Antifungal compounds and uses thereof

Abstract

The application discloses an antifungal compound, and a structural general formula is selected from one of the following structures: in the application, the compound A4 avoids different activities of different configurations, has higher water solubility, and has the same biological activity and mechanism as JIB-04, and can effectively reduce the cost of isomers in drug research.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to an antifungal compound and its application. Background Technology

[0002] Invasive fungal infections (deep fungal infections) are increasingly becoming a global public health problem, with Candida, Aspergillus, Cryptococcus, and Pneumocystis accounting for 90% of the pathogens causing these infections. Currently, only four classes of drugs are used as first-line treatments for deep fungal infections: azoles (fluconazole, ketoconazole); polyenes (amphotericidal B); echinocandins (caspofungin, anidulafungin); and 5-fluorocytosine. The toxic side effects and increasing drug resistance of these drugs limit their clinical application. Therefore, there is an urgent need to develop novel antifungal drugs.

[0003] Phenotypic screening is an effective method for discovering novel lead compounds. Histone methyltransferases (HMTs) and histone demethylases (HDMs) are important epigenetic proteins that are highly conserved in eukaryotic cells. HMT / HDM inhibitors have been reported in the literature for their anti-tumor, anti-parasitic, and anti-SARS-CoV-2 effects. Currently available small molecule inhibitors of HMTs / HDMs have diverse structural types, and some have entered clinical trials. Fungi are eukaryotes, and their transcriptional processes are regulated by modifications such as histone methylation, acetylation, and phosphorylation. These modifications have a significant impact on fungal drug resistance and virulence factors.

[0004] Fungal HMTs / HDMs exhibit high intraspecific homology and similarity to human HMTs / HDMs, indicating that human HMTs / HDM inhibitors can be structurally modified to obtain fungal-specific HMTs / HDM inhibitors. More importantly, histone methylation is associated with histone modifications such as DNA methylation and ubiquitination, suggesting that combining inhibitors of multiple histone modification sites for fungal infection treatment could be a novel research approach. The difference between fungal and human HMTs / HDMs lies in the fact that in fungi, one histone methylation marker corresponds to one corresponding histone methyltransferase, resulting in a relatively simple interaction. This contrasts sharply with the complex relationships between multiple histone methylation markers and various HMTs / HDMs in higher eukaryotes, offering the potential for developing specific antifungal drugs. Furthermore, literature reports that fungal HMTs / HDMs play a crucial role in sporulation, providing a theoretical basis for the antibacterial or bactericidal effects of fungal HMTs / HDM inhibitors. The reported fungal HMTs / HDMs are as follows:

[0005] Research on the mechanisms of histone modification in fungi is mainly based on two yeast-type fungi—Saccharomyces cerevisiae and Schizosaccharomyces pombe—and one filamentous fungus—Neurospora crassa. Although these three fungi do not represent the entire fungal kingdom, these studies are of great significance for drug research on pathogenic fungi.

[0006] Similar to human HMTs, fungal HMTs are mainly divided into two categories containing SET and DOT1 domains. Currently, there are few reports on the action sites, enzyme structures, and inhibitors of fungal HMTs. However, as eukaryotes with high homology, the potential inhibitory effects of human HMT inhibitors on fungi, especially pathogenic fungi, represent a potential source of novel antifungal drugs.

[0007] No LSD1 class HDMs have been found in fungi. Five HDMs have been reported, and their sites of action and names are shown in Table 1. Currently, no fungal HDM inhibitors have been reported in the literature.

[0008] Table 1. Reported fungal HDMs

[0009] name Site of action mammalian homologous enzymes Jhd1 H3K36me2 / 1 JHDM1A Jhd2 / Yjr119C H3K4me2 / 3 JARID Rph1 H3K36me2 / 3 JMJD2A, JHDM3A Gis1 unknow JMJD2A, JHDM3A Ecm5 unknow No reports found

[0010] The classification of fungal histone methylation and demethylation is not yet fully understood. Fungal histone methylation and demethylation processes are involved in various biological processes, including heterochromatin formation and transcriptional regulation. Current research on HMTs / HDMs mainly focuses on anti-tumor research, and whether HMTs / HDMs are novel antifungal targets remains unknown. Summary of the Invention

[0011] The purpose of this invention is to provide an antifungal compound.

[0012] Another object of the present invention is to provide the use of the compound in the preparation of an antifungal medicament.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0014] A first aspect of the present invention provides an antifungal compound having a structural formula selected from one of the following:

[0015]

[0016]

[0017] in,

[0018] R1 is selected from hydrogen and halogens (fluorine, chlorine, bromine, iodine);

[0019] R2 is selected from hydrogen and halogens (fluorine, chlorine, bromine, iodine);

[0020] R3 is selected from hydrogen and halogens (fluorine, chlorine, bromine, iodine);

[0021] R4 is selected from hydrogen and halogens (fluorine, chlorine, bromine, iodine);

[0022] R5 is selected from hydrogen and halogens (fluorine, chlorine, bromine, iodine);

[0023] X is selected from C and N;

[0024] R6 is selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine), C1-C10 alkyl, C1-C10 alkoxy, and -CF3;

[0025] R7 is selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine), C1-C10 alkyl, C1-C10 alkoxy, and -CF3;

[0026] R8 is selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine), C1-C10 alkyl, C1-C10 alkoxy, and -CF3;

[0027] R9 is selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine), C1-C10 alkyl, C1-C10 alkoxy, and -CF3;

[0028] R 10 Selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine), C1-C10 alkyl, C1-C10 alkoxy, -CF3, -CN; R 11 Selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine), C1-C10 alkyl, C1-C10 alkoxy, -CF3, -CN; R 12 Selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine), C1-C10 alkyl, C1-C10 alkoxy, -CF3, -CN; R 13 Selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine), C1-C10 alkyl, C1-C10 alkoxy, -CF3, -CN; R 14 Selected from hydrogen and halogens (fluorine, chlorine, bromine, iodine);

[0029] R 15 Selected from hydrogen and halogens (fluorine, chlorine, bromine, iodine);

[0030] R 16 Selected from hydrogen and halogens (fluorine, chlorine, bromine, iodine);

[0031] R 17 Selected from hydrogen and halogens (fluorine, chlorine, bromine, iodine);

[0032] Y is selected from O and S;

[0033] R18 Selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine), C1-C10 alkyl, C1-C10 alkoxy, -CF3;

[0034] R 19 Selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine), C1-C10 alkyl, C1-C10 alkoxy, -CF3;

[0035] R 20 Selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine), C1-C10 alkyl, C1-C10 alkoxy, -CF3;

[0036] R 21 Selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine), C1-C10 alkyl, C1-C10 alkoxy, -CF3;

[0037] R 22 Selected from hydrogen and C1-C10 alkyl groups;

[0038] R 23 Selected from hydrogen and C1-C10 alkyl groups;

[0039] R 24 Selected from hydrogen and C1-C10 alkyl groups;

[0040] R 25 Selected from hydrogen and C1-C10 alkyl groups;

[0041] Excluding compounds JIB-04 and Z-JIB-04,

[0042]

[0043] Preferably, in the antifungal compound,

[0044] R1 is selected from hydrogen, fluorine, chlorine, and bromine;

[0045] R2 is selected from hydrogen, fluorine, chlorine, and bromine;

