A kanamycin-chenodeoxycholic acid conjugate and its preparation method and application

Abstract

The invention discloses a kanamycin chenodeoxycholic acid conjugate and its preparation method and application, and relates to the technical field of drug preparation. The preparation method is: 1) acetonitrile and tetrahydrofuran are dissolved in chenodeoxycholic acid solution, 2N-hydroxysuccinimide and dicyclohexylcarbodiimide are added, and the mixture is stirred at room temperature for 16 hours under argon gas conditions; the precipitated solid is collected by filtration to obtain a white solid product A; 2) the white solid product A and kanamycin prepared in step 1) are dissolved in N, N-dimethylformamide, and K2CO3 and water are added thereto, and the mixture is stirred at room temperature for 16 hours under argon gas conditions; the reaction is purified by HPLC to obtain a kanamycin chenodeoxycholic acid conjugate. The antibiotic drug synthesized by the present invention shows significant advantages in combating drug-resistant bacteria.

Description

Technical Field

[0001] The present invention relates to the technical field of drug preparation, and more particularly to a kanamycin-chenodeoxycholic acid conjugate and a preparation method and application thereof. Background Art

[0002] Currently, methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common strains of antibiotic-resistant bacteria, resistant to a variety of β-lactam antibiotics, including methicillin, penicillin, carbapenems, cephalosporins and their derivatives. Due to its wide distribution and multidrug resistance, MRSA has become a worrying superbug. The epidemiological characteristics of MRSA are the continuous emergence of epidemic strains, and its high morbidity and mortality pose a great threat to human health. MRSA can form a protective biofilm, which makes it difficult for antimicrobial agents to penetrate, thereby generating drug resistance. Biofilms are composed of microbial communities that attach to surfaces and are supported by the structure of an extracellular polymer matrix. This matrix contains one or more extracellular polysaccharides, proteins, and DNA, and plays an important role in the persistence of bacterial infections. The biofilms formed by pathogens can survive high concentrations of antibiotics, leading to treatment failure and recurrence of infection. Most chronic infections in humans are due to the presence of bacterial biofilms. The biofilms formed by MRSA contribute to its persistence and spread in various environments. Furthermore, biofilms are less susceptible to conventional antimicrobial agents due to limited permeability and enzymes in the extracellular matrix that destroy antimicrobials.

[0003] Aminoglycoside antibiotics are a class of antibiotics composed of amino sugars and aminocyclitols, including kanamycin, gentamicin, streptomycin, etc. They mainly inhibit bacterial growth and reproduction by interfering with bacterial protein synthesis, and have a bactericidal effect on certain bacteria. Kanamycin is a type of aminoglycoside antibiotic that has antibacterial effects on a variety of bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, etc. However, kanamycin also has some disadvantages and application limitations: kanamycin may cause damage to organs during metabolism and excretion in the body, thereby producing ototoxicity and nephrotoxicity. This damage may be caused by the accumulation of drugs in the body or excessive use. And with the widespread use of antibiotics, bacterial resistance to kanamycin has gradually increased, resulting in a decrease in therapeutic efficacy. Kanamycin resistance refers to the reduction or loss of bacterial sensitivity to kanamycin, resulting in the inability of the drug to effectively inhibit bacterial growth and reproduction. Kanamycin resistance is mainly due to the fact that bacteria produce one or more enzymes that can destroy kanamycin, which can reduce the concentration of the drug in the bacteria, thereby reducing its antibacterial effect.

[0004] Therefore, providing a kanamycin-chenodeoxycholic acid conjugate that can specifically kill drug-resistant bacteria and a preparation method and application thereof is an issue that needs to be urgently addressed by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention provides a kanamycin-chenodeoxycholic acid conjugate and a preparation method and application thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A kanamycin-chenodeoxycholic acid conjugate, the chemical structure of the conjugate is:

[0008]

[0009] A method for preparing a kanamycin-chenodeoxycholic acid conjugate comprises the following steps:

[0010] 1) dissolving chenodeoxycholic acid in acetonitrile and tetrahydrofuran, adding 2N-hydroxysuccinimide and dicyclohexylcarbodiimide, mixing and stirring at room temperature for 16 hours under argon gas; collecting the precipitated solid by filtration to obtain a white solid product A;

[0011] 2) The white solid product A prepared in step 1) and kanamycin were dissolved in N,N-dimethylformamide, and K2CO3 and water were added thereto. After mixing, the mixture was stirred at room temperature under argon for 16 hours; the reaction was purified by HPLC to obtain a kanamycin-chenodeoxycholic acid conjugate.

