Echinocandin compounds, their preparation and use

Abstract

The present invention relates to echinocandin compounds, their preparation method and use. Specifically, the present invention discloses echinocandin B deoxy analogs and their biosynthesis method. The echinocandin B deoxy analogs have good antifungal activity.

Description

[Technical field]

[0001] The present invention is in the fields of biosynthesis and medicine, specifically, it relates to echinocandin B deoxy analogs, their preparation methods and uses. [Background technology]

[0002] Echinocandins are natural cyclic lipopeptide compounds discovered in the 1970s. Their chemical structure includes a cyclic hexapeptide backbone and fatty acid side chain fragments (linked to the hexapeptide backbone via amide bonds). Echinocandins have antifungal activity, and their mechanism of action is to inhibit the synthesis of fungal cell walls by noncompetitively inhibiting β-(1,3)-D-glucan synthase. Echinocandins are a representative example of echinocandins. To date, new antifungal drugs such as anidulafungin and rezafungin have been semisynthetically developed based on echinocandin B.

[0003] Research has shown that echinocandin B deoxy analogs have the same antibacterial activity as echinocandin B. At present, the preparation methods of echinocandin B deoxy analogs are divided into chemical synthesis and biosynthesis. Chinese patent CN1298410A discloses the conversion of mulundocandin to its C4-homotyrosine monodeoxy analogs by hydrogenolysis using Raney nickel under neutral conditions. US patent application 222157 discloses the conversion of mulundocandin to its C4-homotyrosine monodeoxy analogs by hydrogenolysis using trifluoroacetic acid, NaCNBH 3 or NaBH(OAc) 3It has been disclosed that pure echinocandin B is reduced to monodeoxy analogues using . The above method is a chemical method, and requires the conversion using a pure product, so the conversion efficiency is low, the cost is high, and the reagents used are harmful to the human body. Chinese patent application CN101993477A discloses a method for producing echinocandin B deoxy analogues by mixing and reacting the fermentation solution of echinocandin B with an acidic solution to produce deoxy analogues. This method produces a large amount of echinocandin B deoxy analogue components, so the conversion efficiency is low and the purification cost is high. A method for producing echinocandin B deoxy analogues by fermentation and culturing a mutant strain in which oxygenase genes (e.g., ecdG or ecdH) are knocked out has also been reported, but the echinocandin B deoxy analogues obtained by this method have a very complicated composition, many by-products, low conversion efficiency, and high purification cost, which are disadvantageous for industrialization. Therefore, there is a strong need for a method for specifically producing certain echinocandin B deoxy analogs with high conversion efficiency, and for using this method to discover and produce echinocandin B deoxy analogs having good antifungal activity. Summary of the Invention [Problem to be solved by the invention]

[0004] In order to solve the above problems, the present inventors have conducted intensive research and discovered a biosynthetic method capable of specifically synthesizing the desired echinocandin B deoxy analogues. Such a method is advantageous for industrialization because it has high conversion efficiency, the components of the reaction product are relatively simple, and there are few impurities. In addition, the present inventors have discovered that the novel echinocandin B deoxy analogues produced by such a method have good antifungal activity. Furthermore, compound derivatives obtained by further modifying the chemical structures of these novel echinocandin B deoxy analogues also show good antifungal activity. [Means for solving the problem]

[0005] Thus, in one aspect, the present invention provides echinocandin compounds represented by general formula (I), or esters, stereoisomers, prodrugs, solvates and pharma- ceutically acceptable salts thereof: [ka] “During the ceremony, R 1 , R 5 , R 6 may be the same or different and are each independently selected from H or OH; R 2 are H, OH, -X 1 -(alkylene-X 2 ) m -Alkylene-X 3 , -X 1 -(alkylene-X 2 ) n -CH[CH 2 (X 4 -alkylene) p -X 5 ] 2 where X is 1 , X 2 , X 4 is selected from -O-, -NH-, and -S-; X 3 , X 5 OR a , N.R. a R b , N.R. a R b R c where R a , R b , R c are each independently selected from H, alkyl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and cycloalkylalkyl, wherein the aryl, heteroaryl, heterocyclyl, and cycloalkyl are each independently selected from hydroxy, alkoxy, amino, alkyl, alkenyl, alkynyl, halogen, cyano, nitro, mercapto, -S-alkyl, -S(O)-alkyl, -SO 2 -Alkyl, NR c R d , COOR e ,CONRc R d and R c , R d , R e are each independently selected from H, alkyl; or R a , R b , R c any two of R are linked to form a heterocycle; or a , R b , R c One of the following is NR a R b R c is bonded to a carbon atom on the alkylene to form a heterocycle; m, n, and p are integers of 0 to 5; R 3 is selected from H, alkyl; R 4 , R 8 may be the same or different and are each independently selected from H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl groups, wherein the alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl groups are selected from halogen, cyano, nitro, mercapto, -S-alkyl, -S(O)-alkyl, -SO 2 -Alkyl, NR c R d , COOR e ,CONR c 'R d ' may be substituted with R c , R d , R e , R c ', R d each ' is independently selected from H, alkyl; R 7 H, OH, halogen, -OSO 3 H, or -alkylene-NR f R g Selected from R f , R g are each independently selected from H, alkyl; R 9 is H or fatty acyl; however, R 1 , R 2 , R 5 , R 6 at least one of is H; R c ' and R d ' is not at the same time H; R 1 , R 2 , R 5 is H and R 6 If R is OH, 3 is not methyl; R 1 , R 6 is OH and R 2 , R 5 If H, then R 3 is not methyl; R 1 , R 2 , R 6 is OH and R 5 If H, then R 3 is not methyl; R 1 , R 5 , R 6 is OH and R 2 If H, then R 3 is not methyl."

[0006] R 2 are H, OH, -O-(C 1-3 Alkylene-O) m -C 1-3 Alkylene-X 3 , -NH-(C 1-3 Alkylene-O) m -C 1-3 Alkylene-X 3 , -O-(C 1-3 Alkylene-NH m -C 1-3 Alkylene-X 3 , -NH-(C 1-3 Alkylene-NH m -C 1-3 Alkylene-X 3 , -O-(C 1-3 Alkylene-O) n-CH[CH 2 (OC 1-3 Alkylene) p -X 5 ] 2 , -NH-(C 1-3 Alkylene-O) n -CH[CH 2 (OC 1-3 Alkylene) p -X 5 ] 2 , -O-(C 1-3 Alkylene-O) n -CH[CH 2 (OC 1-3 Alkylene) p -X 5 ] 2 , -O-(C 1-3 Alkylene-NH n -CH[CH 2 (NH-C 1-3 Alkylene) p -X 5 ] 2 , -NH-(C 1-3 Alkylene-NH n -CH[CH 2 (NH-C 1-3 Alkylene) p -X 5 ] 2 Selected from X 3 , X 5 OR a , N.R. a R b , N.R. a R b R c where R a , R b , R c are each independently selected from H, alkyl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and cycloalkylalkyl, wherein the aryl, heteroaryl, heterocyclyl, and cycloalkyl are each independently selected from hydroxy, alkoxy, amino, alkyl, alkenyl, alkynyl, halogen, cyano, nitro, mercapto, -S-alkyl, -S(O)-alkyl, -SO 2 -Alkyl, NR c R d , COORe ,CONR c R d and R c , R d , R e are each independently selected from H, alkyl; or R a , R b , R c any two of R are linked to form a heterocycle; or a , R b , R c One of the following is NR a R b R c is bonded to a carbon atom on the alkylene to which it is bonded to form a heterocycle; and m, n, and p are integers of 0 to 5, or an ester, stereoisomer, prodrug, solvate, or pharma- ceutically acceptable salt thereof.

[0007] R 2 are H, OH, -O-(C 1-3 Alkylene-O) m -C 1-3 Alkylene-X 3 , -NH-(C 1-3 Alkylene-O) m -C 1-3 Alkylene-X 3 , -O-(C 1-3 Alkylene-NH m -C 1-3 Alkylene-X 3 , -NH-(C 1-3 Alkylene-NH m -C 1-3 Alkylene-X 3 , -O-(C 1-3 Alkylene-O) n -CH[CH 2 (OC 1-3 Alkylene) p -X 5 ] 2 , -NH-(C 1-3 Alkylene-O) n -CH[CH 2 (OC 1-3 Alkylene) p -X 5 ] 2, -O-(C 1-3 Alkylene-O) n -CH[CH 2 (OC 1-3 Alkylene) p -X 5 ] 2 , -O-(C 1-3 Alkylene-NH n -CH[CH 2 (NH-C 1-3 Alkylene) p -X 5 ] 2 , -NH-(C 1-3 Alkylene-NH n -CH[CH 2 (NH-C 1-3 Alkylene) p -X 5 ] 2 Selected from X 3 , X 5 OR a , N.R. a R b , N.R. a R b R c where R a , R b , R c are independently H, C 1-6 Alkyl, C 7-20 Aryl alkyl, C 7-20 Heteroarylalkyl, C 7-20 Heterocyclyl group alkyl, C 7-20 cycloalkylalkyl, wherein the aryl, heteroaryl, heterocyclyl, and cycloalkyl are selected from hydroxy, C 1-6 Alkoxy, Amino, C 1-6 Alkyl, C 1-6 Alkenyl, C 1-6 Alkynyl, halogen, cyano, nitro, mercapto, -SC 1-6 Alkyl, -S(O)-C 1-6 Alkyl, -SO 2 -C 1-6 Alkyl, NR c R d , COOR e ,CONR c R dand R c , R d , R e are independently H, C 1-6 alkyl; or R a , R b , R c any two of R are linked to form a heterocycle; or a , R b , R c One of the following is NR a R b R c is bonded to a carbon atom on the alkylene to which it is bonded to form a heterocycle; and m, n, and p are integers of 0 to 5, or an ester, stereoisomer, prodrug, solvate, or pharma- ceutically acceptable salt thereof. [ka] or an ester, stereoisomer, prodrug, solvate or pharma- ceutically acceptable salt thereof.

[0008] R 4 , R 8 may be the same or different, and each independently represents H, C 1-6 Alkyl, C 1-6 Alkenyl, C 1-6 Alkynyl, C 6-14 Aryl, C 6-14 Heteroaryl, C 6-14 Cycloalkyl, C 6-14 heterocyclyl groups, 1-6 Alkyl, C 1-6 Alkenyl, C 1-6 Alkynyl, C 6-14 Aryl, C 6-14 Heteroaryl, C 6-14 Cycloalkyl, C 6-14 Heterocyclyl groups include halogen, cyano, nitro, mercapto, -SC 1-6 Alkyl, -S(O)-C 1-6 Alkyl, -SO 2 -C 1-6 Alkyl, NR c Rd , COOR e ,CONR c 'R d ' may be substituted with R c , R d , R e , R c ', R d ' are independently H, C 1-6 alkyl, or an ester, stereoisomer, prodrug, solvate, or pharma- ceutically acceptable salt thereof.

[0009] R 4 , R 8 may be the same or different, and each independently represents H, C 1-6 alkyl, 1-6 Alkyl is halogen, cyano, nitro, mercapto, -SC 1-6 Alkyl, -S(O)-C 1-6 Alkyl, -SO 2 -C 1-6 Alkyl, NR c R d , COOR e ,CONR c 'R d ' may be substituted with R c , R d , R e , R c ', R d ' are independently H, C 1-6 alkyl, or an ester, stereoisomer, prodrug, solvate, or pharma- ceutically acceptable salt thereof.

[0010] R 9 is C 6-50 The compounds are fatty acyl and may contain unsaturated alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl groups, or esters, stereoisomers, prodrugs, solvates and pharma- ceutically acceptable salts thereof. [ka] or an ester, stereoisomer, prodrug, solvate or pharma- ceutically acceptable salt thereof.

[0011] however, R 1 , R 2 , R 5 , R 6 at least two of are H; R c ' and R d ' is not at the same time H; R 1 , R 2 , R 5 is H and R 6 If R is OH, 3 is not methyl; R 1 , R 6 is OH and R 2 , R 5 If H, then R 3 is not methyl; R 1 , R 2 , R 6 is OH and R 5 If H, then R 3 is not methyl; R 1 , R 5 , R 6 is OH and R 2 If H, then R 3 is not methyl, or esters, stereoisomers, prodrugs, solvates and pharma- ceutically acceptable salts thereof.

[0012] however, R 1 , R 2 , R 5 , R 6 at least three of are H; R c ' and R d ' is not at the same time H; R 1 , R 2 , R 5 is H and R 6 If R is OH, 3is not methyl; R 1 , R 6 is OH and R 2 , R 5 If H, then R 3 is not methyl; R 1 , R 2 , R 6 is OH and R 5 If H, then R 3 is not methyl; R 1 , R 5 , R 6 is OH and R 2 If H, then R 3 is not methyl, or esters, stereoisomers, prodrugs, solvates and pharma- ceutically acceptable salts thereof.

