Low-toxicity amphotericin B derivative based on 16-position carboxyl modification as well as preparation method and application of low-toxicity amphotericin B derivative
A low-toxic amphotericin B derivative is synthesized via a single-step modification of the 16-carboxyl group, addressing toxicity issues and enhancing clinical applicability by reducing hemolytic and renal toxicity while maintaining antifungal efficacy.
Patent Information
- Application Number
- CN202411323689.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-23
- Publication Date
- 2024-12-31
AI Technical Summary
Existing amphotericin B derivatives suffer from high hemolytic, nephrotoxic, and acute toxicity, with complex synthesis processes and low yields, limiting their clinical application.
A low-toxic amphotericin B derivative is synthesized through a single chemical reaction by modifying the 16-carboxyl group with 2-cyclopentaneethylamine, 2-cyclopentaneethylamine hydrochloride, or 2-cyclohexylethylamine, using mild reaction conditions and a simple post-processing method.
The derivative exhibits significantly reduced hemolytic, renal, and acute toxicity while maintaining effective antifungal activity, with improved synthesis efficiency and yield.
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Figure CN119219717A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of drug preparation, and relates to a low-toxic amphotericin B derivative based on 16-carboxyl modification, and a preparation method and application thereof. Background Art
[0002] Amphotericin B is a polyene macrolide antibiotic produced by Streptomyces nodosus. It has a broad antifungal spectrum and is the first choice for the treatment of deep fungal infections such as Candida albicans, Cryptococcus neoformans, Aspergillus, and Mucor. Amphotericin B preparations used clinically include common amphotericin B for injection (sodium deoxycholate solubilized), liposomes (AmBisome), lipid complexes (Abelcet), and sodium cholesterol sulfate complexes (Amphotec). Since the launch of amphotericin B in 1958, drug-induced renal toxicity has not been effectively resolved.
[0003] In order to improve the hemolytic toxicity, nephrotoxicity and acute toxicity of amphotericin B, amphotericin B derivatives of various structures have been synthesized. The amphotericin B derivatives reported in the existing literature have many synthesis steps (more than three steps), low yield (less than 30%) and insufficiently mild reaction conditions. So far, no amphotericin B derivative products have been marketed for clinical application. Based on a large number of studies, we found that by coupling 2-cyclopentaneethylamine, 2-cyclopentaneethylamine hydrochloride, 2-cyclohexylethylamine or 2-cyclohexylethylamine hydrochloride with the 16-carboxyl group on the amphotericin B molecule, a new type of amphotericin B derivative can be synthesized in only one chemical reaction. The reaction conditions are mild, the post-processing is simple, and the product yield is high. Compared with amphotericin B, this new amphotericin B derivative has significantly reduced hemolytic toxicity, renal cell toxicity and acute toxicity, while maintaining good antifungal activity. Summary of the invention
[0004] In view of this, one of the objects of the present invention is to provide a low-toxic amphotericin B derivative based on 16-carboxyl modification; a second object of the present invention is to provide a method for preparing a low-toxic amphotericin B derivative based on 16-carboxyl modification; a third object of the present invention is to provide an application of a low-toxic amphotericin B derivative based on 16-carboxyl modification in drugs for treating fungal infectious diseases; a fourth object of the present invention is to provide a pharmaceutical composition comprising a low-toxic amphotericin B derivative based on 16-carboxyl modification.
[0005] In order to achieve the above object, the present invention provides the following technical solutions:
[0006] 1. A low-toxic amphotericin B derivative based on 16-carboxyl modification, characterized in that the structural formula of the derivative is shown in the figure below:
[0007]
[0008] Among them, R is
[0009] Preferably, R is cyclopentyl or cyclohexyl.
[0010] Further preferably, the structural formula of the derivative is as follows:
[0011]
[0012] 2. The method for preparing the low-toxic amphotericin B derivative based on the modification of the 16-carboxyl group, the reaction formula of the method is:
[0013]
[0014] Preferably, the method comprises the following steps:
[0015] Dissolve amphotericin B, amino compounds and organic bases in N,N-dimethylformamide, stir at 20-40°C for 5-30 minutes under N2 protection, then add PyBOP, and continue stirring at 20-40°C for 1-6 hours under N2 protection. Pour the reaction solution into methyl tert-butyl ether for precipitation, collect the product by centrifugation, add acetone to fully disperse, pour into methyl tert-butyl ether for precipitation, collect the product by centrifugation, repeat the "dispersion-precipitation" operation 3 times, collect the product by filtration, and vacuum dry to obtain a yellow powdered amphotericin B derivative.
