CN111875653A

CN111875653A - Disulfide bond-containing amphotericin B derivative and application thereof

Info

Publication number: CN111875653A
Application number: CN202010798860.XA
Authority: CN
Inventors: 梁振江; 谭回; 俞玉明; 高明
Original assignee: Shenzhen Childrens Hospital
Current assignee: Shenzhen Childrens Hospital
Priority date: 2020-08-11
Filing date: 2020-08-11
Publication date: 2020-11-03

Abstract

The invention discloses a disulfide bond-containing amphotericin B derivative, which widens the range of the existing antibacterial compound and can be continuously optimized as a lead compound. Meanwhile, the compound has good antibacterial activity, and the antibacterial activity of the compound is better than that of amphotericin B; compared with amphotericin B, the amphotericin B has lower blood toxicity and cytotoxicity, and can reduce the toxic and side effects of amphotericin on human bodies while maintaining the antibacterial activity.

Description

Disulfide bond-containing amphotericin B derivative and application thereof

Technical Field

The invention relates to disulfide-bonded amphotericin B derivatives and their use as antifungal agents.

Background

With the rapid development of social economy, the living standard of people is increasingly improved, and meanwhile, the life health of human beings is also threatened more and more. The use of a large amount of medicines can save human life and 'culture' some super germs which are more and more difficult to kill, and the germs have strong drug resistance to the existing medicines; the super pathogen is not specific to a certain pathogen, but generally refers to bacteria having resistance to multiple antibiotics, and its accurate name should be "multi-drug resistant bacteria". Such germs have a strong resistance to antibiotics, and can escape the danger of being killed, so that human life can be greatly threatened. The superbacteria that are currently of particular interest are mainly: methicillin-resistant staphylococcus aureus (MRSA), multidrug-resistant streptococcus pneumoniae (MDRSP), vancomycin enterococcus (VRE), multidrug-resistant mycobacterium tuberculosis (MDR-TB), multidrug-resistant acinetobacter baumannii (MRAB), and newly discovered escherichia coli and klebsiella pneumoniae that carry NDM-1 gene, and the like. Since superbacteria pose a great risk to human health due to the fact that most antibiotics do not work against them, extensive attention has been paid to the development and application of related drugs.

In recent years, the incidence of fungal infection of opportunistic deep organs is increasing and also becoming more and more serious due to the rapid increase of immune-compromised people, the onset of malignant tumors, malignant blood diseases, acquired immune deficiency syndrome, SARS, diabetes, severe burns and the like, the wide use of broad-spectrum antibiotics and immunosuppressants, and the development of new technologies such as catheter, intubation and organ transplantation. The incidence rate of deep fungal infection in the population is about 11% -40%, and the fatality rate is 40%. Deep fungal infections are much less prevalent than superficial fungal infections, but deep fungal infections are more alarming because of their very high mortality rate, with approximately 150 million people dying from deep fungal infections each year. Of all the mortality reports associated with fungi, more than 90% are caused by species belonging to one of four: cryptococcus, candida, aspergillus, and pneumocystis. Furthermore, the epidemiological data of fungal infections are very poor and fungal infections are often misdiagnosed, since we greatly underestimate the risk of deep fungal infections.

The current antibacterial drugs are mainly classified according to their structures: triazole, polyene, echinocandin, nikkomycin, fluorouracil, beta-1, 3-D-glucan synthase inhibitor, mannoprotein synthesis inhibitor, and the like. Despite the introduction of new antifungal drugs, such as the next generation azoles and echinocandins, polyene macrolides remain the most effective broad spectrum antifungal drugs available clinically. The most representative of the medicine is amphotericin B (amphotericin B) (AMB), which is the first choice medicine for treating deep fungal infection caused by various fungi and is the last line of defense against fungal attack currently in human beings; the antibacterial agent has broad-spectrum activity and extremely low drug resistance, and is the 'gold standard' for treating deep fungal infection clinically.

