CN112175029A

CN112175029A - Antifungal macrocyclic polyene compounds

Info

Publication number: CN112175029A
Application number: CN201910586224.8A
Authority: CN
Inventors: 刘力
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-07-01
Filing date: 2019-07-01
Publication date: 2021-01-05

Abstract

The gamma, omega and v type novel compounds of the macrocyclic polyene antifungal amphotericin B have less hygroscopicity, better storage stability, are more beneficial to the quality control of medicaments and preparations and the like, and are suitable for preparing infection: cryptococcosis, North American blastomycosis, disseminated candidiasis, coccidioidomycosis, histoplasmosis, mucormycosis, sporotrichosis, aspergillosis, chromomycosis, skin fungal infection after burning, respiratory Candida, aspergillosis or cryptococcal infection, fungal corneal ulcer, thermokeratoderma, enterovirus infection, anti-depression and the like.

Description

Antifungal macrocyclic polyene compounds

Technical Field

The invention relates to the technical field of medicines, and particularly provides a compound with a novel molecular formula and a novel molecular structure, which has better stability, and a macrocyclic polyene antifungal drug amphotericin B, and a pharmaceutical composition thereof.

Background

Amphotericin B (Amphotericin B, CAS:1397-89-3, molecular formula C)₄₇H₇₃NO₁₇M-924.08) is a macrocyclic polyene broad-spectrum antifungal drug produced by streptothricin nodosum, is the first choice drug for treating most deep fungal infections, is suitable for deep fungal infections caused by sensitive fungi and progressive disease progression patients, such as septicemia, endocarditis, meningitis (cryptococcus and other fungi), abdominal infections (including those related to dialysis), lung infections, urinary tract infections, endophthalmitis and the like, and can also treat Visceral Leishmaniasis (VL). More specifically, amphotericin B is indicated for cryptococcosis, nystonosis in north america, disseminated candidiasis, coccidioidomycosis, histoplasmosis, mucomycosis caused by mucor, aspergillus, Absidia, Endochromyces, Raney-dung etc., sporotrichosis caused by Sporothrix schenckii, aspergillosis caused by Aspergillus fumigatus etc., and for external use in the treatment of pigmented mycoses, cutaneous fungal infections after burning, Candida, Aspergillus or Cryptococcus infections, fungal corneal ulcers, but most notably adverse reactions such as electrolyte disturbances, blood cell destruction and toxicity to the kidney, heart and liver etc.

Among the modes of amphotericin B administration are mainly: intravenous administration: when intravenous drip is started, 1-5 mg or 0.02-0.1 mg/kg once according to body weight is tried to be administered, then 5mg is added every day or every other day according to tolerance conditions of patients, the maximum daily dose of an adult does not exceed 1mg/kg, the administration is carried out for 1 time every day or every 1-2 days, the accumulated total amount is 1.5-3.0 g, the treatment course is 1-3 months, and the period can be as long as 6 months according to disease conditions and disease types. A small dose is suitable for sensitive fungal infection, namely 20-30 mg for one time.

Intrathecal administration: 0.05-0.1 mg for the first time, then gradually increased to 0.5mg each time, the maximum amount is not more than 1mg once, and the medicine is administrated 2-3 times per week, and the total amount is about 15 mg. When the medicine is administered intrathecally, the medicine should be administered simultaneously with a small dose of dexamethasone or hydrocortisone succinate, and the liquid medicine should be repeatedly diluted by cerebrospinal fluid and slowly injected while being diluted to reduce adverse reaction.

Local application: 5-10 mg of the aerosol is dissolved into 0.2-0.3% solution for application by adults every time when the aerosol is inhaled; when the product is inhaled by ultrasonic atomization, the concentration of the product is 0.01-0.02%, the product is inhaled 2-3 times a day, and 5-10 ml of the product is inhaled each time; when the bladder is continuously flushed, 5mg of amphotericin B is added into 1000ml of sterilized water for injection every day, and the mixture is flushed at the speed of injecting 40ml of amphotericin B into the water for injection every hour for 5-10 days.

Some published documents report the synthesis and analysis of amphotericin B, liposome preparations, microspheres, nanoparticles, various dosage forms, pharmacological toxicological clinic, combined administration, synergistic attenuation effects of combined administration, etc. (patent document: US 4177265; purification process of deep fungal infection treatment drug, CN 102746352; method for purifying and crystallizing amphotericin B, CN 1053794; zuoyian, huangjunqin, pond-king stomach, etc., application research of macroporous resin in amphotericin B bulk drug purification process [ J ]. shanghai medicine, 2012,33(09): 13-15; research of purifying amphotericin B by schuman, mukumi, macroporous resin adsorption method [ J ]. Shandong electric university report, 2005(03): 62-63; painting aspiration, richardui, Shaohui, Shao Wei, shawei, crystallization process research [ J ]. fine and special chemicals of crude amphotericin B, 2007(12) 8-10; HPLC determination of the contents of Huanghong, enemy threne, amphotericin B [ J ]. J.Chinese J.antibiotics, 2001(01): 43-45.; rodong, Malexiu, Okinawa, et al, HPLC assay for amphotericin B content [ J ]. J. pharmaceutical practice, 1998(03):166- "168"; a preparation method of amphotericin B liposome, CN 107412165; amphotericin B polypeptide hydrogel drug delivery systems for the treatment of fungal infections, CN 107375939; an amphotericin B vaginal expansion suppository and its preparation method, CN 108310364; the application of amphotericin B and derivatives thereof in preparing rapid anti-depression drugs, CN 108079004; an antifungal pharmaceutical composition, CN 107812011; a preparation method of amphotericin B liposome, CN 107412165; an anticancer medicinal composition containing Ganoderma extract and amphotericin B, CN 106474164; a formulation of cholesterol-containing liposomal amphotericin for the treatment of fungal infections, CN 106361696; cochleates prepared using soy phosphatidylserine, CN 105050409; a medicament for inhibiting enterovirus 71, CN 104606215; an amphotericin B nano-composite and its preparing process, CN 104856931; amphotericin B lipid complex for injection and its preparation method, CN 101797264; a composition comprising amphotericin B, CN 101442989; amphotericin B structured emulsion, CN 1447682; an aqueous composition of amphotericin B, CN 1505519; pharmaceutical formulations with insoluble active substance, CN 1741789; reference 2, Wangwei amphotericin B treatment of kala-azar random comparisons of daily and alternate day dosing [ J ] foreign medicine (Parasitisis Subdivision), 1995(05): 234-; luxin, Sunworu, Gao Wei, etc., Meta analysis of the preventive effect of inhaled amphotericin B on invasive pulmonary aspergillosis [ J ]. J.Med.Med.Med.J.Med.Med.J.Med.11 (01): 28-36.; chenhong, Wenhai, Xuhong, Xueke, etc., amphotericin B and itraconazole [ J ] in vitro combined drug sensitivity test analysis, J.Chinese and Western medicine J.Dermatology 2011,10(03): 145-147; \37154experiment dosage of liposome of Xiujin, Liuli and amphotericin B causes serious cardiovascular adverse reaction [ J ] adverse drug reaction journal, 2007(01) 53-54; meihe Kun, Wang Dong, Bai nan, etc., amphotericin B nephrotoxicity literature measurement analysis [ J ] Chinese New drug and clinical journal, 2014,33(12): 924-928; document 3, Zelixian, King Juan, Sun Li, amphotericin B long-circulating liposome lyophilized preparation process research [ J ] pharmaceutical and clinical research, 2007(05) 384-386; development of new formulations of liupei, aurantii and amphotericin B and clinical research progress [ J ] chinese medicine guide, 2011,8(19):6-7+ 17.; guo Huili, Meixing, Ligao, amphotericin B lipid dry powder inhalant prescription screening and evaluation of powder properties [ J ] pharmaceutical guidance, 2009,28(02): 225-; pensheng, dianlinghui, Yangxianghe, et al,. preparation and release degree investigation of amphotericin B cubic liquid crystal precursor capsules [ J ]. J.J.Pharmacology, 2012,32(11): 817-820.; ). Patent documents CN 1029617C and CN 102382158B only report the range of water content of amphotericin B products, but neither report whether the resulting products contain other solvents or solvent compounds, and there are necessarily numerous specific compounds of formula or solvent compounds of formula of markush in any range of substances or substances within a range of substances, and the above synthetic methods have some problems.

The stability of the raw material medicine is the basis for ensuring the stability and good preparation performance of the pharmaceutical preparation, and is the basis for ensuring the curative effect and preventing unexpected adverse reactions caused by the instability of the medicine, the most stable form of the raw material medicine is continuously pursued in pharmacology, the pharmacology is an experimental science, the molecular form with good stability is unpredictable in advance, and a plurality of cases for seeking medicine molecules with better stability exist in the development history of the medicine in the world, so that the continuous development and progress of the pharmacology are objectively promoted. As unknown to many professionals, some bulk drugs produced industrially for years have great differences in stability among different batches of raw materials fed into the same batch of raw materials even in the same workshop and the same equipment under the same process flow, some bulk drugs can only keep stability for about 3 months even under specified conditions, some quality control indexes have obvious changes in different degrees, which causes confusion or trouble problems, various potential threat problems continue, and even though the products or bulk drugs are recorded by pharmacopoeias of various countries for a long time, the products or bulk drugs have great unexpected risks to patients in clinical application. From the actual situation, although amphotericin B has been on the market for many years, there still exist problems in the stability of amphotericin B, such as large hygroscopicity, reduced storage life or excessive increase of related substances, and these problems have not been solved for many years, and the general or even most professionals cannot see or imagine the problems! This has led us to seek solutions to the problem of stability of compounds. Although valuable results are expected to be obtained by obtaining changes of crystal forms and the like through some experiments on the basis of unchanged compound molecular formulas, obvious improvement is not obtained, and different problems still exist.