[0046] R3 is selected from hydrogen, fluorine, chlorine, and bromine;

[0047] R4 is selected from hydrogen, fluorine, chlorine, and bromine;

[0048] R5 is selected from hydrogen, fluorine, chlorine, and bromine;

[0049] X is selected from C and N;

[0050] R6 is selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, tert-butyl, n-butyl, isobutyl, methoxy, ethoxy, tert-butoxy, n-propoxy, and -CF3;

[0051] R7 is selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, tert-butyl, n-butyl, isobutyl, methoxy, ethoxy, tert-butoxy, n-propoxy, and -CF3;

[0052] R8 is selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, tert-butyl, n-butyl, isobutyl, methoxy, ethoxy, tert-butoxy, n-propoxy, and -CF3;

[0053] R9 is selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, tert-butyl, n-butyl, isobutyl, methoxy, ethoxy, tert-butoxy, n-propoxy, and -CF3;

[0054] R 10 Selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, tert-butyl, n-butyl, isobutyl, methoxy, ethoxy, tert-butoxy, n-propoxy, -CF3, -CN;

[0055] R 11 Selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, tert-butyl, n-butyl, isobutyl, methoxy, ethoxy, tert-butoxy, n-propoxy, -CF3, -CN;

[0056] R 12 Selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, tert-butyl, n-butyl, isobutyl, methoxy, ethoxy, tert-butoxy, n-propoxy, -CF3, -CN;

[0057] R 13 Selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, tert-butyl, n-butyl, isobutyl, methoxy, ethoxy, tert-butoxy, n-propoxy, -CF3, -CN;

[0058] R 14 Selected from hydrogen, fluorine, chlorine, and bromine;

[0059] R 15 Selected from hydrogen, fluorine, chlorine, and bromine;

[0060] R 16 Selected from hydrogen, fluorine, chlorine, and bromine;

[0061] R 17 Selected from hydrogen, fluorine, chlorine, and bromine;

[0062] Y is selected from O and S;

[0063] R 18 Selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, tert-butyl, n-butyl, isobutyl, methoxy, ethoxy, tert-butoxy, n-propoxy, -CF3;

[0064] R 19Selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, tert-butyl, n-butyl, isobutyl, methoxy, ethoxy, tert-butoxy, n-propoxy, -CF3;

[0065] R 20 Selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, tert-butyl, n-butyl, isobutyl, methoxy, ethoxy, tert-butoxy, n-propoxy, -CF3;

[0066] R 21 Selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, tert-butyl, n-butyl, isobutyl, methoxy, ethoxy, tert-butoxy, n-propoxy, -CF3;

[0067] R 22 Selected from hydrogen, methyl, ethyl, n-propyl, tert-butyl, and n-butyl;

[0068] R 23 Selected from hydrogen, methyl, ethyl, n-propyl, tert-butyl, and n-butyl;

[0069] R 24 Selected from hydrogen, methyl, ethyl, n-propyl, tert-butyl, and n-butyl;

[0070] R 25 Selected from hydrogen, methyl, ethyl, n-propyl, tert-butyl, and n-butyl.

[0071] Most preferably, the antifungal compound is selected from one of the following structures:

[0072]

[0073]

[0074] A second aspect of the invention provides the use of the said compound in the preparation of an antifungal medicament. The compound further includes compounds JIB-04 and Z-JIB-04.

[0075]

[0076] The fungi were selected from Cryptococcus neoformans H99, Candida auricularia 0029, Candida auricularia ATCC 15448, and Candida auricularia 0030.

[0077] A third aspect of the invention provides the use of the compound in the preparation of fungal histone demethylase inhibitors.

[0078] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0079] This invention optimizes the structure of JIB-04 based on the principles of group substitution and conformational restriction, and studies the overall structure-effectiveness relationship.

[0080] In vitro antifungal activity data showed that compounds A2, A3, A4, and A5 had the same antifungal activity as JIB-04. Chemically, compounds A4 and A5 have no isomers, avoiding the problem of different activities in later experiments due to variations in the configurations of the compounds. Compound A4 exhibits significantly improved water solubility, which is beneficial for in vivo studies. In vivo mouse experiments showed that both JIB-04 and compound A4 possessed excellent in vivo antifungal activity against Cryptococcus neoformans H99 and Candida auris 0029. At a dose of 5 mg / kg, compound A4 showed superior in vivo activity against Cryptococcus neoformans H99 in a mouse infection model compared to JIB-04 and fluconazole. Furthermore, both JIB-04 and compound A4 exhibited good inhibitory activity against the fungal demethylase H3K27me3, indicating that these compounds are novel fungal demethylase inhibitors. The compounds of this invention can be used in the preparation of antifungal drugs.

[0081] In this invention, compound A4 avoids the problem of different activities for different configurations, has high water solubility and biological activity, and its mechanism of action is the same as that of JIB-04, which can effectively reduce the cost of isomers in drug research. Attached Figure Description

[0082] Figure 1 This is a schematic diagram of the time-growth curves of the compound on Cryptococcus neoformans H99 and Candida auris 0029.

[0083] Figure 2 This is a schematic diagram illustrating the inhibitory effect of the compound on biofilm formation of Cryptococcus neoformans H99 and Candida auris 0029.

[0084] Figure 3 This is a schematic diagram illustrating the inhibitory effects of different compounds on the capsule formation of Cryptococcus neoformans H99.

[0085] Figure 4 This is a schematic diagram showing the effects of different compounds on the sterol content of Cryptococcus neoformans H99 and Candida auris 0029.

[0086] Figure 5 This is a schematic diagram illustrating the effects of the compound on the cell cycle of Cryptococcus neoformans H99 and Candida auris 0029.

[0087] Figure 6 This is a schematic diagram of the brain bacterial load in a mouse model of deep Cryptococcus neoformans H99 infection with JIB-04 and compound A4.

[0088] Figure 7This is a schematic diagram of the kidney bacterial load in a mouse model of deep infection with Candida auris 0029 by JIB-04 and compound A4. Detailed Implementation

[0089] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0090] All reagents used in this invention were chemically pure or analytically pure. Thin-layer chromatography (TLC) was performed using GF254 silica gel plates (Qingdao Ocean Chemical Co., Ltd., China), and column chromatography was performed using 300-400 mesh silica gel (Qingdao Ocean Chemical Co., Ltd., China). A Bruker AVANCE 300 or Bruker AVANCE 600 (Bruker, Germany) was used for NMR analysis, with TMS as the internal standard and CDCl3 or DMSO-d6 as the solvent. Chemical shifts (δ) and coupling constants (J) are expressed in ppm and Hz, respectively. Mass spectrometry analysis was performed using an API-3000LC-MS mass spectrometer.

[0091] Example 1

[0092] This invention screened commercially available HMTs / HDMs inhibitors for their antifungal activity, selecting *Candida albicans* SC5314 (C. alb. SC5314), drug-resistant *Candida albicans* 0304103 (C. alb. 0304103), and *Cryptococcus neoformans* H99 (C. neo. H99) for in vitro antifungal activity testing. JIB-04 exhibited excellent antifungal activity (MIC) against *Candida albicans* SC5314, drug-resistant *Candida albicans* 0304103, and *Cryptococcus neoformans* H99. 80 =0.5–1 μg / mL), JIB-04 exists in two configurations, Z and E (the E configuration is named "JIB-04", and the Z configuration is named "Z-JIB-04"). JIB-04 is an inhibitor of HDMs, and it has inhibitory activity against a variety of HDMs and is effective in vivo, while the Z configuration has no inhibitory activity against HDMs.

