of the present invention comprises the following steps: firstly, synthesizing a novel cryptotanshinone derivative; then uniformly mixing the derivative with fosfomycin according to the weight ratio; and finally, preparing the mixture into a dosage form suitable for administration, such as tablets, capsules, injections, etc.

[0011] The synthesis of novel cryptotanshinone derivatives can be carried out through specific chemical reactions, such as substitution, addition, or condensation reactions. The specific synthesis route and reaction conditions can be determined through experimental optimization.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] In the present invention, the cryptotanshinone derivative has stronger antibacterial activity and better stability, and can significantly improve the therapeutic effect when used in combination with fosfomycin.

[0014] In the present invention, the combined antibacterial drugs are reasonably matched, the drugs are stable in the body, and the generation of drug resistance is reduced.

[0015] The preparation method of the present invention is simple, feasible and suitable for industrial production.

[0016] The combined antibacterial drug of the present invention can be used to treat various Gram-positive bacterial infectious diseases, especially fosfomycin-resistant Staphylococcus aureus infections, and has important clinical significance. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present invention will be described clearly and completely below.

[0018] A cryptotanshinone derivative and fosfomycin combined antibacterial drug, wherein the cryptotanshinone derivative is obtained by introducing a functional group at a specific position of cryptotanshinone, and the weight ratio of the cryptotanshinone derivative to fosfomycin is 1:(10 ～ 150).

[0019] The cryptotanshinone derivative is one of the following structural formulas:

[0020]

[0021] Antimicrobial drugs are used to treat Gram-positive bacterial infections, especially fosfomycin-resistant Staphylococcus aureus infections.

[0022] Treatment is given orally, by injection, or topically.

[0023] A method for preparing a combined antibacterial drug of a cryptotanshinone derivative and fosfomycin comprises the following steps:

[0024] Step 1: Synthesis of cryptotanshinone derivatives;

[0025] Step 2: uniformly mixing the novel cryptotanshinone derivative and fosfomycin according to the weight ratio;

[0026] Step 3: Prepare the mixture into a dosage form suitable for administration.

[0027] In step one, the synthesis is carried out through a specific chemical reaction, including substitution, addition or condensation reaction. Furthermore, the cryptotanshinone derivatives in the above formulas 1, 2 and 3 are all prepared by the same preparation method as the invention patent with application number: 202010386889.7.

[0028] Example 1: The cryptotanshinone derivative obtained above and fosfomycin were mixed uniformly in a weight ratio of 1:10, and then an excipient accounting for 10% of the total weight of the cryptotanshinone derivative and fosfomycin was added, and finally deionized water accounting for 4% of the total weight of the cryptotanshinone derivative, fosfomycin and excipients was added, and the mixture was placed in a chemical centrifugal mixer and stirred at a stirring temperature of 22°C, wherein the excipient is a filler, and the filler is pregelatinized starch;

[0029] After stirring for 2 hours, the mixture was placed on a flat plate in a cold storage set at -1°C, frozen for 3 hours, taken out, and then added to a granulator to prepare the medicine.

[0030] Example 2: The cryptotanshinone derivative obtained above and fosfomycin were mixed uniformly in a weight ratio of 1:150, and then an excipient accounting for 10% of the total weight of the cryptotanshinone derivative and fosfomycin was added, and finally deionized water accounting for 4% of the total weight of the cryptotanshinone derivative, fosfomycin and excipients was added, and the mixture was placed in a chemical centrifugal mixer and stirred at a stirring temperature of 22°C, wherein the excipient is a filler, and the filler is pregelatinized starch;

[0031] After stirring for 2 hours, the mixture was placed on a flat plate in a cold storage set at -1°C, frozen for 3 hours, taken out, and then added to a granulator to prepare the medicine.

[0032] Example 3: The cryptotanshinone derivative obtained above and fosfomycin were mixed uniformly in a weight ratio of 1:9, and then an excipient accounting for 10% of the total weight of the cryptotanshinone derivative and fosfomycin was added, and finally deionized water accounting for 4% of the total weight of the cryptotanshinone derivative, fosfomycin and excipients was added, and the mixture was placed in a chemical centrifugal mixer and stirred at a stirring temperature of 22°C, wherein the excipient was a filler, and the filler was pregelatinized starch;

[0033] After stirring for 2 hours, the mixture was placed on a flat plate in a cold storage set at -1°C, frozen for 3 hours, taken out, and then added to a granulator to prepare the medicine.