[0013] R 1 , R 5 , R 6 is H; R 2 is H or -X 1 -(alkylene-X 2 ) m -Alkylene-X 3 , -X 1 -(alkylene-X 2 ) n -CH[CH 2 (X 4 -alkylene) p -X 5 ] 2 where X 1 , X 2 , X 4 is selected from -O-, -NH-, and -S-; X 3 , X 5 OR a , N.R. a R b , N.R. a R b R c where R a , R b , R care each independently selected from H, alkyl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and cycloalkylalkyl, wherein the aryl, heteroaryl, heterocyclyl, and cycloalkyl are each independently selected from hydroxy, alkoxy, amino, alkyl, alkenyl, alkynyl, halogen, cyano, nitro, mercapto, -S-alkyl, -S(O)-alkyl, -SO 2 -Alkyl, NR c R d , COOR e ,CONR c R d and R c , R d , R e are each independently selected from H, alkyl; or R a , R b , R c any two of R are linked to form a heterocycle; or a , R b , R c One of the following is NR a R b R c is bonded to a carbon atom on the alkylene to which it is bonded to form a heterocycle; and m, n, and p are integers of 0 to 5, or an ester, stereoisomer, prodrug, solvate, or pharma- ceutically acceptable salt thereof.

[0014] R 1 , R 2 , R 5 , R 6 is H, or an ester, stereoisomer, prodrug, solvate or pharma- ceutically acceptable salt thereof. R 1 , R 2 , R 5 , R 6 is H; R 3 , R 4 , R 8 are each independently H or methyl; R 7 is H, OH, or -OSO 3 H; [ka] or an ester, stereoisomer, prodrug, solvate or pharma- ceutically acceptable salt thereof.

[0015] The compound, wherein said compound is represented by the formula: or an ester, stereoisomer, prodrug, solvate or pharma- ceutically acceptable salt thereof. [ka] "During the ceremony, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 is as defined herein."

[0016] The compound, wherein said compound is represented by the formula: or an ester, stereoisomer, prodrug, solvate or pharma- ceutically acceptable salt thereof. [ka] "During the ceremony, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 is as defined herein."

[0017] The compound, wherein said compound is represented by the formula: or an ester, stereoisomer, prodrug, solvate or pharma- ceutically acceptable salt thereof. [ka] "During the ceremony, R 1 , R 3 , R 4 , R 5 , R 6 , R 7, R 8 , R 9 is as defined herein."

[0018] The compound, wherein said compound is represented by the formula: or an ester, stereoisomer, prodrug, solvate or pharma- ceutically acceptable salt thereof. [ka] "During the ceremony, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 is as defined herein."

[0019] The compound, wherein said compound is represented by the formula: or an ester, stereoisomer, prodrug, solvate or pharma- ceutically acceptable salt thereof. [ka] "During the ceremony, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 is as defined herein."

[0020] The compound, wherein said compound is represented by the formula: or an ester, stereoisomer, prodrug, solvate or pharma- ceutically acceptable salt thereof. [ka] "During the ceremony, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8is as defined herein."

[0021] The compound, wherein said compound is represented by the formula: or an ester, stereoisomer, prodrug, solvate or pharma- ceutically acceptable salt thereof. [ka] "During the ceremony, R 1 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 is as defined herein."

[0022] The compound, wherein said compound is represented by the formula: or an ester, stereoisomer, prodrug, solvate or pharma- ceutically acceptable salt thereof. [ka]

[0023] In another aspect, the invention relates to pharmaceutical compositions comprising said compounds, or esters, stereoisomers, prodrugs, solvates and pharma- ceutically acceptable salts thereof. The pharmaceutical composition further comprises another antifungal agent. In another aspect, the invention relates to an echinocandin biosynthetic gene cluster comprising two or more of the genes ecdA, ecdI, ecdK and htyE.

[0024] The echinocandin biosynthetic gene cluster comprises the genes ecdA, ecdI and htyE. The echinocandin biosynthetic gene cluster includes the genes ecdA, ecdI, ecdK and htyE. The echinocandin biosynthetic gene cluster consists of the genes ecdA, ecdI and htyE. The echinocandin biosynthetic gene cluster consists of the genes ecdA, ecdI, ecdK and htyE. The echinocandin biosynthetic gene cluster further comprises one or more of the genes ecdG, ecdH, or htyF. The echinocandin biosynthetic gene cluster consists of the genes ecdA, ecdI, ecdG and htyE. The echinocandin biosynthetic gene cluster consists of the genes ecdA, ecdI, ecdG, htyE and htyF. In another aspect, the present invention relates to a genetically engineered transformed strain comprising the echinocandin biosynthetic gene cluster according to any one of the preceding claims.

[0025] The genetically engineered transformed strains include a genetically engineered transformed strain deposited on November 2, 2021 at the China Typical Culture Depository (Wuhan University, Wuhan, China) with the accession number CCTCC M 20211360 and the name Aspergillus nidulans LO8030-5.1, a genetically engineered transformed strain deposited on February 28, 2022 at the China Typical Culture Depository (Wuhan University, Wuhan, China) with the accession number CCTCC M 2022168 and the name Aspergillus nidulans LO8030-4.2.1, and a genetically engineered transformed strain deposited on February 28, 2022 at the China Typical Culture Depository (Wuhan University, Wuhan, China) with the accession number CCTCC M 2022168 and the name Aspergillus nidulans LO8030-4.2.1. the genetically engineered transformed strain having the accession number CCTCC M 2023155 and the name Aspergillus nidulans LO8030-4.2.1.5 deposited on February 20, 2023 at the China Typical Culture Depository (Wuhan University, Wuhan, China) under the accession number CCTCC M 2023156 and the name Aspergillus nidulans LO8030-4.2.1.9 deposited on February 20, 2023 at the China Typical Culture Depository (Wuhan University, Wuhan, China) under the accession number CCTCC M 2023156 and the name Aspergillus nidulans LO8030-4.2.1.9.

[0026] In another aspect, the present invention relates to a method for producing the aforementioned compound, comprising fermenting a genetically engineered transformed strain described in any one of the above to express a target product, and then extracting and separating the fermentation culture to obtain the compound. The method includes fermenting the genetically engineered Aspergillus nidulans LO8030-5.1, LO8030-4.2.1, LO8030-4.2.1.5 or LO8030-4.2.1.9 in an XSMM fermentation medium for 5 to 14 days; separating the fungus body and the fungus liquid, extracting them with methanol or ethyl acetate respectively, and concentrating them under vacuum, and combining them to obtain a total extract; and separating the total extract by silica gel column chromatography and high performance liquid chromatography in order to obtain a pure product.

[0027] The above production method, wherein the eluent for the silica gel column chromatography is dichloromethane / methanol (gradient elution). The aforementioned production method, wherein the eluent for the high performance liquid chromatography is methanol / water, and the elution ratio (volume ratio) is 85:15. In addition, since the compounds of the present invention were obtained by biosynthetic methods, the structures of the compounds of the present invention are known to those skilled in the art. In this case, the compounds of the present invention can also be produced by conventional organic synthesis methods. For example, the amino acid [ka] The compound of the present invention can be produced by conventional polypeptide synthesis methods (including liquid phase synthesis and solid phase synthesis) using the above as reaction raw materials. In the reaction process, if necessary, active groups such as amino and carboxy of the above amino acids that are not involved in the reaction can be temporarily protected with a normal protecting group, and after the reaction is completed, the protecting group can be removed to carry out the next reaction. For specific protecting groups, refer to "Protective Groups in Organic Synthesis (Green's Protective Groups in Organic Synthesis 5th Edition)".

[0028] In one embodiment, the method for preparing the echinocandin compound represented by general formula (I) provided by the present invention further comprises the steps of: Step 1: The linoleoyl group of the compound of formula (I') is removed under the catalysis of echinocandin B deacylase to give the compound of formula (I''). [ka]

[0029] Step 2: Under alkaline conditions, the compound of formula (I'') is reacted with R 9 -X to give a compound of formula (I). [ka] "During the ceremony, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 is as defined herein, and R 9 is a fatty acyl and X is a leaving group, including but not limited to halogens, active esters, and acid anhydrides."

[0030] In one specific embodiment, the method provided herein comprises the steps of: Step 1: Under the catalysis of echinocandin B deacylase, the linoleoyl group of echinocandin E (compound 2) or echinocandin F (compound 1) is removed to give compound 2' or compound 1', respectively. [ka]

[0031] Step 2: Compound 2' or compound 1' is reacted with R 9 By reacting with -X, a compound represented by the following formula is obtained. [ka]

[0032] In a further embodiment, the method for preparing the echinocandin compound of general formula (I) provided by the present invention further comprises the steps of: Optionally, in the presence of a condensing agent, a compound of formula (I''') is reacted with R 2 Reaction of the aryl halide with an activated ester of H gives the compound of formula (I). [ka] "During the ceremony, R 1 , R 3 , R 4 , R 5 , R6 , R 7 , R 8 , R 9 is as defined herein, and R 2 -X 1 -(alkylene-X 2 ) m -Alkylene-X 3 , -X 1 -(alkylene-X 2 ) n -CH[CH 2 (X 4 -alkylene) p -X 5 ] 2 where X 1 , X 2 , X 4 is selected from -O-, -NH-, and -S-; X 3 , X 5 OR a , N.R. a R b , N.R. a R b R c Selected from R a , R b , R c are each independently selected from H, alkyl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and cycloalkylalkyl, wherein the aryl, heteroaryl, heterocyclyl, and cycloalkyl are each independently selected from hydroxy, alkoxy, amino, alkyl, alkenyl, alkynyl, halogen, cyano, nitro, mercapto, -S-alkyl, -S(O)-alkyl, -SO 2 -Alkyl, NR c R d , COOR e ,CONR c R d and R c , R d , R e are each independently selected from H, alkyl; or R a , R b , R c any two of R are linked to form a heterocycle; or a , R b, R c One of the following is NR a R b R c is bonded to a carbon atom on the alkylene to form a heterocycle; m, n, and p are integers from 0 to 5.

[0033] In another aspect, the present invention provides a method for the treatment or prevention of a fungal infection comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention. In another aspect, the invention relates to the use of said compound in the manufacture of a medicament for the treatment or prevention of a disease associated with a fungal infection. The fungus may be selected from the group consisting of Aspergillus (e.g., Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger, and Aspergillus terreus), Blastomyces dermatitidis, Candida (e.g., Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida krusei, and Candida guilliermondii), Coccidioides immitis, , Cryptococcus (e.g., Cryptococcus neoformans, Cryptococcus albicans, and Cryptococcus laurentii), Histoplasma capsulatum var. capsulatum, Histoplasma capsulatum var. duboisi, Paracoccidioides braziliensis, Sporothrix schenckii, Absidia corymbifera, Rhizomucor pusillus, and Rhizopus arizus.

[0034] The method or use is characterized in that the disease associated with a fungal infection is selected from one or more of: tinea capitis, tinea corporis, tinea pedis, onychomycosis, periungual mycosis, tinea discolor, thrush, vaginal candidiasis, respiratory tract candidiasis, biliary candidiasis, esophageal candidiasis, urinary tract candidiasis, systemic candidiasis, mucosal / cutaneous candidiasis, aspergillosis, mucormycosis, paracoccidioidomycosis, North American blastomycosis, histoplasmosis, coccidioidomycosis, sporotrichosis, fungal sinusitis, and chronic sinusitis. Effect of the Invention

[0035] The beneficial effects of the present invention are as follows: The present inventors designed a series of biosynthetic gene clusters for echinocandin B deoxy analogs and expressed them in a heterologous host to specifically produce the desired echinocandin B deoxy analogs. Compared with conventional techniques, this method has a simpler product composition, a higher conversion efficiency, fewer reaction impurities, and is easier to separate and purify, making it more favorable for industrialization. The genetically engineered fungus has a simpler composition of secondary metabolites in the fermentation product and a higher yield compared to the wild-type echinocandin B producing strain. The yield of echinocandin B deoxy analogues produced by the genetically engineered transformant strain of Aspergillus nidulans of the present invention is significantly higher than the yield of echinocandin B produced by the wild-type echinocandin B producing strain.