[0093]

[0094] JIB-04 and Z-JIB-04 are both reported compounds, purchased from Shanghai Taoshu Biotechnology Co., Ltd. Their NMR spectra for different configurations have been reported in the literature, primarily based on the fact that Z-JIB-04 forms intramolecular hydrogen bonds, resulting in an increased (NH) chemical shift in the hydrogen spectrum (the synthetic route of the compound is referenced below: A small molecule modulates Jumonjihistone demethylase activity and selectively inhibits cancer growth. Nat. Commun. 2013; 4: 2035.).

[0095] Therefore, this invention tested JIB-04 and Z-JIB-04 against Candida auris, and the results are shown in Table 2. JIB-04 exhibits excellent inhibitory activity (MIC) against naturally resistant Candida auris. 80 =0.06-2 μg / mL), and its activity against the tested strains was superior to that of fluconazol (FLC). Z-JIB-04 had weak antifungal activity against Candida auris, and its antifungal activity was inferior to that of JIB-04.

[0096] Table 2. In vitro antifungal activity (MIC) of JIB-04 and Z-JIB-04 80 (μg / mL)

[0097] strains serial number Z-JIB-04 JIB-04 FLC Candida auris 0030 >64 2 >64 Candida auris 0029 >64 2 >64 Candida auris ATCC 15448 16 0.125 16

[0098] JIB-04 exhibits good in vitro antifungal activity; however, this compound has been reported to possess antitumor, antischistosomiasis, and anti-SARS-CoV-2 activities, and has poor water solubility (0.54 μg / mL). To further discover novel antifungal lead compounds with independent intellectual property rights, this invention optimizes the structure of JIB-04.

[0099] The configuration confirmation data of compounds A7 and A8, A9 and A10, and A12 and A13 prepared in the embodiments of the present invention are mainly based on the difference in the chemical shift of intramolecular hydrogen bonds in the NMR spectra. The hydrogen spectra of compounds with different configurations show obvious differences.

[0100] Example 2

[0101] Synthesis of Class A compounds

[0102] (E)-5-chloro-2-(2-(phenyl(pyridin-2-yl)methylene)hydrazyl)pyridine (lead compound JIB-04)

[0103] Phenyl(pyridin-2-yl) methyl ketone (0.916 g, 5 mmol) and 5-chloro-2-hydrazinopyridine (0.718 g, 5 mmol) were dissolved in 35 mL of isopropanol. Concentrated sulfuric acid (25 mmol) was slowly added dropwise to the reaction solution. After stirring at 50 °C for 0.5 h, saturated sodium bicarbonate solution was slowly added to adjust the pH to 6-7. The precipitate was collected by filtration and subjected to silica gel column chromatography (CH2Cl2 / MeOH = 100:1, V / V) to give the target compound JIB-04 (378 mg, yellow solid, yield 24.5%).

[0104] Preparation of compounds A1-A13:

[0105] Following the preparation method of compound JIB-04, 5-chloro-2-hydrazinopyridine was replaced with 3-chloro-2-hydrazinopyridine to obtain compound A1 with a yield of 9.8%.

[0106] Following the preparation method of compound JIB-04, 5-chloro-2-hydrazinopyridine was replaced with 5-methoxy-2-hydrazinopyridine to obtain compound A2 with a yield of 67.7%.

[0107] Following the preparation method of compound JIB-04, phenyl(pyridin-2-yl) methyl ketone was replaced with (4-chlorophenyl)(2-pyridinyl) methyl ketone to obtain compound A3 with a yield of 67.3%.

[0108] Following the preparation method of compound JIB-04, 5-chloro-2-hydrazinopyridine was replaced with 5-methoxy-2-hydrazinopyridine, and phenyl(pyridin-2-yl)methyl ketone was replaced with 2-dipyridyl ketone to obtain compound A4 with a yield of 37.3%.

[0109] Following the preparation method of compound JIB-04, 5-chloro-2-hydrazinopyridine was replaced with 6-hydrazinonitrile, and phenyl(pyridin-2-yl)methyl ketone was replaced with 2-dipyridyl ketone to obtain compound A5 with a yield of 30.8%.

[0110] Following the preparation method of compound JIB-04, 5-chloro-2-hydrazinopyridine was replaced with p-chlorophenylhydrazine to obtain compound A6 with a yield of 45.5%.

[0111] Following the preparation method of compound JIB-04, 5-chloro-2-hydrazinopyridine was replaced with 4-(trifluoromethyl)phenylhydrazine to obtain compound A7(Z) with a yield of 16.5%.

[0112] Following the preparation method of compound JIB-04, 5-chloro-2-hydrazinopyridine was replaced with 4-(trifluoromethyl)phenylhydrazine to obtain compound A8(E) with a yield of 31.8%.

[0113] Following the preparation method of compound JIB-04, 5-chloro-2-hydrazinopyridine was replaced with (4-fluorophenyl)hydrazine to obtain compound A9(Z) with a yield of 4.7%.

[0114] Following the preparation method of compound JIB-04, 5-chloro-2-hydrazinopyridine was replaced with (4-fluorophenyl)hydrazine to obtain compound A10(E) with a yield of 27.0%.

[0115] Following the preparation method of compound JIB-04, 5-chloro-2-hydrazinopyridine was replaced with 4-(trifluoromethyl)phenylhydrazine, and phenyl(pyridin-2-yl)methyl ketone was replaced with 2-dipyridyl ketone to obtain compound A11 with a yield of 92.0%.

[0116] Following the preparation method of compound JIB-04, 5-chloro-2-hydrazinopyridine was replaced with (4-chlorophenyl)hydrazine, and phenyl(pyridin-2-yl)methyl ketone was replaced with (4-chlorophenyl)(2-pyridinyl)methyl ketone to obtain compound A12(Z) with a yield of 6.3%.

[0117] Following the preparation method of compound JIB-04, 5-chloro-2-hydrazinopyridine was replaced with (4-chlorophenyl)hydrazine, and phenyl(pyridin-2-yl)methyl ketone was replaced with (4-chlorophenyl)(2-pyridinyl)methyl ketone to obtain compound A13(E) with a yield of 3.8%.

[0118]

[0119] Reagents and conditions: (a) H2SO4 (conc.), 50℃, isopropanol, 0.5h, yield: 3.8-92.0%.

[0120] Example 3

[0121] Synthesis of Class B compounds:

[0122]

[0123] Reagents and conditions: (a) InI3, THF, 120℃, 12h, yield: 38.4%.

[0124] Preparation of compound B series

[0125] N-(5-methylpyridin-2-yl)-2-phenyl-2-(pyridin-2-yl)acetamide (B1)

[0126] 2-Phenylacetic-2-(pyridin-2-yl)methyl acetate (100 mg, 3.6 mmol, 1.0 eq) and 2-amino-5-methylpyridine (77 mg, 7.2 mmol, 2.0 eq) were dissolved in 20 mL of tetrahydrofuran, and then indium triiodide (36 mg, 0.72 mmol) was added. The mixture was stirred at 120 °C for 12 h, cooled, and quenched with 20 mL of saturated ammonium chloride solution. The mixture was extracted with ethyl acetate (20 mL × 3), and the organic phases were combined. The mixture was washed with saturated sodium chloride solution (20 mL × 3), dried with anhydrous sodium sulfate, and suspended under reduced pressure. The solution was then subjected to silica gel column chromatography (PE / EA = 5:1, V / V) to give a white solid, compound B1 (42 mg, yield 38.4%).