[0034] Example 4: The cryptotanshinone derivative obtained above and fosfomycin were mixed uniformly in a weight ratio of 1:151, and then an excipient accounting for 10% of the total weight of the cryptotanshinone derivative and fosfomycin was added, and finally deionized water accounting for 4% of the total weight of the cryptotanshinone derivative, fosfomycin and excipients was added, and the mixture was placed in a chemical centrifugal mixer and stirred at a stirring temperature of 22°C, wherein the excipient is a filler, and the filler is pregelatinized starch;

[0035] After stirring for 2 hours, the mixture was placed on a flat plate in a cold storage set at -1°C, frozen for 3 hours, taken out, and then added to a granulator to prepare the medicine.

[0036] Example 5: The cryptotanshinone derivative obtained above and fosfomycin were mixed uniformly in a weight ratio of 1:40, and then an excipient accounting for 10% of the total weight of the cryptotanshinone derivative and fosfomycin was added, and finally deionized water accounting for 4% of the total weight of the cryptotanshinone derivative, fosfomycin and excipients was added, and the mixture was placed in a chemical centrifugal mixer and stirred at a stirring temperature of 22°C, wherein the excipient is a filler, and the filler is pregelatinized starch;

[0037] After stirring for 2 hours, the mixture was placed on a flat plate in a cold storage set at -1°C, frozen for 3 hours, taken out, and then added to a granulator to prepare the medicine.

[0038] Example 6: The cryptotanshinone derivative obtained above and fosfomycin were mixed uniformly in a weight ratio of 1:39, and then an excipient accounting for 10% of the total weight of the cryptotanshinone derivative and fosfomycin was added, and finally deionized water accounting for 4% of the total weight of the cryptotanshinone derivative, fosfomycin and excipients was added, and the mixture was placed in a chemical centrifugal mixer and stirred at a stirring temperature of 22°C, wherein the excipient is a filler, and the filler is pregelatinized starch;

[0039] After stirring for 2 hours, the mixture was placed on a flat plate in a cold storage set at -1°C, frozen for 3 hours, taken out, and then added to a granulator to prepare the medicine.

[0040] Example 7: The cryptotanshinone derivative obtained above and fosfomycin were mixed uniformly in a weight ratio of 1:41, and then an excipient accounting for 10% of the total weight of the cryptotanshinone derivative and fosfomycin was added, and finally deionized water accounting for 4% of the total weight of the cryptotanshinone derivative, fosfomycin and excipients was added, and the mixture was placed in a chemical centrifugal mixer and stirred at a stirring temperature of 22°C, wherein the excipient is a filler, and the filler is pregelatinized starch;

[0041] After stirring for 2 h, it was taken and placed in a flat plate and then placed in a cold storage. The temperature of the cold storage was set at -1°C and frozen for 3 hours. Then it was taken out and added to a granulator to prepare the medicine.

[0042] Comparative Example 1: Cryptotanshinone and fosfomycin were mixed evenly according to a weight ratio of 1:10, and then an auxiliary material accounting for 10% of the total weight of cryptotanshinone and fosfomycin was added. Finally, deionized water accounting for 4% of the total weight of cryptotanshinone, fosfomycin and the auxiliary material was added, and it was placed in a chemical centrifugal stirrer for stirring. The stirring temperature was controlled at 22°C. The auxiliary material was a filler, and the filler was pre-gelatinized starch.

[0043] After stirring for 2 h, it was taken and placed in a flat plate and then placed in a cold storage. The temperature of the cold storage was set at -1°C and frozen for 3 hours. Then it was taken out and added to a granulator to prepare the medicine.

[0044] Comparative Example 2: Cryptotanshinone and fosfomycin were mixed evenly according to a weight ratio of 1:150, and then an auxiliary material accounting for 10% of the total weight of cryptotanshinone and fosfomycin was added. Finally, deionized water accounting for 4% of the total weight of cryptotanshinone, fosfomycin and the auxiliary material was added, and it was placed in a chemical centrifugal stirrer for stirring. The stirring temperature was controlled at 22°C. The auxiliary material was a filler, and the filler was pre-gelatinized starch.

[0045] After stirring for 2 h, it was taken and placed in a flat plate and then placed in a cold storage. The temperature of the cold storage was set at -1°C and frozen for 3 hours. Then it was taken out and added to a granulator to prepare the medicine.