[0036] The echinocandin compounds prepared according to the present invention show good antifungal activity and are expected to be developed as antifungal drugs. According to research, the loss of hydroxyl groups on the ornithine and homotyrosine residues of echinocandin B leads to an instability in the structure of echinocandin B. On the other hand, the echinocandin B deoxy analogs of the present invention, particularly echinocandin E and echinocandin F, have higher chemical stability and are more favorable for subsequent chemical derivatization than the cyclic hexapeptide structure of echinocandin B, although neither the homotyrosine nor the ornithine residues in the structure are hydroxylated. [Brief description of the drawings]

[0037] [Figure 1]A schematic diagram of constructing a plasmid by ligating multiple fragments by yeast homologous recombination is shown. "F1-F3" are multiple segments of one target gene or multiple target genes; the plasmid backbone includes E. coli replication origin "Ori", E. coli ampicillin resistance selection marker "AmpR", yeast replication origin "2μ", and yeast uracil auxotrophy selection marker "URA". [Diagram 2] A schematic diagram of transformation of the Ecd / Hty gene cluster into Aspergillus nidulans LO8030. [Diagram 3] 1 shows a map of plasmid pYX1 and a schematic diagram of the first transformation for constructing a gene expression cassette. [Figure 4] 1 shows a map of plasmid pYX5 and a schematic diagram of the second transformation to construct a gene expression cassette. [Diagram 5] 1 shows a map of plasmid pYX9 and a schematic diagram of the third transformation to construct a gene expression cassette. [Figure 6] 1 shows a map of the control plasmid pYX14 and a transformation scheme for constructing a gene expression cassette. [Figure 7] A map of plasmid pYX10 and a schematic diagram of the fourth transformation are shown. [Figure 8] A map and transformation scheme of the control plasmid pYX12 are shown. [Figure 9] Sequencing and alignment of the biosynthetic genes inserted into the genetically engineered transformant strain LO8030-5.1 of Aspergillus nidulans (a: ecdA; b: ecdI; c: ecdK; d: htyE) are shown. [Figure 10] Figure 2 shows transcriptional analysis of biosynthetic genes in the genetically engineered transformant strain LO8030-5.1 of Aspergillus nidulans. [Figure 11]HPLC and LC-MS analysis of metabolites of transformed strains (LO8030-4.1, LO8030-4C.1, LO8030-5C.1, LO8030-5.1) are shown. (A) HPLC tracer (222 nm) of metabolites of each transformed strain. (B) MS data of compounds 1 and 2. "Exp" represents the m / z value from the experiment; "The" represents the m / z value expected for tetradeoxy compounds. [Figure 12] FIG. 1 shows a schematic diagram of the biosynthesis of echinocandin B deoxy analogs according to the present invention. [Figure 13] A map of plasmid pJQ10 and a transformation scheme are shown. [Figure 14] FIG. 14A shows the peak area-concentration standard curve of Compound 2 (Echinocandin E), and FIG. 14B shows the peak area-concentration standard curve of Compound 1 (Echinocandin F). [Figure 15] The growth of Aspergillus nidulans LO8030 and its genetically engineered transformants (a.LO8030, b.LO8030-1.3, c.LO8030-2.2, d.LO8030-4.1, e.LO8030-5.1) on GMM supplemented with appropriate additives is shown. [Figure 16] FIG. 16A shows the H NMR spectrum of compound 1 (CD3OD, 600 MHz), and FIG. 16B shows the C NMR spectrum of compound 1 (CD3OD, 150 MHz). [Figure 17] The 1H-13C HSQC spectrum (CD3OD, 600 MHz) of compound 1 is shown. [Figure 18] The 1H-1H COSY spectrum (CD3OD, 600 MHz) of compound 1 is shown. [Figure 19] The 1H-13C HMBC spectrum (CD3OD, 600 MHz) of compound 1 is shown. [Figure 20] FIG. 20A shows the H NMR spectrum of compound 2 (CD3OD, 600 MHz), and FIG. 20B shows the C NMR spectrum of compound 2 (CD3OD, 150 MHz). [Figure 21]The 1H-13C HSQC spectrum (CD3OD, 600 MHz) of compound 2 is shown. [Figure 22] The 1H-1H COSY spectrum (CD3OD, 600 MHz) of compound 2 is shown. [Diagram 23] The 1H-13C HMBC spectrum (CD3OD, 600 MHz) of compound 2 is shown. [Figure 24] The 1H-1H COSY and major HMBC correlation peaks of compound 1 and compound 2 (a: compound 1, b: compound 2) are shown. [Diagram 25] 1 shows a map of plasmid pJQ5 and a transformation scheme. [Figure 26] 1 shows a map of plasmid pJQ9 and a transformation scheme. [Figure 27] The 1H-1H COSY spectrum (CD3OD, 600 MHz) of compound 3 (echinocandin G) is shown. [Figure 28] The 1H-13C HMBC spectrum (CD3OD, 600 MHz) of compound 3 is shown. [Figure 29] The 1H-1H COSY spectrum (CD3OD, 600 MHz) of compound 4 (Echinocandin H) is shown. [Diagram 30] The 1H-13C HMBC spectrum (CD3OD, 600 MHz) of compound 4 is shown. [Diagram 31] The 1H-1H COSY and major HMBC correlation peaks of compounds 3 and 4 (a: compound 3; b: compound 4) are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] definition Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms must be clear, but in the event of possible ambiguity, the definitions provided herein shall take precedence over any dictionary or extrinsic definitions. In this specification, the term "ester" refers to an ester formed by an esterification reaction between OH in the compound represented by formula (I) and an organic acid, an inorganic acid, etc. This ester can be hydrolyzed under acidic or alkaline conditions to generate the corresponding acid or alcohol.

[0039] As used herein, the term "stereoisomers" refers to compounds that have the same chemical composition and connectivity but differ in the spatial orientation of their atoms, which orientations cannot be interconverted by rotation of a single bond. "Stereoisomers" includes "diastereomers" and "enantiomers." "Diastereomers" refers to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of one another. Diastereomers differ in physical properties such as melting points, boiling points, spectroscopic properties and reactivity. Mixtures of diastereomers can be separated by high-resolution analytical procedures (e.g., crystallization, electrophoresis and chromatography). "Enantiomers" refers to two stereoisomers of a compound that are non-superimposable mirror images of one another.

[0040] As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound of the present invention. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, hydrogen sulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and bishydroxynaphthalene salts; or ammonium salts (e.g., primary amine salts, secondary amine salts, tertiary amine salts, quaternary ammonium salts), metal salts (e.g., sodium salts, potassium salts, calcium salts, magnesium salts, manganese salts, iron salts, zinc salts, copper salts, lithium salts, aluminum salts).

[0041] As used herein, the term "pharmacologically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with other ingredients contained in the formulation and / or the mammal being treated therewith. As used herein, the term "treatment" refers to therapeutic treatment and prophylactic or preventative or preventative measures aimed at preventing or alleviating (relieving) undesirable pathological changes or conditions.For purposes of the present invention, beneficial or desirable clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, delay or slowing of disease progression, improvement or alleviation of disease state, and remission (whether partial or total), whether detectable or undetectable.

[0042] As used herein, the term "therapeutically effective amount" refers to an amount of a compound of the present invention that is capable of (i) treating or preventing a disease or condition described herein, (ii) ameliorating or eliminating one or more diseases or conditions described herein, or (iii) preventing or delaying the onset of one or more symptoms of a disease or condition described herein.

[0043] As used herein, the term "alkyl" refers to a linear or branched saturated hydrocarbon group having 1 to 20 carbon atoms. Preferably, the alkyl is an alkyl having 1 to 12 carbon atoms. More preferably, the alkyl is an alkyl having 1 to 6 carbon atoms. Most preferably, the alkyl is an alkyl having 1 to 4 carbon atoms. Specific examples of alkyl include, but are not limited to, methyl, ethyl, 1-propyl (n-propyl), 2-propyl (isopropyl), 1-butyl (n-butyl), 2-methyl-1-propyl (isobutyl), 2-butyl (sec-butyl), 2-methyl-2-propyl (tert-butyl), 1-pentyl (n-pentyl), 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, 1-nonyl, 1-decyl, and the like.

[0044] As used herein, the term "alkylene" refers to a divalent alkyl as defined above. As used herein, alkylene refers to an alkyl group having 1 to 10 carbon atoms (i.e., C 1-10 alkylene), 1 to 8 carbon atoms (i.e., C 1-8 alkylene), 1 to 6 carbon atoms (i.e., C 1-6 alkylene) or 1 to 4 carbon atoms (i.e., C 1-4 alkylene). As used herein, the term "alkenyl" refers to an alkyl group containing at least one carbon-carbon double bond and having 2 to 20 carbon atoms (i.e., C 2-20 alkenyl), 2 to 8 carbon atoms (i.e., C 2-8 alkenyl), 2 to 6 carbon atoms (i.e., C 2-6 alkenyl) or 2 to 4 carbon atoms (i.e., C 2-4Alkenyl refers to a hydrocarbon group having the substituent (alkenyl). Specific examples of alkenyl include vinyl, propenyl, and butadienyl (including 1,2-butadienyl and 1,3-butadienyl).

[0045] As used herein, the term "alkynyl" refers to an alkynyl group containing at least one carbon-carbon triple bond and having 2 to 20 carbon atoms (i.e., C 2-20 alkynyl), 2 to 8 carbon atoms (i.e., C 2-8 alkynyl), 2 to 6 carbon atoms (i.e., C 2-6 alkynyl) or 2 to 4 carbon atoms (i.e., C 2-4 The term "alkynyl" refers to a hydrocarbon group having one triple bond and one double bond. As used herein, the term "halogen" refers to fluorine, chlorine, bromine and iodine. As used herein, the term "cycloalkyl" refers to a monovalent saturated carbocyclic group. Preferably, cycloalkyl is a 3- to 8-membered monocyclic group. More preferably, cycloalkyl is a 3- to 6-membered monocyclic group. Specific examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

[0046] As used herein, the term "aryl" refers to a single all-carbon aromatic ring system or a fused all-carbon polycyclic ring system in which at least one ring is aromatic and the aryl has 6-20 carbon atoms, 6-14 carbon atoms, 6-12 carbon atoms, or 6-10 carbon atoms. Aryl includes phenyl. Aryl also includes fused polycyclic ring systems (e.g., ring systems containing 2, 3, or 4 rings) having about 9-20 carbon atoms, in which at least one ring is aromatic and the other rings may or may not be aromatic (i.e., carbocyclic). Such fused polycyclic ring systems may be substituted with one or more (e.g., 1, 2, or 3) oxo groups on any carbocyclic portion of the fused polycyclic ring system. The rings of the fused polycyclic ring system may be connected to each other via fused, spirocyclic, and bridged bonds when allowed by valence requirements. Note that, as defined above, the connection point of the fused polycyclic ring system may be at any position on the ring system, including the aromatic or carbocyclic portions of the rings. Exemplary aryls include phenyl, indenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, anthryl, and the like.

[0047] As used herein, the term "heteroaryl" refers to a 5-6 membered aromatic ring having at least one atom other than carbon (an atom selected from oxygen, nitrogen and sulfur) in the ring. "Heteroaryl" also includes fused polycyclic ring systems having 8-16 atoms and at least one such aromatic ring. Fused polycyclic ring systems are further described below. Thus, "heteroaryl" includes a single aromatic ring of about 1-6 carbon atoms and about 1-4 heteroatoms selected from oxygen, nitrogen and sulfur. The sulfur and nitrogen atoms may be present in oxidized form, provided the ring is aromatic. Exemplary heteroaryl ring systems include, but are not limited to, pyridyl, pyrimidinyl, oxazolyl or furyl. "Heteroaryl" also includes fused polycyclic ring systems (e.g., ring systems containing two or three rings) in which the heteroaryl defined above is fused with one or more rings selected from heteroaryl (e.g., forming naphthyridinyl, such as 1,8-naphthyridinyl), heterocycle (e.g., forming 1,2,3,4-tetrahydronaphthyridinyl, such as 1,2,3,4-tetrahydro-1,8-naphthyridinyl), carbocycle (e.g., forming 5,6,7,8-tetrahydroquinolyl) and aryl (e.g., forming indazolyl) to form a fused polycyclic ring system. Thus, a heteroaryl (single aromatic ring or fused polycyclic ring system) has 1-15 carbon atoms and about 1-6 heteroatoms in the heteroaryl ring. Such fused polycyclic ring systems may be substituted with one or more (e.g., 1, 2, 3, or 4) oxo groups on the carbocyclic or heterocyclic portions of the fused rings. The rings of the fused polycyclic ring system can be linked together via fused, spirocyclic and bridged bonds when allowed by valence requirements. It is understood that the individual rings of the fused polycyclic ring system may be linked to each other in any order. It is understood that the attachment point of the fused polycyclic ring system (as defined above for heteroaryl) can be at any position of the fused polycyclic ring system, including the heteroaryl, heterocyclic, aryl or carbocyclic portion of the fused polycyclic ring system (.It is understood that the point of attachment of the heteroaryl group or heteroaryl group-fused polycyclic ring system can be at any suitable atom of the heteroaryl group or heteroaryl group-fused polycyclic ring system, including carbon atoms and heteroatoms (e.g., nitrogen). Exemplary heteroaryls include, but are not limited to, pyridyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrazolyl, thienyl, indolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, furyl, oxadiazolyl, thiadiazolyl, quinolyl, isoquinolyl, benzothiazolyl, benzoxazolyl, indazolyl, quinoxalinyl, quinazolinyl, 5,6,7,8-tetrahydroisoquinolyl, benzofuryl, benzimidazolyl, thiaindenyl, pyrrolo[2,3-b]pyridyl, quinazolinyl-4(3H)-one, triazolyl, 4,5,6,7-tetrahydro-1H-indazolyl, and 3b,4,4a,5-tetrahydro-1H-cyclopropane[3,4]cyclopentane[1,2-c]pyrazolyl.