[0012] Furthermore, the volume mass ratio of acetonitrile: tetrahydrofuran: chenodeoxycholic acid: 2N-hydroxysuccinimide: dicyclohexylcarbodiimide is 5 mL: 25 mL: 1.96 g: 0.69 g: 1.2 g;

[0013] The volume mass ratio of the white solid product A: kanamycin: N,N-dimethylformamide: K2CO3: water is 489 mg: 484 mg: 5 mL: 276 mg: 5 mL.

[0014] Application of kanamycin-chenodeoxycholic acid conjugate in the preparation of anti-MRSA drugs.

[0015] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects of the present invention are:

[0016] The antibiotic drugs synthesized by the present invention show significant advantages in combating drug-resistant bacteria. Through sophisticated chemical synthesis, these drugs can improve the ability to kill drug-resistant bacteria in a targeted manner, effectively addressing the increasingly serious problem of drug resistance. At the same time, the research and development process also focuses on reducing the toxicity of the two synthetic substances to ensure that the drugs can reduce adverse effects on the human body while exerting their therapeutic effects, thereby achieving safe and effective treatment. This achievement is of great significance for improving the effect of antibacterial treatment and protecting the health of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0018] Figure 1 This is a physical picture of a white powdery mixture;

[0019] Figure 2 Four chemical synthesis pathways for the mixture;

[0020] Figure 3 It is the chemical synthesis route of kanamycin chenodeoxycholic acid conjugate;

[0021] Figure 4 is a chromatogram of light yellow solid compound B;

[0022] Figure 5 This is a physical picture of light yellow solid compound B;

[0023] Figure 6 is the confirmation of the amide bond position, where Figure 6 A is the nuclear magnetic hydrogen spectrum of the synthetic drug K-CDCA. Figure 6 B is the nuclear magnetic hydrogen spectrum of the synthetic raw material kanamycin, Figure 6 C is the H-NMR spectrum of the synthetic raw material chenodeoxycholic acid;

[0024] Figure 7 are the inhibition zones of K-CDCA, K, and M-KCDCA;

[0025] Figure 8 This is the inhibition curve of K-CDCA against MRSA (ATCC33591);

[0026] Fig. 9 The cytotoxicity of K, CDCA, and K-CDCA. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Example 1

[0029] In the previous study of the present invention, chenodeoxycholic acid was used to prepare chenodeoxycholic acid chloride, and the acyl chloride was reacted with the amino group in kanamycin to obtain a white powder mixture ( Figure 1 ). A mixture of substitution products on different amino groups of kanamycin. If acyl chloride is used, it may react with the amino group or the hydroxyl group. The mixture should contain at least 4 kinds of substitution products. Figure 2 ).

[0030] The present invention further experimentally studies and determines the specific reaction of EDC / NHS with a specific amino group, and the steps are as follows:

[0031] 1. ACN acetonitrile (5 mL) and THF tetrahydrofuran (25 mL) were dissolved in a solution of 1-chenodeoxycholic acid (1.96 g, 5.0 mmol), and then 2N-hydroxysuccinimide (0.69 g, 6.0 mmol) and DCC dicyclohexylcarbodiimide (1.2 g, 6.0 mmol) were added. The reaction mixture was stirred at room temperature under argon for 16 hours. The precipitated solid was collected by filtration to obtain a white solid product A (2.0 g, 81%).

[0032] 2. The above white solid product A (489 mg, 1.0 mmol) and kanamycin (484 mg, 1.0 mmol) were dissolved in N, N-dimethylformamide DMF (5 mL), and K2CO3 (276 mg, 2.0 mmol) and water (5 mL) were added thereto. The reaction mixture was stirred at room temperature under argon for 16 hours. The reaction was purified by HPLC to obtain a light yellow solid compound B (53 mg, 6% yield);

[0033] Chemical synthesis routes Figure 3 , chromatogram see Figure 4 .