[0046] Prepare materials

[0047] Strain

[0048] The clinical isolate strain #122 of Staphylococcus aureus resistant to fosfomycin (SA#122) was from the strains previously preserved in the laboratory of Chengdu Medical College. The MIC value of this strain against fosfomycin was 2048 μg / mL, and the fosfomycin-resistant gene glpT was positive.

[0049] Experimental animals

[0050] SPF-grade C57BL / 6J mice, male, 6 - 8 weeks old, with an average weight of about 20 g. They were purchased from Beijing Huafukang Biotechnology Co., Ltd., and the animal license number was SCXK (Beijing) 2019 - 0008. They were raised in the school animal house, and the bedding, feed and water were replaced at fixed times. The mice were adaptively raised for 7 days before the experiment. All animal operations and care in this study strictly complied with the relevant regulations formulated by the Experimental Animal Protection and Ethics Committee, and were approved by the Experimental Ethics Committee of Chengdu Medical College.

[0051] Experimental methods

[0052] Take out the glycerol bacteria of fosfomycin-resistant Staphylococcus aureus (SA#122) stored in the laboratory earlier from the -80℃ refrigerator, place it on ice to dissolve naturally, dip the glycerol bacteria with an inoculation loop and streak it on the TSA solid agar plate, place it in a constant temperature incubator at 37℃ for 12 hours overnight, pick a single colony in 5mL TSB medium, and then place it in a tabletop constant temperature shaker at 37℃ and 200rpm for overnight culture. Then, the bacterial solution and culture medium were expanded into TSB medium at a volume ratio of 1:100. When the bacteria grew to the logarithmic growth phase, centrifuge it at 8000rpm and 4℃ for 10min, discard the supernatant to collect the bacterial precipitate, wash it with PBS, and then resuspend it with sterile saline to make the bacterial solution concentration 2.0×10 8 CFU / mL, 2.0×10 9 CFU / mL, 2.0×10 10 CFU / mL, 3.0×10 10 CFU / mL and 4.0×10 10 CFU / mL, and stored in a -20℃ refrigerator for future use.

[0053] One hundred healthy male mice were randomly divided into nine groups and acclimated for 7 days. Each mouse was then lightly anesthetized with ether, and the amplitude of its chest vibration was observed. When breathing became stable, 100 μL of sterile saline was instilled into the left nostril of the control group using a micropipette. The remaining five groups received an equal amount of bacterial solution of the corresponding concentration. The lungs of the mice were gently stroked to facilitate natural inhalation of the solution. The mice were then returned to their cages to lie flat. After they awoke naturally, they were continued to be housed. Survival and health were observed every 12 hours for 96 hours. The appropriate bacterial solution concentration for modeling was selected by comparing the mortality rate of mice in each group, and the appearance of the mice's lungs was observed to determine the success of the model.

[0054] Prepare the required fosfomycin solution and bacterial suspension, select 100 healthy male mice and randomly divide them into 9 groups. Each group of mice was given preventive medication for 3 days, and then 100 μL (3×10 9 The 100mg CFU (500mg / mL) bacterial liquid was used to construct a pneumonia model. The pills prepared in Examples 1 to 7 and Comparative Examples 1 to 2 were divided by weight in units of 0.5 grams, and then the medicine was powdered, and 10 grams of normal saline was then added and stirred. The control group was subcutaneously injected with an equal amount of normal saline, and the positive control group mice were gavaged with an equal amount of linezolid solution. Since linezolid is orally administered once every 12 hours during human treatment of drug-resistant Staphylococcus aureus infection, based on the principle of drug parallel control, each drug was administered once every 12 hours, and the mode of administration and dosage were the same as the first administration, and the survival and clinical manifestations of mice were observed every 12 hours. Finally, the death of mice within 96 hours was counted, and the survival rate was calculated.

[0055] The required cryptotanshinone solution and bacterial suspension were prepared. Mice were divided into 10 groups and the bacterial solution was administered intranasally after prophylactic administration to establish the model. The control group mice were gavaged with an equal volume of sterile saline, while the positive control group was gavaged with an equal volume of linezolid solution every 12 hours, using the same dosing method and dosage as the first administration. The mice were observed for a period of time, and their survival rate within 96 hours was calculated.