[0048] As used herein, the term "heterocyclyl group" refers to a single saturated or partially unsaturated 4-8 membered ring having at least one atom other than carbon atom (atom selected from oxygen, nitrogen and sulfur) in the ring. The term also includes fused polycyclic ring systems having at least one saturated or partially unsaturated ring and 7-12 atoms. Such fused polycyclic ring systems are further described below. Thus, the term includes single saturated or partially unsaturated rings (e.g., 3-, 4-, 5-, 6-, 7- or 8-membered rings) having about 1-7 carbon atoms and about 1-4 heteroatoms selected from oxygen, nitrogen and sulfur in the ring. Such rings may be substituted with one or more (e.g., 1, 2 or 3) oxo groups, and the sulfur and nitrogen atoms may be present in oxidized form. The rings of the fused polycyclic ring system may be linked to each other through fused, spirocyclic and bridged bonds when allowed by valence requirements. It is understood that the individual rings of the fused polycyclic ring system may be connected to each other in any order. It is understood that the attachment point of the fused polycyclic ring system (as defined above for heterocyclyl) can be at any position of the fused polycyclic ring system. It is understood that the attachment point of a heterocyclyl or heterocyclyl fused polycyclic ring system can be at any suitable atom () of the heterocyclyl group, including carbon and nitrogen atoms.Exemplary heterocyclyls include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, homopiperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, tetrahydrofuryl, dihydrooxazolyl, tetrahydropyranyl, tetrahydrothiopyranyl, 1,2,3,4-tetrahydroquinolyl, benzoxazinyl, dihydrooxazolyl, benzodihydropyranyl, 1,2-dihydropyridyl, 2,3-dihydrobenzofuranyl, 1,3-benzodioxanyl, 1,4-benzodioxanyl, spiro[cyclopropane-1,1'-isoindolinyl]-3'-one, isoindolinyl-1-one, 2-oxa-6-azaspiro[3.3]heptyl, imidazolidin-2-one-N-methyl-, and the like. These include, but are not limited to, ethylpiperidine, imidazolidinyl, pyrazolidinyl, butyrolactam group, valerolactam group, imidazolidinone, hydantoin group, dioxolanyl, phthalimido group, 1,4-dioxanyl, thiomorpholinyl, thiomorpholinyl-S-oxide, thiomorpholinyl-S,S-oxide, pyranyl, 3-pyrrolinyl, thiopyranyl, pyrone, tetrahydrothienyl, quinuclidinyl, tropane group, 2-azaspiro[3.3]heptyl, (1R,5S)-3-azabicyclo[3.2.1]octyl, (1s,4s)-2-azabicyclo[2.2.2]octyl, (1R,4R)-2-oxa-5-azabicyclo[2.2.2]octyl, and pyrrolidin-2-one.

[0049] As used herein, the term "optionally substituted" refers to a structure or group that is unsubstituted or that is substituted with one or more specified substituents. Unless otherwise specified, any position of the substituted group may be substituted. As used herein, the term "solvate" refers to an association or complex of one or more solvent molecules with the compound of the present invention. Specific examples of solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. The term "hydrate" refers to a complex in which the solvent molecule is water. As used herein, the term "prodrug" refers to a compound that is susceptible to undergo chemical changes under physiological conditions to provide the compound of the present invention. Also, prodrugs can be converted to the compound of the present invention by chemical or biochemical methods in an ex vivo environment. For example, when the prodrug is placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent, the prodrug can be gradually converted to the compound of the present invention.

[0050] Any compounds or structures described herein are intended to represent unlabeled and isotopically labeled forms of those compounds. These forms of compounds are also referred to as "isotopically enriched analogs". Isotopically labeled compounds have structures described herein except that one or more atoms are replaced by atoms having a selected atomic mass or mass number. Specific examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine and iodine, e.g., 2 H, 3 H, 11 C. 13 C. 14 C. 13 N, 15 N, 15 O. 17 O. 18 O. 31 P, 32 P, 35 S, 18 F, 36 Cl, 123 I and 125 I. Various isotopically labeled compounds of the present disclosure include, for example, those that contain radioactive isotopes therein, e.g. 3 H, 13 C and 14Those incorporating C. Such compounds are synthesized by means well known in the art, for example, by using starting materials in which one or more hydrogens have been replaced with deuterium.

[0051] As used herein, the term "genome" or "genomic DNA" refers to the genetic information of a host organism. The genomic DNA includes all the genetic material of a cell or organism, including nuclear DNA (chromosomal DNA), extrachromosomal DNA and organelle (e.g., mitochondrial) DNA. Preferably, the term "genome" or genomic "DNA" refers to the chromosomal DNA of the nucleus. As used herein, the term "recombinant," when used to identify an organism or cell (e.g., a microorganism), is used to indicate that the organism or cell contains at least one "transgene," "transgenic," or "recombinant" polynucleotide, which is usually subsequently identified.

[0052] As used herein, a polynucleotide that is "exogenous" to an individual organism is a polynucleotide that is introduced into that organism by any means other than sexual mating. As used herein, the term "promoter" refers to a polynucleotide region that initiates transcription of a coding sequence. Promoters are located near the transcription start site of a gene, on the same strand of DNA and upstream (towards the 5' region of the sense strand). Some promoters are active in all contexts within a cell and are therefore constitutive promoters, whereas other promoters are controlled to become active in response to a particular stimulus (e.g., inducible promoters). As used herein, the term "promoter activity" and its grammatical equivalents refer to the degree of expression of a nucleotide sequence operably linked to a promoter whose activity is being measured. Promoter activity can be measured directly by determining the amount of RNA transcript produced, for example, by Northern blot analysis, or indirectly by determining the amount of product encoded by the linked nucleic acid sequence (e.g., a reporter nucleic acid sequence linked to a promoter).

[0053] As used herein, the term "expression cassette" refers to a construct in which a nucleic acid sequence encoding an amino acid sequence to be expressed is operably linked to at least one genetic control element that permits or regulates its expression (i.e., transcription and / or translation). Expression can be, for example, stable or transient, constitutive or inducible. As used herein, the term "plasmid" refers to an extrachromosomal element that generally carries genes that are not part of the cell's core metabolic machinery and usually exists in the form of a circular double-stranded DNA molecule. Such elements can be autonomously replicating sequences, genomic integration sequences, phages, or nucleotide sequences derived from any source; linear, circular, or supercoiled single- or double-stranded DNA or RNA. Generally, a plasmid contains an origin of replication that functions in a host cell (e.g., E. coli), and a selection marker for detecting host cells that contain the plasmid.

[0054] As used herein, the term "ecdA gene" includes a gene sequence having about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the gene sequence having the accession number AFT91378.1; preferably, the term "ecdA gene" is a gene sequence having about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the gene sequence having the accession number AFT91378.1; more preferably, the term "ecdA gene" is a gene sequence having the accession number AFT91378.1.

[0055] As used herein, the term "ecdI gene" includes a gene sequence having about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the gene sequence having the accession number AFT91380.1; preferably, the term "ecdI gene" is a gene sequence having about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the gene sequence having the accession number AFT91380.1; more preferably, the term "ecdI gene" is a gene sequence having the accession number AFT91380.1.

[0056] As used herein, the "ecdK gene" includes a gene sequence having about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the gene sequence having the accession number AFT91382.1; preferably, the "ecdK gene" is a gene sequence having about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the gene sequence having the accession number AFT91382.1; more preferably, the "ecdK gene" is a gene sequence having the accession number AFT91382.1.

[0057] As used herein, "gene htyE" includes a gene sequence having about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the gene sequence having accession number AFT91391.1; preferably, "gene htyE" is a gene sequence having about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the gene sequence having accession number AFT91391.1. More preferably, "gene htyE" is a gene sequence having accession number AFT91391.1.

[0058] As used herein, "gene htyF" includes a gene sequence having about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the gene sequence having accession number AFT91399.1; preferably, "gene htyF" is a gene sequence having about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the gene sequence having accession number AFT91399.1. More preferably, "gene htyF" is a gene sequence having accession number AFT91399.1.

[0059] As used herein, "gene ecdG" includes a gene sequence having about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the gene sequence having the accession number AFT91379.1; preferably, "gene ecdG" is a gene sequence having about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the gene sequence having the accession number AFT91379.1. More preferably, "gene ecdG" is a gene sequence having the accession number AFT91379.1.

[0060] As used herein, "gene ecdH" includes a gene sequence having about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the gene sequence having the accession number AFT91389.1; preferably, "gene ecdH" is a gene sequence having about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the gene sequence having the accession number AFT91389.1. More preferably, "gene ecdH" is a gene sequence having the accession number AFT91389.1.

[0061] As used herein, the term "transformation" refers to the introduction of one or more exogenous polynucleotides into a host cell by the use of physical or chemical methods. As used herein, the term "expression" refers to the process of revealing the information encoded in a gene. If the gene encodes a protein, expression includes transcription of DNA into mRNA, processing of the mRNA (if necessary) into a mature mRNA product, and translation of the mature mRNA into the protein.

[0062] As used herein, the term "host cell" refers to any type of cell susceptible to transformation, transfection, transduction, etc., with a nucleic acid construct or plasmid comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that differs from the parent cell by mutations that occur during replication. Preferably, the host cell is a microorganism. More preferably, the microorganism is a bacterium, yeast, fungus, such as an Ascomycota cell. Bacteria that can be used as host cells of the present invention are preferably selected from Escherichia coli and Bacillus subtilis. Preferably, the fungus is selected from the group consisting of Aspergillus japonicus, Aspergillus niger, Aspergillus oryzae, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus aculeatus, Aspergillus caesiellus, Aspergillus candidus, Aspergillus carneus, Aspergillus clavatus, Aspergillus deflectus, Aspergillus fischerianus, Aspergillus fischerianus, Aspergillus flavus, Aspergillus glaucus, Aspergillus ochraceus, Aspergillus parasiticus, Aspergillus penicilloides, Aspergillus restrictus, Aspergillus sojae, Aspergillus tamariiAspergillus species such as Aspergillus tamari, Aspergillus terreus, Aspergillus ustus, and Aspergillus versicolor; Penicillium aurantiogriseum, Penicillium bilaiae, Penicillium camemberti, Penicillium candidum, Penicillium chrysogenum, Penicillium claviforme, Penicillium commune, and Penicillium crustosum. crustosum, Penicillium digitatum, Penicillium expansum, Penicillium funiculosum, Penicillium glabrum, Penicillium glaucum, Penicillium italicum, Penicillium lacussarmientei, Penicillium marneffei, Penicillium purpurogenum, Penicillium roqueforti, Penicillium stroniferum stoloniferum, Penicillium ulaiense, Penicillium verrucosum, Penicillium viridicatumPreferred yeasts are selected from the genera Saccharomyces, Schizosaccharomyces, Candida and Pichia.

[0063] Pharmaceutical Compositions and Administration In one embodiment, the present invention provides a pharmaceutical composition having antifungal infection activity comprising a compound of general formula (I), an ester, stereoisomer, prodrug, solvate or pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier. Pharmaceutically acceptable carriers can be solid or liquid. Among them, solid carriers can be one or more substances used as excipients, diluents, sweeteners, solubilizers, lubricants, binders, tablet disintegrating agents, stabilizers, preservatives, or encapsulating materials. Liquid carriers can be solvents or liquid dispersion media. Suitable solid carriers include, but are not limited to, for example, cellulose, glucose, lactose, mannitol, magnesium stearate, magnesium carbonate, sodium carbonate, sodium saccharin, sucrose, dextrin, talc, starch, pectin, gelatin, tragacanth, gum arabic, sodium alginate, parabens, methylcellulose, sodium carboxymethylcellulose, low melting wax, cocoa butter, and the like. Suitable liquid carriers include, but are not limited to, water, ethanol, polyols (e.g., glycerin, propylene glycol, liquid polyethylene glycol, and the like), vegetable oils (e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil), glycerides, agar, pyrogen-free water, isotonic saline, Ringer's solution, and mixtures thereof.

[0064] Methods for preparing the pharmaceutical compositions of the present invention are generally known. The pharmaceutical compositions of the present invention are prepared by known methods, including conventional mixing, granulating, tableting, coating, dissolving or lyophilizing processes. In one embodiment, the pharmaceutical compositions of the invention can be administered to a patient in need of treatment by any suitable method, including orally, parenterally (including subcutaneous, intramuscular, intravenous and intradermal), by inhalation spray, topically, rectally, nasally, bucally, vaginally, or via an implanted reservoir. The compositions suitable for oral administration used in the present invention include solid forms such as pills, tablets, caplets, capsules (including immediate release, timed release and sustained release formulations, respectively), granules and powders; and liquid forms such as solutions, syrups, elixirs, emulsions and suspensions.Forms for ocular administration include sterile solutions or ocular delivery devices.Forms for parenteral administration include sterile solutions, emulsions and suspensions.

[0065] Indications and Treatment Methods The compound of the present invention or its ester, stereoisomer or pharma- ceutically acceptable salt, and pharmaceutical compositions containing the compound of the present invention or its ester, stereoisomer or pharma- ceutically acceptable salt, can be used for treating and / or preventing diseases caused by fungal infection. Thus, the present invention further relates to a method for treating and / or preventing diseases caused by fungal infection, comprising administering to a patient in need thereof a therapeutically effective amount of the compound of formula (I) of the present invention or its ester, stereoisomer, prodrug, solvate or pharma-ceutically acceptable salt.