The size of the hygroscopicity of the powder medicine is closely related to the selection of proper packaging and storage conditions, and even the selection of proper preparation process and dosage form. It is a common phenomenon that changes in intrinsic quality occur due to changes in the appearance of a drug, such as agglomeration, deliquescence, discoloration, caused by improper packaging or storage conditions, and therefore, the hygroscopicity of a powdered drug has been examined as a property of the drug by some national pharmacopoeias. When a drug is exposed to air with certain humidity or an auxiliary material containing free water is contained in the same preparation, the drug can cause moisture absorption to change certain properties such as powder flowability, dispersibility, compactibility, tablet hardness and the like, and can also cause caking, deliquescence, stability reduction and even content change of the drug, so that the moisture absorption of the drug is an important characteristic influencing the stability, effectiveness and safety of the drug (bear Jing, rock, Wujian Min, Huchangchang, Tandle, Zealand, and the relationship between the moisture absorption and water solubility of the chemical is analyzed based on non-parametric tests [ J ]. China pharmaceutical journal, 2016,51(20):1786 and 1789 ]. The improvement of the hygroscopicity of the medicine and the physicochemical properties of the medicine, such as stability, melting point, solubility, hygroscopicity, metabolic stability, dispersion rate, dissolution rate, slow release of the medicine, mechanical properties, bioavailability and the like, are the leading edge and the focus of research of international crystal engineering at the present stage (chenjiao, synthesis and characterization of novel medicine co-crystals [ D ]. jilin university, 2011.).

At present, the moisture absorption test of the medicine is added no matter in European pharmacopoeia medicine standard or Chinese pharmacopoeia to examine the quality of the medicine and/or guide the research of innovative medicine or improved medicine.

Although medicinal chemistry and pharmacy are continuously developed practice science, new guiding pharmaceutical progress is continuously developed, pharmaceutical safety is continuously improved to guide the development of medicines and preparations and guide clinical medication, even though pharmacy influences the development of pharmacy, preparations and clinical medication by changing physical states of bulk drugs, however, due to difficulty and uncertainty of synthesis, documents which are not published at home and abroad so far report deep innovative research and precise synthesis of new stable chemical states of amphotericin B, and new compounds with determined chemical molecular formulas and molecular weights or structural formulas, such as omega-type crystal compounds, nu-type compounds and the like, which are easier to prepare or have better stability, and preparation methods and applications thereof.

Thermal analysis methods have important value and status in material science, chemistry or pharmaceutical analysis, etc., and can be used alone to detect or characterize changes in a compound or its polymorphic or crystalline form (lee, thermal analysis, university of qinghua press, first edition 8 months 1987). Differential Thermal Analysis (DTA) is a relatively common analytical method used for both qualitative and quantitative identification of substances, and is used by Barta et al to identify unknown compounds as early as the second international conference on thermal analysis in 1968. The pharmacopoeia of many countries has already collected the differential thermal analysis method, and over a decade ago, the differential thermal analysis method has been widely applied to chemical and pharmaceutical systems.

Disclosure of Invention

It is well recognized in the chemical arts that the presence or absence of a specific solvent compound for a compound is unpredictable, cannot be defined in advance by the Markush formula, and some solvent compounds are highly toxic, some are highly hygroscopic, some are easily weathered, have poor stability, and are not suitable for use as drug substances for pharmaceuticals. The invention is to carry out deep innovative research and accurate synthesis on the new chemical state of amphotericin B by antifungal drugs, which is not to prepare different crystal forms under the same molecular formula but to prepare a novel amphotericin B crystal hydrate with definite chemical structure (the molecular formula is C)₄₇H₇₃NO₁₇·nH₂O，n＝1.75、2、3.5]I.e. new gamma, omega and v compounds of amphotericin B, i.e. new molecular formula and molecular weight of amphotericin B and new molecular structure compounds and their preparation method and use.

In the process of completing the present invention, it was unexpectedly found that although the current literature reports that amphotericin B is the only choice in pharmacology, the present study found that amphotericin B anhydrate is not the best choice in pharmacology, and that its stability is not so good, which may lead to or lead to disqualification of the drug substance during storage or inaccurate levels during preparation of the formulation, etc. Moreover, due to the proximity of the preparation solvents, and more importantly, even during the preparation of crystalline hydrates of amphotericin B, it has been found that gamma-, omega-and ν -compounds of amphotericin B with better stability can be prepared more easily, which has long been overlooked by the skilled person.

Moreover, the invention also finds that the gamma type, omega type and gamma type compounds of the amphotericin B with definite molecular formula are easy to prepare, and the stability can meet the requirement of pharmacy. This reflects the fact that the gamma, omega and gamma compounds of amphotericin B of defined formula are more convenient to produce or use, which has long been ignored by those skilled in the art.

Further, in the preparation of amphotericin B according to the example of patent document CN 1029617C, solid citric acid is required to be dissolved, and at a relatively low temperature, for example, 15 ℃, the time required for the solid citric acid to be dissolved in the example of the document is about 2 to 8 times longer than the time required for the direct reaction of the acid-addition dissolution of the example 1 of the present invention, and at a lower temperature, there is a possibility that crystallization may occur, resulting in a decrease in the purity of the product.

The preparation of amphotericin B as described in example 1 of patent document CN 102382158B requires the use of acetic acid, which results in that the odor of acetic acid is not easily removed from the final product and the odor of acetic acid is more or less entrained in the product. In contrast, in examples 2 and 3 of the literature, hydrochloric acid is used, and industrial hydrochloric acid generally contains ferric ions, so that originally unstable amphotericin B is easily oxidized, and the content or the type of impurities in the product may be increased.

The gamma, omega and gamma compounds of amphotericin B with new molecular formula and new molecular structure obtained by the invention are surprisingly lower in hygroscopicity of amphotericin B containing crystal water than amphotericin B without crystal water, and the deliquescence of anhydrate ensures that air is isolated to prevent adhesion and the like during treatment. The amphotericin B crystal hydrates of the present invention having different novel molecular formulas and novel molecular structures have different advantages in stability and manufacturability. Furthermore, the invention finds that the gamma type, omega type and v type compounds of amphotericin B have better industrial value or medicinal value than amphotericin B.

Surprisingly, the hydrate of the invention has a corresponding endothermic peak under a weight loss platform (under a weight loss curve before about 140 ℃) of a thermogram of a weight loss platform (TG-DSC or TG-DTA), and an amphotericin B compound, an amphotericin B type compound, of a novel molecular formula and a novel compound structure is visually shown on the thermogram. Even if different crystalline forms of the same substance formula are prepared or obtained, they may have real or potential or future significance or value in pharmacology, not to mention that the obtaining of new substances of different formulae of the same drug may have real or potential or future significance or value in pharmacology.

The preparation of the novel amphotericin B compound comprises the following steps:

adding amphotericin B or amphotericin B anhydride or amorphous amphotericin B, dimethyl sulfoxide, dimethyl formamide DMF, dimethyl acetamide, water and/or organic solvent C into a reaction vessel₁-C₆Is selected from methanol, ethanol, isopropanol, n-butanol, propylene glycol, etc., and C₂-C₈Low molecular ether (selected from but not limited to diethyl ether, tetrahydrofuran, isopropyl ether, methyl tetrahydrofuran, etc.), C₂-C₆Controlling the temperature between 0-50 ℃, adding one or more of sodium hydroxide or potassium hydroxide or sodium carbonate or potassium carbonate or trisodium phosphate or organic amine (including but not limited to one or more of pharmaceutically acceptable ethanolamine, diethanolamine, triethanolamine, arginine, lysine, triethylamine, dimethylamine, trimethylamine, meglumine, and glucamine) solution, stirring for dissolving, filtering, adding one or more of lactic acid or dilute sulfuric acid or phosphoric acid solution into the filtrate, adjusting the pH value to between 4.8-6.2, standing below 0 ℃ to fully separate out the solid, filtering, and using organic solvent C₁-C₆Low molecular alcohol of (2), C₂-C₈Low molecular ether of (2), C₃-C₈Low molecular ketone of (2), C₂-C₆Low molecular nitrile of (2), C₂-C₈Low molecular ester of (2), C₁-C₆Washing one or more of the low molecular weight halogenated hydrocarbons for 1-3 times, filtering, and collecting the solid with dimethyl sulfoxide and dimethyl methylAmide DMF, water, C₁-C₆Low molecular alcohol of (2), C₂-C₈Low molecular ether of (2), C₃-C₈Low molecular ketone of (2), C₁-C₆A low molecular weight halogenated hydrocarbon (selected from but not limited to methylene chloride, chloroform, etc.), C₂-C₈Low molecular ester of (2), C₂-C₆One or more of low molecular nitrile and DMF as crystallization solvent, filtering, standing below 10 deg.C to precipitate crystals, filtering, mixing with water or organic solvent C1-C6 low molecular alcohol, C2-C8 lower ether, C3-C8 low molecular ketone, and C3-C8₂-C₈Washing one or more of the low molecular ester and C1-C6 lower halogenated hydrocarbon, filtering, and drying to obtain new amphotericin B compound;