Amphotericin B has a very important role in clinical treatment of fungal infections, but the side effects of amphotericin B are not negligible and the most severe toxic reaction is irreversible nephrotoxicity. In addition, it also has hepatotoxicity and hemolytic toxicity to erythrocytes, and can cause symptoms such as fever, nausea, vomiting, anorexia, etc. These serious side effects severely limit the clinical use of amphotericin B. In order to solve the outstanding problem, researchers all over the world carry out structural modification on amphotericin B in various modes, so that the toxicity of amphotericin B to human cells can be remarkably reduced while the antibacterial activity is maintained. Currently, modification methods can be mainly divided into two types, one is to prepare a novel preparation: the amphotericin B is coated by a single-layer or multi-layer lipid bilayer membrane and is used for increasing the water solubility of the amphotericin B and further increasing the aggregation concentration, thereby reducing the renal toxicity, the hepatotoxicity and the cell hemolysis of the amphotericin B. A variety of amphotericin B liposomes are currently in clinical use and have reduced toxicity in clinical presentation. However, the residual toxicity of the amphotericin B liposome has large influence on human bodies, the amphotericin B liposome has complex manufacturing process, higher cost and rigorous storage conditions, and other auxiliary conditions are required in the using process, so the amphotericin B liposome has higher cost for treatment, and is not suitable for wide clinical application. Another class is chemical modification: a series of amphotericin B derivatives are obtained by modifying each functional group of an amphotericin B molecule, and the derivatives are subjected to relevant tests such as antibacterial activity, renal toxicity, erythrocytolytic toxicity and the like, so that most of the derivatives are relatively kept in antibacterial activity compared with amphotericin B, and the renal cytotoxicity and the erythrocytolytic toxicity are reduced to different degrees.

Jolanta, G, and the like introduce a D-fructose group on the amino position of amphotericin B, so that the water solubility of amphotericin B is greatly increased, and meanwhile, the amphotericin B forms a salt with aspartic acid to improve the water solubility, is not easy to aggregate in water and reduces the toxicity. The in vitro activity was 2 times lower than AMB, but hemolytic toxicity was reduced 125 times. In order to solve the problem of oral administration of amphotericin B, some researchers recently introduced different polysaccharides into the amino group of amphotericin B to greatly improve the water solubility of amphotericin B, and reduce the renal toxicity and hemolytic toxicity to different degrees. However, such modification methods have problems that when the modification is performed using natural polysaccharides such as chitosan, alginic acid, gum arabic, pectin, etc., it is difficult to isolate and purify them due to inconsistency in physical properties such as molecular weight, branching, type of glycosidic bond and solubility, and many of them are contaminated with proteins, and removal of proteins is a key to prevent immune reaction. Later researchers used monosaccharides to synthesize glucan, polygalactose, polymannan and the like, and then the glucan, the polygalactose, the polymannan and the like were covalently coupled with amphotericin B, the polysaccharide and natural polysaccharide have the same effect, and meanwhile, the artificially synthesized polysaccharide has controllable molecular weight, branches and glycosidic bond types, so that the defects of the natural polysaccharide are overcome, and the polysaccharide shows good application prospects. However, the molecular weight of chemically synthesized polysaccharides can be controlled within a certain range, and researchers need to further explore the covalent bonding mode of the polysaccharides with amphotericin B, the release efficiency of the derivatives from amphotericin B, and other problems. Meanwhile, researches show that N atoms capable of being protonated on amphotericin B sugar amine play an important role in antibacterial activity, and the amino modified by polysaccharide can influence the binding capacity of amphotericin B and ergosterol and reduce the antibacterial activity.

The prior art reports that an S44HP derivative is obtained by carrying out structural modification on amphotericin B isomer 28, 29-didehydrostatin A1(S44HP) molecules. S44HP has similar antibacterial activity to amphotericin B and has strong damage to both kidney cells and erythrocytes. The scientific research worker carries out structural modification on S44HP, introduces a molecule of polyhydroxy alcohol at a carboxyl position through amido bond connection, obtains a derivative of S44HP, and carries out related activity tests on the derivative to find that the antibacterial activity of the derivative is relatively maintained compared with amphotericin B and S44 HP. However, tests show that the compound has strong hemolytic toxicity to human erythrocytes and is not suitable for further research and development.

US2017/0042923a1 reports an antifungal molecule conjugate with reduced toxicity, which can not only reduce the toxicity of an antimicrobial agent, but also solve the problem of poor transportation of the drug while maintaining the antibacterial activity.

