CN113121627A

CN113121627A - Amphotericin B methyl ester peptide derivative and preparation method thereof

Info

Publication number: CN113121627A
Application number: CN202011598943.0A
Authority: CN
Inventors: 冯军; 东圆珍; 张喜全; 张金华; 赵文杰; 徐宏江
Original assignee: Shanghai Duomirui Biotechnology Co ltd; Shanghai Institute of Pharmaceutical Industry; Chia Tai Tianqing Pharmaceutical Group Co Ltd
Current assignee: Shanghai Duomirui Biotechnology Co ltd; Shanghai Institute of Pharmaceutical Industry; Chia Tai Tianqing Pharmaceutical Group Co Ltd
Priority date: 2019-12-31
Filing date: 2020-12-29
Publication date: 2021-07-16

Abstract

The invention belongs to the field of medicines, and particularly relates to an amphotericin B methyl ester peptide derivative and a preparation method thereof. The amphotericin B peptide derivative has broad-spectrum and high-efficiency bactericidal effect on drug-resistant bacteria and fungi.

Description

Amphotericin B methyl ester peptide derivative and preparation method thereof

Technical Field

The invention belongs to the field of medicines, relates to modification of amphotericin B methyl esterified peptide derivatives, and a preparation method and application thereof, and particularly relates to a series of amphotericin B methyl esterified peptide derivatives with higher solubility, low toxicity and better antibacterial activity, and synthesis, preparation and application thereof.

Background

Amphotericin B (AMB) is a broad-spectrum antifungal polyene drug suitable for the treatment of the following fungal infections: candidiasis (Candidiasis), Cryptococcosis (Cryptococcosis), Blastomycosis (Blastomycosis), Coccidioidomycosis (Coccidioidomycosis), mucomycosis (mucomycosis) caused by Mucor (Mucor), Sporotrichosis (sporotrichiosis) caused by Sporotrichosis (Sporothrix), aspergillosis (aspergillosis) caused by most aspergillosis (Aspergillus), and the like.

This compound has attracted attention since the isolation of amphotericin B from Streptomyces metabolites in 1955. On the one hand, amphotericin B is the gold standard for clinical treatment of deep fungal infections and systemic infections and is the only effective therapeutic for some fatal systemic fungal infections; on the other hand, at therapeutic dose, amphotericin B has relatively serious toxic and side effects, such as hemolytic toxicity, renal toxicity, nervous system toxicity, etc., and amphotericin B has very poor water solubility, and is absorbed from gastrointestinal tract less and unstably after oral administration, so that clinical application of amphotericin B is greatly limited.

Although researches show that the liposome used as a drug carrier can obviously reduce the toxic and side effects of amphotericin B, the amphotericin B liposome is a novel drug with a targeted drug delivery function, which is prepared by wrapping drug molecules by utilizing vesicles formed by phospholipid bilayer membranes, and has better tolerance compared with a common preparation. However, the amphotericin B liposome preparation has the following disadvantages: firstly, the antibacterial activity of the liposome preparation is inferior to that of amphotericin B, the treatment dosage needs to be increased, secondly, the liposome preparation has higher cost and higher price, thirdly, the liposome is unstable, and fourthly, the liposome preparation does not fundamentally eliminate the toxic and side effects of the amphotericin B, such as renal toxicity and the like.

In view of the characteristics of amphotericin B and the history of chemical structure modification of amphotericin B, the solid phase synthesis polypeptide technology mastered by the inventors was combined: taking amphotericin B as a lead compound, adopting an experimental method of combining a solid phase and a liquid phase to carry out a peptide grafting reaction on amphotericin B to synthesize an amphotericin B peptide derivative, further finishing the methyl esterification modification of the amphotericin B peptide derivative to obtain a series of amphotericin B methyl esterified peptide derivatives, namely the invention is particularly referred to as H- (AEEAc)_n-OH，H-(Gly)_nthe-OH or hydrophilic (n is an integer and ranges from 2 to 10) amphotericin B methyl esterified peptide derivatives formed by AEEAc and Gly strive to improve the water solubility and reduce the toxic and side effects of hemolytic toxicity, nephrotoxicity and the like while retaining the antibacterial activity.

Disclosure of Invention

On the one hand, the invention relates to an amphotericin B methyl esterified peptide derivative, a synthesis preparation method and an application thereof, in particular to the amphotericin B methyl esterified peptide derivative which improves the solubility of amphotericin B, reduces the toxicity of amphotericin B and keeps the antibacterial activity of amphotericin B, and the synthesis preparation method and the application thereof. The amphotericin B methyl esterified peptide derivative is characterized in that: with R₂-AEEAc-OH and R₂Synthesizing R by taking-Gly-OH as a raw material in a solid-phase synthesis mode₂-(AEEAc)_n-OH，R₂-(Gly)_nAnd (3) coupling a series of compounds formed by combining-OH or AEEAc and Gly with amphotericin B through an amido bond, further finishing methyl esterification modification of the amphotericin B peptide derivative, finally removing an amino protecting group, and purifying to obtain the target compound.

In one aspect, the present invention relates to compounds of the general formula [ I ]:

wherein R is₁Is a hydrophilic polymer moiety; r₂Amino protecting groups such as Fmoc and Boc or H; r₃Is H or C_1-4Of a hydrocarbon group or a phenyl group, preferably, R₃Methyl and ethyl, most preferably, methyl; r₄Is OH or H; alternatively, the hydrophilic polymer may be H- (AEEAc) -containing_n-OH monomer H- (AEEAc)_n-an OH polymer moiety, where n is an integer from 2 to 20, preferably n is an integer from 2 to 15, most preferably n is 5; alternatively, the hydrophilic polymer may be H- (Gly)_n-H- (Gly) of OH monomer_n-OH multimers, where n is an integer from 2 to 20, preferably n is an integer from 2 to 15, most preferably n is 5; the hydrophilic polymer may also be a polypeptide moiety comprising Gly and AEEAc through amide bond, wherein the length of the polypeptide moiety is 2-20 peptides, preferably 2-15 peptides, and most preferably 5 peptides.

In some embodiments, the present invention relates to methods for the synthetic preparation of amphotericin B methyl esterified peptide derivatives.

In some embodiments, the invention relates to the derivatives having improved solubility and even further reduced toxicity while retaining antifungal activity.

In some embodiments, the amphotericin B derivatives of the present invention are linked via amide and carboxylic ester linkages.

In some embodiments, the invention relates to water-soluble polypeptide compounds, including but not limited to H- (AEEAc)_n-OH，H-(Gly)_n-OH or a polypeptide consisting of AEEAc and Gly through an amide bond.

In some embodiments, the amphotericin B derivatives of the present invention comprise: (1) a water-soluble polypeptide moiety linked to the sugar amine structure of amphotericin B through a stable amide bond, and (2) a methyl moiety linked to the carboxyl group of amphotericin B through a carboxyl ester bond. The compound can be referred to as amphotericin B methyl esterified peptide derivative (TY-AME), and the water-soluble polypeptide refers to H- (AEEAc)_n-OH，H-(Gly)_n-OH or a polypeptide consisting of AEEAc in combination with Gly, and the water-soluble polypeptide moiety is a moiety formed after removal of-OH and-H from the end of the corresponding polypeptide.

TY-AME is a derivative of amphotericin B which is obtained by esterifying a derivative of the amphotericin B peptide with a methyl group. Wherein T is a polymer comprising AEEAc and its derivatives, Gly and its derivatives, or AEEAc and Gly and its derivatives.

In some preferred embodiments, R₁Selected from- (AEEAc)_n-、-(Gly)_n-、-(AEEAc)_n-Gly-、-(AEEAc-Gly)_n-AEEAc-、-AEEAc-Gly-、-(AEEAc-Gly)_n-、-(Gly-AEEAc)_n-Gly-、-(AEEAc)_n-Gly-(AEEAc)_m-、-(AEEAc)_n-(Gly)_m-and- (Gly)_n-AEEAc-(Gly)_m-, wherein n and m are each independently an integer of 2 to 10.

In some preferred embodiments, R₁Selected from- (AEEAc)_n-、-(AEEAc)_n-Gly-、-(AEEAc-Gly)_n-AEEAc-、-(AEEAc-Gly)_n-、-(Gly-AEEAc)_n-Gly-and- (AEEAc)_n-Gly-(AEEAc)_m-, wherein n and m are each independently an integer of 2 to 10.

In some preferred embodiments, R₁Selected from- (AEEAc)_n-or- (AEEAc-Gly)_n-AEEAc-, wherein n is an integer from 2 to 10. Preferably, n is an integer from 2 to 5, preferably n is 2 or 5.

In some preferred embodiments, R₂Is H.

In one aspect, the TY-AME of the invention has the structure [ II ]:

R₂amino protecting groups such as Fmoc and Boc or H; alternatively, the hydrophilic polypeptide can be T (T is AEEAc, Gly, or a combination of AEEAc and Gly) having a length in the range of 2-10 peptides, preferably 3-7 peptides, and most preferably 5 peptides.

In some embodiments, the TY-AME may comprise hydrophilic polypeptides of varying lengths, and may comprise from 2 to 20 monomers, preferably from 2 to 10 monomers, and most preferably 5 monomers, and in the present invention, TY-AME may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 monomers. The monomer may be AEEAc, Gly or Gly and AEEAc.

In some embodiments, the TY-AME is a TY-AME in which the hydrophilic polypeptide is T, T is of varying length and may comprise 2 to 10 monomers, preferably 3 to 7 monomers, and most preferably 5 monomers, and in the present invention, TY-AME may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 monomers. The monomer may be AEEAc, Gly or Gly and AEEAc.

