Tilmicosin derivative and synthesis method thereof
By introducing acyl and sulfonyl groups into timicocin, the existing problems of antibiotic resistance and gastrointestinal irritation are solved, and more efficient and stable antibacterial effects are achieved.
Patent Information
- Application Number
- CN202510091583.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2024-09-13
- Filing Date
- 2025-01-21
- Publication Date
- 2025-04-11
AI Technical Summary
Existing antibiotics are prone to drug-resistant bacteria during use, resulting in a reduced therapeutic effect, and some drugs are irritating to the gastrointestinal intestines and leading to adverse reactions.
By combining temicocin with acyl and sulfonyl groups, the fat solubility and acid tolerance of the drug are changed, thereby improving the bioactivity and metabolic stability of the drug.
It improves the antibacterial activity and stability of the drug, reduces the irritation to the gastrointestinal intestines, prolongs the action time of the drug, and improves bioavailability.
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Figure CN119798350A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of drug design and synthesis, and specifically relates to a tilmicosin derivative and a synthesis method thereof. Background Art
[0002] Since their discovery nearly 90 years ago, antibiotics have changed the world of modern medicine. Antibiotics have played an important role in fighting deadly and disabling diseases and saved countless lives. However, the misuse of antibiotics poses a risk to public health. As antibiotics are increasingly prescribed and abused, bacteria are developing resistance. Infections caused by resistant bacteria have become a new challenge for anti-infection treatment in the new century and a major threat to human health and life today. Bacterial resistance has become a global concern.
[0003] Since the advent of macrolide antibiotics, it has been a hot topic in clinical application research, and it still occupies a major position in the antibacterial drug market. Erythromycin A was the first to be clinically used. With the increase in demand for therapeutic drugs, its types have also become diversified. It has developed rapidly with its economical, highly absorbable, and extensive disease treatment characteristics, and has a significant therapeutic effect in respiratory departments. However, erythromycin A is irritating and burdens patients with poor gastrointestinal function. Therefore, second-generation drugs such as fluoroerythromycin and azithromycin have also been developed. Compared with the first-generation drugs, the drug properties and pharmacokinetics are more stable, which effectively alleviates the irritation of erythromycin drugs and reduces the occurrence of gastrointestinal adverse reactions. The second-generation drugs have a wider range of clinical applications than the first-generation drugs, involving all disciplines, but with its continuous application and the emergence of multi-drug resistant bacteria, its shortcomings have gradually become apparent, and have been studied and concerned by professionals. The main clinical research and development direction is to reduce adverse reactions such as irritation and improve drug stability and antibacterial ability. Subsequently, third-generation drugs such as telithromycin have emerged, which are better than the second-generation drugs in terms of drug properties and efficacy. Tilmicosin has a strong antibacterial effect and excellent pharmacokinetic characteristics. It can also improve the immunity of animals. In addition, the latest research shows that Tilmicosin has an antiviral effect in the prevention and treatment of individual pig diseases. Moreover, there is no cross-resistance between it and other commonly used antibiotics in clinical practice, and it has achieved remarkable results in the clinical treatment of various animal diseases. Compared with its raw material tylosin, it has a small dosage, lasting effect, small side effects, low body residue, and is safe and non-toxic. It is an economical, safe and effective new veterinary drug commonly used for antibacterial and growth promotion in livestock and poultry. Since acylation can change the lipid solubility of drugs, improve the distribution of drugs in the body, and effectively improve the acid tolerance of drugs, we chose to start with the acylation of hydroxyl groups. At the same time, we also learned that sulfonyl groups have unique physical and chemical properties. The introduction of sulfonyl groups can regulate the solubility and acidity and alkalinity of small molecules. Sulfonyl groups can provide two hydrogen bond receptors. The reasonable introduction of sulfonyl groups can increase the biological activity of compounds by increasing the hydrogen bond interaction between small molecules and targets. Sulfonyl groups have a relatively stable structure. The introduction of sulfonyl groups can improve the metabolic stability of drugs by blocking the metabolic sites, prolonging their duration of action, and improving their bioavailability, thereby improving the pharmacokinetic properties of small molecules. Therefore, we plan to introduce acyl and sulfonyl groups at the same time and carry out subsequent research on synthetic drugs. Summary of the invention
[0004] The present invention uses tilmicosin as a substrate, and aims to make the target compound exert antibacterial activity by non-covalently binding with RNA. Acylation can change the lipid solubility of the drug, improve the distribution of the drug in the body, and effectively improve the acid tolerance of the drug.