In summary, although there are many reports on structural modification of amphotericin B, there is still a need to develop new amphotericin B derivatives that can reduce renal toxicity, erythrocytic hemolytic toxicity, etc. while maintaining antibacterial activity, and can solve the problem of poor water solubility of amphotericin B.

Disclosure of Invention

In order to overcome the technical problems of nephrotoxic hepatotoxicity, hemolytic toxicity to erythrocytes and the like of amphotericin B in the prior art, in a first aspect, the invention provides a disulfide bond-containing amphotericin B derivative shown as a formula I:

or a pharmaceutically acceptable salt thereof.

In a second aspect of the invention, there is provided a process for the preparation of a compound of formula I as hereinbefore described, the reaction scheme being as follows:

the method comprises the following reaction steps:

1) adding amphotericin B and Fmoc-Osu into a reaction solvent, taking anhydrous DMF (60mL) and anhydrous methanol as a mixed solvent, stirring and dissolving, then dropwise adding pyridine, reacting at room temperature in a dark place, and after the reaction is finished, carrying out post-treatment to obtain a compound 1;

2) adding 2- (pyridine-2-yl disulfanyl) ethane-1-amine and the compound 1 into DMF under stirring at room temperature, then adding HATU and triethylamine, stirring the obtained reaction mixture at room temperature, diluting with EtOA after the reaction is finished, washing with water and brine, drying an organic layer, removing the solvent under reduced pressure, and carrying out flash column chromatography on the obtained residue to obtain a compound 2;

3) adding the compound 2 into an aqueous solution of NaOH at room temperature under stirring until the substances are dissolved, then adding 1-amino-2-methylpropane-2-thiol, then adding another aqueous solution of NaOH, stirring the obtained reaction mixture at room temperature, diluting the reaction mixture with EtOA after the reaction is finished, washing an organic layer with water and brine, drying, removing the solvent under reduced pressure, and directly using the obtained crude solid in the next step without further purification;

and (2) adding the prepared solid into a reaction bottle, stirring and dissolving the solid by using N, N-dimethylformamide as a solvent, adding piperidine, dropwise adding the reaction solution into a cold ether solution after the reaction is finished, separating out a solid product, and purifying by column chromatography to obtain the target product, namely the compound shown in the formula I.

Preferably, the molar ratio of amphotericin B to Fmoc-Osu in step 1) is: 1 (2-3), preferably 1:2-2.5, most preferably 1: 2.25;

the molar ratio of the compound 1 and the 2- (pyridin-2-yldisulfanyl) ethane-1-amine in step 2) is 1:0.5 to 2, preferably 1:0.5 to 1.5, most preferably 1: 1;

the molar ratio of the compound 2 to the 1-amino-2-methylpropane-2-thiol in the step 3) is as follows: 1 (0.8-1.2), preferably 1: 1.

Another aspect of the present invention provides a pharmaceutical composition, which comprises a compound represented by formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

Another aspect of the invention pertains to the use of a compound of formula I, and pharmaceutically acceptable salts thereof, or a pharmaceutical composition comprising the same, in the manufacture of a medicament for the control or treatment of fungal infections;

preferably, the fungal infection is caused by a pathogenic fungus from a strain of yeast, filamentous fungi or candida. In particular, the fungal infection is caused by a pathogenic fungus from a strain of Candida albicans, Candida tropicalis or Candida krusei.

Defining:

in certain embodiments, the pharmaceutically acceptable form is a pharmaceutically acceptable salt, which is well known in the art. Examples of pharmaceutically acceptable salts are forms which form salts with compounds such as hydrochloric, hydrobromic, phosphoric, sulfuric, perchloric, acetic, oxalic, maleic, tartaric, citric, succinic or malonic, acetic, propionic, glycolic, pyruvic, oxalic, lactic, trifluoroacetic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicylic acid and the like.

"pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coating agents, isotonic and absorption delaying agents and the like. Pharmaceutically acceptable carriers or excipients do not destroy the pharmacological activity of the disclosed compounds and are non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the compound. The use of such media and agents for pharmaceutically active substances is well known in the art.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention provides a disulfide bond structure modified amphotericin B derivative, and the obtained novel disulfide bond-containing amphotericin B ester derivative widens the range of the existing antibacterial compound and can be continuously optimized as a lead compound;

(2) the compound has good antibacterial activity, and the antibacterial activity of the compound is better than that of amphotericin B;

(3) compared with amphotericin B, the compound of the invention has lower blood toxicity and cytotoxicity, and can reduce the toxic and side effects of amphotericin on human bodies while maintaining antibacterial activity.