In another aspect, the invention relates to the formula [ II]Or a derivative thereof, wherein: r₂-T-is selected from:

H-(AEEAc)₅-

H-AEEAc-Gly-AEEAc-Gly-AEEAc-，

H-AEEAc-Gly-，

H-Gly-Gly-，

H-AEEAc-AEEAc-Gly-，

H-(Gly)5-，

H-AEEAc-AEEAc-Gly-Gly-Gly-，

H-Gly-Gly-AEEAc-Gly-Gly-，

H-Gly-AEEAc-Gly-AEEAc-Gly-，

H-AEEAc-AEEAc-Gly-AEEAc-AEEAc-，

H-AEEAc-Gly-AEEAc-Gly-AEEAc-Gly-，

H-Gly-AEEAc-Gly-AEEAc-Gly-AEEAc-，

H-Gly-AEEAc-Gly-AEEAc-Gly-AEEAc-Gly-，

H-(AEEAc)₂-，

H-(AEEAc)₁₀-，

or H- (AEEAc)₉-Gly-。

In some embodiments, the compound of the invention is a compound of the formula or a derivative thereof,

in some embodiments, the compound of the invention is a compound of formula [ III ] or a derivative thereof,

wherein: H-T-is selected from: h- (AEEAc)₂-、H-(AEEAc)₉-Gly-or H- (AEEAc)₁₀-。

In some embodiments, the present invention relates to a method of making the above compound or a pharmaceutically acceptable salt thereof, preferably, the method comprises: (1) performing solid-phase synthesis on resin to obtain polypeptide, cracking the obtained polypeptide product with weak acid, filtering, performing rotary evaporation, adding a first organic solvent for dissolution, performing rotary evaporation, precipitating with a second organic solvent, and drying to obtain polypeptide containing an amino protecting group; (2) activating the polypeptide containing the amino protecting group, and then reacting the polypeptide with amphotericin B in an anhydrous solvent in the presence of a catalytic amount of alkali; (3) carrying out methyl esterification reaction on carboxyl of amphotericin B; and optionally, (4) removing the amino protecting group from the polypeptide moiety comprising the amino protecting group.

The weak acid described in step (1) includes, but is not limited to, trifluoroethanol, and the weak acid may be formulated with dichloromethane at a ratio of 1:4(V/V) as a weak acid solution.

The first organic solvent in step (1) may be selected from DCM and THF.

The second organic solvent in step (1) may be selected from ethyl ether, isopropyl ether, and methyl tert-butyl ether.

The anhydrous solvent in the step (2) is selected from DMF and DMSO.

The base in the step (2) includes but is not limited to N, N-Diisopropylethylamine (DIEA).

The methyl esterification reagent in the step (3) includes but is not limited to monoiodomethane (CH)₃I) Dimethyl sulfate, diazomethane or dimethyl carbonate, preferably monoiodomethane (CH)₃I)。

In step (4), the amino protecting group is removed using a removing agent selected from the group consisting of: piperidine (PIP) solution, preferably 10% to 40% by weight PIP in DMF, more preferably 20% to 25% by weight PIP in DMF.

In some embodiments, the present invention relates to pharmaceutical compositions comprising the above compounds or pharmaceutically acceptable salts thereof.

In some embodiments, the present invention relates to a method of preventing and/or treating a fungal infection in a subject in need thereof, wherein a therapeutically effective amount of the above compound or a pharmaceutically acceptable salt thereof is administered to the subject. In a preferred embodiment, the fungal infection is selected from the group consisting of infections by: such as Cryptococcus (Cryptococcus), Blastomyces dermatitidis (Blastomyces dermatitidis), Candida albicans (Candida albicans), Candida krusei (Candida krusei), Candida parapsilosis (Candida parapsilosis), Coccidioides immitis (Coccidioides immitis), Mucor (Mucor), Sporotrichomyces schenckii (Sporotrichschinkii), and Aspergillus fumigatus (Aspergillus fumigatus). For example, the subject has Cryptococcosis (Cryptococcosis), Blastomycosis (Blastomycosis), Candidiasis (Candidiasis), Coccidioidomycosis (Coccidioidomycosis), mucomycosis (mucormycosis) caused by mucorales, Sporotrichosis (sporotrichiosis) caused by Sporotrichosis (Sporothrix), aspergillosis (aspergillosis) caused by most aspergillosis (Aspergillus).

In some embodiments, the present invention relates to the use of the above-described compounds or pharmaceutically acceptable salts thereof for the manufacture of a medicament, such as an antifungal medicament. Preferably, the fungus is selected from, for example, cryptococcus, blastomyces dermatitidis, candida albicans, candida krusei, candida parapsilosis, coccidioidomycosis, mucor, sporothrix schenckii and aspergillus fumigatus. Preferably, the above-mentioned compounds of the invention or pharmaceutically acceptable salts thereof have comparable antifungal activity, and have improved solubility, and may even further have reduced toxicity, compared to amphotericin B.

In some embodiments, the present invention relates to the above compounds or pharmaceutically acceptable salts thereof for use in the prevention and/or treatment of fungal infections. Preferably, the fungal infection is selected from the group consisting of infections by: such as Cryptococcus, Blastomyces dermatitidis, Candida albicans, Candida krusei, Candida parapsilosis, Coccidioides immitis, Mucor, Sporothrix schenckii or Aspergillus fumigatus. Further preferably, the above compound or a pharmaceutically acceptable salt thereof is used for treating a disease caused by fungal infection selected from the group consisting of: cryptococcosis, blastomycosis, candidiasis, coccidioidomycosis, mucormycosis caused by mucorales, sporotrichosis caused by sporotrichosis, aspergillosis caused by most aspergillosis. Preferably, the above-mentioned compounds of the invention or pharmaceutically acceptable salts thereof have comparable antifungal activity, and have improved solubility, and may even further have reduced toxicity, compared to amphotericin B.

At present, the main pathogenic bacteria of deep fungal infection are still Candida albicans, and the drug resistance phenomenon is the most prominent, so that the prevention and treatment of the deep fungal infection of the Candida albicans are also the key points in the research field of antifungal infection, and the compound has a good bacteriostatic effect on the Candida albicans.

The compounds referred to above may be in the form of pharmaceutically acceptable salts.

The compound can be used as an effective pharmaceutical ingredient of an oral preparation; can also be used as an effective pharmaceutical ingredient for injection, such as intravenous injection, subcutaneous injection, intramuscular injection and the like; can also be used as effective medicinal component for topical application.

The compound can be prepared into a pharmaceutically effective dosage unit by the existing pharmaceutical technology, and the form of the pharmaceutically effective dosage unit can be oral administration, tablets, capsules, liquid and other dosage forms.

The medicinal component can be made into water-containing preparation with water content of not less than 50%.

Oral formulations may be in the form of liquids, suspensions, powders, tablets, capsules and the like; tablets containing various excipients (e.g., calcium carbonate, calcium phosphate, etc.) may also be formulated as disintegrating formulations.

The drug components can be released in a controlled manner, including sustained or rapid release, and the controlled release dosage of the relevant drug component can be achieved by known pharmaceutical techniques.

The pharmaceutical composition may comprise 0.1-95% TY-AME (TY-AME by weight), preferably 1-70%.

FIG. 3 is a schematic diagram of the chemical synthesis of TY-AME, which includes the first, second, third and fourth steps below.

The first step involves solid phase synthesis of the polypeptide R₂T and activation of the series of polypeptides, and the invention provides a synthetic method of the series of polypeptides. In some aspects, the method comprises: synthesizing the series of polypeptides. In some embodiments, the series of polypeptides can be prepared using solid phase synthesis techniques, including:

(1) solid phase synthesis of the polypeptide on a resin;

(2) and (2) cracking the product obtained in the step (1) with weak acid, filtering, performing rotary evaporation, adding a proper amount of organic solvent to dissolve the polypeptide, performing rotary evaporation again, repeating the steps for 2-3 times, and finally precipitating with the organic solvent and drying to obtain the target polypeptide.

Optionally, the step (1) comprises the steps of:

(a) soaking resin, feeding (the first amino acid and high steric hindrance base), determining a resin substitution value, removing an amino protecting group, washing, monitoring, coupling amino acid, monitoring, washing, removing the amino protecting group, sequentially coupling the rest amino acids until the last amino acid is obtained, and then washing without removing the amino protecting group; the amino-protecting group is a chemical group introduced for protecting an amino group participating in a condensation reaction. The amino protecting group includes, but is not limited to, t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Z) or 9-fluorenyl-methylcarbonyl (Fmoc), preferably 9-fluorenyl-methylcarbonyl (Fmoc), and the protecting group includes, but is not limited to, those which can be appropriately selected depending on the circumstances.

The feeding in the step (a) is to weigh an appropriate amount of R₂-AEEAc-OH or R₂-Gly-OH and an appropriate amount of a sterically hindered base is taken up, dissolved in DCM, for example 10ml of LCM, and then introduced into the reactor.

The high steric base reagent includes, but is not limited to, N-Diisopropylethylamine (DIEA).

The resin substitution value of step (a) is determined by taking an appropriate amount of resin coupled with the first amino acid in each of two EP tubes, drying and weighing (W), adding an amino protecting group removing agent (e.g., 1mL of 20% PIP/DMF) to perform a reaction (e.g., 30min) to remove the amino protecting group, taking the deprotecting solution (e.g., 100. mu.L), adding an organic solvent (e.g., 10mL of DMF; dilution factor S, e.g., 101-fold), and determining the absorbance value A at 301nm (also referred to herein as "A301"), wherein the substitution value (SD) is expressed by the formula: SD is a301 × S/(7800 × W), and W is in g.

The liquid phase environment of step (a) is selected from the group consisting of: dimethylformamide (DMF), Dichloromethane (DCM), N-methylpyrrolidone (NMP), preferably DCM and DMF.

In the step (a), an amino protecting group removing agent is required to be added, wherein the amino protecting group removing agent is piperidine (PIP) solution with the concentration of 10-40% (PIP/DMF), and the removing time is 20-50 min; preferably, the concentration is 20-25% (PIP/DMF), and the removal time is 25-35 min.

The coupling of the amino acid in step (a) requires the addition of a coupling reagent consisting of: a carbodiimide type reagent or a benzotriazole onium salt type reagent and 1-hydroxybenzotriazole (HOBt).

The carbodiimide type reagent includes, but is not limited to, Dicyclohexylcarbodiimide (DCC), Diisopropylcarbodiimide (DIC), or N-diaminopropyl-N-Ethylcarbodiimide (EDC).

The benzotriazol onium salt type reagent includes, but is not limited to, 2- (1H-benzotriazol L-1-yl) -1,1,3, 3-tetramethyluronium tetrafluoroborate (TBTU), O-benzotriazole-N, N' -tetramethyluronium Hexafluorophosphate (HBTU), benzotriazole-1-oxytris (dimethylamino) phosphonium hexafluorophosphate (BOP), or benzotriazole-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP).

The coupling reagent is preferably Diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt), or 2- (1H-benzotriazol L-1-yl) -1,1,3, 3-tetramethyluronium tetrafluoroborate (TBTU) and 1-hydroxybenzotriazole (HOBt), and further preferably DIC (diisopropylcarbodiimide) and 1-hydroxybenzotriazole (HOBt).

The "monitoring" in said step (a) is to monitor the condensation reaction of the polypeptide using ninhydrin detection.