[0005] The design idea of the present invention is: the present invention starts with the acylation of hydroxyl groups, and at the same time, it is noted that the sulfonyl group has unique physical and chemical properties, and the introduction of the sulfonyl group can adjust the solubility and acidity and alkalinity of small molecules; the sulfonyl group can provide two hydrogen bond receptors, and the reasonable introduction of the sulfonyl group can increase the biological activity of the compound by increasing the hydrogen bond interaction between the small molecule and the target; the sulfonyl group structure is relatively stable, and the introduction of the sulfonyl group can improve the metabolic stability of the drug by blocking the easily metabolized site, prolong its action time, and improve its bioavailability, thereby improving the pharmacokinetic properties of the small molecule. Its chemical reaction formula is: .
[0006] The specific technical solution of the present invention is: A tilmicosin derivative, characterized in that the derivative has the structural formula: ; Wherein, R1 is one of the following chemical formulas: ; R2 is one of the following chemical formulas: .
[0007] A method for synthesizing a tilmicosin derivative, characterized in that it comprises the following steps: 1) Add tilmicosin to a 0.5 mol / L trifluoroacetic acid aqueous solution and react at 100 °C for 5 h to obtain decyclized tilmicosin; 2) reacting with the corresponding sulfonyl chloride under alkaline conditions provided by KOH at room temperature for more than 8 hours to obtain the corresponding sulfonylated product; 3) The above product was reacted with anhydride in dichloromethane at room temperature for 24 h to obtain the corresponding acylated product.
[0008] Compared with the prior art, the advantages of the present invention are: 1) The tilmicosin derivatives of the present invention change the lipid solubility of the drug by introducing acyl and sulfonyl groups, improve the distribution of the drug in the body, and can effectively improve the acid tolerance of the drug.
[0009] 2) However, since Tilmicosin contains multiple hydroxyl groups, site selectivity is one of the difficulties. We selected different substituents to replace them by taking advantage of the strength of the hydroxyl activity and connected them to the corresponding sites. After the synthesis was completed, biological experiments such as UV-visible absorption spectroscopy and in vitro antibacterial tests also verified its damage to genetic material and antibacterial effect. BRIEF DESCRIPTION OF THE DRAWINGS
[0010] Figure 1 is the T-1 UV-visible absorption spectrum; Figure 2 It is the T-16 UV-visible absorption spectrum. DETAILED DESCRIPTION
[0011] Example 1
[0012] (1) Weigh trifluoroacetic acid (1.425 g, 0.0125 mol) and dissolve it in 25 mL of water. Weigh tilmicosin (0.5 g, 0.575 mmol) and dissolve it in the above aqueous solution. Heat to 100 °C and maintain this temperature for 5 h. After the reaction is completed, cool it. After cooling, add NaHCO3 to adjust the pH to neutral. Extract with dichloromethane three times, combine the organic layers, dry the organic layers with anhydrous magnesium sulfate, and remove the solvent of the organic layer in vacuo to obtain a light yellow amorphous solid with a yield of 70.26%.
[0013] (2) Dissolve cyclotrimicosin (0.1 g, 0.144 mmol) in 20 ml of ethyl acetate, add methylsulfonyl chloride (0.024 g, 0.21 mmol) and react for 10 minutes, then add an appropriate amount of KOH to provide an alkaline environment, and stir the mixture at room temperature for more than 8 h. After the reaction is completed, add 20 ml of water, separate the liquids, extract the aqueous phase with ethyl acetate three times, combine the organic phases, wash twice with saturated brine, and remove the solvent under reduced pressure to obtain a yellow foamy product with a yield of 80.51%.
[0014] (3) The above product (0.1 g, 0.13 mmol) was dissolved in 15 ml of dichloromethane, acetic anhydride (0.04 g, 0.39 mmol) was added, and the mixture was stirred at room temperature for 24 h. After the reaction was completed, the solvent was removed in vacuo to obtain a yellow foamy solid (T-1) with a yield of 90.5%.
[0015] Example 2
[0016] (1) Weigh trifluoroacetic acid (1.425 g, 0.0125 mol) and dissolve it in 25 mL of water. Weigh tilmicosin (0.5 g, 0.575 mmol) and dissolve it in the above aqueous solution. Heat to 100 °C and maintain this temperature for 5 h. After the reaction is completed, cool it. After cooling, add NaHCO3 to adjust the pH to neutral. Extract with dichloromethane three times, combine the organic layers, dry the organic layers with anhydrous magnesium sulfate, and remove the solvent of the organic layer in vacuo to obtain a light yellow amorphous solid with a yield of 70.26%.