wherein amphotericin B (weight g) and water, or C1-C6 low molecular weight alcohol, or C2-C8 lower ether (selected from but not limited to diethyl ether, tetrahydrofuran, isopropyl ether, methyl tetrahydrofuran, etc.), or C2-C6 lower nitrile (selected from but not limited to acetonitrile, propionitrile, etc.), C2-C6₁-C₆A low molecular weight halogenated hydrocarbon (selected from but not limited to methylene chloride, chloroform, etc.), C₂-C₈One or more of organic solvents such as low molecular ester, dimethyl sulfoxide, dimethylformamide, dimethylacetamide and the like in a weight-volume ratio is (are) generally: 1 (g): 5 to 100(ml), preferably in the ratio: 1 (g): 6-60 (ml); the volume ratio of water to the organic solvent used in the crystallization or recrystallization is generally 1: 1-300, preferably the ratio is: 1: 1 to 80.

The crystallization or recrystallization solvent of the novel amphotericin B compound is selected from one or more of water, acetonitrile, methanol, ethanol, isopropanol, n-butanol, acetone, ethyl acetate, butyl acetate, ethyl formate, diethyl ether, tetrahydrofuran, isopropyl ether, dichloromethane, chloroform, dimethyl sulfoxide, dimethyl formamide and the like; the amphotericin B crystal or recrystallization solvent is preferably one or more of water, acetonitrile, methanol, ethanol, isopropanol, n-butanol, propylene glycol, acetone, tetrahydrofuran, ethyl acetate, diethyl ether, isopropyl ether, dichloromethane, chloroform, dimethyl sulfoxide and dimethylformamide. In the preparation method of the invention, one or more of dimethyl sulfoxide, dimethyl formamide and the like are firstly used or mixed with alkali or acid to dissolve amphotericin B crystal hydrate or acid or mixed with alkali to neutralize, and then the organic solvent mentioned in the invention is used for crystallizing.

The carbon number of the organic solvent lower alcohol or low molecular alcohol in the present invention is defined as C1-C6 (i.e., alcohol of 1-6 carbon atoms), such as methanol, ethanol, isopropanol, etc.; the carbon atom number of the lower ether or low molecular ether is defined as C2-C8, such as diethyl ether, dibutyl ether, tetrahydrofuran, etc.; the lower halogenated hydrocarbon has carbon number defined as C1-C6, and includes dichloromethane, dichloroethane, chloroform, etc.; the number of carbon atoms of the lower ester is defined as C2-C8, and includes methyl acetate, butyl acetate, ethyl formate, etc., unless otherwise specified as a lower ester of formic acid; the C3-C8 low molecular ketone is defined as ketone with 3-8 carbon atoms, including acetone, butanone, isohexanone, etc.; the labeling method for the number of carbon atoms of any of the compounds described as "lower or low molecular" appears once in the text, and the number of carbon atoms of any of the other unlabeled compounds of the same class described as "lower or low molecular" is consistent with the number already indicated herein.

The product of the present invention may be dried at various temperatures (e.g., between 20-70 c), for drying times (e.g., 0.5 hours to several days), or in ambient conditions with other drying agents (including silica gel, phosphorus pentoxide, anhydrous calcium chloride, anhydrous sodium sulfate, etc.), or using atmospheric or reduced pressure. The drying temperature is preferably from 25 to 55 ℃.

The methods for detecting amphotericin B and novel compounds of amphotericin B in the present invention or examples can be referred to the methods under the section of amphotericin B in the second pharmacopoeia of china, 2015 edition. The moisture content of the novel amphotericin B compound or amphotericin B in the present invention is measured by the karl fischer method with dimethyl sulfoxide and DMF as solvents, with reference to the measurement of moisture content (2015 version chinese pharmacopoeia general convention 0832 first method 1).

The content of amphotericin B or a preparation thereof including an injection and a method for measuring the reference (a reference method (refer to the method of amphotericin B in the second part of the Chinese pharmacopoeia 2015 edition and the injection) and the chromatographic conditions of a high performance liquid chromatography detection method are that a chromatographic column is a KROMASIL 4.6mm multiplied by 250mm,5 mu m and C18 column, the detection wavelength is 383nm, the column temperature is 25 ℃, and the measuring method of amphotericin B in the second part of the Chinese pharmacopoeia 2015 edition is referred to for mobile phase, flow rate, sample injection amount and the like.

Powder X-ray diffraction can generally be used to characterize and/or identify polymorphic forms, for which the modifier "about" is used before the peak is reported when characterizing and/or identifying. This is common practice in the field of solid state chemistry in view of the inherent variation of the peak. Typical accuracy of the 2 theta X-axis values of the peaks of the powder pattern spectrum is on the order of + -0.2 deg. 2 theta, therefore, a powder X-ray diffraction peak occurring at "about 8.0 deg. 2 theta" means that the peak may be between 7.8 deg. 2 theta and 8.2 deg. 2 theta when measured on most X-ray diffractometers. The change in peak intensity is a result of how the individual crystals are oriented in the sample container relative to the external X-ray source, the orientation effect not providing structural information about the crystals. In one aspect, the present invention provides compounds of different novel molecular formulas and novel molecular structures of amphotericin B.

In another aspect, the invention provides different novel molecular formulas and novel molecular structures and methods for their preparation.

In another aspect, the invention provides a pharmaceutical composition comprising any one or more of the novel amphotericin B compounds prepared by the process of the invention, and one or more pharmaceutically acceptable excipients.

The present invention further provides a process for the preparation of a pharmaceutical formulation comprising combining any one or more of the novel compound formulations of amphotericin B prepared by the process of the present invention or with at least one or pharmaceutically acceptable excipients.

The invention further provides the use of the novel compound of amphotericin B in the preparation of a medicament or a pharmaceutical composition for treatment and the like, which has the same use as amphotericin B.

The amphotericin B new compound provided by the invention is more stable, and is beneficial to the preparation of medicines, quality control and clinical medication safety.

The new crystalline drug substance also expands the repertoire of materials available to formulation scientists for designing, for example, pharmaceutical dosage forms of the drug substance with a target release profile or other desired characteristic, the construction of libraries of compounds of the drug substance being of great importance, not only for comparative research purposes, etc., there is a need in the art for new crystals of amphotericin B or new crystalline hydrates of amphotericin B.

The novel compound of amphotericin B provided by the invention is a crystal substance, is convenient to filter and dry, and has higher stability than an amphotericin B anhydride.

The novel amphotericin B compound can be used for treating fungal infection or viral infection in combination with other drugs and the like.

The compound can be used for preparing medicines for treating or preventing cryptococcosis, North American blastomycosis, disseminated candidiasis, coccidioidomycosis, histoplasmosis, mucormycosis caused by mucor, aspergillus, Absidia, endophytic and frogmatophytosis, sporotrichosis caused by Sporothrix schenckii, aspergillosis caused by aspergillus fumigatus, chromomycosis, skin fungal infection after burning, respiratory candida infection, aspergillus or cryptococcus infection, fungal corneal ulcer, enterovirus infection, heat black disease, depression and the like.

The novel amphotericin B compound can be used for preparing a pharmaceutical composition with an antifungal synergist or an anti-toxin agent, and the pharmaceutical composition consists of an effective dose of the antifungal synergist, an effective dose of the novel amphotericin B compound and other pharmaceutically acceptable auxiliary materials. The antifungal synergist or the toxin reducing agent is selected from one or more of ganoderma lucidum extract, cinnamic acid, epigallocatechin gallate, curcumin, hyaluronic acid, lentinan and the like.

For example, the invention relates to an antifungal pharmaceutical composition and a medical application thereof, wherein the active ingredients of the pharmaceutical composition are as follows: (1) pharmaceutically acceptable antifungal agents such as fluconazole, itraconazole, ketoconazole, miconazole, voriconazole, hydroxytitaconazole, posaconazole, lafuticonazole and caspofungin; (2) novel amphotericin B compounds; wherein the weight ratio of (1) to (2) is selected from, but not limited to, 200: 1 to 1: 5.

The novel compound of amphotericin B of the invention is used for preparing compound pharmaceutical compositions for combined administration with other antifungal drugs.

The general route of administration of the novel amphotericin B compounds is selected from, but not limited to, oral or intravenous drip or topical: the usage amount can be similar to that of amphotericin B.

The hygroscopicity of the medicine is an important content for investigating the stability of the medicine and a necessary work in the process of medicine research, the selection of a more reasonable form of a compound as a raw material medicine of the medicine is one of the contents of medicine research, and the pharmacology always tends to select a compound with better stability as a raw material medicine, which is closely related to the safe, effective and accurate manufacture of the medicine in clinic and the controllable quality. The advantages of the novel amphotericin B compounds of the present invention are also shown below: the novel amphotericin B compound of the present invention is storage stable. The comparison study of the hygroscopicity test of the amphotericin B crystal hydrate and the anhydrous sample shows that the amphotericin B crystal hydrate and the anhydrous sample have the advantages.