[0066] Diseases caused by fungal infections include, but are not limited to, one or more of tinea capitis, tinea corporis, tinea pedis, onychomycosis, periungual mycosis, tinea discolor, thrush, vaginal candidiasis, respiratory tract candidiasis, biliary candidiasis, esophageal candidiasis, urinary tract candidiasis, systemic candidiasis, mucosal and cutaneous candidiasis, aspergillosis, mucormycosis, paracoccidioidomycosis, North American blastomycosis, histoplasmosis, coccidioidomycosis, sporotrichosis, fungal sinusitis, and chronic sinusitis.

[0067] The dosage is determined by various factors, including the age, weight and condition of the patient, and the route of administration. The exact dosage is determined based on the judgment of the treating physician. The actual dosage and duration of administration of the active ingredient in the pharmaceutical composition of the present invention may be varied to obtain the amount of active ingredient described below, which is an amount that effectively achieves the desired therapeutic response for a particular patient, composition and administration form, but is not toxic to the patient. The pharmaceutical composition of the present invention is usually administered in an amount sufficient to reduce or eliminate symptoms associated with fungal infection.

[0068] A preferred dosage of the drug is the maximum amount that the patient can tolerate without causing severe or unacceptable side effects. Exemplary dosage ranges include 0.01 mg to 250 mg / day, 0.01 mg to 100 mg / day, 1 mg to 100 mg / day, 10 mg to 100 mg / day, 1 mg to 10 mg / day, and 0.01 mg to 10 mg / day. A preferred dosage of the drug is the maximum amount that the patient can tolerate without causing severe or unacceptable side effects. In the examples, the drug is administered at a concentration of about 10 μg to about 100 mg / kg body weight / day, about 0.1 to about 10 mg / kg body weight / day, or about 1.0 mg to about 10 mg / kg body weight / day.

[0069] In one embodiment, a therapeutically effective dosage results in a serum concentration of drug of from about 0.1 ng / ml to about 50-100 mg / ml. These pharmaceutical compositions should typically provide a dosage of from about 0.001 mg to about 2000 mg of compound per kg body weight per day. For example, the ranges of systemic doses administered to a human patient include 1-10 mg / kg, 20-80 mg / kg, 5-50 mg / kg, 75-150 mg / kg, 100-500 mg / kg, 250-750 mg / kg, 500-1000 mg / kg, 1-10 mg / kg, 5-50 mg / kg, 25-75 mg / kg, 50-100 mg / kg, 100-250 mg / kg, 50-100 mg / kg, 250-500 mg / kg, 500-750 mg / kg, 750-1000 mg / kg, 1000-1500 mg / kg, 10 The dosage may be 1500-2000 mg / kg, 5 mg / kg, 20 mg / kg, 50 mg / kg, 100 mg / kg, 500 mg / kg, 1000 mg / kg, 1500 mg / kg, or 2000 mg / kg. Pharmaceutical unit dosage forms are prepared to provide from about 1 mg to about 5000 mg (e.g., from about 100 mg to about 2500 mg) of the compound or combination of required ingredients per unit dosage form. Preferred unit dosage preparations are those containing a daily dose or unit, daily sub-dose, or an appropriate amount thereof, of the administered ingredients as discussed herein. The present invention will be further described below with reference to specific examples, but the present invention is not limited thereto. Those skilled in the art can make various modifications and adjustments based on the teachings of the present invention without departing from the spirit and scope of the present invention. EXAMPLES

[0070] material Strains: The strains used in this invention are shown in Table 1. Among them, the triple auxotrophic chassis strain Aspergillus nidulans LO8030 (pyrG-, riboB-, pyroA-) has a clean metabolic background and low need for precursors by knocking out eight of the most highly expressed biosynthetic gene clusters (BGCs) genes (see Chiang YM et al., (2013) An efficient system for heterologous expression of secondary metabolite genes in Aspergillus nidulans, Journal of the American Chemical Society 135, 7720-7731). Considering the number of genes to be introduced and the huge number of gene fragments encoding the core biosynthetic enzymes, one of the challenges for the heterologous expression of fungal BGCs is to assemble and introduce the BGC genes into heterologous chassis strains.

[0071] Media: Solid glucose basal medium (GMM) Formulation: 10g glucose, 6g NaNO per liter of medium 3 , 0.52g KCl, 0.52g MgSO 4 7H 2 O, 1.52 g KH 2 PO 4 , 0.022g ZnSO 4 7H 2 0, 0.011 g H 3 BO 3 , 0.005 g MnCl 2 4H 2 0, 0.0016 g FeSO 4 7H 2 0, 0.0016 g CoCl 2 5H 2 0, 0.0016 g CuSO 4 5H 2 O, 0.0011 g (NH 4 ) 6 Mo 7 O24 4H 2 O, 0.05g Na 4 Contains EDTA, 16 g agar powder, and NaOH (to adjust pH to 6.5).

[0072] Preparation process: 120g NaNO 3 , 10.4g KCl, 10.4g MgSO 4 7H 2 O, 30.4g KH 2 PO 4 Prepare 1 L of 20× Nitrate Salts containing: 2.2 g ZnSO 4 7H 2 O, 1.1 g H 3 BO 3 , 0.5 g MnCl 2 4H 2 0, 0.16 g FeSO 4 7H 2 0, 0.16 g CoCl 2 5H 2 0, 0.16 g CuSO 4 5H 2 O, 0.11 g (NH 4 ) 6 Mo 7 O 24 4H 2 O, 5g Na 4 Prepare 100 mL of trace elements containing EDTA; then, for each liter of GMM liquid medium, take 50 mL of 20x Nitrate Salts, 1 mL of trace elements, 10.0 g of glucose, 16.0 g of agar powder, and add them to 800 mL of ddHO. 2 O was added, the pH was adjusted to 6.5 with NaOH, the volume was increased to 1 liter, and the mixture was sterilized in an autoclave at 121°C for 20 minutes. The mixture was cooled to 60°C and dispensed onto plates, which were then cooled and solidified at room temperature to obtain a solid glucose basal medium.

[0073] Liquid glucose basal medium: Formulation: 10g glucose, 6g NaNO per liter of medium 3 , 0.52g KCl, 0.52g MgSO 4 7H2 O, 1.52 g KH 2 PO 4 , 0.022g ZnSO 4 7H 2 0, 0.011 g H 3 BO 3 , 0.005 g MnCl 2 4H 2 0, 0.0016 g FeSO 4 7H 2 0, 0.0016 g CoCl 2 5H 2 0, 0.0016 g CuSO 4 5H 2 O, 0.0011 g (NH 4 ) 6 Mo 7 O 24 4H 2 O, 0.05g Na 4 Contains EDTA, NaOH (adjust pH to 6.5).

[0074] Preparation process: 120g NaNO 3 , 10.4g KCl, 10.4g MgSO 4 7H 2 O, 30.4g KH 2 PO 4 Prepare 1 L of 20× Nitrate Salts containing: 2.2 g ZnSO 4 7H 2 O, 1.1 g H 3 BO 3 , 0.5 g MnCl 2 4H 2 0, 0.16 g FeSO 4 7H 2 0, 0.16 g CoCl 2 5H 2 0, 0.16 g CuSO 4 5H 2 O, 0.11 g (NH 4 ) 6 Mo 7 O 24 4H 2 O, 5g Na 4100 mL of trace elements containing EDTA was prepared; Then, for each liter of GMM liquid medium, take 50 mL of 20x Nitrate Salts, 1 mL of trace elements, 10.0 g of glucose, and add 800 mL of ddHO. 2 O was added, the pH was adjusted to 6.5 with NaOH, the volume was increased to 1 liter, and the mixture was sterilized in an autoclave at 121°C for 20 minutes and cooled to room temperature to obtain a liquid glucose basal medium.

[0075] Culture conditions: Emericella rugulosa NRRL 11440 and Aspergillus nidulans LO8030 strains were stored in glycerol stocks at -80°C and were activated in solid glucose basal medium (GMM) at 37°C (Shimizu et al. (2001) Genetic involvement of a cAMP-dependent protein kinase in a G protein signaling pathway regulating morphological and chemical transitions in Aspergillus nidulans, Genetics 157, 591-600) and supplemented with nutrients as necessary. For pyrG auxotrophy, GMM was supplemented with 0.56 g / L uracil and 1.26 g / L uridine; for pyroA auxotrophy, 1 mg / L pyridoxine hydrochloride; and for riboB auxotrophy, 2.5 mg / L riboflavin. Saccharomyces cerevisiae BJ5464 was used to construct plasmids by yeast homologous recombination (Yin WB, et al., (2013). Discovery of cryptic polyketide metabolites from dermatophytes using heterologous expression in Aspergillus nidulans, ACS Synth Biol 2, 629-634.), and E. coli DH5α was used to amplify the plasmids.

[0076] [Table 1]

[0077] a In the description of Aspergillus nidulans LO8030 transformants, the genotype parts that are the same as LO8030 are omitted. b 1.1 / 1.3 / 1.6 represent three parallel transformants generated in the first transformation into Aspergillus, and the genotypes of the three transformants are the same; 2.2 / 2.4 / 2.5, 4.1 / 4.2 / 4.3 and 5.1 / 5.2 / 5.3 / 5.4 / 5.5 / 5.6 represent different parallel transformants generated in the second, third and fourth transformation, respectively. 1 Yin, WB, Chooi, YH, Smith, AR, Cacho, RA, Hu, Y., White, TC, and Tang, Y. (2013) Discovery of cryptic polyketide metabolites from dermatophytes using heterologous expression in Aspergillus nidulans, ACS Synth Biol 2, 629-634. 2 Chiang, YM, Ahuja, M., Oakley, CE, Entwistle, R., Asokan, A., Zutz, C., Wang, CC, and Oakley, BR (2016) Development of Genetic Dereplication Strains in Aspergillus nidulans Results in the Discovery of Aspercryptin, Angewandte Chemie (International ed. in English) 55, 1662-1665.

[0078] Example 1 Plasmid Construction To extract genomic DNA (gDNA), Emericella rugulosa NRRL 11440 and Aspergillus nidulans LO8030 were cultured in liquid GMM at 37°C for 24 h, supplementing nutrient deficiencies as necessary. gDNA was extracted from Emericella rugulosa NRRL 11440 according to standard methods (Sambrook, J., and Russell, DW (2001) Molecular cloning: a laboratory manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Then, the echinocandin biosynthetic gene cluster (BGC) genes (shown in Table 3) were cloned by PCR (PCR procedure is shown in Table 2) using high-fidelity DNA polymerase Primestar HS / MAX (Takara, Japan) with Emericella rugulosa NRRL 11440 gDNA as a template. The non-ribosomal polypeptide synthetase (NRPS) gene ecdA of the echinocandin biosynthetic gene cluster (BGC) was divided into four fragments ecdA1-4 (each fragment was about 6-7 kb) and used for PCR cloning. Among them, the ecdA1 fragment contains base pairs at positions 1-5831, the ecdA2 fragment contains base pairs at positions 5648-11563, the ecdA3 fragment contains base pairs at positions 10591-16084, and the ecdA4 fragment contains base pairs at positions 14938-21910. All PCR primers used in the present invention are shown in Table 4.

[0079] [Table 2]

[0080] [Table 3]

[0081] [Table 4] TIFF2025511851000024.tif236164TIFF2025511851000025.tif116167

[0082] Based on the description by Yin et al. (Discovery of cryptic polyketide metabolites from dermatophytes using heterologous expression in Aspergillus nidulans, ACS Synth Biol (2013) 2, 629-634.), a plasmid was constructed in the budding yeast BJ5464 by yeast homologous recombination (Figure 1), and the constructed plasmid can generate a gene expression cassette for transformation into Aspergillus (shown in Table 5). The E. coli origin of replication (Ori), ampicillin resistance selection marker (Amp R The plasmid backbone, containing the pYH-yA-riboA gene (GenBank:M77789), the yeast replication origin 2μ (2 micron / 2 micron ori / 2 mu), and the uracil auxotrophic selection marker URA (GenBank:X75459), was derived from the plasmid pYH-yA-riboA (Wang, PM et al. (2016). "TrpE feedback mutants reveal roadblocks and conduits toward increasing secondary metabolism in Aspergillus fumigatus. " Fungal genetics and biology 89: 102-113.).

[0083] [Table 5]

[0084] Example 2 Preparation of Aspergillus nidulans transgenic strain LO8030-5.1 Several fragments of the plasmid were first ligated by double-joint PCR (DJ-PCR) (Yu et al. (2004) Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi, Fungal genetics and biology : FG & B 41, 973-981.), and then the plasmid was constructed by yeast homologous recombination to obtain the gene expression cassette. As shown in Figure 2, the heterologous gene expression cassettes of ecdA and ecdI were integrated into the yA gene site of Aspergillus nidulans LO8030 by three rounds of transformation.