[0017] (2) Dissolve cyclotrimicosin (0.1 g, 0.144 mmol) in 20 ml of ethyl acetate, add methylsulfonyl chloride (0.024 g, 0.21 mmol) and react for 10 minutes, then add an appropriate amount of KOH to provide an alkaline environment, and stir the mixture at room temperature for more than 8 h. After the reaction is completed, add 20 ml of water, separate the liquids, extract the aqueous phase with ethyl acetate three times, combine the organic phases, wash twice with saturated brine, and remove the solvent under reduced pressure to obtain a yellow foamy product with a yield of 80.51%.
[0018] (3) The above product (0.1 g, 0.117 mmol) was dissolved in 15 ml of dichloromethane, and propionic anhydride (0.045 g, 0.35 mmol) was added. The mixture was stirred at room temperature for 24 h. After the reaction was completed, the solvent was removed in vacuo to obtain a yellow foamy solid (T-2) with a yield of 88.3%.
[0019] Example 3
[0020] (1) Weigh trifluoroacetic acid (1.425 g, 0.0125 mol) and dissolve it in 25 mL of water. Weigh tilmicosin (0.5 g, 0.575 mmol) and dissolve it in the above aqueous solution. Heat to 100 °C and maintain this temperature for 5 h. After the reaction is completed, cool it. After cooling, add NaHCO3 to adjust the pH to neutral. Extract with dichloromethane three times, combine the organic layers, dry the organic layers with anhydrous magnesium sulfate, and remove the solvent of the organic layer in vacuo to obtain a light yellow amorphous solid with a yield of 70.26%.
[0021] (2) Dissolve cyclotrimicosin (0.1 g, 0.144 mmol) in 20 ml of ethyl acetate, add p-toluenesulfonyl chloride (0.041 g, 0.21 mmol) and react for 10 minutes, then add an appropriate amount of KOH to provide an alkaline environment, and stir the mixture at room temperature for more than 8 hours. After the reaction is completed, add 20 ml of water, separate the liquids, extract the aqueous phase with ethyl acetate three times, combine the organic phases, wash twice with saturated brine, and remove the solvent under reduced pressure to obtain a yellow foamy product with a yield of 82.31%.
[0022] (3) The above product (0.1 g, 0.117 mmol) was dissolved in 15 ml of dichloromethane, and acetic anhydride (0.036 g, 0.35 mmol) was added. The mixture was stirred at room temperature for 24 h. After the reaction was completed, the solvent was removed in vacuo to obtain a yellow foamy solid (T-16) with a yield of 90.5%.
[0023] Example 4
[0024] (1) Weigh trifluoroacetic acid (1.425 g, 0.0125 mol) and dissolve it in 25 mL of water. Weigh tilmicosin (0.5 g, 0.575 mmol) and dissolve it in the above aqueous solution. Heat to 100 °C and maintain this temperature for 5 h. After the reaction is completed, cool it. After cooling, add NaHCO3 to adjust the pH to neutral. Extract with dichloromethane three times, combine the organic layers, dry the organic layers with anhydrous magnesium sulfate, and remove the solvent of the organic layer in vacuo to obtain a light yellow amorphous solid with a yield of 70.26%.
[0025] (2) Dissolve cyclotrimicosin (0.1 g, 0.144 mmol) in 20 ml of ethyl acetate, add p-toluenesulfonyl chloride (0.041 g, 0.21 mmol) and react for 10 minutes, then add an appropriate amount of KOH to provide an alkaline environment, and stir the mixture at room temperature for more than 8 hours. After the reaction is completed, add 20 ml of water, separate the liquids, extract the aqueous phase with ethyl acetate three times, combine the organic phases, wash twice with saturated brine, and remove the solvent under reduced pressure to obtain a yellow foamy product with a yield of 82.31%.
[0026] (3) The above product (0.1 g, 0.117 mmol) was dissolved in 15 ml of dichloromethane, and propionic anhydride (0.045 g, 0.35 mmol) was added. The mixture was stirred at room temperature for 24 h. After the reaction was completed, the solvent was removed in vacuo to obtain a yellow foamy solid (T-17) with a yield of 82.43%.
[0027] Example 5: In vitro antibacterial experiment The minimum inhibitory concentration (MIC) of T-1, T-2, T-16, and T-17 against several Gram-positive and Gram-negative bacteria was determined by the broth dilution method (CLSI 2003) recommended by the Clinical Laboratory Standards Institute (CLSI). The experiment set up T-1 group, T-2 group, T-16 group, T-17 group, and Tilmicosin group. The five drugs were diluted into 25-60 μg / ml solution with sterile water, and various bacterial solutions were diluted into 10 5 ~10 6 ml -1. Take a 96-well polystyrene microplate sterilized by ultraviolet light, add 120 μL of diluted bacterial solution to the first well of each row with a pipette, and add 100 μL of diluted bacterial solution to the 2nd to 12th wells in sequence. Add only 100 μL of drug solution to the first well of the last row as a drug control, and add only 100 μL of bacterial solution to the second well as a bacterial solution control. Take 80 μL of the dilution solution to the first well, mix it with a pipette, and then draw 100 μL into the second well. Dilute to the 12th well in the same way, draw 100 μL from the 12th well and discard it. Place it in an incubator at 37 °C and observe it after 16 to 20 hours. The lowest concentration of sterile growth is the MIC. The quality control strain used in this test is Staphylococcus aureus ATCC25923. The reference value of the minimum inhibitory concentration MIC of tilmicosin recommended by CLSI is 1 to 4 μg / ml. The test result is acceptable only if the MIC value is within this range. Otherwise, the batch of data is invalid and the test should be repeated after the cause is found.