[0034] 3.HPLC purification conditions are:

[0035] Sample name: MC22-173-109;

[0036] Instrument model: Waters 2767 / 2545 / 2489;

[0037] Preparative column: Welch Ultimate XB-C4 (filler 70 g) 10 um;

[0038] Mobile phase A: 0.1% TFA in water;

[0039] Mobile phase B: CH3CN;

[0040] Wavelength: 254nm / 214nm;

[0041] Flow rate: 30mL / min;

[0042] Column temperature: RT;

[0043] Retention time: 9.77min

[0044] Table 1: Gradient conditions

[0045] Time (min) A(％) B(％) 0.00 80 20 1 73 27 12 60 40 18 5 95 20 95 5

[0046] 4. Purity identification: HPLC purity identification is 98.552%

[0047] Table 2: Chromatographic parameters of light yellow solid

[0048] Peak Retention time min Peak width Peak height area Signal-to-Noise Ratio Area percentage % 1 6.831 0.126 103774 440337 1157.84 98.552 2 7.211 0.361 355 4000 3.97 0.892 3 7.415 0.270 275 2468 3.07 0.552 total 446805 100.000

[0049] The results showed that the drug purity of the synthesized product could reach 98.552%, and the product was a pale yellow solid (see Figure 5 ). The molecular weight of the drug is 859.07. The carboxyl group of kanamycin and the amino group of chenodeoxycholic acid undergo amide bond condensation. The combined position is at the expected position (see Figure 6 ). Figure 6 A is the nuclear magnetic hydrogen spectrum of the synthetic drug K-CDCA. Figure 6 B is the nuclear magnetic hydrogen spectrum of the synthetic raw material kanamycin, Figure 6 C is the H NMR spectrum of the synthetic raw material chenodeoxycholic acid. By comparing the H NMR spectra of 4B, 4C and 4A, the types and numbers of hydrogen atoms, the positions of the linking bonds and other information of the synthetic compound K-CDCA were determined.

[0050] Disk diffusion method drug sensitivity test to verify the efficacy of kanamycin-chenodeoxycholic acid drug steps:

[0051] The inhibition zone method was used to verify the antibacterial activity of kanamycin (K), kanamycin-chenodeoxycholic acid (K-CDCA), and a mixture of kanamycin and chenodeoxycholic acid (M-KCDCA) against MRSA at the same molar concentration. MRSA (ATCC33591) No. 4 and clinically isolated MRSA Nos. 1 to 3 were diluted to 1×10 7CFU / mL of bacterial solution, prepare sterile paper slips, and drop K and K-CDCA, M-KCDCA solutions of the same molar concentration on each paper slip to ensure that the paper slips fully absorb the solution. Use a sterile applicator to evenly spread the diluted MRSA bacterial solution on the surface of the culture medium. Use sterile tweezers to gently place the drug-containing paper slip on the culture medium coated with the bacterial solution. Make sure that the paper slip fits tightly against the surface of the culture medium to avoid bubbles. Place the culture dish in a 37°C constant temperature incubator and culture for 18 hours. After the culture is completed, observe the inhibition zone around the paper slip. Compare the sizes of the inhibition zones of different paper slips to evaluate the antibacterial activity of different drugs against MRSA. The results showed that at the same molar concentration, the inhibition zone of K-CDCA was larger than that of K and M-KCDCA (see Figure 7 ).

[0052] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of K, K-CDCA, and M-KCDCA against MRSA (ATCC33591) were determined by microdilution method. The specific operation is as follows:

[0053] Preparation of drug solution: accurately weigh 1mmol of K, K-CDCA, and M-KCDCA and dissolve them in 1ml of MH medium. In a clean bench, dilute K, K-CDCA, and M-KCDCA with MH medium in a 2-fold ratio to obtain the original solution, 2 -1 ×Original solution, 2 -2 ×Original solution, 2 -3 ×Original solution, 2 -4 ×Original solution, 2 -5 ×Original solution, 2 -6 ×Original solution, 2 -7 ×Original solution, 2 -8 ×Original solution, 2 -9 ×Original solution, 2 -10 ×The original solution is kept for future use.

[0054] Determination of MIC value: The experimental groups were K group, K-CDCA group, M-KCDCA group, negative control group, and positive control group. First, add the diluted drug solution of each concentration to the 96-well plate, 100 μL per well, and set up 3 parallel wells for each experimental group; the negative control group was 200 μL MH culture medium; the positive control group was 100 μL MH culture medium. Subsequently, 100 μL of 1.0x10 6 CFU / mL of MRSA bacterial solution, then mix thoroughly, and operate in a sterile environment. After incubation in a 37℃ constant temperature incubator for 18-20h, use an enzyme reader to read the OD600 When OD 600 When it is less than 0.10, it can be regarded as no bacterial proliferation, and the minimum concentration without bacterial reproduction is the MIC.