[0016] Further preferably, the molar ratio of amphotericin B, amino compound, organic base and PyBOP in the step is 1:1.1-3:1.1-3:1.1-3;
[0017] The amino compound is any one of 2-cyclopentaneethylamine, 2-cyclopentaneethylamine hydrochloride, 2-cyclohexylethylamine or 2-cyclohexylethylamine hydrochloride;
[0018] The organic base is triethylamine or N,N-diisopropylethylamine;
[0019] The mass volume ratio of amphotericin B and N,N-dimethylformamide is 10:0.4-1, mg / ml;
[0020] The volume ratio of N,N-dimethylformamide to methyl tert-butyl ether is 1:8-15;
[0021] The mass volume ratio of amphotericin B to acetone is 10:0.5-2, mg / ml;
[0022] The volume ratio of the acetone to methyl tert-butyl ether is 1:3-10.
[0023] 3. Use of the above-mentioned low-toxic amphotericin B derivatives based on modification of the 16-carboxyl group in the preparation of drugs for inhibiting fungal growth or treating fungal infectious diseases.
[0024] 4. A pharmaceutical composition comprising the above-mentioned low-toxic amphotericin B derivative based on modification of the 16-carboxyl group.
[0025] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is any one of distearoylphosphatidylethanolamine-polyethylene glycol, soybean lecithin or egg yolk lecithin.
[0026] Preferably, the pharmaceutical composition is in the form of an injection.
[0027] The beneficial effects of the present invention are:
[0028] 1. The present invention discloses a low-toxic amphotericin B derivative based on 16-position carboxyl modification. The present invention uses 2-cyclopentaneethylamine, 2-cyclopentaneethylamine hydrochloride, 2-cyclohexylethylamine or 2-cyclohexylethylamine hydrochloride to chemically modify the 16-position carboxyl of the amphotericin B molecule, and designs a novel amphotericin B derivative, which is significantly superior to amphotericin B in terms of hemolytic toxicity, renal cell toxicity and acute toxicity, and still has good antifungal activity, and has good application prospects in inhibiting fungal growth or treating fungal infectious diseases.
[0029] 2. The present invention also discloses a method for preparing a low-toxic amphotericin B derivative based on 16-carboxyl modification. The preparation method is simple and can synthesize the amphotericin B derivative in only one step of chemical reaction. The reaction conditions are mild, the post-treatment is simple, and the product yield is high. It has certain innovation in the design and synthesis of amphotericin B derivatives.
[0030] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:
[0032] Figure 1 is the structural formula of the low-toxic amphotericin B derivative based on the modification of the 16-carboxyl group of the present invention;
[0033] Figure 2 is the NMR image of the amphotericin B derivative I-1 prepared in Example 1;
[0034] Figure 3 is the NMR image of the amphotericin B derivative I-2 prepared in Example 2;
[0035] Figure 4 It is the hemolysis rate test result of in vitro hemolysis test;
[0036] Figure 5 The results of Cryptococcal fungal load experiments in the lungs and brains of infected mice;
[0037] Figure 6 These are the results of the Candida albicans fungal load experiment in the kidneys of infected mice. DETAILED DESCRIPTION
[0038] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments only illustrate the basic concept of the present invention in a schematic manner, and the following embodiments and features in the embodiments can be combined with each other without conflict.