Detailed Description

The present invention will be described in detail with reference to examples. In the present invention, the following examples are intended to better illustrate the present invention and are not intended to limit the scope of the present invention. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

1) Amphotericin B (1.033g, 1.12mmol) and Fmoc-Osu (0.852g, 2.52mmol) were weighed and charged into a 250mL round-bottomed flask, and after dissolving with super-dry DMF (60mL) and methanol (30mL) as a mixed solvent with stirring, pyridine (99%, 0.85mL) was added dropwise. The reaction was carried out under nitrogen atmosphere at room temperature in the dark for 24 hours. The reaction progress was monitored by thin layer chromatography and after completion of the reaction, most of the solvent was removed by distillation under reduced pressure. It was then added dropwise to a cold solution of diethyl ether (200mL) to precipitate a pale yellow solid, which was collected by centrifugation or suction filtration under reduced pressure. Flash column chromatography gave 1.05g of compound 1, yield: 81.8 percent; m/z: 1145.5[ M-H⁺]。

2) Compound 1(575mg, 0.5mmol) and 2- (pyridin-2-yl-disulfanyl) ethan-1-amine (93mg, 0.5mmol) were dissolved in 10mL of anhydrous THF, HATU (228mg,0.6mmol) was added, 2 drops of triethylamine were added, and the mixture was reacted at room temperature for 18h under anhydrous conditions by drying nitrogen for 0.5h, followed by dilution with EtOAc and washing with water and brine after the reaction was completed. The organic layer was dried (Na2SO4) and concentrated under reduced pressure. The residue obtained is chromatographed on silica gel (CH)₂Cl₂MeOH) gave 383mg of compound 2, yield: 58.3 percent; m/z: 1313.4[ M-H⁺]。

3) Compound 2(131mg, 0.1mmol) was added to 2mL of aqueous NaOH (5%) solution at room temperature with stirring until the material was dissolved, followed by addition of 1-amino-2-methylpropane-2-thiol (10.5mg, 0.1mmol) followed by another 2mL of aqueous NaOH (5%) solution, stirring the resulting reaction mixture at room temperature for 6h, after completion of the reaction diluted with EtOA, the organic layer was washed with water, brine, dried, and the solvent was removed under reduced pressure, and the resulting crude solid was used in the next step without further purification;

taking the preparedThe crude product was charged into a 25mL round-bottom flask and dissolved with stirring in N, N-dimethylformamide (4 mL). Piperidine (0.8mL) was added and the reaction progress was monitored by thin layer chromatography, and after completion of the reaction, the reaction was added dropwise to a cold ether (100mL) solution to precipitate a solid product. By column chromatography (DCM: MeOH: H)₂O ═ 20:10:1) purification gave 46.2mg of the desired product in yield: 42.5 percent; m/z: 1085.4[ M-H⁺]。

¹H-NMR(400MHz,CD₃OD:CDCl₃6.72-5.91(m,14H),5.63-5.21(m,2H),4.96-4.11(m,7H),3.96-3.53(m,5H),3.49-3.24(m,7H),2.82-2.17(m,4H),2.13-1.29(m,18H),1.27(s,3H),1.22(s 3H),1.19(d, J-6.3 Hz,3H),1.13(d, J-6.3 Hz,3H),1.02(d, J-7.1 Hz,3H), 2H less H

Example 2 in vitro Activity assay

The in vitro antifungal activity was determined according to standard methods (National Committee for clinical Laboratory Standards) using serial dilution method in 96-well microplates in the buffer medium RPMI 1640pH 7.0. At a wavelength λ of 531nm (A)₅₃₁) Here, the optical density of the cell suspension was determined using a microplate reader. On the basis of the obtained results, a graph was prepared showing the relationship between the a531 value and the concentration of the test compound. From these graphs, IC50 values were read, which are interpolated concentrations of test compounds at which values a₅₃₁The value is exactly A of the control sample₅₃₁50% of the value. In addition, the MIC value, which is the lowest concentration of test compound, at which value A₅₃₁The value is measured A for the control sample₅₃₁At most 20% of the value.