[0127] Example 4

[0128] Synthesis of Class C compounds

[0129]

[0130] Reagents and conditions: (a) t-BuLi, -78℃, THF, pentane, yield: 29.4%; (b) 150℃, yield: 19.2%

[0131] 2-Pyridylbenzoic acid (8)

[0132] 20 mL of tert-butyllithium (13 eq) was added to a dry container and pre-cooled to -78 °C. Then, 2-bromopyridine (3.78 g, 24 mmol, 12 eq) was dissolved in 20 mL of tetrahydrofuran and slowly added to the container. The mixture was stirred for 0.5 h. Phthalic anhydride (4.14 g, 28 mmol, 14 eq) was dissolved in 20 mL of tetrahydrofuran and quickly added to the container. The mixture was stirred for 2 h. The pH was adjusted to 3 with 1 N hydrochloric acid. The mixture was extracted with dichloromethane (40 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (30 mL × 3). The mixture was dried with anhydrous sodium sulfate and evaporated to dryness under reduced pressure. The solution was then subjected to silica gel column chromatography (PE / EA = 2:1, V / V) to obtain intermediate 8 (1.58 g, white solid, yield 29.4%). 1 H NMR(600MHz,DMSO-d6)δ12.96(s,1H),8.54(d,J=4.7Hz,1H),8.13(d,J=7.8Hz,1H),8.08-8.00(m,1H ),7.94(d,J=7.6Hz,1H),7.78-7.68(m,1H),7.69-7.61(m,1H),7.61-7.55(m,1H),7.53-7.43(m,1H).

[0133] 2-(5-chloropyridin-2-yl)-4-(pyridin-2-yl)o-phthalazin-1(2H)-one (C1)

[0134] Intermediate 8 (0.4 g, 1.8 mmol, 1 eq) was mixed with 5-chloro-2-hydrazinopyridine (0.26 g, 1.8 mmol, 1 eq) under solvent-free conditions and heated to 150 °C for 1 h. The mixture was then subjected to silica gel column chromatography (PE / EA = 2:1, V / V) to obtain the target compound C1 (0.28 g, white solid, yield 47.1%).

[0135] Example 5

[0136] Synthesis of Class D compounds

[0137]

[0138] Reagents and conditions: (a) Triethylamine, DCM, rt, yield: 86.3%. (b) Fe, NH4Cl, EtOH, H2O. (c) THF, rt, total yield of b to c: 85.7-87.7%. (d) Pyridine, Cu, CuCl, KOAc, yield: 54.9-65.9%.

[0139] N-(2-Nitrophenyl)pyridine-2-amine (11)

[0140] 2-Nitrobenzenesulfonyl chloride (5.3 g, 24 mmol, 1.2 eq) and 2-aminopyridine (0.9 g, 20 mmol, 1 eq) were dissolved in 30 mL of dichloromethane. Triethylamine (2.4 g, 24 mmol, 1.2 eq) was added dropwise under ice bath conditions, and the mixture was stirred at room temperature for 2 h. After the reaction was complete, the dichloromethane was removed by rotary evaporation under reduced pressure. Then, 30 mL of water was added, and the mixture was extracted with ethyl acetate (40 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (30 mL × 3), dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The solution was then subjected to silica gel column chromatography (PE / EA = 15:1, V / V) to give intermediate 11 (4.4 g, red solid, yield 86.3%). 1 H NMR(600MHz,DMSO-d6)δ9.68(s,1H),8.23-8.17(m,1H),8.18-8.16(m,1H),8.08-8.03( m,1H),7.74-7.60(m,2H),7.15-7.06(m,2H),6.98-6.89(m,1H).HRMS(ESI):calculated for C 11 H 10 N3O2[M+H] +216.0768, found[M+H] + 216.0774.

[0141] 1-(pyridin-2-yl)-1,3-dihydro-2H-benzo[d]imidazol-2-one intermediate (14a)

[0142] Intermediate 11 (2.2 g, 10 mmol, 1 eq) was added to 50 mL of 70% ethanol along with iron powder (1.68 g, 30 mmol, 3 eq) and ammonium chloride (2.7 g, 50 mmol, 5 eq). The mixture was stirred at 100 °C for 3 h, quenched with saturated sodium bicarbonate solution, filtered, and extracted with ethyl acetate (50 mL × 3). The organic phases were combined, evaporated to dryness under reduced pressure, and subjected to silica gel column chromatography (petroleum ether / ethyl acetate, 15:1, V / V) to obtain intermediate 12 (1.8 g, white solid, 97% yield).

[0143] Intermediate 12 was dissolved in 40 mL of tetrahydrofuran with carbonyl diimidazole (1.3 g, 8.1 mmol, 1.5 eq). The mixture was stirred at room temperature for 1.5 h under nitrogen protection. The tetrahydrofuran was removed by decomposition under reduced pressure. The solution was dissolved in 5 mL of methanol. Excess saturated sodium bicarbonate solution was added. After the solid was completely precipitated, the mixture was filtered to give 0.98 g of intermediate 14a (white solid, yield 85.7%). 1 H NMR(600MHz,DMSO-d6)δ8.61-8.59(m,1H),8.12-8.08(m,1H),8.03-7.97(m,1H),7.94-7.9 1(m,1H),7.39-7.35(m,1H),7.14-7.08(m,2H),7.08-7.04(m,1H).HRMS(ESI):calculated for C 12 H9N3O[M+H] + 212.0818, found[M+H] + 212.0828.

[0144] 1-(4-methylpyridin-2-yl)-3-(pyridin-2-yl)-1,3-dihydro-2H-benzo[d]imidazol-2-one (D1)

[0145] Intermediate 14a (0.21 g, 1 mmol, 1 eq), 2-bromo-4-methylpyridine (0.26 g, 1.5 mmol, 1.5 eq), copper powder (31 mg, 0.5 mmol, 1.5 eq), copper chloride powder (49 mg, 0.5 mmol, 0.5 eq), and potassium acetate (294 mg, 3 mmol, 3 eq) were added to 30 mL of pyridine and stirred at 120 °C for 24 h. After the reaction was complete, the mixture was filtered, evaporated to dryness under reduced pressure, and 20 mL of water was added. The mixture was extracted with dichloromethane (30 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The solution was then subjected to silica gel column chromatography (PE / EA, 15:1, V / V) to give 0.17 g of compound D1 (white solid, yield 57.59%).

[0146] Following the preparation method of compound D1, 2-bromo-4-methylpyridine was replaced with 2-bromo-5-chloropyridine to obtain compound D2, with a yield of 66.0%.

[0147] Following the preparation method of compound D1, carbonyl diimidazole was replaced with thiocarbonyl diimidazole to obtain compound 14b. Then, compound 14a in the preparation method of compound D1 was replaced with compound 14b, and 2-bromo-4-methylpyridine was replaced with 2-bromo-5-chloropyridine to obtain compound D3, with a yield of 54.9%.

[0148] 1-(pyridin-2-yl)-1,3-dihydro-2H-benzo[d]imidazol-2-thione (14b)

[0149] White solid (yield 86%) 1 H NMR(600MHz,DMSO-d6)δ8.66-8.61(m,1H),8.10-8.07(m,1H),8.04-7.99(m,1H),7.48-7 .43(m,1H),7.27-7.23(m,1H),7.23-7.19(m,1H),7.14-7.09(m,1H),7.04-6.98(m,1H).