[0039] Example 1
[0040] A low-toxic amphotericin B derivative based on 16-carboxyl modification with a structural formula of I-1 is prepared. The structural formula of I-1 is:
[0041]
[0042] The specific steps include:
[0043] 100 mg of amphotericin B was weighed and dissolved in 9 ml of N,N-dimethylformamide, 33 mg (2.0 eq.) of 2-cyclopentaneethylamine hydrochloride and 33 mg (3.0 eq.) of triethylamine were added and stirred to completely dissolve, and then 113 mg (2.0 eq.) of PyBOP was added after stirring at 30° C. for 30 min under N2 protection, and stirring was continued at 35° C. for 4 h under N2 protection, the reaction solution was poured into 90 ml of methyl tert-butyl ether for precipitation, the product was collected by centrifugation, 10 ml of acetone was added to fully disperse, and then poured into 60 ml of methyl tert-butyl ether for precipitation, the product was collected by centrifugation, the "dispersion-precipitation" operation was repeated 3 times, the product was collected by filtration, and vacuum dried to obtain a yellow powder, which is the amphotericin B derivative with structural formula I-1, with a yield of 84%. 1 H NMR (400MHz, DMSO-d6) spectrum is attached Figure 2 shown.
[0044] Example 2
[0045] A low-toxic amphotericin B derivative based on 16-carboxyl modification with a structural formula of I-2 is prepared. The structural formula of I-2 is:
[0046]
[0047] The specific steps include:
[0048] 100 mg of amphotericin B was weighed and dissolved in 10 ml of N,N-dimethylformamide. 39 mg (2.2 eq.) of 2-cyclohexylethylamine hydrochloride and 33 mg (3.0 eq.) of triethylamine were added and stirred to dissolve completely. After stirring at 30° C. for 30 min under N2 protection, 124 mg (2.2 eq.) of PyBOP was added. Stirring was continued at 35° C. for 5 h under N2 protection. The reaction solution was poured into 95 ml of methyl tert-butyl ether for precipitation. The product was collected by centrifugation. 12 ml of acetone was added to fully disperse the product. The product was poured into 70 ml of methyl tert-butyl ether for precipitation. The product was collected by centrifugation. The "dispersion-precipitation" operation was repeated 3 times. The product was collected by filtration and dried in vacuo to obtain a yellow powder, which is the amphotericin B derivative with structural formula I-2. The yield was 86%. Its 1H NMR (400 MHz, DMSO-d6) spectrum is shown in the attached figure. Figure 3 shown.
[0049] Example 3
[0050] The performance of the low-toxic amphotericin B derivatives based on 16-carboxyl modification prepared in Examples 1 and 2 was tested:
[0051] 1. In vitro hemolysis test
[0052] Amphotericin B was dissolved in DMSO to obtain a concentrated solution with a concentration of 5 mM, and diluted with a 5% glucose aqueous solution to obtain a drug solution with a concentration of 10 to 400 μM. Amphotericin B derivatives with structural formulas I-1 and I-2 were dissolved in DMSO to obtain a concentrated solution with a concentration of 50 mM, and diluted with a 5% glucose solution to obtain drug solutions of different concentrations. 500 μl of 2% red blood cell suspension, 450 μl of 0.9% sodium chloride solution and 50 μl of drug solution were drawn, so that the final concentration of amphotericin B in the red blood cell suspension was 0.5 to 20 μM, and the final concentration of amphotericin B derivatives in the red blood cell suspension was 1 to 200 μM. Two groups of controls were set up at the same time, the positive control was 500 μl of 2% red blood cell suspension and 500 μl of purified water, and the negative control was 500 μl of 2% red blood cell suspension and 500 μl of physiological saline solution. After mixing the above solutions, place them in a 37±0.5℃ thermostat for 1 and 3 hours respectively, then centrifuge at 2000rpm for 10 minutes, observe the hemolysis phenomenon, and simultaneously take the supernatant to measure the absorbance value (Abs) at 540nm. According to the calculated hemolysis rate, data fitting was performed to obtain the EH50 values corresponding to amphotericin B and its derivatives, that is, the concentration of the corresponding drug when the hemolysis rate reaches 50%. Figure 4 It can be seen that the EH50 values of the amphotericin B derivatives with structural formulas I-1 and I-2 are significantly higher than that of AMB, indicating that the hemolytic toxicity of the derivatives is significantly reduced.