[0055] Determination of MBC value: Take 100 μL of bacterial solution from the well without bacterial proliferation and spread it on the plate. After continuing to culture in a 37°C constant temperature incubator for 24 hours, the lowest concentration without colony formation is the MBC value. The results show that the MIC molar concentration of K-CDCA drug to inhibit MRSA (ATCC33591) is more than 8 times lower than that of K and M-KCDCA.

[0056] Table 3: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of K, K-CDCA or M-KCDCA

[0057]

[0058] The inhibition curve of K-CDCA against MRSA (ATCC33591)

[0059] Take 8 50ml disposable shaking tubes and adjust the concentration of MRSA solution to 1.0x10 4 CFU / mL, inoculated into MH medium at a 10% inoculation ratio, the total volume of the system was 30ml, kanamycin-chenodeoxycholic acid was added respectively, and the final concentrations of kanamycin-chenodeoxycholic acid were 2MIC, MIC, and 1 / 2MIC, respectively. A control group without drug was set up at the same time. After the system configuration is completed, the shaking tube is in a semi-closed state, air is allowed to enter, and then placed in a constant temperature shaker at 37°C and 180r / min for 48h. At 2h, 4h, 6h, 8h, 10h, 12h, 24h, and 48h of incubation, aseptically sample and use a multifunctional microplate reader to measure OD 600 , and record, the experiment was repeated three times. 600 The value is the vertical axis and the time is the horizontal axis, and the bacterial growth curve is drawn. The results show that 2MIC and MIC of kanamycin-chenodeoxycholic acid can significantly inhibit the proliferation of bacteria, but when the concentration drops to 1 / 2MIC, the antibacterial effect of kanamycin-chenodeoxycholic acid is not as significant as MIC and 2MIC (see Figure 8 ).

[0060] Cytotoxicity of K, CDCA, and K-CDCA

[0061] Mouse macrophages were cultured at 1×10 5 / mL was inoculated in a 96-well plate. After it adhered to the wall and grew, K, CDCA, K-CDCA and M-KCDCA containing drug concentrations of 1mg / L, 0.5mg / L, 0.25mg / L, 0.125mg / L, 0.0625mg / L, and 0.0317mg / L were added respectively. Each group had 5 parallel wells, and a blank control group without drug addition and a cell-free group without inoculated cells were set up at the same time. After that, each group of drugs was co-incubated with mouse macrophages for 72h. After the incubation, the drug solution was discarded, and after rinsing with sterile PBS 3 times, a cell culture medium containing 10% CCK-8 (DMEM / F12 containing 15% FBS and 1% double antibody) was added. After 30min of color development, the absorbance at 450nm was detected by a multifunctional microplate reader.

[0062] According to the formula:

[0063] Cell activity was calculated by cell activity = (OD value of the experimental group - OD value of the cell-free group) / (OD value of the blank group - OD value of the cell-free group).

[0064] The results showed that the cytotoxicity of K-CDCA was significantly lower than that of CDCA and M-KCDCA at each concentration tested (see Fig. 9 ).

[0065] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A kanamycin-chenodeoxycholic acid conjugate, characterized in that: The chemical structure of the conjugate is:

2. A method for preparing the kanamycin-chenodeoxycholic acid conjugate according to claim 1, characterized in that: The following steps are involved: 1) dissolving chenodeoxycholic acid in acetonitrile and tetrahydrofuran, adding N-hydroxysuccinimide and dicyclohexylcarbodiimide, mixing and stirring at room temperature for 16 hours under argon gas; collecting the precipitated solid by filtration to obtain a white solid product A; 2) The white solid product A prepared in step 1) and kanamycin were dissolved in N,N-dimethylformamide, and K2CO3 and water were added thereto. After mixing, the mixture was stirred at room temperature under argon for 16 hours; the reaction was purified by HPLC to obtain a kanamycin-chenodeoxycholic acid conjugate.

3. The preparation method according to claim 2, characterized in that: Step 1) the volume mass ratio of acetonitrile: tetrahydrofuran: chenodeoxycholic acid: N-hydroxysuccinimide: dicyclohexylcarbodiimide is 5 mL: 25 mL: 1.96 g: 0.69 g: 1.2 g; Step 2) The volume mass ratio of the white solid product A: kanamycin: N,N-dimethylformamide: K2CO3: water is 489 mg: 484 mg: 5 mL: 276 mg: 5 mL.

4. Use of the kanamycin-chenodeoxycholic acid conjugate according to claim 1 in the preparation of anti-MRSA drugs.

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