[0150] The chemical structural formulas, NMR and MS data of the compounds of this invention are detailed in Table 3.

[0151] Table 3: NMR and MS data of the compounds of this invention

[0152]

[0153]

[0154]

[0155]

[0156]

[0157] Example 1

[0158] Materials and instruments: culture media, experimental consumables, fungal culture methods and fungal cell sources

[0159] YEPD medium: Dissolve 8g yeast extract, 16g D-glucose, and 16g peptone in 800mL triple-distilled water, dispense into portions, autoclave at 121℃ for 15min, cool, and store at 4℃ for later use.

[0160] RPMI 1640 medium: Dissolve 20g of RPMI 1640 medium powder, 69g of MOPS, 4g of sodium bicarbonate and 5.4g of sodium hydroxide in 2000mL of triple-distilled water. After filtration through 0.45μm and 0.22μm microporous membranes, dispense into containers and store at 4℃ for later use.

[0161] PBS buffer: Dissolve 8.0g sodium chloride, 12g disodium hydrogen phosphate, 0.2g potassium chloride and 0.24g potassium dihydrogen phosphate in 1000mL of triple-distilled water, dispense into containers, autoclave at 120℃ for 15min, and store at room temperature for later use.

[0162] SDA solid medium: Dissolve 10g peptone, 40g glucose and 20g agar in 1 liter of triple-distilled water, autoclave at 120℃ for 15 minutes, cool to about 50℃ and pour into cell culture dishes. After solidification, store at 4℃ for later use.

[0163] DMEM medium: Add 5% serum and 0.1% penicillin antibiotics to commercially available DMEM medium, mix well and set aside.

[0164] Experimental consumables: sample loading tank, EP tube, 96-well plate, glass tube.

[0165] Fungal culture: Remove fungal cells from the -80℃ freezer, and place 10 μL in 1 mL of YEPD medium for initial activation at 30℃ for 24 hours. Then, take 10 μL of the activated bacterial suspension and place it in 1 mL of YEPD medium for 16 hours. Plate the cells onto an SDA plate. Take a single colony and place it in 1 mL of YEPD medium for 16 hours of activation. At this point, the fungus has reached the late exponential growth stage. Centrifuge (3000 rpm, 1 min) and discard the supernatant. Wash three times with PBS buffer before use (unless otherwise specified, all fungi used in subsequent experiments are late exponential growth fungi washed three times with PBS buffer).

[0166] In vitro antifungal activity assay: Following the standard protocol M27-A3 (2017) published by the Clinical and Laboratory Standards Institute (CLSI), fungal cells were diluted to 1×10⁻⁶ in RPMI 1640 medium. 3 Cells / mL: 100 μL of diluted bacterial suspension was added to each well of a 96-well plate. The test compound was added to the bacterial suspension in a 2:1 dilution, with an initial concentration of 64 μg / mL. No drug treatment was used as a negative control, and RPMI 1640 medium was used as a blank control. After incubating the 96-well plates at 35°C for a certain period (72 h for Cryptococcus, 48 ​​h for other strains), the OD values ​​were read using a microplate reader. 630 For each compound, three parallel determinations were performed, and the average value was taken. The lowest inhibitory concentration (MIC) with an inhibition rate greater than 80% was used as the MIC. 80 value.

[0167] In vitro antifungal activity and structure-activity relationship of the compounds prepared in this invention

[0168] Since compound JIB-04 exhibits the best activity against Cryptococcus neoformans and good activity against naturally resistant Candida auris, this invention selected three fungi—Cryptococcus neoformans H99, Candida auris 0029, and Candida auris ATCC15448—for in vitro antifungal activity testing. The results are shown in Table 4.

[0169] Table 4. In vitro antifungal activity (MIC) of JIB-04 derivatives 80 (μg / mL)

[0170]

[0171]

[0172]

[0173] Based on Table 4, the structure-activity relationship of the compounds of this invention against Cryptococcus neoformans H99 is summarized as follows:

[0174] The effects of ring group substitutions on the antifungal activity of JIB-04 were investigated in Class A compounds. A slight decrease in antifungal activity (A1, MIC) occurred when the position of the chlorine atom in the C ring of JIB-04 changed from position 5 to position 3. 80 =1 μg / mL), indicating that the substituent at the 5-position of the C ring has good activity. Replacing the chlorine atom in the C ring of JIB-04 with a methoxy group maintained its antifungal activity (A2, MIC). 80=0.5 μg / mL), indicating that whether the group at the 5-position of the C ring is an electron-withdrawing or electron-donating group has no effect on its antifungal activity. Introducing a chlorine atom at the 4-position of the B ring of JIB-04 maintains its antifungal activity (A3, MIC). 80 =0.5 μg / mL), indicating that the introduction of chemical groups into the B ring has no effect on its activity. Replacing the B ring of JIB-04 with a pyridine ring derivative does not change the Z / E configuration, and its antifungal activity is maintained. In this case, replacing the substituent at the 5-position of the C ring with a methoxy or cyano group has no effect on its antifungal activity (A4, A5, MIC). 80 =0.5 μg / mL). Replacing the C ring of JIB-04 with a benzene ring significantly reduced its antifungal activity (A6, MIC). 80 =32 μg / mL), at this time, the derivative obtained by replacing the chlorine atom at position 4 of the C ring with a trifluoromethyl group actually has some antifungal activity in the Z configuration (A7, MIC). 80 =32 μg / mL) while the E configuration had no antifungal activity (A8); the derivatives obtained by replacing the chlorine atom in the C ring with a fluorine atom had the same antifungal activity in both configurations, but both were lower (A9, A10, MIC). 80 =32 μg / mL); the derivative with the B ring replaced by a pyridine ring showed no configurational change and exhibited superior antifungal activity (A11>A6). In this case, the two configurations of derivatives obtained by introducing a chlorine atom at the 4-position of the B ring showed similar but lower activities (A12, A13, MIC). 80 =32 μg / mL). The above results indicate that the position and electronic properties of the substituents on the ring have little effect on the antifungal activity of JIB-04. However, the pyridine ring of the C ring is essential for JIB-04 to maintain its antifungal activity. The presence of a pyridine ring in the B ring or the introduction of substituents into the B ring has no effect on the antifungal activity of JIB-04.

[0175] The effects of hydrazone substitution on the antifungal activity of JIB-04 were investigated in Group B compounds. When the hydrazone group was replaced with an amide bond, the antifungal activity significantly decreased (B1, MIC). 80 =32μg / mL), indicating that the hydrazone structure is essential to maintain the antifungal activity of JIB-04.

[0176] For compounds of types C and D, the effect of conformational restriction strategies on the antifungal activity of JIB-04 was investigated by linking the amino group to the benzene ring. Linking the amino group to the benzene ring with a carbonyl group resulted in derivatives with no conformational change, but their antifungal activity was significantly reduced (C1, MIC). 80 =64 μg / mL). Replacing the hydrazone structure with a urea or thiourea group and linking it to a benzene ring completely resulted in the loss of antifungal activity (D1-D3). These results indicate that excessively increasing molecular rigidity reduces the antifungal activity of JIB-04.