The sequentially coupled amino acids in the step (a) refer to the amino acids that are connected one by one from the C-terminus to the N-terminus according to the amino acid sequence of the polypeptide.

The weak acid of step (2) includes, but is not limited to, trifluoroethanol, and the weak acid may be mixed with dichloromethane in a ratio of 1:4(V/V) was prepared as a weak acid solution.

Another part of the first reaction is the activation of a polypeptide containing an amino protecting group, wherein the polypeptide T is a 2-10 peptide, preferably a 3-7 peptide, and most preferably a 5 peptide. Activation of the carboxyl group of the polypeptide includes: an activated ester method, a symmetric anhydride method, an azide method and the like, and a milder activated ester method is preferred; activating reagents used: a carbodiimide type reagent or a benzotriazol onium salt type reagent and 1-hydroxybenzotriazole (HOBt) or succinimide (HOSU). The carbodiimide type reagent includes, but is not limited to, Dicyclohexylcarbodiimide (DCC), Diisopropylcarbodiimide (DIC), or N-diaminopropyl-N-Ethylcarbodiimide (EDC). The benzotriazolium salt type reagents include, but are not limited to, 2- (1H-benzotriazol L-1-yl) -1,1,3, 3-tetramethyluronium tetrafluoroborate (TBTU), O-benzotriazole-N, N' -tetramethyluronium Hexafluorophosphate (HBTU), benzotriazole-1-oxytris (dimethylamino) phosphonium hexafluorophosphate (BOP) or benzotriazole-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP), preferably Diisopropylcarbodiimide (DIC) and Hydroxysuccinimide (HOSU); the solvent in the liquid phase system used for activation is preferably an organic solvent, including DMF, DMSO, DCM, THF, etc., preferably THF tetrahydrofuran. The amino protecting group includes, but is not limited to, t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Z) or 9-fluorenyl-methylcarbonyl (Fmoc), preferably 9-fluorenyl-methylcarbonyl (Fmoc); activation of the ester at 30 ℃ for 1-2 hours, a catalytic amount of a sterically hindered base is added to facilitate completion of the activation, including but not limited to N, N-Diisopropylethylamine (DIEA); and removing the organic solvent by rotary evaporation to obtain the compound 2.

In the second step, compound 2 is reacted with amphotericin B in an anhydrous solvent such as DMF, DMSO at room temperature for 1-2 hours. The reaction requires protection from light, preferably, a catalytic amount of a high steric hindrance base is added to facilitate completion of the reaction, and the high steric hindrance base reagent includes, but is not limited to, N-Diisopropylethylamine (DIEA); compound 4 can be obtained, and compound 4 can be prepared and purified by semi-preparative RP-HPLC.

In the third step, the carboxyl of amphotericin B is subjected to methyl esterification reaction, and the methyl esterification reagent includes but is not limited to monoiodomethane (CH)₃I) Dimethyl sulfate, diazomethane or dimethyl carbonate, preferably monoiodomethane (CH)₃I) Compound 5 can be obtained.

A fourth step of subjecting the polypeptide containing an amino protecting group including, but not limited to, t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Z) or 9-fluorenyl-methylcarbonyl (Fmoc), preferably 9-fluorenyl-methylcarbonyl (Fmoc) to a deamination protectant; removing amino protecting group by adding a removing agent of the amino protecting group, wherein the removing agent of the amino protecting group is piperidine (PIP) solution with the concentration of 10-40% (PIP/DMF), and the removing time is 20-50 min; preferably, the concentration is 20-25% (PIP/DMF), and the removal time is 25-35 min. Compound 6 (i.e., TY-AME) is obtained.

Alternatively, 5-deoxyamphotericin B, i.e., R of amphotericin B, can be used as a starting material in place of amphotericin B₄The position has-OH changed into-H; the related 5-deoxyamphotericin B can be obtained synthetically.

Particularly beneficial is that in order to meet the quality requirement of medical application, the preparation method of the amphotericin B methyl esterified peptide derivative provided by the invention can further comprise a purification step. The purification method employed includes, but is not limited to, reverse phase chromatography or ion exchange chromatography, preferably reverse phase chromatography.

The in vitro antibacterial activity of the amphotericin B methyl esterified peptide derivative can be identified by measuring the Minimum Inhibitory Concentration (MIC) of the amphotericin B methyl esterified peptide derivative. The american clinical laboratory standards committee (NCCLS) recommended that Minimal Inhibitory Concentrations (MICs) of each antibacterial agent be determined using a broth dilution method using a modified RPMI-1640 medium. Amphotericin B was used as a positive control. In vitro activity determination shows that the amphotericin B methyl esterified peptide derivative provided by the invention has better anti-candida albicans activity.

The in vivo antifungal drug efficacy evaluation shows that the compound, particularly DMR086, has antifungal activity obviously superior to AMB, has lower drug toxicity than AMB, can be administrated at high dose, prolongs the survival time of a patient and improves the survival rate. In particular, the compounds of the present invention all function to inhibit candida albicans ATCC 10231. The high-dose compound can remarkably improve the survival time and the survival rate of mice in a specific time, and has the efficacy of treating candida albicans infection.

Definition of

The following terms used in the present application have the following meanings, unless otherwise specified. A particular term should not be considered as ambiguous or unclear without special definition, but rather construed according to ordinary meaning in the art. When a trade name appears herein, it is intended to refer to its corresponding commodity or its active ingredient.

The terms "optionally" or "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, ethyl is "optionally" substituted with halo, meaning that ethyl may be unsubstituted (CH)₂CH₃) Monosubstituted (e.g. CH)₂CH₂F) Polysubstituted (e.g. CHFCH)₂F、CH₂CHF₂Etc.) or completely substituted (CF)₂CF₃). It will be appreciated by those skilled in the art that any substituent or substituents will not be incorporated into any radical containing one or more substituentsSubstitutions or substitution patterns which are not possible and/or cannot be synthesized.

C as used herein_m-nMeaning that the moiety has m-n carbon atoms. For example, "carbon_3-10Cycloalkyl "means that the cycloalkyl group has 3 to 10 carbon atoms. "carbon_0-6Alkylene "means that the alkylene group has 0 to 6 carbon atoms, and when alkylene has 0 carbon atom, the group is a bond.

Numerical ranges herein refer to each integer in the given range. E.g. "C_1-6By "is meant that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.

When any variable (e.g., R) occurs more than one time in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 2R, then there are separate options for each R.

The term "halo" or "halogen" refers to fluorine, chlorine, bromine and iodine.

The term "acyl" refers to a-CO-group.

The term "carboxyl" refers to the-COOH group.

The term "hydroxy" refers to an-OH group.

The term "amino" refers to the group-NH₂A group.

The term "alkyl" refers to a group of formula C_nH_2n+1A hydrocarbon group of (1). The alkyl group may be linear or branched. For example, the term "hydrocarbon_1-6Alkyl "refers to a monovalent straight or branched chain aliphatic group containing 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, 3-dimethylpropyl, hexyl, 2-methylpentyl, and the like). Similarly, the alkyl portion (i.e., alkyl) of alkoxy, alkylamino, dialkylamino, alkylsulfonyl and alkylthio groups have the same definitions as above.

The term "alkoxy" refers to-O-alkyl. The term "alkylthio" refers to-S-alkyl. The term "alkylamino" refers to-NH (alkyl).

The term "cycloalkyl" refers to a carbon ring that is fully saturated and may exist as a single ring, fused ring, or spiro ring. Unless otherwise indicated, the carbocycle is typically a3 to 10 membered ring, preferably a3 to 8 membered ring. Non-limiting examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl (bicyclo [2.2.1] heptyl), bicyclo [2.2.2] octyl, adamantyl, and the like.

The term "aryl" refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated pi-electron system. For example, the aryl group can have 6 to 20 carbon atoms, 6 to 14 carbon atoms, or 6 to 12 carbon atoms. Non-limiting examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, and the like.

The term "heteroaryl" refers to a monocyclic or fused polycyclic ring system containing at least one ring atom selected from N, O, S, the remaining ring atoms being C, and having at least one aromatic ring. Preferred heteroaryls have a single 4-to 8-membered ring, especially a 5-to 8-membered ring, or have multiple fused rings containing 6 to 14, especially 6 to 10 ring atoms. Non-limiting examples of heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, isoindolyl, and the like.

The term "substituted" means that any one or more hydrogen atoms on a particular atom is replaced with a substituent, so long as the valence of the particular atom is normal and the substituted compound is stable. When the substituent is oxo (i.e., ═ O), meaning that two hydrogen atoms are substituted, oxo does not occur on the aryl.

The term "pharmaceutically acceptable" is intended to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As the pharmaceutically acceptable salt, for example, a metal salt, an ammonium salt, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or acidic amino acid, and the like can be mentioned. Non-limiting examples of metal salts include, but are not limited to, salts of alkali metals, such as sodium, potassium, and the like; salts of alkaline earth metals such as calcium, magnesium, barium, and the like; aluminum salts, and the like. Non-limiting examples of salts with organic bases include, but are not limited to, salts with trimethylamine, triethylamine, pyridine, picoline, 2, 6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and the like. Non-limiting examples of salts with inorganic acids include, but are not limited to, salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like. Non-limiting examples of salts with organic acids include, but are not limited to, salts with formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, malic acid, maleic acid, tartaric acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like. Non-limiting examples of salts with basic amino acids include, but are not limited to, salts with arginine, lysine, ornithine, and the like. Non-limiting examples of salts with acidic amino acids include, but are not limited to, salts with aspartic acid, glutamic acid, and the like.

The term "pharmaceutical ingredient" refers to a formulation of one or more compounds of the present application or salts thereof with excipients, diluents, or carriers generally accepted in the art for delivering biologically active compounds to an organism (e.g., a human). The purpose of the pharmaceutical composition is to facilitate administration of the compounds of the present application to an organism.

The term "pharmaceutically acceptable excipient, diluent, or carrier" refers to those excipients, diluents, or carriers that do not significantly irritate the organism and do not impair the biological activity and performance of the active compound. Suitable carriers, diluents and excipients are well known to those skilled in the art and include materials such as carbohydrates, waxes, water soluble and/or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like.

The term "polypeptide" refers to a compound formed by more than 2 amino acids linked by peptide bonds, such as, but not limited to, 2 peptides, 3 peptides, 4 peptides, 5 peptides, 6 peptides, 7 peptides, 8 peptides, 9 peptides, 10 peptides, 15 peptides, 20 peptides.