1. Wicking test

The amphotericin B crystal hydrate of the present invention is more favorable for stable storage. Samples of the novel compound amphotericin B and anhydrate were subjected to the hygroscopicity test: respectively taking about 5g of amphotericin B anhydrate and about 5g of each new compound of the invention, placing the amphotericin B anhydrate and the new compound of the invention in a dry constant-weight surface dish, precisely weighing, placing in an experimental box with the temperature of about 25 +/-2 ℃ and the relative humidity of about 50 +/-5%, respectively sampling in test 0h and test 8h, and calculating the percentage of moisture-attracting weight increase, wherein the result shows that the anhydrate moisture-attracting rate has significant difference compared with the corresponding new compound of the amphotericin B of the invention, the new compound of the invention can be stored stably and better, and the result is shown in table 1.

TABLE 1 moisture wicking test results

The moisture absorption test result shows that the difference of the anhydrous moisture absorption property of the novel amphotericin B compound is very significant compared with the difference of the novel amphotericin B compound, the novel compound of the invention is observed to have no efflorescence phenomenon in the experimental process, and the novel amphotericin B compound can better and stably respond to the change of drying temperature, humidity and the like from two different angles, and is more beneficial to stable storage.

The administration dose of amphotericin B itself is very low, the effective dose is highly closely related to the dose of severe toxic reaction, and the accuracy of the preparation dose appears to be very important. The novel amphotericin B compound is different from deliquescence of anhydrous substances, so that air is isolated during treatment to prevent adhesion and the like, and the novel compound has good sliding property, so that the operability in the preparation process is improved, the dosage is more accurate in the preparation process, the yield of raw material medicines or preparations is improved, the unqualified products are prevented, the scrapping loss is forced to occur in the production process, or the unqualified products flow into the market.

The hygroscopicity experiment shows that the amphotericin B anhydride has lower storage stability than the amphotericin B new compound, but the pharmaceutical science always tends to select a compound with better stability as a raw material medicine, and obviously, the experiment shows that the amphotericin B new compound is a more reasonable better selection of the medicine form of the raw material medicine.

2. Comparative experiment for quality controllability of preparation

The raw material medicaments play an important role in controlling the quality of the preparation in the preparation process of the preparation. Because the content of each preparation cannot have absolute equality due to the existence of errors such as tablet weight in the preparation process or loading amount in the filling process, the marked amount of 90-110% of the main drug of the amphotericin B preparation for injection in the drug standard is used as the qualified index for controlling the content of the preparation. Nevertheless, the condition of formulations based on amphotericin B, at the same time, necessarily creates problems of content uniformity of the formulation. However, especially when the raw material drug is fed by 95% of the labeled amount of the main drug, if the raw material drug absorbs moisture to cause inaccurate sample weighing, the content of the finished product of the preparation must be controlled between 90% and 110% of the legal labeled amount of the main drug, and because the operation error in the manufacturing process cannot be avoided, the error is amplified, which leads to the great increase of the rejection rate of the finished product. The method comprises the steps of exposing raw material medicines (raw materials prepared by an embodiment method and amphotericin B anhydrate) with determined content of main drug amphotericin B of different sources in RH52 +/-5% and 25 +/-2 ℃ in the air for 3 hours, then respectively weighing samples to prepare qualified samples (the content specification is amphotericin B5 mg/unit) with the labeled amount of 90-110% according to the method of the embodiment 8 method of the specification, and when the raw material medicines are fed by 100% of the labeled amount of the main drug, for example, when the same weight of amphotericin B is accurately weighed according to absolute anhydrate calculation to prepare 200 unit preparations, the rest auxiliary materials and the mixture ratio are the same. The final preparation product is randomly sampled for 50 injections respectively to determine the content of the main drug of amphotericin B, the main drug content is disqualified when the content of the main drug exceeds 90-110% of the marked amount (5 mg/injection), and the experimental results are shown in the following table 2.

TABLE 2 comparative experimental results on the controllability of the preparation

Preparation samples	Percent defective of content in product
		EXAMPLE 8 method sample (example 1 method starting material)	4％
EXAMPLE 8 method sample (example 2 method starting material)	4％
		EXAMPLE 8 method sample (example 3 method starting material)	6％
EXAMPLE 8 method sample (example 4 method starting material)	2％
		EXAMPLE 8 method sample (amphotericin B anhydrate starting material)	12％

The method comprises the steps of exposing raw material medicines (raw materials prepared by an embodiment method and amphotericin B anhydrate) with determined content of amphotericin B in different sources in RH52 +/-5% and 25 +/-2 ℃ in the air for 3 hours, then respectively weighing samples to prepare qualified samples (the content specification is amphotericin B10 mg/unit) with the marked amount of 90-110% according to the method of the embodiment 9 method of the specification, and when the raw material medicines are fed by 95% of the marked amount of the main material medicines, precisely weighing 0.95 g of the main material medicine of the amphotericin B with the same weight according to the calculation of absolute anhydrate to prepare 200 unit preparations, the rest auxiliary materials and the mixture ratio are the same. The final preparation product was randomly sampled by 50 injections to determine the content of the main drug, and the main drug content exceeding 90-110% of the labeled amount (10 mg/injection) was determined as unqualified, and the experimental results are shown in Table 3 below.

TABLE 3 comparative experimental results on the controllability of the preparation

The experimental results show that for the same preparation process, the product reject ratio of the amphotericin B anhydrous serving as the raw material feeding greatly exceeds that of the novel amphotericin B compound, obviously, the novel amphotericin B compound is more beneficial to the quality controllability in preparation manufacturing and the improvement of the qualified rate of finished preparations.

The novel amphotericin B compound of the present invention has the following uses: the compound or the amphotericin B can be used for preparing solid preparations, suppositories, injections, microemulsions, nano preparations, emulsions, lotions, ointments or creams or gels (including ophthalmic gels and the like), transdermal preparations, external preparations, aerosols, inhalants and pharmaceutically acceptable preparations, wherein the injections are selected from but not limited to lyophilized powder injections for injection, small-volume injections, sterile subpackaged powder injections, large infusion preparations, injectable liposome preparations or injectable microsphere preparations, microemulsion preparations for injection, nano preparations for injection and the like, and the large infusion preparations are selected from but not limited to sodium chloride injections, bottled or bagged large infusions, double-chamber instant large infusions, non-PVC solid-liquid double-chamber instant large infusions, non-PVC multilayer co-extruded films to prepare instant large infusions and other pharmaceutically acceptable large preparations; the solid preparation is selected from, but not limited to, tablets, capsules, liposome-containing preparations, and the like.

The novel compound of amphotericin B or the pharmaceutical composition thereof is used for preparing pharmaceutically acceptable solid preparations, and the used auxiliary materials can comprise fillers, lubricants, binders, disintegrants, antioxidants, emulsifiers, preservatives or stabilizers and the like.

Can be used for preparing pharmaceutically acceptable tablet (including enteric coated tablet, rapidly disintegrating tablet, effervescent tablet, vaginal effervescent tablet, etc.), capsule (including enteric coated capsule, delayed release capsule), etc., and can contain pharmaceutically acceptable adjuvants or carriers, such as filler such as starch, modified starch, lactose, microcrystalline cellulose, cyclodextrin, sorbitol, mannitol, calcium phosphate, amino acids, etc.; pharmaceutically acceptable disintegrants such as starch, modified starch, microcrystalline cellulose, croscarmellose, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, surfactants (sodium lauryl sulfate, etc.); pharmaceutically acceptable wetting agents and binders such as gelatinized starch, methyl cellulose, sodium carboxymethyl cellulose, ethyl cellulose, low-substituted hydroxypropyl cellulose, polyvinylpyrrolidone, alginic acid and salts thereof, and the like; pharmaceutically acceptable lubricants and glidants, such as stearic acid, magnesium stearate, polyethylene glycol 4000-; pharmaceutically acceptable sweetening agents and flavoring agents, such as aspartame, sodium cyclamate, saccharin sodium, sucralose, edible flavoring agents, etc.

An injection of a novel compound amphotericin B, prepared by:

preparing sterile subpackaged powder injection: the packaging is carried out using sterile raw materials as is customary.

The large infusion preparation comprises large infusion in bottle or bag, large infusion in dual-chamber instant type, large infusion in non-PVC solid-liquid dual-chamber instant type, and large infusion in instant type prepared by non-PVC multilayer co-extrusion film, and can be prepared by conventional method.

The preparation method of the freeze-dried powder injection preparation comprises the following steps: taking amphotericin B crystal hydrate, adding pharmaceutically acceptable cosolvent, freeze-drying supporting agent or auxiliary agent, stabilizing agent and water for injection, stirring to dissolve, adjusting pH to 7.2-8.0 by pharmaceutically acceptable acid and alkali if necessary, adding 0.005-0.5% (W/V) of activated carbon, stirring for 15-45 min, filtering, replenishing water, performing sterile filtration, subpackaging according to 5-20 mg/bottle, freeze-drying and plugging to obtain a finished product.