[0177] The structure-activity relationship of the compounds of this invention against Candida auris 0029 and Candida auris ATCC 15448 is similar to that of Cryptococcus neoformans H99.

[0178] The above results indicate that the pyridine-hydrazone-pyridine structure is a key essential group for these compounds to maintain their antifungal activity.

[0179] Water solubility test of the compounds in this invention: Among the compounds of this invention, compounds A4 and A5 maintain their antifungal activity and show no configurational change. Compounds A4 and A5 were selected for further research. First, a water solubility test was conducted. A standard curve was prepared using 0.1 mg / mL and 0.5 mg / mL standard solutions of the compounds using high-performance liquid chromatography (HPLC). The concentration and solubility of the compounds were calculated after testing saturated aqueous solutions using the same method. The results are shown in Table 5. Compared with JIB-04, the water solubility of compound A4 is significantly improved, being 147 times that of JIB-04, and the water solubility of compound A5 is 7 times higher than that of JIB-04. Therefore, compound A4 was selected for further research in this invention.

[0180] Table 5: Water solubility (μg / mL) of JIB-04, compound A4, and compound A5

[0181] compound solubility Increase multiplier JIB-04 0.54 A4 79.20 147 A5 3.93 7

[0182] The compound of this invention was tested using a time-growth curve: fungal cells in the late stage of exponential growth were diluted to 1×10⁻⁶ in RPMI 1640 medium. 5 The bacterial suspension was analyzed by adding different concentrations of the drug to the cells / mL, with the group without the compound serving as a negative control. The suspension was placed in an incubator (200 rpm, 30°C), and 100 μL of the bacterial suspension was taken at set times (0, 4, 8, 12, 24, 48, and 72 h). A standard curve was calculated using a bacterial suspension of known concentrations, and the number of fungal cells in the suspension was plotted using GraphPad Prism 8.3.

[0183] Time-growth curves are important indicators for evaluating the inhibitory effects of compounds on fungal growth. This invention selected JIB-04 and the preferred compound A4 to study their inhibitory effects on the growth of Cryptococcus neoformans H99 and Candida auris 0029. The results are as follows... Figure 1 As shown, Figure 1This is a schematic diagram of the time-growth curves of the compounds against Cryptococcus neoformans H99 and Candida auris 0029. Figure A shows the time-growth curve of the compound against Cryptococcus neoformans, and Figure B shows the time-growth curve of the compound against Candida auris 0029. Figure A contains three graphs, with the top, middle, and bottom showing the time-growth curves of JIB-04, compound A4, and fluconazole against Cryptococcus neoformans, respectively. Figure B also contains three graphs, with the top, middle, and bottom showing the time-growth curves of JIB-04, compound A4, and fluconazole against Candida auris 0029, respectively. The results show that JIB-04 and compound A4 effectively inhibited the growth of Cryptococcus neoformans H99 at both low concentrations (4 μg / mL) and high concentrations (16 μg / mL), while fluconazole showed only a slight inhibitory effect at high concentrations (16 μg / mL). Figure 1 (As shown in Figure A). JIB-04 and compound A4 exhibited similar inhibitory effects on the growth of Candida auris 0029. Compound A4 completely inhibited its growth at a concentration of 16 μg / mL, while fluconazole did not inhibit the growth of Candida auris 0029. Figure 1 (As shown in Figure B). The above results indicate that the preferred compound A4 has an inhibitory effect comparable to JIB-04 in in vitro tests inhibiting the growth of Cryptococcus neoformans H99 and Candida auris 0029, and is superior to fluconazole.

[0184] Biofilm formation inhibition experiment: Fungal cells in the late stage of exponential growth were diluted to 1×10⁻⁶ in RPMI 1640 medium. 6 After obtaining cells / mL, add to 96-well plates (100 μL per well), incubate at 37°C for 3 h to allow fungal cell adhesion, then discard the culture medium, add 100 μL of the compound solution diluted with RPMI 1640, incubate at 37°C for 24 h, then discard the supernatant, add 150 μL of XTT solution (0.5 mg / mL, containing 1 μM menadione) dissolved in PBS, incubate for 3 h, and then read the OD value. 490 ).

[0185] Biofilms are important virulence factors for fungal pathogenicity and drug resistance in organisms. This invention selected JIB-04 and preferred compound A4 to study the inhibitory effects on biofilm formation of Cryptococcus neoformans H99 and Candida auris 0029. The results are as follows... Figure 2 As shown, Figure 2This is a schematic diagram illustrating the inhibitory effects of the compounds on biofilm formation of *Cryptococcus neoformans* H99 and *Candida auris* 0029. In the diagrams, ns, no significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs DMSO. A is a schematic diagram illustrating the inhibitory effects of the compounds on biofilm formation of *Cryptococcus neoformans* H99, and B is a schematic diagram illustrating the inhibitory effects of the compounds on biofilm formation of *Candida auris* 0029. A contains three diagrams, from left to right: JIB-04, compound A4, and fluconazole, illustrating the inhibitory effects on biofilm formation of *Cryptococcus neoformans* H99. B also contains three diagrams, from left to right: JIB-04, compound A4, and fluconazole, illustrating the inhibitory effects on biofilm formation of *Candida auris* 0029. The results in the figure show that fluconazole at 4 μg / mL inhibited biofilm formation in Cryptococcus neoformans H99, while JIB-04 and compound A4 inhibited biofilm formation in Cryptococcus neoformans H99 at lower concentrations (0.25 μg / mL). Figure 2 (As shown in Figure A). Fluconazole had no inhibitory effect on biofilm formation of *Candida auris* 0029. JIB-04 significantly inhibited biofilm formation of *Candida auris* 0029 at a concentration of 16 μg / mL, but the inhibition rate was less than 50%, while compound A4 inhibited biofilm formation of *Candida auris* 0029 at a concentration of 4 μg / mL, and the inhibition rate was higher than 50% at 16 μg / mL. Figure 2 (As shown in Figure B). The above results indicate that both JIB-04 and compound A4 can significantly inhibit fungal biofilm formation, with better inhibitory effects than fluconazole. Furthermore, compound A4 exhibits stronger inhibitory effects on the biofilm formation of Candida auris 0029.

[0186] Capsule formation inhibition experiment: Fungal cells in the late stage of exponential growth were diluted to 1×10⁻⁶ in DMEM medium (containing 10% FBS). 6 After the cells / mL were collected, 15 mL of the mixture was added to a centrifuge tube and incubated in a CO2 incubator at 37°C for 24 h to allow capsule formation. After 24 hours of treatment with different drugs, 10 μL of Indian ink was added, mixed well, and observed and photographed using a laser confocal microscope.

[0187] The capsule is a virulence factor unique to Cryptococcus neoformans and plays an important role in the pathogenicity and drug resistance of the fungus. This invention selected JIB-04 and the preferred compound A4 to study the inhibitory effect on capsule formation of Cryptococcus neoformans H99. The results are as follows... Figure 3 As shown, Figure 3This diagram illustrates the inhibitory effects of different compounds on capsule formation in Cryptococcus neoformans H99. A shows microscopic images of the effects of fluconazole, JIB-04, and compound A4 on the capsule formation of Cryptococcus neoformans H99. B shows an analysis of the capsule thickness of Cryptococcus neoformans H99 after treatment with fluconazole, JIB-04, and compound A4. B contains three figures: left, center, and right, representing overall cell size, capsule volume, and cell membrane volume, respectively. The results show that fluconazole at a concentration of 4 μg / mL only partially inhibits capsule formation in Cryptococcus neoformans H99, while JIB-04 and compound A4 at concentrations of 1-2 μg / mL completely inhibit capsule formation without affecting the cell size of Cryptococcus neoformans H99. Figure 3 The above results indicate that JIB-04 and compound A4 have a good inhibitory effect on the virulence factors of Cryptococcus neoformans H99.