Throughout this specification, unless otherwise indicated, the terms "comprises, comprising and including" mean that the elements, components and steps may be included in addition to those listed.

The term "AEEAc" refers to 2- (2- (2-aminoethoxy) ethoxy) acetic acid. The term "Gly" refers to glycine.

The present application also includes isotopically-labeled compounds of the present application, which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the present application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine and chlorine, such as respectively²H、³H、¹¹C、¹³C、¹⁴C、¹³N、¹⁵N、¹⁵O、¹⁷O、¹⁸O、³¹P、³²P、³⁵S、¹⁸F、123I、¹²⁵I and³⁶cl, and the like.

Certain isotopically-labelled compounds of the present application (e.g. with³H and¹⁴c-labeled ones) can be used in compound and/or substrate tissue distribution assays. Tritiated (i.e. by tritiation)³H) And carbon-14 (i.e.¹⁴C) Isotopes are particularly preferred for their ease of preparation and detection. In addition, heavier isotopes are used (such as deuterium (i.e., deuterium)²H) Substitution may provide certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements), and thus may be preferred in certain circumstances. Positron emitting isotopes, such as¹⁵O、¹³N、¹¹C and¹⁸f can be used in Positron Emission Tomography (PET) studies to determine substrate occupancy. In general, the following procedures, similar to those disclosed in the schemes and/or examples below, can be followed by co-locationIsotopically labeled compounds of the present application are prepared by substituting an isotopically unlabeled reagent with a biotin labeling reagent.

The application also includes the following scheme:

1. a compound represented by the formula [ I ] or a pharmaceutically acceptable salt thereof:

wherein R is₁Selected from hydrophilic polymer moieties;

R₂selected from amino protecting groups or H;

R₃is selected from H or C_1-4A hydrocarbyl or phenyl group;

R₄is OH or H;

alternatively, the hydrophilic polymer moiety may be H- (Gly)_n-OH or H- (AEEAc)_n-a fraction of OH multimers, wherein n is an integer from 1 to 20, preferably n is an integer from 2 to 15, most preferably n is 5;

alternatively, the hydrophilic polymer portion may be a polypeptide portion consisting of Gly and AEEAc via a peptide bond, the length of the polypeptide portion being 2-20 peptides, preferably 2-15 peptides, most preferably 5 peptides;

alternatively, the amino protecting group is Fmoc or Boc.

2. The compound of item 1 or a pharmaceutically acceptable salt thereof, wherein R₁Is selected from- (Gly)_n-、-(AEEAc)_n-、-(AEEAc)_n-Gly-、-(AEEAc-Gly)_n-AEEAc-、-AEEAc-Gly-、-(AEEAc-Gly)_n-、-(Gly-AEEAc)_n-Gly-、-(AEEAc)_n-Gly-(AEEAc)_m-、-(AEEAc)_n-(Gly)_m-and- (Gly)_n-AEEAc-(Gly)_m-, wherein n and m are each independently an integer of 2 to 10.

3. The compound of

item

1 or 2, or a pharmaceutically acceptable salt thereof, wherein R₁Selected from- (AEEAc)_n-Gly-、-(AEEAc-Gly)_n-AEEAc-、-(AEEAc)_n-、-(AEEAc-Gly)_n-、-(Gly-AEEAc)_n-Gly-and- (AEEAc)_n-Gly-(AEEAc)_m-, wherein n and m are each independently an integer of 2 to 10.

4. The compound according to any one of items 1 to 3 or a pharmaceutically acceptable salt thereof, wherein R₂Is H.

5. The compound of any one of items 1 to 4 or a pharmaceutically acceptable salt thereof, wherein R₃Is methyl.

6. A compound of any one of items 1-5, or a pharmaceutically acceptable salt thereof, having the structure of formula [ II ]:

wherein R is₂Fmoc, Boc or H;

alternatively, T is a polymer moiety comprising AEEAc or a derivative thereof, Gly or a derivative thereof, or a combination polypeptide or derivative thereof consisting of AEEAc and Gly, where T is in the range of 2-10 peptides, preferably 3-7 peptides, and most preferably 5 peptides in length.

7. The compound of item 6 or a pharmaceutically acceptable salt thereof, wherein R₂-T-is selected from:

H-(AEEAc)₅-

H-AEEAc-Gly-AEEAc-Gly-AEEAc-，

H-AEEAc-Gly-，

H-Gly-Gly-，

H-AEEAc-AEEAc-Gly-，

H-(Gly)₅-，

H-AEEAc-AEEAc-Gly-Gly-Gly-，

H-Gly-Gly-AEEAc-Gly-Gly-，

H-Gly-AEEAc-Gly-AEEAc-Gly-，

H-AEEAc-AEEAc-Gly-AEEAc-AEEAc-，

H-AEEAc-Gly-AEEAc-Gly-AEEAc-Gly-，

H-Gly-AEEAc-Gly-AEEAc-Gly-AEEAc-，

H-Gly-AEEAc-Gly-AEEAc-Gly-AEEAc-Gly-，

H-(AEEAc)₂-，

H-(AEEAc)₁₀-，

or H- (AEEAc)₉-Gly-。

8. The compound of any one of claims 1-7, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:

9. a compound according to any one of items 1 to 5, or a pharmaceutically acceptable salt thereof, having the structure of formula [ III ]:

wherein H-T-is selected from: h- (AEEAc)₂-、H-(AEEAc)₉-Gly-or H- (AEEAc)₁₀-。

10. A process for preparing a compound of any one of claims 1-9, or a pharmaceutically acceptable salt thereof, comprising: (1) performing solid-phase synthesis on resin to obtain polypeptide, cracking the obtained polypeptide product with weak acid, filtering, performing rotary evaporation, adding a first organic solvent for dissolution, performing rotary evaporation, precipitating with a second organic solvent, and drying to obtain polypeptide containing an amino protecting group; (2) activating the polypeptide containing the amino protecting group, and then reacting the polypeptide with amphotericin B in an anhydrous solvent in the presence of a catalytic amount of alkali; (3) carrying out methyl esterification reaction on carboxyl of amphotericin B; and optionally, (4) removing the amino protecting group from the polypeptide moiety comprising the amino protecting group.

11. A pharmaceutical composition comprising a compound of any one of claims 1-9, or a pharmaceutically acceptable salt thereof.

12. Use of a compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament, preferably an antifungal medicament.

13. The use of item 12, wherein the compound or pharmaceutically acceptable salt thereof has comparable antifungal activity and improved solubility compared to amphotericin B.

14. The use according to

item

12 or 13, wherein the fungus is selected from the group consisting of, for example, cryptococcus, blastomyces dermatitidis, candida albicans, candida krusei, candida parapsilosis, coccidioidomycosis, mucor, sporothrix schenckii and aspergillus fumigatus.

15. A method of preventing and/or treating a fungal infection in a subject in need thereof, wherein a therapeutically effective amount of a compound of any one of items 1-9, or a pharmaceutically acceptable salt thereof, is administered to the subject.

16. The method of item 15, wherein the fungal infection is selected from the group consisting of: cryptococcus (Cryptococcus), Blastomyces dermatitidis (Blastomyces dermatitidis), Candida albicans (Candida albicans), Candida krusei (Candida krusei), Candida parapsilosis (Candida parapsilosis), Coccidioides immitis (Coccidioides immitis), Mucor (Mucor), Sporotrichomyces (Sporotrichschenckii), and Aspergillus fumigatus (Aspergillus fumigatus).

The compounds of the present application may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations thereof with other chemical synthetic methods, and equivalents thereof well known to those skilled in the art, with preferred embodiments including, but not limited to, the examples of the present application.

The chemical reactions of the embodiments herein are carried out in a suitable solvent that is compatible with the chemical changes of the present application and the reagents and materials required therefor. In order to obtain the compounds of the present application, it is sometimes necessary for a person skilled in the art to modify or select the synthesis steps or reaction schemes based on the existing embodiments.

All patents, patent applications, and other established publications are herein expressly incorporated by reference for the purpose of description and disclosure. These publications are provided solely for their disclosure prior to the filing date of the present application. All statements as to the date of these documents or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates of these documents or the contents of these documents.

The following examples represent only one aspect of the present invention, and are not intended to limit the subject matter of the invention.

Drawings

FIG. 1 shows the chemical structure of amphotericin B.

FIG. 2 shows the overall chemical structure of TY-AME comprising T and methyl groups.

FIG. 3 depicts the general scheme for TY-AME chemical synthesis.

FIG. 4 is a chemical structural diagram of target products DMR005-JZH, DMR078-JZH and DMR 086.

FIG. 5 is a time sterilization curve for DMR005-JZH, DMR078-JZH, and DMR 086.

FIG. 6 is an absorption spectrum of DMR005-JZH, DMR078-JZH, DMR086 and AmB. FIG. 6A shows absorption spectra of DMR005-JZH, DMR078-JZH, DMR086, and AmB (12.7. mu.g/mL) in methanol; FIG. 6B shows absorption spectra of DMR005-JZH, DMR078-JZH, DMR086, and AmB (12.7. mu.g/mL) in PBS buffer; FIG. 6C shows the absorption spectra of DMR005-JZH, DMR078-JZH, DMR086, and AmB (62.4. mu.g/mL) in PBS buffer.

FIG. 7 shows the results of Candida albicans ATCC10231 on the minimal lethal concentration of mice.

FIG. 8 is a graph of the effect of DMR005-JZH, DMR078-JZH, DMR086, and AmB on mouse survival.

Detailed description of the preferred embodiments

The first embodiment is as follows: preparation and purification of DMR005-JZH

Hydrophilic polypeptide structure: Fmoc-AEEAc-AEEAc-AEEAc-AEEAc-AEEAc-OH

DMR005-JZH Structure:

(1) materials and reagents

2-CTC resin, substitution value 0.945 mmol/g.

The amino acids are: Fmoc-AEEAc-OH

Synthesizing a reagent: HATU, DMF, DCM, DIEA, piperidine, iodomethane.

(2) Instrument for measuring the position of a moving object

CS-BIO type polypeptide synthesizer, Waters600 semi-preparative high performance liquid chromatograph, Beckman centrifuge and Buchi rotary evaporator.