[0085] Specifically, the gene expression cassette used in the first transformation was derived from the plasmid pYX1 (Figure 3). The gene expression cassette sequentially contains the xylose-inducible promoter xylp (GenBank:HM640258) from the pSK plasmid (Hartmann, T. et al. (2010). "Validation of a self-excising marker in the human pathogen Aspergillus fumigatus by employing the beta-rec / six site-specific recombination system." Appl Environ Microbiol 76(18): 6313-6317.), the first and second fragments of ecdA, ecdA1-2, and the auxotrophic marker gene riboB (AfriboB) from Aspergillus fumigatus (Nayak, T. et al. (2006). "A versatile and efficient gene-targeting system for Aspergillus nidulans." Genetics 172(3): 1557-1566.), and both sides of the gene cassette are the gene yA (O'Hara, E. B. and W. Timberlake (1989). "Molecular characterization of the Aspergillus nidulans yA locus." Genetics 121(2): 249-254.) The approximately 1 kb DNA fragments yA-up and yA-down upstream and downstream were obtained. First, the ecdA1 and ecdA2 fragments were linked by double joint PCR to obtain ecdA1-2. The templates and primers for PCR cloning of each fragment are shown in Table 6, and the specific primer sequences used are shown in Table 4. After amplifying each fragment by PCR (Table 6), gel cutting and purification were performed, and then the fragments were mixed and transformed into yeast BJ5464 to obtain yeast transformants. The yeast plasmid was then extracted using a yeast plasmid extraction kit (OMEGA Bio-tek), and the plasmid was transformed into E. coli competent cells to perform plasmid amplification and PCR detection.The primer pair detected the boundary of each fragment, and the target fragment of the desired size was obtained, and the plasmid pYX1 was successfully constructed (Figure 3). The plasmid pYX1 was cleaved with the restriction enzyme EcoRI, and the gene expression cassette with a size of 17.9 kb was retrieved using a DNA fragment selection and purification kit (BioMag, China). Protoplast generation, transformation, screening and verification of the transformed strains were performed according to Ma et al. ((2018) Rational design for heterologous production of aurovertin-type compounds in Aspergillus nidulans, Appl Microbiol Biotechnol 102, 297-304) and Bok et al. ((2004) LaeA, a regulator of secondary metabolism in Aspergillus spp, Eukaryotic cell 3, 527-535.). Three transformed strains carrying xylp-ecdA1-2-AfriboB at the yA locus were obtained and named LO8030-1.1, 1.3 and 1.6, respectively. One of them, LO8030-1.3, was used as the parent strain for the next transformation (Figure 3).

[0086] [Table 6]

[0087] The gene expression cassette used in the second transformation was derived from the plasmid pYX5 (Figure 4). The gene expression cassette contains the third fragment of ecdA3, the auxotrophic marker gene pyrG (AppyrG) from A. parasiticus (GenBank: EU817656), the promoter gpdAp encoding the glyceraldehyde-3-phosphate dehydrogenase gene from A. nidulans (Lim, FY et al. (2012). "Genome-based cluster deletion reveals an endocrocin biosynthetic pathway in Aspergillus fumigatus." Appl Environ Microbiol 78(12): 4117-4125.), ecdI, and a DNA fragment of about 1 kb downstream of the yA gene, yA-down. Here, ecdA3 has a homologous sequence of about 1 kb with ecdA2, and this homologous sequence is used as a fragment of the homologous region for transforming Aspergillus and homologous recombination. After amplifying each fragment by PCR (Table 7), gel cutting and purification were performed, and the fragments were mixed and transformed into yeast BJ5464 to obtain yeast transformants. The yeast plasmid was extracted using a yeast plasmid extraction kit (OMEGA Bio-tek), and the plasmid was transformed into E. coli competent cells for plasmid amplification and PCR detection. When the boundary of each fragment was detected with the primer pair, the target fragment of the desired size was obtained, and the construction of plasmid pYX5 was successful (Figure 4).Plasmid pYX5 was digested with restriction enzyme NotI, and the gene expression cassette with a size of 11.7 kb was retrieved using a DNA fragment selection and purification kit (BioMag, China). Protoplast generation, transformation, screening and verification of the transformed strains were performed according to Ma et al. ((2018) Rational design for heterologous production of aurovertin-type compounds in Aspergillus nidulans, Appl Microbiol Biotechnol 102, 297-304) and Bok et al. ((2004) LaeA, a regulator of secondary metabolism in Aspergillus spp, Eukaryotic cell 3, 527-535.). Three transformants in which xylp-ecdA1-3-AppyrG-gpdAp-ecdI was inserted at the yA locus were obtained and named LO8030-2.2, 2.4 and 2.5, respectively. One of them, LO8030-2.2, was used as the parent strain for the next transformation (Figure 4).

[0088] [Table 7]

[0089] The gene expression cassette used in the third transformation was derived from the plasmid pYX9 (Figure 5). The gene expression cassette contained the fourth fragment of ecdA, ecdA4, the auxotrophic marker gene riboB (AfriboB) from Aspergillus fumigatus (Nayak, T. et al. (2006). "A versatile and efficient gene-targeting system for Aspergillus nidulans." Genetics 172(3): 1557-1566.), and the promoter of the glyceraldehyde-3-phosphate dehydrogenase-encoding gene gpdAp from Aspergillus nidulans (A. nidulans) (Lim, FY et al. (2012). "Genome-based cluster deletion reveals an endocrocin biosynthetic pathway in Aspergillus fumigatus." Appl Environ Microbiol 78(12): 4117-4125.). Here, ecdA4 has a homologous sequence of about 1 kb with ecdA3, and this homologous sequence is used as a fragment of the homologous region for transforming Aspergillus and homologous recombination. After amplifying each fragment by PCR (Table 8), gel cutting and purification were performed, and the fragments were mixed and transformed into yeast BJ5464 to obtain yeast transformants. The yeast plasmid was extracted using a yeast plasmid extraction kit (OMEGA Bio-tek), and the plasmid was transformed into E. coli competent cells for plasmid amplification and PCR detection. When the boundary of each fragment was detected with the primer pair, the target fragment of the desired size was obtained, and the construction of plasmid pYX9 was successful (Figure 5).Plasmid pYX9 was digested with restriction enzyme NotI, and a gene expression cassette with a size of 10.7 kb was recovered using a DNA fragment selection and purification kit (BioMag, China). Protoplast generation, transformation, screening and verification of the transformed strains were performed according to Ma et al. ((2018) Rational design for heterologous production of aurovertin-type compounds in Aspergillus nidulans, Appl Microbiol Biotechnol 102, 297-304) and Bok et al. ((2004) LaeA, a regulator of secondary metabolism in Aspergillus spp, Eukaryotic cell 3, 527-535.). Three transformed strains in which xylp-ecdA-AfriboB-gpdAp-ecdI was inserted at the yA locus were obtained and named LO8030-4.1, 4.2, and 4.3, respectively. One of them, LO8030-4.1, was used as the parent strain for the next transformation (Figure 5).

[0090] [Table 8]

[0091] A control plasmid pYX14 was constructed to obtain an expression cassette containing xylp, AfriboB and gpdAp (Figure 6). After amplifying each fragment by PCR (Table 9), gel cutting and purification were performed, and the fragments were mixed and transformed into yeast BJ5464 to obtain yeast transformants. The yeast plasmids were then extracted using a yeast plasmid extraction kit (OMEGA Bio-tek), and the plasmids were transformed into E. coli competent cells for plasmid amplification and PCR detection. When the boundaries of each fragment were detected with the primer pair, the target fragments of the desired size were obtained, and the construction of plasmid pYX14 was successful (Figure 6). Plasmid pYX14 was digested with restriction enzyme NotI, and a gene expression cassette with a size of 7.5 kb was retrieved using a DNA fragment selection and purification kit (BioMag, China). Protoplasts of Aspergillus nidulans LO8030 were generated, transformed, and the transformants were screened and verified according to Ma et al. ((2018) Rational design for heterologous production of aurovertin-type compounds in Aspergillus nidulans, Appl Microbiol Biotechnol 102, 297-304) and Bok et al. ((2004) LaeA, a regulator of secondary metabolism in Aspergillus spp, Eukaryotic cell 3, 527-535.). A control transformant with xylp-gpdAp-AfriboB inserted at the yA locus was obtained and named LO8030-4C.1 (Figure 6).

[0092] [Table 9]

[0093] The "E. coli-yeast-Aspergillus" shuttle plasmid pYX10 was constructed to express the oxygenase genes ecdK and htyE in the transformed strain LO8030-4.1 (Figure 7). The plasmid contains the E. coli origin of replication (Ori), an ampicillin resistance selection marker (Amp), and a genomic DNA fragment encoding the ECdK and htyE genes. R) (GenBank: M77789), yeast replication origin 2μ (2 micron / 2 micron ori / 2 mu), and uracil auxotrophic selection marker URA (GenBank: X75459) were included as a plasmid backbone. The plasmid also contained the htyE gene (amyBp-htyE) controlled by promoter amyBp, and the promoter gpdAp (Lim, FY et al. (2012). "Genome-based cluster deletion reveals an endocrocin biosynthetic pathway in Aspergillus fumigatus." Appl Environ Microbiol 78(12): The ecdK gene (gpdAp-ecdK) controlled by the chromosome 4117-4125.), the auxotrophic marker gene pyrG (AppyrG) from Aspergillus parasiticus (GenBank: EU817656), and the Aspergillus autonomously replicating sequence AMA1 from Aspergillus nidulans (Storms, R. et al. (2005). "Plasmid vectors for protein production, gene expression and molecular manipulations in Aspergillus niger." Plasmid 53(3): 191-204.) were included. After amplifying each fragment by PCR (Table 10), gel cutting and purification were performed, and the fragments were mixed and transformed into yeast BJ5464 to obtain yeast transformants, and yeast plasmids were extracted using a yeast plasmid extraction kit (OMEGA Bio-tek), and the plasmids were transformed into E. coli competent cells for plasmid amplification and PCR detection. By detecting the boundaries of each fragment with the primer pair, a target fragment of the desired size was obtained, and the plasmid pYX10 was successfully constructed (Figure 7).According to Ma et al. ((2018) Rational design for heterologous production of aurovertin-type compounds in Aspergillus nidulans, Appl Microbiol Biotechnol 102, 297-304) and Bok et al. ((2004) LaeA, a regulator of secondary metabolism in Aspergillus spp, Eukaryotic cell 3, 527-535.), we generated protoplasts of Aspergillus nidulans LO8030-4.1, transformed them, and screened and verified the transformed strains. We obtained a series of transformed strains carrying the plasmid pYX10 in the cells, which were named LO8030-5.1 to 5.6, respectively, and one of them, LO8030-5.1, was taken for further studies. In addition, a control plasmid pYX12 was constructed. The difference between this plasmid and pYX10 is that it does not have the ecdK and htyE genes. After amplifying each fragment by PCR according to Table 11, the transformation of yeast BJ5464, generation of protoplasts of Aspergillus nidulans LO8030-4.1, transformation, screening and verification of the transformed strains were carried out in the same manner as above. A transformed strain carrying the control plasmid pYX12 was obtained, and the control transformed strain was named LO8030-5C.1 (Figure 8).

[0094] [Table 10]

[0095] [Table 11]

[0096] The whole genome of the genetically transformed strain LO8030-5.1 was sequenced by Illumina HiSeq 2000, and it was found that no mutations occurred in the four gene cluster genes (ecdA, ecdI, ecdK and htyE) expressed in the transformed strain LO8030-5.1 (shown in Figure 9), and each gene cassette was not randomly inserted into the chromosome. This indicates that high-fidelity DNA polymerase can effectively prevent gene mutations that are prone to occur by PCR cloning of large fragments, and also proves the accuracy and reliability of homologous recombination and integration of the gene expression cassette into the chassis strain Aspergillus nidulans LO8030.

[0097] Example 3 RNA preparation and semi-quantitative RT-PCR Aspergillus nidulans transgenic strain LO8030-5.1 and its control LO8030-5C.1 were cultured in XSMM medium under the same conditions as those for fermentative production of echinocandins, and the fungal cells were harvested. The volume was divided into 2 parts per formula. XSMM medium was derived from liquid GMM medium, except that the carbon source glucose (10 g / L) in GMM was replaced with xylose (20 g / L) and starch (20 g / L). Total RNA was extracted using Trizol® Reagent (Ambion) and DNase I (Promaga). cDNA was synthesized using PrimeScript RT Kit (TaKaRa) and 100 ng of DNase I-treated RNA. Semiquantitative RT-PCR was performed in 25 μL of reaction mixture. The mixture contained 1 μL of diluted cDNA (10-fold diluted) as template and 12.5 μL of 2×Es Taq MasterMix (Cwbio, China). The internal control was the histone 2B gene (H2B) and the primers used for each specific gene are shown in Table 4. The results showed that all four transformed genes (ecdA, ecdI, htyE and ecdK) were transcribed to various degrees. Also, expression of ecdG, ecdH and htyF was not detected, indicating that these genes were not transformed into LO8030-5.1. The RT-PCR results are shown in Figure 10.