[0188] Sterol content analysis: 100 μL of fungi in the late exponential growth stage was added to 50 mL of LYPED medium. DMSO was used as a blank control, fluconazole as a positive control, and different concentrations of compounds were used as treatment groups. After incubation at 30 °C on a shaker for 24 hours, the supernatant was removed by centrifugation (3000 rpm, 2 min). The mixture was washed three times with PBS. 10 mL of saponifying agent (85% ethanol solution containing 15% NaOH) was added to the precipitate. After saponification in an oven at 80 °C for 3 h, the mixture was extracted with petroleum ether (4 mL × 3). The petroleum ethers were combined and washed with 4 mL of water. The mixture was heated to dryness in a water bath at 60 °C. The residue was dissolved in 400 μL of cyclohexane, centrifuged, and the supernatant was collected. The sterol content was analyzed using gas chromatography-mass spectrometry (GC-MS). The molecular fragments of each peak in the GC-MS spectrum were matched with the corresponding sterol compounds in the National Institute of Standards and Technology (NIST) database to identify the structure of the sterol components.

[0189] Ergosterol is a major component for fungal cell membrane integrity. This invention investigated the changes in sterol content in Cryptococcus neoformans H99 and Candida auris 0029 after treatment with JIB-04 and the preferred compound A4. The results are as follows... Figure 4 As shown, Figure 4This is a schematic diagram showing the effects of different compounds on the sterol content of Cryptococcus neoformans H99 and Candida auris 0029. In the diagram, 1 represents ergosterol; 2 represents Ergosta-14,22-dien-3-ol; 3 represents fungisterol; 4 represents obustifoliol; 5 represents stigmasterol; 6 represents lanosterol; 7 represents an unknown sterol; 8 represents fenestrated sterol; 9 represents cycloartane-3β,25-diol-ol; and 10 represents an unknown sterol. Specifically, A shows the effect of different compounds on the sterol content of Cryptococcus neoformans H99, B shows the effect of different compounds on the sterol content of Candida auris 0029, and C shows the analysis of the main sterol content of Cryptococcus neoformans H99 and Candida auris 0029 after compound treatment. The results indicate that for Cryptococcus neoformans H99 (… Figure 4 As shown in Figure A), the ergosterol content in the blank control group was 44.17%. After treatment with fluconazole, the ergosterol content decreased to 36.5%, which is consistent with the mechanism of action of azole drugs. Under the same concentration conditions (4 μg / mL), JIB-04 reduced the ergosterol content to 13.8%, which is consistent with the MIC of JIB-04 against Cryptococcus neoformans. 80 The values ​​were consistently low; however, at 4 μg / mL, the ergosterol content in Cryptococcus neoformans H99 increased to 44.04% after treatment with compound A4, with increased levels of sterols 5 and 9, and decreased levels of sterol 3; at 8 μg / mL, the ergosterol content in Cryptococcus neoformans H99 was 30.5%, and the increasing trend of sterols 5 and 9 was more pronounced. These results indicate that the ergosterol synthesis pathway in Cryptococcus neoformans H99 is disrupted after treatment with compound A4.

[0190] For Candida auris 0029 ( Figure 4 As shown in Figure B, the ergosterol content in the blank control group was 100%, and there was no change in ergosterol content after fluconazole treatment, which is consistent with the natural resistance of Candida auris 0029 to azole drugs. At a concentration of 8 μg / mL, JIB-04 increased the content of unknown sterol 10 to 28.58% while decreasing the ergosterol content to 52.45%. Unlike the effect of JIB-04, after treatment with compound A4, the content of sterol 2 increased to 48.98% while decreasing the ergosterol content to 41.69%. Although the ergosterol content of Candida auris 0029 decreased at different concentrations of compound A4, it did not show a concentration-dependent trend, and the changes in the sterol components of Candida auris 0029 varied at different concentrations of compound A4. The above experimental results indicate that JIB-04 and compound A4 affect fungal ergosterol synthesis, but the mechanisms of action differ among different fungi.

[0191] Cell cycle experiment: Fungi in the late stage of exponential growth were diluted with YPED medium to 5×10⁻⁶. 5Cells / mL, 20 mL of bacterial suspension was added to a 50 mL centrifuge tube, with DMSO as a blank control, fluconazole as a positive control, and different concentrations of compounds as treatment groups. After incubation at 30℃ for 24 hours on a shaker, the cells were centrifuged (4000 rpm, 5 min) to remove the supernatant. The cells were washed three times with PBS, and the wet fungal weight was weighed. For every 200 mg of fungus, 6 mL of snailase buffer, 500 μL of snailase, and 24 μL of 2-mercaptoethanol were added, and the cells were incubated at 30℃ with shaking for 1 h to disrupt the fungal cell wall. After washing three times with PBS, an appropriate amount of 75% ethanol was added and the cells were fixed at 4℃ for 8 h. After washing with PBS, the cells were stained with 50 μg / mL PI for 30 min, and then analyzed by flow cytometry.

[0192] Histone methylation pathways affect cholesterol synthesis, which in turn affects cell cycle changes. This invention further investigated the effects of JIB-04 and compound A4 on the fungal cell cycle, with results as follows: Figure 5 As shown, Figure 5 This diagram illustrates the effects of the compound on the cell cycle of *Cryptococcus neoformans* H99 and *Candida auris* 0029. In diagram A, the compound affects the cell cycle of *Cryptococcus neoformans* H99, and in diagram B, the compound affects the cell cycle of *Candida auris* 0029. The results indicate that for *Cryptococcus neoformans* H99 (… Figure 5 As shown in Figure A), after treatment with JIB-04, the proportion of cells in the G2 phase decreased to 1.7%, while the proportion of cells in the S phase increased to 66.3%; after treatment with compound A4, the proportion of cells in the G2 phase decreased to 1.1%, while the proportion of cells in the S phase increased to 86.4%. These results indicate that JIB-04 and compound A4 inhibited the transition of Cryptococcus neoformans H99 strain from the S phase to the G2 phase, causing fungal cells to remain in the S phase and exert their antifungal effect, with compound A4 showing a stronger inhibitory effect. For Candida auris 0029 (… Figure 5 As shown in Figure B), after treatment with JIB-04, the proportion of cells in the S phase decreased to 10.9%, while the proportion of cells in the G1 phase increased to 60.8%; after treatment with compound A4, the proportion of cells in the S phase decreased to 4.2%, while the proportion of cells in the G1 phase increased to 68.4%. These results indicate that JIB-04 and compound A4 inhibited the transition from the G1 phase to the S phase in Candida auris strain 0029, causing fungal cells to remain in the G1 phase and exert their antifungal effect, with compound A4 showing a stronger inhibitory effect. These results also indicate that treatment with JIB-04 and compound A4 affects the fungal cell cycle, but the effects of JIB-04 and compound A4 differ for different fungi.