(3) Operating procedure (taking 1g of resin as an example)

a. Solid phase chemical synthesis of polypeptides

Weighing 1.00g of 2-CTC resin, placing the 2-CTC resin in a reactor of a polypeptide synthesizer, adding 10mLDCM, soaking for 1h, weighing 2-3 times of Fmoc-AEEAc-OH and absorbing 4-6 times of DIEA, adding the mixture into 10mLDCM, dissolving, putting the mixture into the reactor, reacting for two hours at room temperature (above 25 ℃ or longer than the temperature, or prolonging the reaction time), namely, coupling the first amino acid onto the resin, washing the resin for 6 times by DCM, and determining the substitution value (SD) of the resin at the moment; then adding 10ml of 20% PIP/DMF solution, mixing for 30min to remove an amino protecting group, washing the resin with DMF for 6 times, coupling a second amino acid, weighing three times of Fmoc-AEEAc-OH and HATU and absorbing six times of DIEA, adding 10ml of DMF to dissolve, reacting, wherein the reaction temperature is room temperature, the reaction progress is monitored by ninhydrin reaction, and the reaction is completed when colorless is monitored, and washing the resin with DMF for 6 times. Then, the coupling reaction of the next amino acid can be continued according to the method for coupling the second amino acid, and the cycle is repeated until all the amino acids are coupled.

b. Cracking and precipitation

After the synthesis of the polypeptide is finished, the wet weight is weighed. Adding a cracking reagent according to the ratio of 5mL of the cracking reagent to 1g of resin, stirring and reacting for 1 hour at room temperature, filtering to a 25mL rotary evaporation bottle, evaporating the liquid at 40 ℃, adding 10mL of LDCM into the rotary evaporation bottle, further rotary evaporating the liquid to dryness, repeating the operation for 2-3 times, finally adding 3mL of LDCM, dissolving the cut polypeptide, transferring the solution to a 50mL centrifugal tube, adding 40mL of diethyl ether, placing the centrifugal tube in a refrigerator at-20 ℃ for 20min, centrifuging, drying in vacuum, and weighing the crude peptide.

c. Liquid phase reaction for preparing amphotericin B methyl esterified peptide derivative

Weighing dried crude peptide 0.1mmol, HOSU 0.095mmol, DIC0.095mmol, and adding 5ml of THF (25 deg.C, reacting for 2 hr), and rotary evaporating to remove THF; adding 5mLDMSO to dissolve a reaction product, adding 0.095mmol of amphotericin B and 0.2mmol of DIEA, and reacting at room temperature for 1-2 hours; 1.5mmol of CH are added continuously₃I and 0.2mmol LDIEA for 2 hours at room temperature; and finally, adding 4ml of 20% PIP/DMF solution, and reacting for 10-20 min to obtain a crude product.

The crude product was purified by semi-preparative RP-HPLC as follows.

1 purification of

A chromatographic column: nano Micro C18 preparation column (10 mm. times.250 mm,10 μm)

Flow rate: 5ml/min

Detection wavelength: 409nm

Mobile phase: phase A: 1% HAC/Water

Phase B: 1% HAC/acetonitrile

The gradient elution procedure is shown in table 1.

TABLE 1 gradient elution procedure

The collected product was analyzed by Agilent 1260HPLC under the following conditions

A chromatographic column: YMC-pack ODS-AQ C18 analytical column (4.6 mm. times.250 mm,5 μm)

Flow rate: 1ml/min

Detection wavelength: 215,383 and 405nm

Mobile phase: phase A: 0.1% TFA/water

Phase B: 0.1% TFA/acetonitrile

The gradient elution procedure is shown in table 2.

TABLE 2 gradient elution procedure

Collecting the target component with purity of more than 90%, rotary steaming, and freeze drying. Molecular weight confirmation by ESI-MS, M/Z ═ 1663(M + H)⁺In line with the theoretical molecular weight.

Example two: preparation and purification of DMR078-JZH

Hydrophilic polypeptide structure: Fmoc-AEEAc-Gly-AEEAc-Gly-AEEAc-OH

DMR078-JZH structure:

(1) materials and reagents

2-CTC resin, substitution value 0.945 mmol/g.

The amino acids are: Fmoc-AEEAc-OH and Fmoc-Gly-OH

Synthesizing a reagent: HATU, DMF, DCM, DIE, piperidine, iodomethane.

(2) Instrument for measuring the position of a moving object

(3) Operating procedure (taking 1g of resin as an example)

a. Solid phase chemical synthesis of polypeptides

Weighing 1.00g of 2-CTC resin, placing the 2-CTC resin in a reactor of a polypeptide synthesizer, adding 10mLDCM, soaking for 1h, weighing 2-3 times of Fmoc-AEEAc-OH and absorbing 4-6 times of DIEA, adding the mixture into 10mLDCM, dissolving, putting the mixture into the reactor, reacting for two hours at room temperature (above 25 ℃ or longer than the temperature, or prolonging the reaction time), namely, coupling the first amino acid onto the resin, washing the resin for 6 times by DCM, and determining the substitution value (SD) of the resin at the moment; then adding 10ml of 20% PIP/DMF solution, mixing for 30min to remove an amino protecting group, washing the resin with DMF for 6 times, coupling a second amino acid, weighing three times of Fmoc-Gly-OH and HATU and absorbing six times of DIEA, adding 10ml of DMF to dissolve, reacting, monitoring the reaction process by ninhydrin reaction at room temperature, and washing the resin with DMF for 6 times if the reaction is finished if the reaction is monitored to be colorless. Then, the coupling reaction of the next amino acid can be continued according to the method for coupling the second amino acid, and the cycle is repeated until all the amino acids are coupled.

b. Cracking and precipitation

c. Preparation of amphotericin B peptide derivatives by liquid phase reaction

Weighing dried crude peptide 0.1mmol, HOSU 0.095mmol, and DIC0.095mmol, adding 5ml THF (25 deg.C, reacting for 2 hr), and rotary evaporating to remove THF; adding 5mLDMSO to dissolve a reaction product, adding 0.095mmol of amphotericin B and 0.2mmol of DIEA, and reacting at room temperature for 1-2 hours; 1.5mmol of CH3I and 0.2mmol of DIEA were added and reacted at room temperature for 2 hours; and finally, adding 4ml of 20% PIP/DMF solution, and reacting for 10-20 min to obtain a crude product.

The crude product was purified by semi-preparative RP-HPLC as follows.

1 purification of

Flow rate: 5ml/min

Detection wavelength: 409nm

Mobile phase: phase A: 1% HAC/Water

Phase B: 1% HAC/acetonitrile

The gradient elution procedure is shown in table 3.

TABLE 3 gradient elution procedure

Flow rate: 1ml/min

Detection wavelength: 215,383 and 405nm

Mobile phase: phase A: 0.1% TFA/water

Phase B: 0.1% TFA/acetonitrile

The gradient elution procedure is shown in table 4.

TABLE 4 gradient elution procedure

Collecting the target component with purity of more than 90%, rotary steaming, and freeze drying. Molecular weight confirmation by ESI-MS, M/Z-1487 (M + H)⁺In line with the theoretical molecular weight.

Example three: preparation and purification of DMR086

Hydrophilic polypeptide structure: Fmoc-AEEAc-AEEAc-OH

DMR086 structure:

(1) materials and reagents

2-CTC resin, substitution value 0.945 mmol/g.

Amino acids: Fmoc-AEEAc-OH,

synthesizing a reagent: HATU, DMF, DCM, DIEA, piperidine, iodomethane.

(2) Instrument for measuring the position of a moving object

(3) Operating procedure (taking 1g of resin as an example)

a. Solid phase chemical synthesis of polypeptides

Weighing 1.00g of 2-CTC resin, placing the resin in a reactor of a polypeptide synthesizer, adding 10mL of DCM, soaking for 1h, weighing 2-3 times of Fmoc-AEEA-OH and absorbing 4-6 times of DIEA, adding the mixture into 10mL of DCM for dissolving, putting the mixture into the reactor for reaction at room temperature (25 ℃ and above, otherwise, prolonging the reaction time) for two hours, namely, coupling the first amino acid onto the resin, then washing the resin for 6 times by the DCM, and determining the substitution value (SD) of the resin at the moment; then adding 10mL of 20% PIP/DMF solution, mixing for 30min to remove an amino protecting group, washing the resin with DMF for 6 times, coupling a second amino acid, weighing three times of Fmoc-AEEA-OH and HATU and absorbing 6 times of DIEA, adding 10mL of DMF to dissolve, reacting, monitoring the reaction process by ninhydrin reaction at room temperature, and finally washing the resin with DCM for 6 times.

b. Cracking and precipitation

After the synthesis of the polypeptide is finished, the wet weight is weighed. Adding a cracking reagent according to the ratio of 5mL of the cracking reagent to 1g of resin, stirring and reacting for 1h at room temperature according to the ratio of TFE: DCM (1: 4) (V: V), filtering to a 25mL rotary evaporation bottle, evaporating the liquid at 40 ℃, adding 10mL of DCM into the rotary evaporation bottle, further rotary evaporating the liquid to dryness, repeating the rotary evaporation for 3 times, drying in vacuum, and weighing the crude peptide.

c. Preparation of amphotericin B peptide derivatives by liquid phase reaction

Weighing dried crude peptide 0.1mmol, HOSU 0.095mmol, and DIC 15 μ L, adding 5mL THF (25 deg.C, reacting for 2h), and rotary evaporating to remove THF; adding 5mL of DMSO to dissolve a reaction product, adding 0.095mmol of amphotericin B and 0.2mmol of DIEA, and reacting at room temperature for 1-2 h; 1.5mmol of CH are added continuously₃I and 0.2mmol DIEA are reacted for 2 hours at room temperature; and finally, adding 4mL of 20% PIP/DMF solution, and reacting for 10-20 min to obtain a crude product.

The crude product was purified by semi-preparative RP-HPLC as follows.

1 purification of

Flow rate: 5mL/min

Detection wavelength: 409nm

Mobile phase: phase A: 1% HAC/Water

Phase B: 1% HAC/acetonitrile

The gradient elution procedure is shown in table 3.

TABLE 3 gradient elution procedure

Flow rate: 1mL/min

Detection wavelength: 215,383 and 405nm

Mobile phase: phase A: 0.1% TFA/water

Phase B: 0.1% TFA/acetonitrile

The gradient elution procedure is shown in table 4.

TABLE 4 gradient elution procedure

Collecting the target component with purity of more than 90%, rotary steaming, and freeze drying. The molecular weight was confirmed by ESI-MS that M/Z is 1228[ M + H ]]⁺In line with the theoretical molecular weight.