[0098] Example 4 Fermentation and HPLC / MS Analysis Aspergillus nidulans genetically transformed strains LO8030-4.1 and LO8030-5.1 and their controls LO8030-4C.1 and LO8030-5C.1 were cultivated in XSMM fermentation medium. XSMM medium is derived from liquid GMM medium, only the carbon source glucose (10 g / L) in GMM was replaced by xylose (20 g / L) and starch (20 g / L). LO8030-5.1 and LO8030-5C.1 medium was supplemented with 1 mg / L pyridoxine hydrochloride, and LO8030-4.1 and LO8030-4C.1 medium was supplemented with 1.26 g / L uracil, 1.26 g / L uridine, and 100 mg / L 4R-Me-L-proline. Since the BGC biosynthetic gene cluster transformed in the present invention does not contain the htyA-D genes, L-homotyrosine (100 mg / L) was added to the medium of the present invention for the synthesis of deoxyechinocandins. Furthermore, linoleic acid (300 mg / L) was added to the XSMM fermentation medium to promote the production of echinocandins. 100 mL of the above XSMM medium was placed in a 500 mL flask and fermented for 12 days at 25°C and 180 rpm. The total was 3 parts per batch.

[0099] The cells were then separated from the culture by filtration. The cells were immersed in methanol and subjected to ultrasonic extraction three times for 30 minutes each time. The culture was extracted three times with an equal volume of ethyl acetate, the organic phases were combined, and the solvent was removed under vacuum. The extract was dissolved in 1 mL of methanol, filtered through a 0.22 μm filter membrane, and used for HPLC and LC-MS analysis. 20 μL of the filtrate was subjected to reverse phase high performance liquid chromatography (RP-HPLC) (Agilent 1260). 18 -MS-II chromatography column (4.6 × 250 mm, 5 μm) was used, the flow rate was 1 mL / min, the column temperature was 35 °C, and the eluent was 10 to 100% MeOH-H 2 O (v / v) (linear gradient), time was 60 min. Chromatographic peaks were detected at a wavelength of 222 nm.

[0100] Agilent UPLC-Triple-TOF 5600 + LC-MS analysis was performed using a Waters ACQUITY UPLC HSS SB-C LC / MS system (with positive mode electrospray ionization). 18 A chromatography column (100 × 2.1 mm, 1.7 μm) was used, with the eluent being 5 to 100% aqueous methanol + 0.1% formic acid (linear gradient) at a flow rate of 0.4 mL / min. HPLC analysis revealed no new obvious peaks in LO8030-4.1 compared to the control strain LO8030-4C.1 (shown in FIG. 11). Compared to the control strain LO8030-5C.1, the HPLC spectrum of LO8030-5.1 showed two new peaks at retention times of 57.3 and 57.8 minutes (shown in FIG. 11). The molecular weights of these two peaks were then determined by LC-MS. Compound 1: TOF-ESIMS, m / z 982.5841 [M+H] + , m / z 1004.5668 [M+Na] + ;Compound 2:TOF-ESIMS,m / z 996.6004 [M+H] + , m / z 1018.5823 [M+Na] + (Figure 11).

[0101] Example 5 Nuclear Magnetic Resonance Analysis of Compound 2 (Echinocandin E) and Compound 1 (Echinocandin F) After the transformant LO8030-5.1 was fermented in 10 L of XSMM medium, the fermentation liquid and cells were extracted three times with equal volumes of ethyl acetate and methanol, respectively. The organic phases were combined and distilled under reduced pressure to obtain a crude extract (2.20 g). The extract was separated on a silica gel column, and the mobile phase was CH 2 Cl 2 / MeOH (50:1, 20:1, 10:1, 5:1, 2:1, 1:1). Initial identification of echinocandins was achieved by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). They were then further purified by semi-preparative high-performance liquid chromatography using a COSMOCIL C18-MS-II chromatography column (10 × 250 mm, 5 μm) and MeOH / H2O as the mobile phase. 2 O (85:15) and the flow rate was 3.0 mL / min. The separated compounds 1 and 2 were then subjected to CD 3 The sample was dissolved in OD and subjected to nuclear magnetic resonance analysis using a JNM-ECZ600R / S1 spectrometer. 1 H, 13 C and 2D( 1 H- 13 C HSQC, 1 H- 13 C HMBC and 1 H- 1 The nuclear magnetic resonance spectrum of HCOSY was recorded. 1 H NMR and 13 C NMR data is shown in Table 12.

[0102] [Table 12] TIFF2025511851000034.tif84162

[0103] Compound 1 (echinocandin F) has the same structure as L-homotyrosine except for the loss of the methyl group at the 3S-OH-4S-Me-L-proline residue. 3 / C 4 In L-ornithine, the hydroxyl group is lost and the C 4 / C 5The structural features of compound 2 (echinocandin E), including the loss of a hydroxyl group, are the same as those of compound 2 (echinocandin E) (shown in FIG. 12). Of the gene cluster (ecdA, ecdI, ecdK, htyE), ecdK is involved in the formation of 3S-OH-4S-Me-L-proline. Therefore, it is speculated that the reason why the transformed bacterium LO8030-5.1 produces echinocandin F is that ecdK does not function in the biosynthetic process of the compound, i.e., the biosynthetic gene cluster of echinocandin F is (ecdA, ecdI, htyE). This speculation was verified in Examples 6 and 7.

[0104] Example 6 Plasmid Construction and Preparation of Aspergillus nidulans Transformant Strain LO8030-4.2.1 The "E. coli-yeast-Aspergillus" shuttle plasmid pJQ10 was constructed to express the oxygenase gene htyE in the transformed strain LO8030-4.2 (Figure 13). This plasmid contains the E. coli origin of replication (Ori), an ampicillin resistance selection marker (Amp), and a genomic DNA fragment encoding the htyE gene. RIn addition to the plasmid backbone of the yeast replication origin 2μ (2 micron / 2 micron ori / 2 mu) and the uracil auxotrophic selection marker URA (GenBank: X75459), it further contains the htyE gene controlled by the promoter amyBp (amyBp-htyE), the auxotrophic marker gene pyrG (AppyrG) from Aspergillus parasiticus (GenBank: EU817656), and the Aspergillus autonomously replicating element AMA1 from Aspergillus nidulans (Storms, R. et al. (2005). "Plasmid vectors for protein production, gene expression and molecular manipulations in Aspergillus niger." Plasmid 53(3): 191-204.). After amplifying each fragment by PCR (Table 13), gel cutting and purification were performed, and the fragments were mixed and transformed into yeast BJ5464 to obtain yeast transformants. The yeast plasmid was extracted using a yeast plasmid extraction kit (OMEGA Bio-tek), and the plasmid was transformed into E. coli competent cells to amplify the plasmid and perform PCR detection. When the boundary of each fragment was detected with the primer pair, the target fragment of the desired size was obtained, and the construction of plasmid pJQ10 was successful (Figure 13). Protoplast generation, transformation, screening and validation of transformed strains of Aspergillus nidulans LO8030-4.2 were performed according to Ma et al. ((2018) Rational design for heterologous production of aurovertin-type compounds in Aspergillus nidulans, Appl Microbiol Biotechnol 102, 297-304) and Bok et al. ((2004) LaeA, a regulator of secondary metabolism in Aspergillus spp, Eukaryotic cell 3, 527-535.).A series of transformants carrying the plasmid pJQ10 in their cells were obtained, and one of the transformants was selected and designated LO8030-4.2.1 for further study.

[0105] [Table 13]

[0106] Example 7 Fermentation and Analysis The Aspergillus nidulans gene transformant LO8030-4.2.1 was cultured in XSMM medium, and 1 mg / L pyridoxine hydrochloride was added to the medium. L-homotyrosine (100 mg / L) and linoleic acid (300 mg / L) were also added to the XSMM fermentation medium. After fermenting the transformant LO8030-4.2.1 in 10 L of XSMM medium for 12 days, the fermentation liquid and the fungus were extracted three times with equal volumes of ethyl acetate and methanol, respectively. The organic phases were combined and distilled under reduced pressure to obtain a crude extract. It was confirmed that the transformant LO8030-4.2.1 produced echinocandin F by the analytical method of Example 4.

[0107] Example 8 Quantitative analysis of echinocandin E and echinocandin F in fermentation products According to the fermentation method described above, the transformed strains LO8030-5.1 and LO8030-4.2.1 were fermented in three bottles in parallel (200 mL / bottle), and 10-fold volume of methanol was added to the fermented product (including the cells and the cell solution) of each bottle, and after precipitation, the mixture was centrifuged and filtered through a filter membrane, and then used as a sample for HPLC quantitative analysis. The analysis conditions for ODS-HPLC were isocratic elution with 80% methanol / water, a flow rate of 1 mL / min, and a detection wavelength of 220 nm. The reference substances for quantification were echinocandin E and echinocandin F separated above. Solutions of different concentrations of the reference substances were prepared, and the HPLC peak areas of the solutions of each concentration were measured under the condition that the injection amount was constant, and standard curves (Figures 14A and 14B) were plotted. According to the standard curves, the yields of echinocandin E and echinocandin F in LO8030-5.1 fermentation were 10 mg / L and 6 mg / L, respectively, and the yield of echinocandin F in LO8030-4.2.1 fermentation was calculated to be 7 mg / L.

[0108] Example 9 Plasmid Construction and Preparation of Aspergillus nidulans Transgenic Strain LO8030-4.2.1.5 The "E. coli-yeast-Aspergillus" shuttle plasmid pJQ5 was constructed to express the oxygenase gene ecdG in the transformed strain LO8030-4.2.1 (Figure 25). This plasmid contains the E. coli origin of replication (Ori), an ampicillin resistance selection marker (Amp), and a genomic DNA fragment encoding the ecdG gene. RIn addition to the plasmid backbone of the yeast replication origin 2μ (2 micron / 2 micron ori / 2 mu) and the uracil auxotrophic selection marker URA (GenBank:X75459), it further contains the ecdG gene controlled by the promoter amyBp (amyBp-ecdG), the auxotrophic marker gene pyroA4 from Aspergillus fumigatus (GenBank:CP097567.1), and the Aspergillus autonomously replicating element AMA1 from Aspergillus nidulans (Storms, R. et al. (2005). "Plasmid vectors for protein production, gene expression and molecular manipulations in Aspergillus niger." Plasmid 53(3): 191-204.). After amplifying each fragment by PCR (Table 14), gel cutting and purification were performed, and the fragments were mixed and transformed into yeast BJ5464 to obtain yeast transformants. The yeast plasmid was extracted using a yeast plasmid extraction kit (OMEGA Bio-tek), and the plasmid was transformed into E. coli competent cells to amplify the plasmid and perform PCR detection. When the boundary of each fragment was detected with the primer pair, the target fragment of the desired size was obtained, and the construction of plasmid pJQ5 was successful (Figure 25). Protoplast generation, transformation, screening and validation of transformed strains of Aspergillus nidulans LO8030-4.2.1 were performed according to Ma et al. ((2018) Rational design for heterologous production of aurovertin-type compounds in Aspergillus nidulans, Appl Microbiol Biotechnol 102, 297-304) and Bok et al. ((2004) LaeA, a regulator of secondary metabolism in Aspergillus spp, Eukaryotic cell 3, 527-535.).A series of transformants harboring the plasmid pJQ5 in their cells were obtained, and one of the transformants was selected and designated LO8030-4.2.1.5 for further study.

[0109] [Table 14]

[0110] Example 10 Fermentation and Analysis Aspergillus nidulans genetically transformed strain LO8030-4.2.1.5 was cultivated in XSMM medium, and L-homotyrosine (100 mg / L) and linoleic acid (300 mg / L) were added to the medium. After fermenting the transformed strain LO8030-4.2.1.5 in 10 L of XSMM medium for 12 days, the fermentation liquid and fungal cells were extracted three times with equal volumes of ethyl acetate and methanol, respectively. The organic phases were combined and distilled under reduced pressure to obtain a crude extract. The extract was separated on a silica gel column, and the mobile phase was replaced with CH 2 Cl 2 / MeOH (50:1, 20:1, 10:1, 5:1, 2:1, 1:1). Initial identification of echinocandins was achieved by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). They were then further purified by semi-preparative high-performance liquid chromatography using a COSMOCIL C18-MS-II chromatography column (10 × 250 mm, 5 μm) and MeOH / H2O as the mobile phase. 2 O (85:15) at a flow rate of 3.0 mL / min. Separation afforded a novel detetraoxyechinocandin, compound 3, which was named echinocandin G (ECG). TOF-ESIMS m / z 998.5800 [M+H] + , m / z 1020.5626 [M+Na] + (Figure 26).

[0111] The separated compound 3 was 3 The sample was dissolved in OD and subjected to nuclear magnetic resonance analysis using a JNM-ECZ600R / S1 spectrometer. 1 H, 13 C and 2D( 1H- 13 C HSQC, 1 H- 13 C HMBC and 1 H- 1 The nuclear magnetic resonance spectrum of HCOSY was recorded. 1 H NMR and 13 C NMR data is shown in Table 15.