The freeze-drying supporting agent or auxiliary agent is selected from one or more of sodium dehydrocholate, sodium cholesteryl sulfate, xylitol, sorbitol, mannitol, invert sugar, maltose, dextran, maltitol, lactitol, erythritol, sodium chloride, sodium lactate, sodium sulfate and the like. Sorbitol includes one or more of D-sorbitol, anhydrous sorbitol or sorbitol hemihydrate, 1 water sorbitol or instant sorbitol, etc.

Pharmaceutically acceptable excipients also include, but are not limited to: solubilizer (which can include surfactant) selected from polyoxyethylene sorbitan monooleate, tween-80, vitamin E succinic acid polyethylene glycol ester (vitamin E TPGS), glycerol-polyethylene glycol oxystearate, PEG-32 glyceryl palmitostearate, sodium lauryl sulfate, sorbitan monolaurate, polyethylene glycol 400-, sodium dehydrocholate, sodium cholesteryl sulfate, pharmaceutically acceptable Lewis acid or Lewis base, amino acid or medicinal salt thereof, pharmaceutically acceptable alcohol, pharmaceutically acceptable polyol, poloxamer 407, poloxamer 188, azone, laurocapram, cyclodextrin or cyclodextrin pharmaceutically acceptable derivative.

The heat source removing and sterilizing mode can be activated carbon heat source removing with the addition of 0.005-3% of the preparation amount, microporous filter membrane sterilizing and hot-pressing sterilizing, and ultrafiltration sterilizing and heat source removing can also be adopted. In the ultrafiltration method, the ultrafiltration filter can be flat plate type, roll type, tubular type, hollow fiber type or round box type, and the like, preferably roll type and hollow fiber type ultrafiltration filters, most of heat-generating substances and bacteria are removed by adopting a filter membrane with the intercepted relative molecular mass of 5-30 ten thousand, and then residual heat sources are removed by adopting an ultrafiltration membrane with the intercepted relative molecular mass of 3000-60000, and preferably the ultrafiltration membrane with the intercepted relative molecular mass of 6000-20000.

The pharmaceutically acceptable pH regulator can be pharmaceutically acceptable inorganic acid or organic acid, inorganic base or organic base, or generalized Lewis acid or base, and can contain one or more of hydrochloric acid, phosphoric acid, propionic acid, acetic acid and acetate, such as sodium acetate, etc., lactic acid and lactic acid medicinal salt, citric acid medicinal salt, sodium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, phosphate, tartaric acid and medicinal salt thereof, borax, boric acid, succinic acid, hexanoic acid, adipic acid, fumaric acid, maleic acid, polyhydroxy carboxylic acid and medicinal salt, such as glucuronic acid, gluconic acid, lactobionic acid, malic acid, threonic acid, glucoheptonic acid, etc.

The pharmaceutically acceptable antioxidant and stabilizer thereof is selected from but not limited to sulfurous acid, sulfite, bisulfite, pyrosulfite, dithionite, thiosulfate, organic sulfur compound thiourea, glutathione, dimercaprol, thioglycolic acid and salts, thiolactic acid and salts, thiodipropionic acid and salts, phenol compounds such as gallic acid and salts, caffeic acid and salts, ferulic acid and salts, di-tert-butyl-p-phenol, 2, 5-dihydroxybenzoic acid and salts, salicylic acid or salts; ascorbic acid and salts thereof, erythorbic acid and salts thereof, nicotinamide, tartaric acid, nitrate, phosphate, acetic acid medicinal salts, citrate, EDTA and EDTA salts, including one or more of EDTA disodium, EDTA tetrasodium or hydrates thereof, calcium edetate sodium salts (including sodium calcium edetate or sodium calcium 2 hydrate, sodium calcium edetate 4 hydrate), N-di (2-hydroxyethyl) glycine and the like.

The novel amphotericin B compound can be used for preparing liposome and pharmaceutically acceptable preparations thereof, comprises the novel amphotericin B compound, neutral phospholipid and cholesterol and/or negatively charged phospholipid and/or long-circulating phospholipid, and the raw material amount can be as follows according to each 1000mL of liposome solution:

wherein: the molar ratio of the neutral phospholipid to the cholesterol is 1: 2-1: 0.1, the molar ratio of the neutral phospholipid to the negatively charged phospholipid is 5: 5-5: 0.1, and the weight ratio of the amphotericin B new compound to the phospholipid is 0.01: 1-1: 3.

The preferred amount of starting material per 1000mL of liposome solution is:

the liposome includes, but is not limited to, neutral phospholipid selected from soybean lecithin, yolk lecithin, hydrogenated soybean lecithin, hydrogenated yolk lecithin, distearic acid lecithin or distearic acid lecithin.

The negatively charged phospholipid contained in the liposome is selected from, but not limited to, dimyristoyl phosphatidylglycerol, dilauroyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, dimyristoyl phosphatidic acid, distearoyl phosphatidic acid, dilauroyl phosphatidic acid, dipalmitoyl phosphatidic acid, dioleoyl phosphatidylserine, or dilinoleoyl phosphatidylinositol.

The liposome comprises long-circulating phospholipid material selected from, but not limited to, PEG-DSPE formed by reaction of polyethylene glycol (PEG) with different molecular weights and distearoyl phosphatidyl ethanolamine (DSPE).

The liposome is prepared by the following method and steps:

respectively weighing neutral phospholipid, negatively charged phospholipid, long-circulating phospholipid, cholesterol or cholesterol sodium sulfate according to the formula ratio, dissolving in a eggplant-shaped bottle by using solvents such as dimethyl sulfoxide or chloroform, performing reduced pressure evaporation on a rotary evaporator to remove the solvents such as dimethyl sulfoxide or chloroform, forming a uniform lipid film on the wall of the bottle, adding sodium lactate or sodium citrate buffer solution with the pH value of 7.0-7.5 into the lipid film, shaking until the lipid film is completely hydrated, and preparing blank liposome by using a micro extruder. Adding a pH regulator solution, regulating the pH to 7.2-8.0, mixing the blank liposome solution with a prescription amount of the amphotericin B new compound sucrose solution, and placing in a water bath at 55-75 ℃ for heat preservation for 15-30min to obtain the amphotericin B liposome.

The preparation method of the liposome can also be prepared by the following steps:

respectively weighing neutral phospholipid, negatively charged phospholipid, long-circulating phospholipid, cholesterol or cholesterol sodium sulfate according to the formula ratio, dissolving in a eggplant-shaped bottle by using solvents such as dimethyl sulfoxide or chloroform, and removing the solvents such as chloroform by reduced pressure evaporation on a rotary evaporator to form a uniform lipid film on the wall of the bottle. Adding a proper amount of pharmaceutically acceptable salt solution into a lipid film, shaking until the lipid film is completely hydrated, preparing blank liposome by using a miniature extruder, eluting the blank liposome solution with 5-15% of sucrose solution, passing through a Sephadex G50 gel column or replacing an external water phase by a dialysis method (the dialysate is 5-15% of sucrose solution), adding the amphotericin B new compound sucrose solution with the prescription amount, mixing, and placing in a water bath at 55-75 ℃ for heat preservation for 15-30min to obtain the amphotericin B.

The entrapment rate of the liposome prepared by the invention is more than 80%, the liposome is round and smooth in surface and is obtained by measuring the particle size through the observation of a projection electron microscope, the distribution of the liposome is narrow, and the particle size is about 80-150 nm.

The amphotericin B liposome injection comprises a liposome membrane and amphotericin B embedded in a water phase of the liposome membrane, wherein the drug-to-lipid ratio (w/w) of the amphotericin B to the liposome membrane is 0.01: 1-1: 3.

The liposome membrane contains but is not limited to soybean hydrogenated lecithin (HSPC), cholesterol or cholesterol sodium sulfate, polyethylene-ethylene glycol-distearoyl phosphatidyl ethanolamine (DSPE-PEG2000) and the like.

The liposome membrane may be composed of soybean hydrogenated lecithin for injection (HSPC), cholesterol or and sodium cholesterol sulfate and polyethylene-ethylene glycol-distearoyl phosphatidyl ethanolamine (DSPE-PEG 2000).

The liposome provided by the invention can effectively avoid phagocytosis of a reticuloendothelial system, the stability and the encapsulation rate are improved, the leakage rate is reduced, and the systemic circulation time of the liposome is obviously prolonged. The liposome has remarkably increased concentration in focal tissue, reduced concentration in organs such as heart bone marrow, and reduced toxicity.

Generally, when the novel amphotericin B compound or pharmaceutical composition thereof of the present invention is administered intravenously, it can be dissolved in a volume of 50ml or more of sodium chloride injection or 5% glucose injection and the like and then instilled.

Drawings

FIG. 1 is a thermogram spectrum of amphotericin B2 hydrate (example 1)

FIG. 2 is a powder X-ray diffraction pattern of amphotericin B2 hydrate (example 1)

FIG. 3 is a thermogram spectrum of amphotericin B1.75 hydrate (example 3)

FIG. 4 is a thermogram spectrum of amphotericin B3.5 hydrate (example 4)

Detailed Description

Other than in the examples, and where otherwise indicated, all numbers expressing quantities of ingredients used in the specification and claims are to be understood as being modified in all instances by the term "about", and thus, unless otherwise indicated, the numerical parameters set forth in this specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure, and at the very least, and are not intended to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations. The numerical values set forth in the specific examples are reported as precisely as possible, and any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing.