Example four: preparation and purification of other amphotericin B methyl esterified peptide derivatives containing T

Amino acid sequence: is 2-10 peptide containing AEEAc, Gly or AEEAc and Gly

The structure of the product is as follows:

synthesizing amphotericin B methyl esterified peptide derivative of H-T-AME, wherein the synthesis method refers to a compound DMR005-JZH or DMR078-JZH, namely, the solid phase synthesis method is adopted to synthesize Fmoc-T (T is AEEAc-OH, Gly-OH or hydrophilic polypeptide formed by combining AEEAc-OH and Gly-OH, and the length range of the peptide is 2-10); preparing Fmoc-T-Osu; ③ Fmoc-T-Osu modifies the amino group of amphotericin B; fourthly, iodomethane modifies carboxyl of Fmoc-T-AMB, piperidine removes Fmoc, and finally the amphotericin B methyl esterified peptide derivative crude product of H-T-AME is obtained. Purifying the crude product by RP-HPLC; according to the general formula [ III ] of the compounds, Table 5 lists the abbreviations and relative molecular masses of the compounds:

abbreviation and relative molecular weight of the Compounds of Table 5

Name (R)	Relative molecular mass
		H-(AEEAc)₂-AME	1227
H-(AEEAc)₉-Gly-AME	2300
		H-(AEEAc)₁₀-AME	2387

Example five: determination of the solubility of amphotericin B peptide derivatives

Amphotericin B and amphotericin B methyl esterified peptide derivative solubility was measured in the present invention patent using double distilled water, and the results are shown in table 6:

TABLE 6 results of solubility measurement

Compound (I)	Solubility (mg/mL)
		Amphotericin B (AMB)	<0.001
Amphotericin B methyl ester (AME)	<0.001
		H-AEEAc-Gly-AME	<0.5
H-AEEAc-AEEAc-AME	<2
		H-5G-AME	<0.5
DMR005-JZH	>60
		DMR078-JZH	>60
DMR086	>15
		H-(AEEAc)₉-Gly-AME	>100
H-(AEEAc)₁₀-AME	>100

Example six: stabilization of amphotericin B methyl esterified peptide derivatives

Three unstable sites of the parent structure of AMB-C₁₃Hemiketal structure, seven conjugated double bond structures and C₁₉The beta-glycosidic bond, so that amphotericin B is regulated in pharmacopoeia to be stored in a dark and refrigerated manner. The amphotericin B methyl esterified peptide derivative prepared by the method is modified on the amphotericin B structure, so that a similar degradation path also exists. First, in methanol solution (room temperature 12h), amphotericin B methyl esterification peptide derivative C₁₃The hemiketal structure will be methylated; ② at low pH (such as TFA), m/z 801 impurity is easily generated, and C is presumed to be₁₉Cleavage of the beta-glycosidic bond of (a); ③ under the condition of lightproof room temperature, the water solution conjugated hepta-olefinic bond is easy to oxidize, and under the condition of low temperature (-20 ℃) lightproof, the compound conjugated hepta-olefinic bond is only slightly oxidized, so the prepared amphotericin B methyl esterified peptide derivative is stored by adopting the lightproof and refrigeration mode.

Example seven: in vitro antimicrobial Activity assay

The Minimal Inhibitory Concentration (MIC) of each antibacterial agent was determined according to the minimal broth dilution method recommended by the Clinical Laboratory Standards Institute (CLSI), and the culture medium for bacteria was Mueller-Hinton (MH) broth, and the culture medium for Candida albicans and Cryptococcus neoformans was Hyclone modified RPMI-1640 medium.

The method comprises the following specific steps:

(1) preparing an antibacterial medicament stock solution:

accurately preparing amphotericin B methyl esterified peptide derivatives with the concentration of 320 mu g/mL and positive control amphotericin B, and storing in a dark place at-20 ℃ for later use.

(2) Preparation of a culture medium:

the fungus growth culture medium adopts an improved martin culture medium, and the specific preparation method comprises the following steps: the 1L culture medium contains 2% glucose, 0.2% yeast extract powder, 0.5% fish meal peptone, 0.05% magnesium sulfate and 0.1% dipotassium hydrogen phosphate, the corresponding amount of each substance is weighed according to the proportion, after the substances are dissolved in a certain amount of purified water, the volume is determined to be 1L, the pH value is adjusted to 7.2, 2% agar is added, and the high temperature sterilization is carried out for 30min at 121 ℃.

The fungus MIC test adopts Hyclone improved RPMI-1640 culture medium, and the specific preparation method is as follows: weighing 18.00g of glucose, dissolving in a certain amount of purified water, diluting to 500mL, and sterilizing at 115 ℃ for 15 min. In a sterile environment, 500mL of RPMI-1640 medium was added to the sterilized glucose solution, mixed, and stored at 4 ℃ until use.

(3) Preparation of inoculum:

selecting a fungus single colony growing for 48 hours on a plate, transferring the fungus single colony to a slant culture medium of an improved Martin culture medium, incubating for 24 hours at 28 ℃, adjusting the turbidity of the enriched bacterial liquid in the logarithmic phase to reach 0.5 McLeod standard by using a Hyclone improved RPMI-1640 liquid culture medium, wherein the turbidity is equivalent to that the turbidity of the bacterial liquid per milliliter contains 1 multiplied by 10⁶～5×10⁶CFU (colony-forming unit), diluting the bacterial suspension with Hyclone modified RPMI-1640 liquid culture medium at a ratio of 1:20, and diluting at a ratio of 1:50 to obtain the inoculum with a concentration of 1 × 10³～5×10³CFU/mL。

(4) Preparing diluted antibacterial drugs and inoculating bacterial liquid:

taking a 96-well plate, adding 160 μ L of RPMI-1640 liquid culture medium into the 1 st well, adding 100 μ L of RPMI-1640 liquid culture medium into the 2 nd-12 th well, adding 40 μ L of antibacterial drug stock solution (320 μ g/mL) into the 1 st well, mixing, sucking 100 μ L into the 2 nd well, mixing, sucking 100 μ L into the 3 rd well from the 2 nd well, diluting to the 10 th well in a continuous multiple ratio manner, sucking 100 μ L from the 10 th well, discarding, adding 100 μ L of the prepared inoculum into the 1 st-10 th well and the 12 th well to make the final concentration of the bacterial solution of each well be about 2.5 × 10³CFU/mL. The drug concentrations in the 1 st to 10 th wells are 32. mu.g/mL, 16. mu.g/mL, 8. mu.g/mL, 4. mu.g/mL, 2. mu.g/mL, 1. mu.g/mL, 0.5. mu.g/mL, 0.25. mu.g/mL, 0.125. mu.g/mL, 0.0625. mu.g/mL, respectively, the 11 th well is a blank control containing no antibacterial drug and no inoculum, and the 12 th well is a negative control containing no antibacterial drug.

(5) Incubation

And placing the 96-well plate inoculated with the fungi in an air incubator at 28 ℃ for incubation for 40-50 h.

(6) Results

The lowest concentration of the drug without fungal growth was visually observed as the Minimum Inhibitory Concentration (MIC) of the sample. The MIC determination results for each AMB methyl esterified peptide derivative are shown in table 7.

TABLE 7MIC measurement results

Example eight: determination of the time Sterilization Curve

(1) Preparing an antibacterial medicament stock solution:

DMR005-JZH with the concentration of 480 mu g/ml, DMR078-JZH with the concentration of 360 mu g/ml, DMR086 with the concentration of 240 mu g/ml and amphotericin B as a positive control substance with the concentration of 120 mu g/ml are accurately prepared, and the prepared stock solutions are placed in an environment with the temperature of-20 ℃ and are kept in the dark for standby.

(2) Preparation of a culture medium:

the fungus growth culture medium adopts YPD liquid culture medium, and the specific preparation method comprises the following steps: the 1L culture medium contains 2% glucose, 1% yeast extract powder and 2% tryptone, the corresponding amount of each substance is weighed according to the proportion, dissolved in a certain amount of purified water, the volume is determined to 1L, if YPD solid culture medium is prepared, 2% agar is additionally added, and the YPD solid culture medium is sterilized at the high temperature of 121 ℃ for 30 min. The Hyclone improved RPMI-1640 culture medium is adopted for the fungus time sterilization curve test, and the specific preparation method is as follows: weighing 18.00g of glucose, dissolving in a certain amount of purified water, diluting to 500mL, and sterilizing at 115 ℃ for 15 min. In a sterile environment, 500mL of RPMI-1640 medium was added to the sterilized glucose solution, mixed, and stored at 4 ℃ until use.

(3) Preparation of inoculum:

selecting single fungus colony growing on plate for 48 hr, inoculating to YPD liquid culture medium, incubating at 30 deg.C for 20-24 hr, and adjusting turbidity of the enriched bacteria liquid to 0.5 McLeod standard with Hyclone improved RPMI-1640 liquid culture medium, which is equivalent to 1 × 10 per ml⁶～5×10⁶CFU (colony-forming unit), taking the bacterial suspension, and carrying out 1: after 10 dilutions, 1: 10 dilution as inoculum, bacteriaThe suspension concentration corresponds to 1X 10⁴～5×10⁴CFU/mL。

(4) Determination step and incubation of time sterilization curve

A96-well plate was prepared and 180. mu.L each of the prepared inoculum (total 30 empty, containing one well, i.e.3 rows and 10 columns) of 180. mu.L RPMI-1640 liquid medium was added, followed by a 6-fold dilution of DMR005-JZH, DMR078-JZH, DMR086 and amphotericin B (AmB) of 20. mu.L, which was 2-fold higher than the MIC determined for the strain (8, 6,8 and 2. mu.g/ml, respectively) and incubated with shaking in a30 ℃ microplate shaker. Samples were taken at different time points (0, 3, 6, 9, 12, 24h), diluted and plated on YPD solid medium. Colonies were counted after 24 hours incubation at 30 ℃. Untreated candida albicans was used as a blank control and AmB treated candida albicans was used as a positive control.

(5) Results

The results of the time sterilization profiles of AmB, DMR005-JZH, DMR078-JZH, and DMR086 are shown in FIG. 5.