[0056] According to the therapeutic concentration range of fosfomycin and cryptotanshinone explored in the above experiment, they were combined, and 100 healthy mice were selected and randomly divided into 10 groups, namely, groups 1 to 7 of Examples and groups 1 to 2 of Comparative Examples. The model was re-established 3 days after preventive administration. Each group of mice was first gavage-administered with the corresponding medication of each Example and Comparative Example 2 hours after infection with fosfomycin-resistant Staphylococcus aureus; and then subcutaneously injected with the corresponding medication of each Example and Comparative Example once after an interval of 2 hours; the control group mice were first subcutaneously injected at the same time, and then gavage-administered with an equal amount of sterile saline 2 hours later; the positive control group was gavage-administered with an equal amount of linezolid solution, 12 hours / time, with the same dosing method and dosage, and returned to the cage for continued breeding. Their survival status was observed every 12 hours, and the survival rate of the mice within 96 hours was calculated.

[0057] result

[0058] Determine the strain concentration to establish a mouse pneumonia model of Staphylococcus aureus infection resistant to fosfomycin

[0059] Table 1 shows the trends in survival rates of mice treated with different Example and Comparative Example drugs for pneumonia. The survival rates of mice in each study group varied significantly after treatment. The survival rates of mice in Examples 1 to 7 reached over 40%, significantly higher than those in Comparative Example groups 1 and 2, indicating a significant therapeutic effect. The most effective treatment was achieved in Example 5, with a survival rate of 90%.

[0060] Survival rate of infected mice treated with different FOS concentrations

[0061]

[0062] Table 1

[0063] The concentration range of bacterial solution used to construct mouse pneumonia models is very wide, and the pathogenic mechanism of pathogens is closely related to their invasion mode, virulence level, host immune function, and the degree of perfection of the skin and mucosal barrier. Therefore, the construction of animal models of infectious diseases is extremely complex. The optimal bacterial solution concentration for the construction of a mouse pneumonia model infected with fosfomycin-resistant Staphylococcus aureus was determined, and a mouse pneumonia model infected with fosfomycin-resistant Staphylococcus aureus was established; mice were infected by intranasal dripping of different concentrations of fosfomycin-resistant Staphylococcus aureus bacterial solution, and the clinical status and survival of mice infected with each concentration of bacterial solution were observed and analyzed. Finally, the optimal modeling concentration of the SA#122 strain was determined, which is 3×10 10 CFU / ml.

[0064] The results suggest that the combination of cryptotanshinone derivatives and fosfomycin is effective against pneumonia caused by fosfomycin-resistant Staphylococcus aureus alone, and that cryptotanshinone derivatives and fosfomycin exhibit a synergistic effect. Using animal models to simulate human infection, the efficacy and safety of cryptotanshinone derivatives combined with fosfomycin were evaluated in vivo, providing a basis for the clinical application of this drug.

[0065] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention.

[0066] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention.

Claims

1. A cryptotanshinone derivative and fosfomycin combined antibacterial drug, characterized in that: The cryptotanshinone derivative is obtained by introducing a functional group at a specific position of cryptotanshinone, and the weight ratio of the cryptotanshinone derivative to fosfomycin is 1:(10 ～ 150).

2. The antibacterial drug combined with a cryptotanshinone derivative and fosfomycin according to claim 1, characterized in that: The cryptotanshinone derivative is one of the following structural formulas:

3. The antibacterial drug combined with a cryptotanshinone derivative and fosfomycin according to claim 1 or 2, characterized in that: The antibacterial drug is used for treating Gram-positive bacterial infectious diseases, particularly fosfomycin-resistant Staphylococcus aureus infection.

4. The cryptotanshinone derivative and fosfomycin combined antibacterial drug according to claim 3, characterized in that The treatment is carried out orally, by injection or topically.

5. A method for preparing the antibacterial drug of cryptotanshinone derivative and fosfomycin combined with any one of claims 1 to 3, characterized in that: The following steps are involved: Step 1: Synthesis of cryptotanshinone derivatives; Step 2: uniformly mixing the novel cryptotanshinone derivative and fosfomycin according to a weight ratio; Step 3: Prepare the mixture into a dosage form suitable for administration.

6. The method for preparing a combined antibacterial drug of cryptotanshinone derivatives and fosfomycin according to claim 5, characterized in that: The synthesis in step 1 is carried out by specific chemical reactions, including substitution, addition or condensation reactions.

7. Use of a cryptotanshinone derivative according to any one of claims 1 to 3 in combination with fosfomycin as an antibacterial drug in the preparation of a drug for treating fosfomycin-resistant Staphylococcus aureus infection.

Images