[0053] 2. In vitro pharmacodynamic evaluation
[0054] The minimum inhibitory concentration (MIC) of amphotericin B and its derivatives against Cryptococcus (Cryptococcus neoformans H99, Cryptococcus gattii R256) and Candida (Candida parapsilosis ATCC 22019, Candida albicans SC 5314, Candida albicans ATCC 90028, Candida auris and Candida krusei ATCC 6258) was determined by microplate dilution method. The specific method is as follows:
[0055] Take a sterile 96-well plate and add 160 μL of bacterial solution (1.25×10 3 CFU / mL) and 40μl of different concentrations of amphotericin B or its derivative solutions, with a final concentration of 0.0625-8μM. The 96-well plate was placed in a 30±0.5℃ incubator for incubation, taken out for observation after 24 hours, and the absorbance value of each well at a wavelength of 490nm was measured using an ELISA reader. MIC is the minimum drug concentration that completely inhibits the growth of Cryptococcus in a certain well. The MIC determination results of amphotericin B and its derivatives are shown in Table 1.
[0056] Table 1 Evaluation of in vitro antifungal activity of amphotericin B and its derivatives
[0057]
[0058] 3. Evaluation of renal cell toxicity
[0059] The toxicity of the low-toxic amphotericin B derivatives based on the modification of the 16-position carboxyl group prepared in Examples 1 and 2 to the human renal cortical proximal tubule epithelial cells HK-2 was investigated, and the specific method was as follows: HK-2 cells in the logarithmic growth phase were digested and inoculated into a sterile 96-well plate, 160 μl per well, and placed in a cell culture incubator at 37±0.5°C for 24 hours. 40 μl of amphotericin B solutions of different concentrations (concentration in the cell suspension was 1 to 32 μM) or its derivative solutions (concentration in the cell suspension was 2 to 256 μM) were added to each well, and the plates were cultured in a cell culture incubator at 37±0.5°C for 48 hours. Aspirate the culture medium, wash with PBS 2-3 times, then add 200 μl of new culture medium and 20 μl of MTT solution (5 mg / mL), incubate at 37±0.5°C for 4 hours, discard the culture medium, add 150 μl of DMSO, shake to dissolve, place in a microplate reader, measure the OD value at 510 nm, and calculate the cell survival rate. The cell survival rate calculation formula is as follows:
[0060] Cell survival rate (%) = (OD 样品 -OD 空白 ) / (OD 对照 -OD 空白 )×100
[0061] The experimental results showed that amphotericin B had strong renal cell toxicity, killing more than 90% of HK-2 cells at a concentration of 64 μM, while amphotericin B derivatives I-1 or I-2 did not show renal cell toxicity at concentrations as high as 256 μM. This shows that compared with amphotericin B, the renal cell toxicity of amphotericin B derivatives is significantly reduced.
[0062] 4. Acute toxicity test
[0063] Healthy KM mice (weight 18-22g) were randomly divided into groups, with 10 mice in each group. Amphotericin B or its derivatives I-1 or I-2 were prepared into polymer micelle preparations using distearoylphosphatidylethanolamine-polyethylene glycol 2000 as carrier materials, and the preparations were administered by tail vein injection in mice. The symptoms of mice in each group and the deaths within 14 days were observed and recorded. As can be seen from Table 2, when amphotericin B polymer micelle solution was administered at a dose of 5 mg / kg, the mortality rate of mice was 100%. In contrast, when amphotericin B derivatives I-1 or I-2 polymer micelle solution was administered at 60 mg / kg, all mice behaved normally and all survived within 14 days. It can be seen that compared with amphotericin B, the acute toxicity of amphotericin B derivatives is significantly reduced.
[0064] Table 2 Mortality of mice within 14 days at different doses of amphotericin B and its derivatives I-1 or I-2 (%)
[0065]
[0066] 5. In vivo pharmacodynamic evaluation in mice
[0067] The fungal infection model of mice was established by injecting Cryptococcus neoformans into the tail vein. The concentration of 1×10 7 CFU / ml of Cryptococcus neoformans H99 bacterial solution was injected into the mouse tail vein at a dose of 100μl / 20g to establish the model. 24h after infection, amphotericin B derivative I-1 or I-2 polymer micelle solution was injected into the tail vein at a dose of 2mg / kg, once a day for 3 consecutive days. 24h after the third administration, the mice were dissected on a sterile operating table, the lungs and brain tissues were removed, washed with sterilized saline, and the water was dried with filter paper. After adding saline at a mass volume ratio of 1:3 (g / ml) to saline, the homogenate was diluted, 50μl was taken and spread on YPD solid culture medium, placed in a 30℃ incubator for 48h, the fungal growth in each culture plate was observed, and the colony count was performed.