Example nine: in vitro hemolytic Activity assay

1. Materials and reagents

Aseptic defibrinated sheep blood, NaCl, KCl, Na₂HPO₄·12H₂O,KH₂PO₄。

2. Instrumentation and equipment

Allegra X-22R type low-temperature high-speed centrifuge, electronic balance, micropipettor, SHP-150 type biochemical incubator, 96well and microplate reader

3. Experimental methods

(1) Preparation of PBS (phosphate buffered saline) buffer solution

PBS，pH 7.4，1L:

Potassium dihydrogen phosphate (KH)₂PO₄):0.27g

Disodium hydrogen phosphate (Na)₂HPO₄·12H₂O):3.58g

Sodium chloride (NaCl) 8.00g

0.20g of potassium chloride (KCl)

Adding about 800mL of water, stirring thoroughly to dissolve, adding hydrochloric acid to adjust pH to 7.4, and diluting to 1L

(2) Preparation of test substance

A sample of the amphotericin B methyl esterified peptide derivative to be detected was accurately weighed and prepared into a solution to be detected at a concentration of 1.024mg/mL using a PBS buffer solution having a pH of 8.0, and the solution was refrigerated at-20 ℃ for future use. Amphotericin B and amphotericin B methyl ester were weighed out accurately, first made up to 10.24mg/mL in DMSO and then diluted to 1.024mg/mL in PBS buffer pH 7.4.

(3) Processing of sheep blood red blood cells

Taking several milliliters of aseptic defibered sheep blood, adding PBS buffer solution with pH7.4 which is about 10 times of the amount of the aseptic defibered sheep blood, shaking up, centrifuging for 15 minutes at 1500r/min, removing supernatant, washing the precipitated red blood cells for 2-3 times by using the PBS buffer solution with pH7.4 according to the method, and ensuring that the supernatant does not show red. The red blood cells were made up to 2% suspension in PBS buffer, pH7.4, for testing.

(4) Hemolytic toxicity assay procedure

Adding 100 μ L of PBS buffer solution with pH7.4 into the 1 st-11 th well of a 96-well plate, adding 100 μ L of double distilled water into the 12 th well, adding 100 μ L of antibacterial drug stock solution (1024 μ g/mL) into the 1 st well, mixing, sucking 100 μ L to the 2 nd well, sucking 100 μ L to the 3 rd well from the 2 nd well after mixing, diluting to the 10 th well in a continuous multiple ratio, sucking 100 μ L from the 10 th well, discarding, adding 100 μ L of 2% erythrocyte suspension of each prepared inoculum into the 1 st-12 th well, and making the final erythrocyte concentration of each well be about 1 × 10⁸Cells/well. The drug concentration of the control drug amphotericin B in the 1 st to 10 th holes is respectively 256 mug/mL, 128 mug/mL, 64 mug/mL, 32 mug/mL, 16 mug/mL, 8 mug/mL, 4 mug/mL, 2 mug/mL, 1 mug/mL and 0.5 mug/mL, the 11 th hole is a negative control of erythrocyte suspension and PBS, and the 12 th hole is a positive control of erythrocyte suspension and double distilled water. Finally, the mixture was incubated at 37 ℃ for 1 hour.

(5) Incubation

After 1h incubation, the 96well plates were removed, centrifuged at 600r/min for 15 minutes and the supernatant was measured for OD at 540nm using a microplate reader. The hemolysis rate was calculated according to the following formula, wherein the negative control was PBS and the positive control was double distilled water. Hemolysis rate (test OD-negative OD)/(positive OD-negative OD)

(6) Results

The results of in vitro hemolytic toxicity of DMR086, DMR005-JZH, AMB, AME and DMR078-JZH are shown in Table 8.

TABLE 8 in vitro hemolytic toxicity results

Compound (I)	EH(μg/mL)
		AmB	8
AME	16
		DMR005-JZH	>256
DMR078-JZH	>256
		DMR086	>256

EXAMPLE ten DMR005-JZH, AmB, DMR078-JZH and DMR086 spectral properties and self-aggregation characteristics

1. Materials and reagents

NaCl,KCl,Na₂HPO₄·12H₂O,KH₂PO₄,DMSO,CH₃OH。

2. Instrumentation and equipment

Electronic balance, constant temperature water bath, UV1800 UV visible spectrophotometer

3. Experimental methods

(1) Preparation of PBS (phosphate buffered saline) buffer solution

PBS，pH 7.4，1L:

Potassium dihydrogen phosphate (KH)₂PO₄):0.27g

Disodium hydrogen phosphate (Na)₂HPO₄·12H₂O):3.58g

Sodium chloride (NaCl) 8.00g

0.20g of potassium chloride (KCl)

(2) Preparation of test substance

DMR086, DMR005-JZH and DMR078-JZH to be detected are accurately weighed, and are respectively prepared into a solution to be detected with the concentration of 1.28 and the concentration of 2.56mg/mL by using DMSO and a PBS buffer solution with the pH of 7.4, and the solution is refrigerated at the temperature of minus 20 ℃ for standby. AmB was weighed out accurately and dispensed with DMSO at 1.28 and 6.40mg/mL and refrigerated at-20 ℃ until use.

(3) Measurement procedure

First, 20. mu.L aliquots of each DMSO solution were added to 2mL of methanol or 2mL of PBS buffer (eliminating the effect of DMSO), and another 20, 30, 40, and 50. mu.L of 2.56mg/mL solutions of DMR086, DMR005-JZH, and DMR078-JZH, respectively, were added to 2mL of PBS buffer; secondly, the solution is incubated for 30 minutes at 30 ℃; finally, UV-Vis spectral data in the range of 300-430nm were recorded for each sample.

(4) Results

The results of the spectra of AmB, DMR086, DMR005-JZH and DMR078-JZH are shown in FIG. 6. FIG. 6A shows absorption spectra of DMR086, DMR005-JZH and DMR078-JZH and AmB (12.7. mu.g/mL) in methanol; FIG. 6B shows absorption spectra of DMR086, DMR005-JZH and DMR078-JZH and AmB (12.7. mu.g/mL) in PBS buffer; FIG. 6C shows absorption spectra of DMR086, DMR005-JZH and DMR078-JZH and AmB (62.4. mu.g/mL) in PBS buffer.

The literature reports the presence of AMB in PBS buffer in several forms, namely monomeric, soluble and insoluble self-aggregation. And absorbance A₃₄₈/A₄₀₉Has been used as a measure of AmB self-aggregation(s); from the above figures, AmB, DMR086, DMR005-JZH andDMR078-JZH did not self-aggregate in methanol solution (FIG. 6A); at the same concentration (12.7. mu.g/mL), the absorption and spectral shape at 409nm for AmB shows: AmB showed significant self-aggregation, as did DMR086, however, the ratio of DMR005-JZH and DMR078-JZH monomeric forms was high and no self-aggregation was observed (FIG. 6B); at higher concentrations (62.4. mu.g/mL), AmB, DMR086, DMR005-JZH and DMR078-JZH all exhibited significant self-aggregation (FIG. 6C). From the above analysis results, it can be seen that: in solution, DMR086 exists in a more similar form to AmB, and thus DMR086 is superior to DMR005-JZH and DMR 078-JZH. Example eleven: in vitro HEK293T cytotoxicity assay

1. Materials and reagents

HEK293T cells, NaCl, KCl, Na₂HPO₄·12H₂O,KH₂PO₄,MTT,DMEM

2. Instrumentation and equipment

Electronic balance, micropipettor, multifunctional microplate reader, A2 type biosafety cabinet, microplate constant temperature oscillator, CO₂Incubator, centrifuge, inverted microscope, and constant-temperature water bath

3. Experimental methods

(1) Preparation of PBS (phosphate buffered saline) buffer solution

PBS，pH 7.4，1L:

Potassium dihydrogen phosphate (KH)₂PO₄):0.27g

Disodium hydrogen phosphate (Na)₂HPO₄·12H₂O):3.58g

Sodium chloride (NaCl) 8.00g

0.20g of potassium chloride (KCl)

(2) Preparation of test substance

DMR086, DMR005-JZH and DMR078-JZH to be tested were accurately weighed, and prepared into a solution to be tested (using a sterile syringe and a 0.22 μm filter head, filtration was performed in a clean bench) at a concentration of 3.00mg/mL with a PBS buffer solution having a sterile pH of 7.4, and the solution was refrigerated at-20 ℃ for future use. Amphotericin B and amphotericin B methyl ester were weighed out accurately, prepared at 10.24mg/mL in DMSO, and then diluted to 1.024mg/mL in PBS buffer pH7.4 (diluted in sterile PBS buffer pH7.4 on the clean bench).

(3) Determination of cell nephrotoxicity and incubation

Collecting logarithmic phase cells, adjusting cell suspension concentration, adding 100 μ L per well, and plating to adjust cell density to 0.5 × 10⁴Cells/well (marginal wells filled with sterile PBS), 5% CO₂Incubation was performed overnight at 37 ℃. Until the cell monolayer is paved on the bottom of the well (96-well flat bottom plate), discarding the supernatant, adding the medicine with gradient concentration, totally 8 concentration gradients are added, each 100 mu L is provided with 1 multiple wells, 5% CO₂Incubated at 37 ℃ for 24-48 hours and observed under an inverted microscope. The supernatant was discarded, 10. mu.L MTT solution (5mg/mL, i.e., 0.5% MTT) was added to each well, and incubation continued for 4-6 h. The culture medium in the wells was aspirated, 100. mu.L of dimethyl sulfoxide was added to each well, and the mixture was placed on a micropore shaker and shaken at low speed for 10min to dissolve the crystals sufficiently. The absorbance of each well was measured at OD 580nm of an ELISA detector. And setting a zero setting hole (culture medium, MTT and dimethyl sulfoxide), a positive control hole (cells, a drug dissolution medium with the same concentration, the culture medium, MTT and dimethyl sulfoxide) and a 1% dimethyl sulfoxide control hole (cells, the culture medium, MTT and dimethyl sulfoxide).

(4) Results

The results of in vitro nephrotoxicity of DMR086, DMR005-JZH, AmB, AME and DMR078-JZH are shown in Table 9.