[0028] Comps 25923 29213 8739 25922 9027 27853 T-1 4 4 2 4 2 2 T-2 8 8 16 8 8 16 T-16 4 2 2 4 4 4 T-17 8 16 16 8 8 16 TIM 4 4 2 2 4 4 TYL 8 16 8 8 16 8 .
[0029] Four candidate tilmicosin derivatives and different concentrations of tilmicosin and tylosin were cultured with the following various bacterial colonies in an incubator for 16-24 h. The antibacterial results are shown in the table above. The minimum inhibitory concentration of the drug in the well where no bacteria grow is the minimum inhibitory concentration. The experiment was repeated three times to prevent errors. According to the minimum inhibitory concentration data, T-1 and T-16 are more effective, so these two drugs were selected for subsequent characterization.
[0030] 3. UV-Visible Spectroscopy UV-visible absorption spectroscopy was performed in a fixed 200 μM T-1 (T-16) solution, in which ctDNA was titrated from 100 μL to 800 μL in 0.2 M phosphate buffer (PBS, pH 7.4). After each titration, the mixture was incubated at 37 °C for 5 min with gentle stirring and then allowed to stand for 1 min to ensure the stability of the drug-DNA complex. The concentration (c) of the ctDNA solution was determined by UV-visible absorption spectroscopy and calculated according to the Lambert-Beer law A = ε[ctDNA] × c × l, where A is the UV-visible absorbance value and ε[ctDNA] is the molar absorptivity at λ = 260 nm (20 °C) [6600 L•mol −1 •cm −1 ], l is the thickness of the absorption cell (1 cm).
[0031] UV-visible absorption spectroscopy is one of the most commonly used techniques for detecting the interaction between small molecules and DNA. When different concentrations of DNA are added to a fixed concentration compound solution, the changes in the UV-visible spectrum before and after can evaluate the interaction and binding strength between the compound and DNA. The insertion of the measured compound into DNA will reduce the peak intensity of the compound's characteristic absorption peak, which is the hypochromic effect; the coupling of the compound with the base molecular orbital may also lead to a change in the peak position of the characteristic absorption peak, resulting in a red shift or a blue shift.
[0032] As shown in the attached figure, as the concentration of ctDNA increases, the maximum absorption spectrum of the compound blue-shifts and a hypochromic effect occurs. The blue shift may be caused by the change in the vibration frequency of the chemical bonds in the molecule due to the transition of intramolecular electrons, and the appearance of the hypochromic effect proves that the compound can be intercalated with DNA. According to (Eq. 1), the hypochromic rates of the drugs were calculated to be 10.63% and 6.62%, respectively. Combining the spectral data and the Benesi-Hildebrand equation (Eq. 2), the binding constant (Kb) of the small molecule-DNA complex can be obtained.
[0033] (Eq. 1); (Eq. 2).
[0034] Where A0 and A are the absorption values of completely free T-1 or T-16 and the complex, εH and εG-H are the molar extinction coefficients of free T-1 or T-16 and the complex, and [DNA] is the concentration of ctDNA. The binding constants (Kb) of T-1-ctDNA complex and T-16-ctDNA complex are 5.01×104 M -1 and 4.92×104 M -1 Combining the above parameters, the change in Gibbs free energy (ΔG) was calculated according to ΔG = -RT lnKb. The calculated result ∆G < 0 suggested that the embedding between the two drugs and ctDNA was spontaneous.
Claims
1. A tilmicosin derivative, characterized in that: The derivative structural formula is: ； Wherein, R1 is one of the following chemical formulas: ； R2 is one of the following chemical formulas: 。 2. A method for synthesizing a tilmicosin derivative, characterized in that: The steps include: Firstly, a trifluoroacetic acid aqueous solution is used to de-ring tilmicosin to obtain de-ring tilmicosin, which is then reacted with a corresponding sulfonyl chloride under alkaline conditions to obtain a corresponding sulfonylated product, and finally the above product is reacted with various acid anhydrides to obtain a corresponding acylated product.