[0112] [Table 15] TIFF2025511851000038.tif81167

[0113] Example 11 Plasmid construction and preparation of Aspergillus nidulans transgenic strain LO8030-4.2.1.9 The "E. coli-yeast-Aspergillus" shuttle plasmid pJQ9 was constructed to express the oxygenase genes ecdG and htyF in the transformed strain LO8030-4.2.1 (Figure 28). This plasmid contains the E. coli origin of replication (Ori), an ampicillin resistance selection marker (Amp), and a genomic DNA fragment encoding the ECdG and htyF genes. RIn addition to the plasmid backbone of the yeast replication origin 2μ (2 micron / 2 micron ori / 2 mu) and the uracil auxotrophic selection marker URA (GenBank: X75459), it further contains the ecdG gene controlled by the promoter amyBp (amyBp-ecdG), the htyF gene controlled by the promoter gpdAp (gpdAp-htyF), the auxotrophic marker gene pyroA4 from Aspergillus fumigatus (GenBank: CP097567.1), and the Aspergillus autonomously replicating element AMA1 from Aspergillus nidulans (Storms, R. et al. (2005). "Plasmid vectors for protein production, gene expression and molecular manipulations in Aspergillus niger." Plasmid 53(3): 191-204.). After amplifying each fragment by PCR (Table 16), gel cutting and purification were performed, and the fragments were mixed and transformed into yeast BJ5464 to obtain yeast transformants. The yeast plasmid was extracted using a yeast plasmid extraction kit (OMEGA Bio-tek), and the plasmid was transformed into E. coli competent cells to amplify the plasmid and perform PCR detection. When the boundary of each fragment was detected with the primer pair, the target fragment of the desired size was obtained, and the construction of plasmid pJQ9 was successful (Figure 28).According to Ma et al. ((2018) Rational design for heterologous production of aurovertin-type compounds in Aspergillus nidulans, Appl Microbiol Biotechnol 102, 297-304) and Bok et al. ((2004) LaeA, a regulator of secondary metabolism in Aspergillus spp, Eukaryotic cell 3, 527-535.), we generated protoplasts of Aspergillus nidulans LO8030-4.2.1, transformed them, and screened and verified the transformants. We obtained a series of transformants carrying the plasmid pJQ9 in the cells, and one of them was taken and named LO8030-4.2.1.9 for further studies.

[0114] [Table 16]

[0115] Example 12 Fermentation and Analysis The Aspergillus nidulans genetically transformed strain LO8030-4.2.1.9 was cultivated in XSMM medium, and the medium was supplemented with L-homotyrosine (100 mg / L) and linoleic acid (300 mg / L). After fermenting the transformed strain LO8030-4.2.1.9 in 10 L of XSMM medium for 12 days, the fermentation liquid and fungal cells were extracted three times with equal volumes of ethyl acetate and methanol, respectively. The organic phases were combined and distilled under reduced pressure to obtain a crude extract. The extract was separated on a silica gel column, and the mobile phase was replaced with CH 2 Cl 2 / MeOH (50:1, 20:1, 10:1, 5:1, 2:1, 1:1). Initial identification of echinocandins was achieved by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). They were then further purified by semi-preparative high-performance liquid chromatography using a COSMOCIL C18-MS-II chromatography column (10 × 250 mm, 5 μm) and MeOH / H2O as the mobile phase.2 O (85:15) at a flow rate of 3.0 mL / min. The separation afforded a novel detetraoxyechinocandin, compound 4, which was named echinocandin H (ECH). 51 H 79 N 7 O 14 m / z 1014.5770 [M+H] + , m / z 1036.5590 [M+Na] + (Figure 29). The separated compound 4 was 3 The sample was dissolved in OD and subjected to nuclear magnetic resonance analysis using a JNM-ECZ600R / S1 spectrometer. 1 H, 13 C and 2D( 1 H- 13 C HSQC, 1 H- 13 C HMBC and 1 H- 1 The nuclear magnetic resonance spectrum of HCOSY was recorded. 1 H NMR and 13 C NMR data is shown in Table 17.

[0116] [Table 17] TIFF2025511851000041.tif85169

[0117] Example 13 Biological activity measurement The minimum inhibitory concentration (MIC) values ​​of echinocandin E and echinocandin F were determined by broth microdilution method, and the test strain was Candida albicans ATCC 10231. The MIC value is defined as the lowest antibiotic concentration that inhibits the growth of the strain by more than 90%. Amphotericin B was used as the positive control and echinocandin B was used as the reference, and three parallel experiments were performed for each sample. Mueller-Hinton (MH) broth medium (50 mL of 20x Nitrate Salts, 1 mL of trace elements, 10.0 g of glucose in 800 mL of ddHO)2 The mixture was dissolved in 100 water, the pH was adjusted to 6.5, and the volume was increased to 1 L. 16.0 g of agar powder was added and sterilized in an autoclave at 121 °C for 20 minutes. Candida albicans was cultured in a 100 mL flask (purchased from Hangzhou Basebio Biotechnology Co., Ltd.) at 37 °C and 180 rpm for 12 to 16 hours, and the bacterial solution was serially diluted with MH broth medium to a concentration of 10. 6 It was expressed as CFU / mL.

[0118] Sample solutions were prepared as follows. Test samples (amphotericin B, echinocandin B, echinocandin E, echinocandin F) were dissolved in DMSO to prepare a mother solution with a concentration of 40 μg / ml. Sterile MH broth medium and test compounds were added to a 96-well plate so that the total volume of each well was 100 μL, and the final concentrations of the compounds were 2, 1.5, 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05, and 0 μg / ml, respectively. A fixed amount of Candida albicans was inoculated, and DMSO (volume concentration 5%) was used as a negative control. The 96-well plate was placed in a 37°C incubator and incubated for 12 hours. The growth of the pathogens in the wells was observed, and the OD was measured using a microplate reader based on the concentration at which the pathogens were not growing. 600 The bacterial concentration in the liquid was measured, and the MIC value of each sample was determined. The results are shown in Table 13 below.

[0119] [Table 18]

[0120] Experiments have demonstrated that echinocandins E, F, G and H exhibit good antibacterial activity against Candida albicans. This further clearly indicates that the compounds obtained by further appropriately modifying the compounds of the present invention also have good antibacterial activity against Candida albicans. Although the foregoing invention has been described in detail by way of illustration and example for purposes of clarity of understanding, those skilled in the art will readily appreciate that, in light of the teachings of the invention, various changes and modifications may be made without departing from the spirit or scope of the invention, as defined by the appended claims.

Claims

1. An echinocandin compound represented by the general formula (I), or an ester, stereoisomer, prodrug, solvate or pharma- ceutically acceptable salt thereof: 【Chemistry 1】 “During the ceremony, R １ is selected from H; R ２ is selected from H; R ３ is selected from H, alkyl; R ５ , R ６ may be the same or different and are each independently selected from H or OH; R ４ , R ８ may be the same or different and are each independently selected from H, an alkyl, an alkenyl, an alkynyl, an aryl, a heteroaryl, a cycloalkyl, and a heterocyclyl group, wherein the alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl groups are halogen, cyano, nitro, mercapto, -S-alkyl, -S(O)-alkyl, -SO ２ -Alkyl, NR ｃ R ｄ , COOR ｅ , C.O.R. ｃ 'R ｄ ', R ｃ , R ｄ , R ｅ , R ｃ ', R ｄ each ' is independently selected from H, alkyl; R ７ is H, OH, halogen, -OSO ３ H, or -alkylene-NR ｆ R ｇ is selected from R ｆ , R ｇ are each independently selected from H, alkyl; R ９ is H or fatty acyl; however, R ｃ ' and R ｄ ' is not H at the same time; R １ , R ２ , R ５ is H, and R ６ is OH, R ３ is not methyl."

2. R ４ , R ８ may be the same or different, and each independently represents H, C １－６ Alkyl, C １－６ Alkenyl, C １－６ Alkynyl, C ６－１４ Aryl, C ６－１４ Heteroaryl, C ６－１４ Cycloalkyl, C ６－１４ heterocyclyl groups, １－６ Alkyl, C １－６ Alkenyl, C １－６ Alkynyl, C ６－１４ Aryl, C ６－１４ Heteroaryl, C ６－１４ Cycloalkyl, C ６－１４ Heterocyclyl groups include halogen, cyano, nitro, mercapto, -S-C １－６ Alkyl, -S(O)-C １－６ Alkyl, -SO ２ -C １－６ Alkyl, NR ｃ R ｄ , COOR ｅ , C.O.R. ｃ 'R ｄ ', R ｃ , R ｄ , R ｅ , R ｃ ', R ｄ ' are each independently H, C １－６ 2. The compound of claim 1, wherein R is selected from the group consisting of alkyl, aryl, arylsulfates, arylalkyl, arylsulfates ...

3. R ４ , R ８ may be the same or different, and each independently represents H, C １－６ alkyl, １－６ Alkyl is halogen, cyano, nitro, mercapto, -S-C １－６ Alkyl, -S(O)-C １－６ Alkyl, -SO ２ -C １－６ Alkyl, NR ｃ R ｄ , COOR ｅ , C.O.R. ｃ 'R ｄ ', R ｃ , R ｄ , R ｅ , R ｃ ', R ｄ ' are each independently H, C １－６ 2. The compound of claim 1, wherein R is selected from the group consisting of alkyl, aryl, arylsulfates, arylalkyl, arylsulfates ...

4. R ９ is C ６－５０ 2. The compound of claim 1, which is a fatty acyl and may contain unsaturated alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl groups, or esters, stereoisomers, prodrugs, solvates and pharma- ceutically acceptable salts thereof. 【Request 5】 【Chemical 2】 5. The compound according to any one of claims 1 to 4, wherein:

6. however, R ５ , R ６ at least one of is H; R ｃ ' and R ｄ ' is not H at the same time; R １ , R ２ , R ５ is H, and R ６ is OH, R ３ The compound of claim 1, wherein is not methyl, or an ester, stereoisomer, prodrug, solvate or pharma- ceutically acceptable salt thereof.

7. R １ , R ２ , R ５ , R ６ The compound of claim 1, wherein is H, or an ester, stereoisomer, prodrug, solvate or pharma- ceutically acceptable salt thereof.

8. R １ , R ２ , R ５ , R ６ is H; R ３ , R ４ , R ８ are each independently H or methyl; R ７ is H, OH, or -OSO ３ H; 【Chemistry 3】 2. The compound of claim 1, wherein:

9. 2. The compound of claim 1, wherein the compound is represented by the formula: or an ester, stereoisomer, prodrug, solvate or pharma- ceutically acceptable salt thereof. 【Chemistry 4】

10. A pharmaceutical composition comprising a compound according to any one of claims 1 to 9, or an ester, stereoisomer, prodrug, solvate or pharma- ceutically acceptable salt thereof.

11. 11. The pharmaceutical composition of claim 10 further comprising another antifungal agent.

12. An echinocandin biosynthetic gene cluster consisting of the genes ecdA, ecdI and htyE, and optionally ecdG and / or htyF, or consisting of the genes ecdA, ecdI, ecdK and htyE, and optionally ecdG and / or htyF.

13. 13. The echinocandin biosynthetic gene cluster of claim 12, comprising the genes ecdA, ecdI and htyE, comprising the genes ecdA, ecdI, ecdK and htyE, comprising the genes ecdA, ecdI, ecdG and htyE, or comprising the genes ecdA, ecdI, ecdG, htyE and htyF.

14. A genetically engineered transformed strain comprising the echinocandin biosynthetic gene cluster of claim 12 or 13.

15. 15. The genetically engineered transformed strain according to claim 14, which is the transformed strain LO8030-5.1 having the accession number CCTCC M 20211360, the transformed strain LO8030-4.2.1 having the accession number CCTCC M 2022168, the transformed strain LO8030-4.2.1.5 having the accession number CCTCC M 2023155, or the transformed strain LO8030-4.2.1.9 having the accession number CCTCC M 2023156.

16. 10. Use of a compound according to any one of claims 1 to 9 in the manufacture of a medicament for the treatment or prevention of a disease associated with a fungal infection.

17. The fungus may be selected from the group consisting of the genus Aspergillus (e.g., Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger, and Aspergillus terreus), Blastomyces dermatitidis, the genus Candida (e.g., Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida krusei, and Candida guilliermondii), Coccidioides immitis, 17. The use according to claim 16, wherein the bacterial strain is selected from one or more of Cryptococcus (e.g., Cryptococcus neoformans, Cryptococcus albicans and Cryptococcus laurentii), Histoplasma capsulatum var. capsulatum, Histoplasma capsulatum var. duboissi, Paracoccidioides braziliensis, Sporothrix schenckii, Absidia corymbifera, Rhizomucor pusillus and Rhizopus arizus.

18. 17. The use according to claim 16, wherein the disease associated with a fungal infection is selected from one or more of tinea capitis, tinea corporis, tinea pedis, onychomycosis, periungual mycosis, tinea discolor, thrush, vaginal candidiasis, respiratory tract candidiasis, biliary candidiasis, esophageal candidiasis, urinary tract candidiasis, systemic candidiasis, mucosal and cutaneous candidiasis, aspergillosis, mucormycosis, paracoccidioidomycosis, North American blastomycosis, histoplasmosis, coccidioidomycosis, sporotrichosis, fungal sinusitis and chronic sinusitis.

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