[0193] Fungal histone demethylase activity assay:

[0194] Extraction of total fungal nucleoprotein: Take 1 mL of fungi cultured in the late stage of exponential growth and extract total fungal nucleoprotein using the yeast nucleoprotein extraction kit (BB-3168-50T) provided by BestBio. Then, quantify the concentration of fungal nucleoprotein using the protein quantification kit (BB-3401-1) provided by BestBio.

[0195] Fungal demethylase activity assay: The activities of JMJD3 / UTX and JARID demethylases were tested using kits P-3084 and P-3082 provided by EmyJet Scientific. The data were curve-fitted using Graphpad Prime 8.3 to calculate the IC50. 50 value.

[0196] HDMs Inhibitory Activity: HDMs are evolutionarily conserved in eukaryotes. JIB-04 is an enzyme inhibitor of the Jumonji family in human HDMs, and it also inhibits various histone demethylases. This invention extracts total nucleoproteins from Cryptococcus neoformans strain H99 and Candida auris strain 0029 for enzyme inhibition experiments (IC50). 50 The results are shown in Table 6. JIB-04 and compound A4 were found to inhibit the demethylation of H3K27me3 by fungal histone demethylases. The IC50 values ​​of JIB-04 and compound A4 against Cryptococcus neoformans H99 were... 50 The values ​​were 168 nM and 134 nM, respectively, for the IC50 of Candida auris 0029. 50 The values ​​were 95 nM and 12 nM, respectively, indicating that JIB-04 and compound A4 had comparable inhibitory effects on fungal HDMs. These experimental results show that JIB-04 inhibits histone demethylases acting on fungal H3K27me3. However, in more extensive experiments inhibiting fungal histone demethylases, JIB-04 and compound A4 were found to have no inhibitory effect on the demethylation process of H3K4me3 in fungal HDMs, which differs from previous reports of JIB-04 inhibiting multiple histone demethylases.

[0197] Table 6: Inhibitory effects of JIB-04 and compound A4 on Cryptococcus neoformans H99 and Candida auris 0029 strains HDMs

[0198] compound Cryptococcus neoformans H99 (nM) Candida auris 0029 (nM) JIB-04 168 95 A4 134 12

[0199] In vivo activity evaluation experiment

[0200] A mouse model of Cryptococcus neoformans H99 brain infection: ICR female mice (18-22 g, 4-6 weeks old) were fed for 7 days, and each mouse was injected via the tail vein with a concentration of 1×10⁻⁶. 6Cryptococcus neoformans H99 cells / mL. Test compounds were prepared in 98% saline, 1.5% glycerol, and 0.5% Tween 80. The control group was treated with saline. Mice were sacrificed on day 6, and their brains were collected. The mouse brains were homogenized and diluted with 1 mL of saline. The homogenates were then diluted at different ratios and coated onto prepared SDA plates (containing 100 μg / mL chloramphenicol). The plates were incubated at 35°C for 48 h, counted, and significant differences between groups were analyzed using Student's t-test.

[0201] Mouse model of Candida auris 0029 kidney infection: ICR female mice (18-22 g, 4-6 weeks old) were fed for 7 days, and each mouse was administered 5-fluorouracil (5 mg / kg) by gavage to suppress the immune system. The next day, a concentration of 1×10⁻⁶ mg / kg was administered intravenously. 7 Candida auris 0029 cells / mL (0.2 mL). Test compounds were prepared in 98% saline, 1.5% glycerol, and 0.5% Tween 80. The control group was treated with saline. Mice were sacrificed on day 6, and kidneys were harvested. Mouse kidneys were homogenized and diluted with 1 mL of saline. The homogenates were then diluted at different ratios and plated onto prepared SDA plates (containing 100 μg / mL chloramphenicol). The plates were incubated at 35°C for 48 h, counted, and significant differences between groups were analyzed using Student's t-test.

[0202] In vivo activity studies: Since JIB-04 and compound A4 exhibited excellent in vitro antifungal activity, and the in vitro metabolic stability of compound A4 was comparable to that of JIB-04 (results are shown in Table 6), this invention further investigated the in vivo efficacy of JIB-04 and compound A4 in mice. The compounds were examined against a brain infection model of Cryptococcus neoformans H99 and a kidney infection model of Candida auris 0029, respectively, using intraperitoneal injection. For Cryptococcus neoformans H99, the results are as follows: Figure 6 As shown, Figure 6This is a schematic diagram of the bacterial load in the brain of a mouse model of deep Cryptococcus neoformans H99 infection with JIB-04 and compound A4. Where ns, no significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs DMSO. The left figure shows the results of administration of fluconazole, JIB-04, and compound A4 at a dose of 2 mg / kg. The right figure shows the results of administration of fluconazole, JIB-04, and compound A4 at a dose of 5 mg / kg. The results in the figures show that at a dose of 2 mg / kg, both JIB-04 and compound A4 significantly reduced the bacterial load in the mouse brain, with better effects than fluconazole. As the dose increased to 5 mg / kg, JIB-04, due to its lower solubility and severe precipitation, had an effect comparable to that at 2 mg / kg, while compound A4, due to its higher solubility, had a more significant effect. For Candida auris 0029, the results are as follows... Figure 7 As shown, Figure 7 This is a schematic diagram of the nephrotic load of JIB-04 and compound A4 in a mouse model of deep infection with Candida auris 0029. In the diagram, ns, no significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs DMSO. The left graph shows the results of fluconazole, JIB-04, and compound A4 administered at a dose of 2 mg / kg, and the right graph shows the results of fluconazole, JIB-04, and compound A4 administered at a dose of 5 mg / kg. The results showed that although *Candida auris* 0029 was resistant to fluconazole in vitro, fluconazole was effective against it in vivo. At a dose of 2 mg / kg, both JIB-04 and compound A4 significantly reduced the bacterial load in mouse kidneys, exhibiting better efficacy than fluconazole. Unlike the *Cryptococcus neoformans* assay, in the *Candida auris* 0029 assay, the formulation was adjusted to ensure complete dissolution of JIB-04, resulting in more pronounced effects from both JIB-04 and compound A4, comparable to fluconazole. These experimental results indicate that both JIB-04 and compound A4 possess excellent in vivo efficacy. Compound A4, due to its lack of isomers and increased water solubility, is more suitable for in vivo studies.

[0203] Table 6: Mouse liver microsomal stability of JIB-04 and compound A4

[0204] compound <![CDATA[T 1 / 2 (min)]]> CL (mL / min / kg) JIB-04 34.3 159 A4 50.6 108

[0205] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. An antifungal compound, characterized in that, The general structural formula is selected from the following structures: ； in, X is selected from N; R 10 Selected from hydrogen; R 11 Selected from C1~C10 alkoxy groups, -CN; R 12 Selected from hydrogen; R 13 Selected from hydrogen.

2. The antifungal compound according to claim 1, characterized in that, In the antifungal compound, X is selected from N; R 10 Selected from hydrogen; R 11 Selected from methoxy, ethoxy, tert-butoxy, n-propoxy, and -CN; R 12 Selected from hydrogen; R 13 Selected from hydrogen.

3. The antifungal compound according to claim 2, characterized in that, The antifungal compound is selected from one of the following structures: 。 4. The use of the compound according to any one of claims 1 to 3 in the preparation of an antifungal medicament, characterized in that, The fungi were selected from Cryptococcus neoformans H99, Candida auris 0029, and Candida auris ATCC 15448.

5. The use of a compound according to any one of claims 1 to 3 in the preparation of a fungal histone demethylase inhibitor.

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