The hematological toxicity assay was carried out by the serial dilution method according to the known method (Slisz, M., et al., E., J Antibot, 57: 669-Buck 678 (2004)). Human red blood cells were suspended in saline solution to obtain 2X 10⁷Suspension cell density in ml. Appropriate amounts of compound dilutions were added to the cell suspension in the tubes and incubated at 37 ℃ for 30 minutes, followed by centrifugation (4 ℃). After centrifugation of the red blood cell suspension, the concentration of the red blood cell suspension was measured at a wavelength λ 540nm (A)₅₄₀) The absorbance of (b) was measured, and the hemoglobin concentration in the supernatant was measured. At 0.1% Tritone X-100 (control sample)) In the presence, the maximum level of hemolysis is obtained after incubation of the cell suspension. Based on the results obtained, preparation A₅₄₀A plot of the relationship between the values and the concentration of the test compound. From these graphs, interpolated concentrations EH of the compounds are read₅₀Value of A₅₄₀The value is exactly the measurement A of the control sample₅₄₀50% of the value. The maximum concentration of the tested derivative cannot exceed 100. mu.g/ml in order to maintain sufficient solubility under the experimental conditions. At the maximum concentration of the compound, which exhibits particularly low hematological toxicity, it is not possible to determine EH₅₀Value, in this case, hematologic toxicity is assigned EH₅₀> 100. mu.g/ml. The corresponding test results are shown in the following table:

the above table shows that, compared with amphotericin B, the compound of the present invention has improved antibacterial effect, significantly reduced blood toxicity, and good development and application prospects.

Example 3 cytotoxicity test on mammalian cells

Cell lines used for detection: CCRF-CEM-human acute lymphocytic leukemia; HepG 2-epithelial cells of human malignant liver cancer, LLC-PK 1-pig kidney; all cell lines were from commercial purchase.

CCRF-CEM cells were cultured in RPMI 1640+ 10% Fetal Bovine Serum (FBS) medium, LLC-PK1 cells were cultured in medium 199+ 3% FBS medium, and HepG2 cells were cultured in MEM + 10% FBS medium. All media contained 100. mu.g/ml penicillin G and streptomycin. Mixing 1.2X 10⁴The cells/well amount of cells were seeded in 24-well microplates containing the appropriate media and left to stand overnight. Next, test compounds were added in a volume of 10 μ Ι (serial 2x dilution) as a solution in dimethyl sulfoxide (DMSO). Add 10. mu.l DMSO to the control wells. The plate with the cell suspension was incubated at 37 ℃ for 120 hours under an atmosphere of 95%/5% CO 2. After incubation, 200. mu.l of 3- (4, 5-dimethyltriazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) was added to all wellsIn PBS (4mg/m1), and the plate was incubated at 37 ℃ for an additional 4 h. Next, 1ml of DMSO was added to dissolve formazan crystals and the absorption of the solution was measured at a wavelength λ 540nm (a540) using a microplate reader (Victor3, Perkin-Wallac). Based on the obtained results, a graph was prepared showing the relationship between the a540 value and the concentration of the test compound. From these graphs, the IC50 values were read, i.e., the concentration at which the a540 value of the test compound was present was half the a540 value measured in the control sample. The results obtained are shown in the following table:

as can be seen from the above table, the compounds of the present invention have lower toxicity to animal cells and lower side effects relative to amphotericin B.

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt thereof, having the structure:

2. a process for the preparation of a compound of formula I according to claim 1, which reaction scheme is as follows:

3. the method of claim 2, comprising the steps of:

4. The production method according to claim 2 or3, characterized in that:

the molar ratio of the amphotericin B to the Fmoc-Osu in the step 1) is as follows: 1 (2-3), preferably 1:2-2.5, most preferably 1: 2.25;

5. A pharmaceutical composition comprising a compound of formula I as described in any one of claims 1-3, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

6. Use of a compound of claim 1 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of claim 5 in the manufacture of a medicament for the control or treatment of a fungal infection.

7. The use according to claim 6, wherein the fungal infection is caused by a pathogenic fungus from a strain of yeast, filamentous fungi or Candida.

8. The use according to claim 6, wherein the fungal infection is caused by a pathogenic fungus from a Candida albicans strain.