It is noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise, and thus, for example, if reference is made to a composition containing "a compound" includes a mixture of two or more compounds, it is further noted that, unless the context clearly dictates otherwise, the term "or" typically includes "and/or".

As used herein, the term "obtained" or "obtaining" refers to isolating a compound of value at a level of content or purity, including but not limited to, greater than 90%, 95%, 96%, 97%, 98%, and 99%. The content or purity level can be determined by, but is not limited to, high performance liquid chromatography methods or other literature methods specified in pharmacopoeial standards for amphotericin B. The infrared spectrum data of the sample is measured by a Fourier transform infrared spectrometer, and the used instruments comprise a Nexus intelligent Fourier transform infrared spectrometer (Thermo Nicolet) and the like.

The present "solvate" is meant herein to also include crystalline forms of molecules, atoms and/or ions of solvent molecules that penetrate into the crystal structure, which may be in a regular and/or disordered arrangement, and which are solvates of the present invention.

Polymorphism herein refers to crystals having the same chemical composition but differing in the spatial arrangement of the molecules, atoms and/or ions that form the crystal.

The pharmaceutical composition comprises: as used herein, "pharmaceutical composition" refers to a composition of matter that may contain at least one pharmaceutically acceptable adjuvant or carrier.

As used herein, "pharmaceutically acceptable adjuvant or carrier" refers to a pharmaceutically acceptable carrier or vehicle suitable for administration of the compounds provided herein, including any such carriers known to those skilled in the art to be suitable for a particular mode of administration.

In the present invention, the "which" in the pharmaceutically acceptable salt or solvate thereof or the clathrate thereof represents one of them or either of them unless otherwise specified.

In the present invention, unless otherwise specified, "suitable amount" means a preferred or optimum amount or the minimum required amount or mass or weight or volume or the like required for carrying out the present invention.

In the present invention, unless otherwise specified, "such a combination or a combination thereof" means a multi-component mixture of the respective elements described, for example, two, three, four and up to the maximum possible multi-component mixture.

In the present invention, all "parts" and percentages (%) may refer to parts by weight or percent by weight volume, unless otherwise indicated.

When preparing the sterile raw materials, solvents such as sterile water for injection or sterile solvents, or raw and auxiliary materials or packaging materials or facilities are used, and the equipment, facilities and the environment are subjected to cleaning treatment or sterilization.

Amphotericin B anhydrate: the amphotericin B anhydrous substance can be obtained by taking a commercially available amphotericin B anhydrous substance sample for experiment, or taking a commercially available amphotericin B sample or prepared according to a literature method, drying the amphotericin B sample for about 4 hours under high vacuum at about 80 ℃ in a vacuum drying oven, then placing a triangular flask containing sufficient sodium hydroxide in the vacuum drying oven, continuously keeping high vacuum at room temperature for drying for three days to obtain the amphotericin B anhydrous substance, and measuring the moisture content by a Karl Fischer method to be less than or equal to about 0.7 percent.

For a further understanding of the invention, reference will now be made to the preferred embodiments of the present invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the present invention, and not to limit the scope of the claims.

Infrared spectrum: potassium bromide pellets, and the IR spectral data of the samples were determined using an instrument comprising U.S. thermoelectric corporation NICOLET 5700 FTIR Spectrometer, Nexus intelligent fourier transform infrared Spectrometer (Thermo Nicolet), and the like. Name of infrared spectrometer instrument company: the NiCOLET 5700 FTIR Spectrometer of the United states thermoelectric company uses the function of middle infrared 4000-^-1Resolution of 4cm^-1The maximum can reach 0.09cm^-1。

Thermal analysis method

And (3) testing conditions are as follows: setsys 16, Setaram corporation, sample size about 3-10mg, rate of temperature rise: 10K/min, N2 flow rate: 50ml/min, temperature: generally, the temperature is about room temperature to 400 ℃.

Surprisingly, characteristically, the hydrates of the present invention have a corresponding endothermic peak at the weight loss plateau of the thermogram (TG-DTA or TG-DSC) showing the crystalline hydrate of amphotericin B, such as its 2 hydrate, etc.

Powder X-ray diffraction method

Using a D/MX-III A X radiation diffractometer, voltage: about 30-60kv, current: about 30-100mA, scanning speed: 10 DEG/min, a copper target, a wavelength wavelengh (A) of 1.54, a diffraction angle 2 theta, a scanning range of 3-60 DEG, and a powder X-ray diffraction pattern of amphotericin B crystal hydrate is measured, wherein all peak positions are within +/-0.2 DEG 2 theta; or by using D8 Advance X-ray diffractometer of Bruker, Germany, wavelength

1.54, diffraction angle 2 theta, scan range 3-60 deg., and other indices (voltage, current, etc.) approximately as before, the samples were measured.

Figure 2 of example 1 in this specification is corroborated with the following data.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

EXAMPLE 1 preparation of amphotericin B2 hydrate (amphotericin B omega type compound)

In a 250ml flask, 10g of amphotericin B, 8ml of dimethyl sulfoxide, 120ml of methanol and 10ml of isopropanol are added50ml of water, introducing nitrogen for protection, stirring, keeping the temperature at about 30-45 ℃, adding 8% of sodium hydroxide solution, adjusting the pH value to about 7.4, stirring to be completely dissolved, adding a small amount of activated carbon, stirring for about 15 minutes, filtering, adding 60% of lactic acid solution into filtrate, adjusting the pH value to about 5.5, adding 8ml of dichloromethane, stirring, standing at 0-minus 10 ℃, fully precipitating after precipitation, performing suction filtration, washing a small amount of chloroform, water and ethanol for three times, performing suction filtration, spreading the obtained solid, and performing forced air drying for 3 hours at about 50 ℃ in an oven to obtain about 6.5g of yellow solid; and (3) identification: HPLC: the retention time of the HPLC main peak in the content determination is consistent with that of the amphotericin B reference substance; ② referring to the method under the item B of amphotericin in the second pharmacopoeia of 2015 edition, taking the product, adding water to prepare 3% suspension, and measuring according to the method to obtain the pH value of 5.66; the Karl method determines that the water content is 3.86%, and the thermal analysis shows that the platform weight loss is about 3.93% (see figure 1), and the corresponding endothermic peak (DTA) is arranged under the weight loss platform before about 140 ℃, which is in an error range with the result that the sample contains 2 crystal water (the theoretical value is 3.75%); infrared spectrum (v)^KBr _max cm^-1): 3416.7, 3010.2, 2975.5, 2959.2, 2922.8, 2855.1, 1718.4, 1690.2, 1634.5, 1562.4, 1451.0, 1402.1, 1322.6, 1187.0, 1132.0, 1069.3, 1039.4, 1010.0, 887.0, 850.7, 755.1, 693.9; x powder diffraction: a plurality of distinct characteristic peaks (powder X-ray diffraction) measured in the range of 3-60 ° at diffraction angle 2 θ (see figure 3): 4.83, 9.43, 11.36, 11.97, 14.00, 15.19, 17.20, 18.36, 18.995, 20.11, 20.75, 21.32, 21.61, 22.90, 23.43, 25.53, 26.75, 27.62, 32.31, 40.18; amphotericin B content (see methods under the section amphotericin B of pharmacopeia second part of 2015 edition): 1032 IU/mg.

Example 2 preparation of amphotericin B2 hydrate (amphotericin B omega type compound)

Adding 10g of amphotericin B, 10ml of dimethylformamide, 120ml of 95% ethanol, 50ml of acetone and 40ml of water into a 250ml flask, introducing nitrogen for protection, stirring, maintaining the temperature between 35 and 50 ℃, adding a 5% potassium hydroxide solution, adjusting the pH value to about 7.1, stirring for dissolution, adding a small amount of activated carbon, stirring for about 20 minutes, filtering, adding 40% milk into the filtrateAcid solution, adjusting the pH value to about 5.5, adding 5ml of trichloromethane, stirring, standing at 0-minus 10 ℃, fully precipitating the precipitate, performing suction filtration, washing with a small amount of water and ethanol for four times, performing suction filtration, spreading the obtained solid, and performing forced air drying in an oven at 40 ℃ for about 3 hours to obtain 7.3g of yellow crystalline solid; and (3) identification: HPLC: the HPLC peak retention time in normalized assay was consistent with that of amphotericin B anhydrate; ② acidity (refer to the method under the item B of amphotericin in the second pharmacopoeia of 2015 edition), taking the product, adding water to prepare 3% suspension, and determining according to the method that the pH value is 5.62; the Karl method determines that the water content is 3.86%, the thermal analysis is that the platform weight loss is about 3.93%, the corresponding endothermic peak (DTA) is provided under the weight loss platform before about 140 ℃, and the result is in the error range with the result that the sample contains 2 crystal waters (the theoretical value is 3.75%); infrared spectrum (v)^KBr _max cm^-1): the peak positions were substantially identical to those of the infrared spectrum of example 1; amphotericin B content (see methods under the section amphotericin B of pharmacopeia second part of 2015 edition): 1027 IU/mg.