[0068] From the attached Figure 5 It can be seen that compared with the untreated model group, the amphotericin B derivative I-1 or I-2 polymer micelles have obvious antifungal activity and significantly reduce the number of fungi in the lungs and brains of mice. There is no significant difference in the therapeutic effect between the I-2 polymer micelle preparation and the commercially available preparation AmBisome.
[0069] In vivo Candida albicans fungal load experiment in mice: The mouse kidney fungal infection model was established by tail vein injection of Candida albicans ATCC 90028. The entire experimental method was the same as the Cryptococcal fungal load experiment. The results are shown in the attached Figure 6 Compared with the untreated model group, the amphotericin B derivative I-1 or I-2 polymer micelles had significant antifungal activity and significantly reduced the number of fungi in the kidneys of mice, and there was no significant difference in the therapeutic effect with the commercially available preparation AmBisome.
[0070] The above results indicate that the low-toxic amphotericin B derivative based on 16-carboxyl modification prepared by the present invention has good in vivo antibacterial activity.
[0071] In summary, the derivative of the present invention modifies the carboxyl group at the C16 position in the amphotericin B molecule to obtain a novel amphotericin B derivative, which has the characteristics of low nephrotoxicity, low hemolytic effect and high safety compared with amphotericin B. In addition, the present invention also provides a method for preparing the derivative, and the prepared derivative has good in vitro and in vivo antifungal effects and has broad market prospects in the treatment of systemic deep fungal infections.
[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.
Claims
1. A low-toxic amphotericin B derivative based on 16-carboxyl modification, characterized in that: The structural formula of the derivative is shown in the figure below: Among them, R is 2. The low-toxic amphotericin B derivative based on modification of the 16-carboxyl group according to claim 1, characterized in that: The R is a cyclopentyl group or a cyclohexyl group.
3. The low-toxic amphotericin B derivative based on 16-carboxyl modification according to claim 2, characterized in that: The structural formula of the derivative is shown below:
4. The method for preparing a low-toxic amphotericin B derivative based on modification of the 16-carboxyl group according to any one of claims 1 to 3, characterized in that: The reaction formula of the method is:
5. The preparation method according to claim 4, characterized in that: The method comprises the following steps: Dissolve amphotericin B, amino compounds and organic bases in N,N-dimethylformamide, stir at 20-40°C for 5-30 minutes under N2 protection, then add PyBOP, and continue stirring at 20-40°C for 1-6 hours under N2 protection. Pour the reaction solution into methyl tert-butyl ether for precipitation, collect the product by centrifugation, add acetone to fully disperse, pour into methyl tert-butyl ether for precipitation, collect the product by centrifugation, repeat the "dispersion-precipitation" operation 3 times, collect the product by filtration, and vacuum dry to obtain a yellow powdered amphotericin B derivative.
6. The preparation method according to claim 5, characterized in that: The molar ratio of amphotericin B, amino compound, organic base and PyBOP in the step is 1:1.1-3:1.1-3:1.1-3, the amino compound is any one of 2-cyclopentaneethylamine, 2-cyclopentaneethylamine hydrochloride, 2-cyclohexylethylamine or 2-cyclohexylethylamine hydrochloride, the organic base is triethylamine or N,N-diisopropylethylamine, the mass volume ratio of amphotericin B and N,N-dimethylformamide is 10:0.4-1, mg / ml, the volume ratio of N,N-dimethylformamide to methyl tert-butyl ether is 1:8-15, the mass volume ratio of amphotericin B and acetone is 10:0.5-2, mg / ml, and the volume ratio of acetone to methyl tert-butyl ether is 1:3-10.
7. Use of the low-toxic amphotericin B derivative based on modification of the 16-carboxyl group according to any one of claims 1 to 3 in the preparation of a drug for inhibiting fungal growth or treating fungal infectious diseases.
8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the low-toxic amphotericin B derivative based on modification of the 16-carboxyl group according to any one of claims 1 to 3.
9. The composition according to claim 8, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is any one of distearoyl phosphatidylethanolamine-polyethylene glycol, soybean lecithin or egg yolk lecithin.
10. The composition according to claim 9, characterized in that The dosage form of the pharmaceutical composition is injection.