TABLE 9 in vitro nephrotoxicity results

Compound (I)	EH(μg/mL)
		AmB	25.6
AME	187.5
		DMR005-JZH	750
DMR078-JZH	750
		DMR086	750

EXAMPLE twelve in vivo evaluation of antifungal drug efficacy

1. Compound (I)

TABLE 10 in vivo test Compound List

Compound (I)	Molecular weight	Purity%	Others
				AmB	924	89％	Light-sensitive
DMR005-JZH	1662	92％	Light-sensitive
				DMR078-JZH	1486	95％	Light-sensitive
DMR086	1227	96％	Light-sensitive

2. Strains and animals

The strain is as follows: candida albicans ATCC10231 source: shanghai medical institute

Mice: female ICR sources: shanghai Sphere-Bikai laboratory animals Co., Ltd.: 20180006010648(MLD assay), 20180006011159 (pharmacodynamic assay)

3. Test method

3.1 detection of Minimal Lethal Dose (MLD) of Candida albicans to ICR mice

Preparing Candida albicans cultured for 48h into 5 × 10 with physiological saline⁴CFU/mL，2.5×10⁵CFU/mL，5×10⁵CFU/mL，2.5×10⁶CFU/mL，5×10⁶CFU/mL，1×10⁷CFU/mL，2.5×10⁷CFU/mL and 5X 10⁷CFU/mL。

Female ICR mice, 53, 19-22g, were divided into 9 groups of 6 mice each (5 mice in one group). 8 groups of the above were infected by tail vein injection with 200. mu.l of bacterial solutions of different concentrations (a-5X 10)⁴CFU/mL；b-2.5×10⁵CFU/mL；c-5×10⁵CFU/mL；d-2.5×10⁶CFU/mL；e-5×10⁶CFU/mL；f-1×10⁷CFU/mL；g-2.5×10⁷CFU/mL；h-5×10⁷CFU/mL), group 9 was treated as a control group and 200. mu.L of physiological saline (i-0CFU/mL) was injected into the tail vein. Mice were observed daily and the number of deaths recorded for 3 weeks, giving the lowest lethal concentration (MLD).

3.2 in vivo pharmacodynamic assay

According to the MLD test results (see FIG. 7), the minimum bacterial suspension concentration (i.e., the lowest lethal dose) that causes total death of the mice is the infectious dose.

Preparing Candida albicans cultured for 48h into 1 × 10 with physiological saline⁷CFU/ml bacterial suspension. 80 mice were injected 200. mu.l 1X 10 via tail vein⁷The infection was carried out by using Candida albicans at CFU/ml, and the infection was divided into 8 groups (A-H groups), each group containing 10 individuals, and a blank control group containing 10 individuals (group I) which did not undergo infection was provided.

Wherein the group A is a model group, and after 24h of infection, the solvent (5% glucose containing 1% DMSO) is given to the tail vein according to 10 μ l/g, 1 time per day and 4 times continuously; in group B, 1mg/kg of AmB is given to the tail vein at a rate of 10 mul/g 24h after infection, 1 time per day and 4 times continuously; respectively administering 4mg/kg and 16mg/kg of DMR005-JZH to group C and group D according to 10 μ L/g tail vein, 1 time per day, and 4 times continuously; group E and group F were administered 4mg/kg and 16mg/kg of DMR078-JZH, respectively, 1 time daily for 4 consecutive times, according to 10 μ L/g tail vein; the group G and the group H are respectively given 4mg/kg and 16mg/kg of DMR086 according to 10 mu L/G tail vein, 1 time per day and 4 times continuously; group I did not.

After 24d infection, surviving mice were sacrificed by cervical dislocation and dissected. The kidney of the mouse was aseptically taken, and a tissue homogenate was prepared using a tissue homogenizer (Magna Lyser, Roche) by adding 0.9ml of physiological saline to 0.1g of the tissue. The tissue homogenate was diluted to a certain dilution, 0.2mL was added to a dish, and 10mL of a modified Martin agar medium (HB4701, Qingdao Haibo) melted at about 50 ℃ was added. Mixing, standing, solidifying the culture medium, placing into a common air incubator (DHP-9162B, Shanghai-Heng) at 30 deg.C, performing inverted culture for 48h, and calculating the viable bacteria load of mouse tissue.

4. Test results

4.1 MLD detection results

The lowest lethal concentration, MLD, of candida albicans ATCC10231 that caused all deaths in mice was detected by tail vein injection of different concentrations of candida albicans ATCC 10231. It can be seen from FIG. 7 that the concentration of Candida albicans infection is lower than 1X 10⁷At CFU/mL, mice partially died; when in useThe concentration of the bacteria is more than or equal to 1 multiplied by 10⁷CFU/mL mice all died at different times, and the MLD of Candida albicans ATCC10231 to mice was 1X 10⁷CFU/mL。

4.2 in vivo pharmacodynamic assay

4.2.1 viable renal cell count

After 24 days of infection, the kidneys of the mice were aseptically harvested and the tissue viable load was examined by viable count. It can be seen from fig. 8 that DMR005-JZH, DMR078-JZH and DMR086 in the high dose group were superior to those in the corresponding low dose group, but DMR005-JZH in the high dose group was not significantly different from those in the low dose group, whereas DMR078-JZH and DMR086 were significantly different, and DMR086 was more significantly different. In addition, both the low and high dose groups had DMR005-JZH and DMR078-JZH less than 1mg/kg AmB. Although the low dose group also had DMR086 inferior to 1mg/kg AmB, the high dose group had DMR086 superior to 1mg/kg AmB.

4.2.2 Effect of test Compounds on mouse survival

Mice were infected by tail vein injection of candida albicans ATCC10231, and the mice were observed for survival 1 time a day for four consecutive times, starting at 24h post infection. As can be seen from table eleven, mice in the model group that were not treated with the drug began to die at day 9 after infection, and reached 80% mortality by day 14; the 4mg/kg group of mice of DMR005-JZH died from day 11 after infection, the mortality rate reached 80% by day 17 after infection, the survival time of the mice was prolonged but the survival rate was not improved; the mice in the 16mg/kg group of DMR005-JZH die from 12 days after infection, the death rate of the mice is 60 percent from 20 days after infection, compared with the mice in the model group, the survival time is prolonged, and the survival rate is improved by 20 percent; the 4mg/kg group of mice of DMR078-JZH died from 12 days after infection, and the mortality rate reached 80% by 19 days after infection, compared with the model group of mice, the survival time of the mice was prolonged but the survival rate was not improved; the mice in the 16mg/kg group of DMR078-JZH die from the 20 th day after infection, the death rate of the mice is 20%, and compared with the mice in the model group, the survival time of the mice is obviously prolonged, and the survival rate is improved by 60%; the 1mg/kg group of mice of AmB died 19 days after infection, the mortality rate of 20 days after infection was 10%, and compared with the model group of mice, the survival time of the mice was obviously prolonged, and the survival rate was improved by 70%. The 4mg/kg group of mice of the DMR086 died from day 14 after infection, and the mortality rate reached 70% by day 22 after infection, which resulted in prolonged survival time and slightly improved survival rate compared to the model group of mice; the 16mg/kg group of mice of DMR086 did not die, and both survival time and survival rate were significantly prolonged compared to the model group of mice.

TABLE 11 Effect of test Compounds on mouse survival

In summary, 1) DMR005-JZH, DMR078-JZH, and DMR086 all showed concentration dependence with decreasing renal viable cell load with increasing dose; at the same dose, DMR086 was superior to DMR005-JZH and DMR 078-JZH; only the high dose group DMR086 was better than 1mg/kg AmB. 2) The survival time and survival rate of the mice are improved to different degrees along with the increase of the dosage of the DMR005-JZH, the DMR078-JZH and the DMR086, wherein the improvement of the DMR078-JZH and the DMR086 is the most remarkable; in addition, at the same dose, the survival time and survival rate of the DMR086 on the mouse are better than that of the DMR005-JZH and that of the DMR 078-JZH; wherein the high dose group DMR086 had no mice that died better than 1mg/kg AmB.

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention.

The disclosures of all documents cited herein are incorporated by reference herein, to the extent that they provide exemplary, procedural and other details supplementary to those set forth herein.

Claims

wherein R is₁Selected from hydrophilic polymer moieties;

R₂selected from amino protecting groups or H;

R₃is selected from H or C_1-4A hydrocarbyl or phenyl group;

R₄is OH or H;

alternatively, the amino protecting group is Fmoc or Boc.

2. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R₁Is selected from- (Gly)_n-、-(AEEAc)_n-、-(AEEAc)_n-Gly-、-(AEEAc-Gly)_n-AEEAc-、-AEEAc-Gly-、-(AEEAc-Gly)_n-、-(Gly-AEEAc)_n-Gly-、-(AEEAc)_n-Gly-(AEEAc)_m-、-(AEEAc)_n-(Gly)_m-and- (Gly)_n-AEEAc-(Gly)_m-, wherein n and m are each independently an integer of 2 to 10.

3. A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R₁Selected from- (AEEAc)_n-Gly-、-(AEEAc-Gly)_n-AEEAc-、-(AEEAc)_n-、-(AEEAc-Gly)_n-、-(Gly-AEEAc)_n-Gly-and- (AEEAc)_n-Gly-(AEEAc)_m-, wherein n and m are each independently an integer of 2 to 10.

4. A compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein R₂Is H.

5. A compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein R₃Is methyl.

6. The compound according to any one of claims 1-5, or a pharmaceutically acceptable salt thereof, having the structure of formula [ II ]:

wherein R is₂Fmoc, Boc or H;

7. A compound or pharmaceutically acceptable salt thereof according to claim 6, wherein R₂-T-is selected from:

H-(AEEAc)₅-

H-AEEAc-Gly-AEEAc-Gly-AEEAc-，

H-AEEAc-Gly-，

H-Gly-Gly-，

H-AEEAc-AEEAc-Gly-，

H-(Gly)₅-，

H-AEEAc-AEEAc-Gly-Gly-Gly-，

H-Gly-Gly-AEEAc-Gly-Gly-，

H-Gly-AEEAc-Gly-AEEAc-Gly-，

H-AEEAc-AEEAc-Gly-AEEAc-AEEAc-，

H-AEEAc-Gly-AEEAc-Gly-AEEAc-Gly-，

H-Gly-AEEAc-Gly-AEEAc-Gly-AEEAc-，

H-Gly-AEEAc-Gly-AEEAc-Gly-AEEAc-Gly-，

H-(AEEAc)₂-，

H-(AEEAc)₁₀-，

or H- (AEEAc)₉-Gly-。

8. The compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

9. a process for preparing a compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, comprising: (1) performing solid-phase synthesis on resin to obtain polypeptide, cracking the obtained polypeptide product with weak acid, filtering, performing rotary evaporation, adding a first organic solvent for dissolution, performing rotary evaporation, precipitating with a second organic solvent, and drying to obtain polypeptide containing an amino protecting group; (2) activating the polypeptide containing the amino protecting group, and then reacting the polypeptide with amphotericin B in an anhydrous solvent in the presence of a catalytic amount of alkali; (3) carrying out methyl esterification reaction on carboxyl of amphotericin B; and optionally, (4) removing the amino protecting group from the polypeptide moiety comprising the amino protecting group.

10. Use of a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament, preferably an antifungal medicament.