Example 3 preparation of amphotericin B1.75 hydrate (amphotericin B. gamma. type compound)

Adding 10g of amphotericin B into a 250ml flask, adding 5ml of dimethyl sulfoxide, adding 80ml of 95% ethanol, 30ml of isopropanol and 40ml of water, introducing nitrogen for protection, stirring, maintaining the temperature between 35 and 40 ℃, adding 6% sodium hydroxide solution, adjusting the pH value to about 7.5, stirring until the mixture is dissolved, adding a small amount of activated carbon, stirring for about 20 minutes, filtering, adding 40% lactic acid solution into filtrate, adjusting the pH value to about 5.5, adding 5ml of dichloroethane, stirring, standing at 0 to-10 ℃, fully precipitating to obtain a precipitate, performing suction filtration, washing a small amount of water and ethanol for four times, performing suction filtration, spreading the obtained solid, drying in a baking oven at 40 ℃ in vacuum for about 3 hours to obtain about 7.0g of yellow crystalline solid; and (3) identification: HPLC: the HPLC peak retention time in normalized assay was consistent with that of amphotericin B anhydrate; ② acidity (refer to the method under the item B of amphotericin in the second pharmacopoeia of 2015 edition), taking the product, adding water to prepare 3% suspension, and determining according to the method that the pH value is 5.62; the Karl method determines that the water content is 3.41%, and the thermal analysis shows that the platform weight loss is about 3.33% (see figure 3), and the corresponding endothermic peak (DTA) is arranged under the weight loss platform before about 140 ℃, which is in an error range with the result that the sample contains 1.75 crystal water (the theoretical value is 3.30%); amphotericin B content (see methods under the section amphotericin B of pharmacopeia second part of 2015 edition): 1036 IU/mg.

Example 4 preparation of amphotericin B3.5 hydrate (amphotericin B v type compound)

Adding 10g of amphotericin B into a 250ml flask, adding 5ml of dimethyl sulfoxide, adding 100ml of 95% ethanol, 50ml of tetrahydrofuran and 50ml of water, introducing nitrogen for protection, stirring, maintaining the temperature between 50 and 60 ℃, adding 6% sodium hydroxide solution, adjusting the pH value to about 7.3, stirring until the mixture is dissolved, adding a small amount of activated carbon, stirring for about 20 minutes, filtering, adding 20% lactic acid solution into the filtrate, adjusting the pH value to about 5.5, adding 5ml of dichloroethane, stirring, standing at 0 to-10 ℃, fully precipitating the precipitate, performing suction filtration, washing the solid with a small amount of water and methanol for three times, performing suction filtration, spreading the obtained solid to be thin, and performing forced air drying at 35 ℃ in an oven for about 4 hours to obtain 7.2g of yellow crystalline solid; and (3) identification: HPLC: the retention time of the HPLC main peak in the content measurement is consistent with that of the amphotericin B anhydride; ② acidity (refer to the method under the item B of amphotericin in the second pharmacopoeia of 2015 edition), taking the product, adding water to prepare 3% suspension, and measuring according to the method to obtain pH value of 5.64; the moisture content measured by the Karl method is 6.31%, the thermal analysis shows that the plateau weight loss is about 6.37%, and the corresponding endothermic peak (DTA) is provided under the weight loss plateau before about 140 ℃ (see figure 4), which is within the error range of the result that the sample contains 3.5 crystal water (theoretical value is 6.39%). Amphotericin B content (see methods under the section amphotericin B of pharmacopeia second part of 2015 edition): 1013 IU/mg.

EXAMPLE 5 preparation of amphotericin B crystalline hydrate lyophilized powder for injection (prescription: 1000 bottles)

Prescription: amphotericin B crystal hydrate (prepared by the method of example 1 or example 2, the weight of the main drug is calculated by amphotericin B) 10g, deoxysodium cholate 40g, sorbitol 40g, triethanolamine 2g, EDTA disodium 2 hydrate 0.1g, about 1M solution of lactic acid and trisodium phosphate, proper amount, and proper amount of water for injection;

taking amphotericin B crystal hydrate, sorbitol, sodium deoxycholate, triethanolamine and EDTA disodium 2 hydrate according to the prescription amount, putting the amphotericin B crystal hydrate, sorbitol, sodium deoxycholate, triethanolamine and EDTA disodium 2 hydrate into a liquid preparation tank, adding about 1500ml of water for injection, adjusting the pH to 7.5-8.0 by about 1M of lactic acid and trisodium phosphate solution, stirring to dissolve, adding about 2000ml of water for injection to make the volume of the solution reach, adding 0.01% (W/V) of activated carbon, stirring for 30min, filtering by using a 0.22 micron microporous filter membrane, subpackaging according to 10 mg/bottle of the weight of the main drug, freezing at-55 to-45 ℃ for about 4 hours, freezing and drying at-45 to-10 ℃ for about 10 hours in vacuum, drying at-10 ℃ for about 6 hours in vacuum, pressing and rolling an aluminum cap.

EXAMPLE 6 preparation of amphotericin B crystalline hydrate lyophilized powder for injection (prescription: 1000 bottles)

Prescription: 5g of amphotericin B crystal hydrate (prepared by the method of example 1 or example 2, the weight of the main drug is calculated by amphotericin B), 20g of deoxysodium cholate, 50g of mannitol, 3g of arginine, 0.6g of EDTA calcium sodium 4 hydrate, a proper amount of lactic acid and sodium hydroxide solution with about 1M, and a proper amount of water for injection;

taking amphotericin B crystal hydrate, deoxysodium cholate, arginine, mannitol and EDTA calcium sodium in a prescription amount, adding about 2000ml of water for injection, adjusting the pH value to about 7.5 by using about 1M lactic acid and sodium hydroxide solution, stirring to dissolve, adding about 2500ml of water for injection, adding 0.02(W/V) of activated carbon, stirring for 15-30min, filtering by using a 0.22 micron microporous filter membrane, subpackaging according to 5mg of the weight of a main drug per bottle, freezing at-50 to-40 ℃ for about 4 hours, freezing and drying at-45 to-10 ℃ for about 25 hours in vacuum, drying at-10 to-26 ℃ for about 6 hours in vacuum, pressing and rolling an aluminum cap to obtain a finished product.

EXAMPLE 7 preparation of amphotericin B New Compound lyophilized powder for injection (prescription: 1000 bottles)

Prescription: amphotericin B crystal hydrate (prepared by the method of example 1) 10g, sodium deoxycholate 50g, sorbitol 40g, calcium sodium EDTA 4 hydrate 0.2g, meglumine 6g, 2M malic acid solution and 1M sodium hydroxide solution in appropriate amounts, and water for injection to a total amount of 3000ml

The preparation process comprises the following steps: weighing or preparing raw and auxiliary materials according to a prescription, sequentially stirring the components of the prescription amount with a proper amount of fresh water for injection in a liquid preparation tank, adjusting the pH value to 7.8 by using a proper amount of malic acid solution and sodium hydroxide solution, stirring to dissolve, adding the water for injection to the full amount, uniformly stirring and circularly filtering a 0.22 mu m microporous filter membrane, subpackaging the filtrate according to the weight of 10 mg/bottle of the main drug, freezing for about 4 hours at the temperature of-45 to-35 ℃, freezing and drying for about 18 hours at the temperature of-45 to-10 ℃, vacuum drying for about 6 hours at the temperature of-10 to 20 ℃, pressing a plug, and rolling an aluminum cover to obtain a finished product.

EXAMPLE 8 preparation of amphotericin B New Compound lyophilized powder for injection (prescription: 200 bottles)

Prescription: amphotericin B crystal hydrate (prepared by the method of example 2, weight on anhydrous basis) 1g, sodium deoxycholate 4g, disodium hydrogen phosphate 1g, sorbitol 50g, calcium sodium EDTA 4 hydrate 0.05g, appropriate amount of 2M citric acid solution and 1M sodium hydroxide solution, and water for injection added to total amount of 600ml

The preparation process comprises the following steps: weighing or preparing raw and auxiliary materials according to a prescription, sequentially stirring the components of the prescription amount with a proper amount of fresh water for injection in a liquid preparation tank, adjusting the pH value to 7.6 by using a proper amount of citric acid solution and sodium hydroxide solution, stirring to dissolve, adding the water for injection to the full amount, uniformly stirring, filtering twice by using a 0.22 mu m microporous filter membrane, subpackaging the filtrate by using 3ml of injection extraction solution according to 3 ml/branch, freezing at-55 to-40 ℃ for about 4 hours, vacuum freeze-drying at-45 to-10 ℃ for about 16 hours, vacuum-drying at-10 to-25 ℃ for about 6 hours, and pressing and rolling an aluminum cover to obtain a finished product.

EXAMPLE 9 preparation of amphotericin B New Compound lyophilized powder for injection (prescription: 200 bottles)

Prescription: amphotericin B novel compound (prepared by the method of example 4, weight on anhydrous basis) 2g, sodium deoxycholate 7g, disodium hydrogen phosphate 2g, meglumine 3g, sorbitol 50g, calcium sodium EDTA 4 hydrate 0.1g, appropriate amount of 2M malic acid solution and 1M sodium hydroxide solution, and water for injection added to total amount of 600ml

The preparation process comprises the following steps: weighing or preparing raw and auxiliary materials according to a prescription, sequentially stirring the components of the prescription amount with a proper amount of fresh water for injection in a liquid preparation tank, adjusting the pH value to 7.6 by using a proper amount of malic acid solution and sodium hydroxide solution, stirring to dissolve, adding the water for injection to the full amount, uniformly stirring, filtering twice by using a 0.22 mu m microporous filter membrane, filling the filtrate with 3ml of an injection extraction solution into an ampoule according to the volume of 3ml per ampoule, freezing for about 4 hours at the temperature of-50 to-40 ℃, vacuum freeze drying for about 24 hours at the temperature of-45 to-10 ℃, vacuum drying for about 6 hours at the temperature of-10 to 25 ℃, plugging, and rolling an aluminum cover to obtain a finished product.

Example 10: preparation of amphotericin B New Compound Capsule (prescription 1000 granules)

Prescription: amphotericin B New Compound 5g

Microcrystalline cellulose 40g

Magnesium stearate 0.5g

The amphotericin B new compound (prepared by the method of example 1), microcrystalline cellulose and magnesium stearate in the prescribed amounts were sieved through 100 mesh sieve, mixed well and filled into No. 2 capsules.

Example 11: preparation of amphotericin B New Compound Capsule (prescription 1000 granules)

The amphotericin B new compound (prepared by the method of example 2), microcrystalline cellulose, beta-cyclodextrin and magnesium stearate in the prescribed amounts were sieved through a 100 mesh sieve, mixed well and filled into a No. 1 capsule.

Example 12: preparation of amphotericin B New Compound Capsule (prescription 1000 granules)

The formula amount of the amphotericin B new compound (prepared by the method of example 3), epigallocatechin gallate, microcrystalline cellulose and magnesium stearate are sieved by a 100-mesh sieve, mixed evenly and filled into a No. 1 capsule.

EXAMPLE 13 amphotericin B New Compound tablet (prescription 1000 tablet)

The amphotericin B new compound (prepared by the method of example 1), the instant sorbitol, the microcrystalline cellulose, the low-substituted hydroxypropyl cellulose and the magnesium stearate in the prescription amount are sieved by a 100-mesh sieve, mixed uniformly and pressed into large tablets, the tablets are ground and pressed into granules of 18-24 meshes, the magnesium stearate sieved by the 100-mesh sieve is added, mixed uniformly and pressed into tablets.

EXAMPLE 14 preparation of amphotericin B New Compound ointment (size 10 g/stick)

Prescription: 10g of amphotericin B new compound, 8g of arginine, 1g of taurine, 10g of vitamin E succinic acid polyethylene glycol ester, 400012 g of polyethylene glycol, 150ml of water for injection, 1g of EDTA2 sodium 4 hydrate, 30g of stearyl alcohol, 30g of lanolin and 1000g of yellow vaseline;

the preparation method comprises the following steps: taking a prescription amount of the amphotericin B new compound, arginine, taurine, vitamin E polyethylene glycol succinate and EDTA2 sodium 4 hydrate of the invention into a small stainless steel keg with a proper amount of water for injection, heating and stirring the mixture to dissolve the mixture for later use; placing polyethylene glycol 4000, stearyl alcohol, lanoline, and yellow vaseline in a container with a cover, heating to melt, adding sterilized liquid paraffin, and mixing to obtain matrix; when the temperature of the matrix is reduced to about 70-80 ℃, adding the dissolved standby substance containing the new compound of the amphotericin B into the matrix under rapid stirring, stirring while adding, adding yellow Vaseline to full dose, measuring the content, and subpackaging to obtain the finished product.

The present invention has been described in detail with reference to the specific embodiments and examples, but it should be understood that the scope of the present invention is not limited thereto, and it will be apparent to those skilled in the art that various modifications, improvements, substitutions and combinations can be made to the technical solution of the present invention and the embodiments thereof without departing from the spirit and scope of the present invention, and these are within the scope of the present invention. It should be understood, however, that the drawings and detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention as defined by the appended claims.

Claims

1. Novel antifungal compoundsIs characterized in that: the antifungal new compound is amphotericin B crystal hydrate with molecular formula of C₄₇H₇₃NO₁₇·nH₂O，n＝1.75、2、3.5。

2. The antifungal novel compounds in accordance with claim 1, characterized in that: the antifungal new compound is amphotericin B2 hydrate with molecular formula of C₄₇H₇₃NO₁₇·2H₂O。

3. The novel antifungal compound according to claims 1 and 2, wherein: measured by powder X-ray diffractometry, at a diffraction angle 2 theta, in a measurement range of 3-60 DEG, at the following 2 theta values, ranging between + -0.2 DEG, there are corresponding characteristic values: 4.83,9.43, 11.36, 11.97, 14.00, 15.19, 17.20, 18.36, 18.995, 20.11, 20.75, 21.32, 21.61, 22.90, 23.43, 25.53, 26.75, 27.62, 32.31, 40.18.

4. The novel antifungal compound according to any one of claims 1 to 3, wherein: the antifungal compound is used for preparing a pharmaceutical composition containing the antifungal novel compound, and the pharmaceutical composition is used for preparing solid preparations, suppositories, injections, implants, ointments, gels, microemulsions, nano preparations, emulsions, lotions, transdermal preparations and pharmaceutically acceptable preparations, wherein the injections are selected from but not limited to lyophilized powder injections, large infusion solutions, injectable liposome preparations or injectable microsphere preparations or injectable microemulsion preparations, and injectable nano preparations, and the solid preparations are selected from but not limited to tablets, capsules, granules, pellets and vaginal effervescent tablets.

5. The novel antifungal compound or the pharmaceutical composition thereof as described in any one of claims 1 to 4, wherein: for the preparation of a vaccine against the following infections: cryptococcosis, North American blastomycosis, disseminated candidiasis, coccidioidomycosis, histoplasmosis, mucormycosis caused by Mucor, Rhizopus, Absidia, Endochromyces and Rana, sporotrichosis caused by Sporothrix schenckii, aspergillosis caused by Aspergillus fumigatus, chromomycosis, skin fungal infection after burning, Candida respiratory tract infection, Aspergillus or Cryptococcus infection, fungal corneal ulcer, thermomelanosis, enteroviral infection, and treatment or prevention of anti-depression.

6. The process for the preparation of the novel antifungal compounds according to claims 1 to 5, characterized in that: the preparation method comprises the following steps: adding amphotericin B or amphotericin B anhydride or amorphous amphotericin B, dimethyl sulfoxide, dimethyl formamide DMF, dimethyl acetamide, water and/or organic solvent C into a reaction vessel₁-C₆Low molecular alcohol of (2), C₂-C₈Low molecular ether of (2), C₂-C₆Controlling the temperature between 0-50 ℃, adding one or more of sodium hydroxide or potassium hydroxide or sodium carbonate or potassium carbonate or trisodium phosphate or organic amine solution, stirring for dissolving, filtering, adding a proper amount of one or more of lactic acid or dilute sulfuric acid or phosphoric acid solution into the filtrate, adjusting the pH value to between 4.8-6.2, standing below 0 ℃, fully separating out the solid, filtering, and using an organic solvent C₁-C₆Low molecular alcohol of (2), C₂-C₈Low molecular ether of (2), C₃-C₈Low molecular ketone of (2), C₂-C₆Low molecular nitrile of (2), C₂-C₈Low molecular ester of (2), C₁-C₆Washing with one or more of low molecular weight halogenated hydrocarbons for 1-3 times, filtering, and collecting solid with dimethyl sulfoxide, dimethyl formamide DMF, water, and C₁-C₆Low molecular alcohol of (2), C₂-C₈Low molecular ether of (2), C₃-C₈Low molecular ketone of (2), C₁-C₆Low molecular halogenated hydrocarbon of C₂-C₈Low molecular ester of (2), C₂-C₆One or more of low molecular nitrile and DMF as crystallization solvent, filtering, standing below 10 deg.C to precipitate crystals, filtering, mixing with water or organic solvent C1-C6 low molecular alcohol, C2-C8 lower ether, C3-C8 low molecular ketone, and C3-C8₂-C₈Washing one or more of the low molecular ester and C1-C6 lower halogenated hydrocarbon, filtering, and drying to obtain new amphotericin B compound;

wherein, the organic solvent C₁-C₆The low molecular alcohol of (a) is selected from but not limited to methanol, ethanol, isopropanol, butanol, propylene glycol; c₂-C₆The low molecular nitrile of (a) is selected from, but not limited to, acetonitrile, propionitrile; c₂-C₈The low molecular ether of (A) is selected from but not limited to diethyl ether, isopropyl ether, tetrahydrofuran, methyl tetrahydrofuran; c₁-C₆The lower halogenated hydrocarbon is selected from but not limited to dichloromethane and chloroform; c₂-C₈The low molecular ester is selected from but not limited to butyl acetate, ethyl acetate and ethyl formate; c₃-C₈The low molecular ketone is selected from but not limited to acetone, butanone and isohexanone.