A no-donor type antifungal compound and its preparation and application

Abstract

This invention discloses a NO donor-type antifungal compound, its preparation method, and its application. The compound is represented by general formula V. By introducing a nitric oxide (NO) donor fragment into an active azole antibacterial fragment, it achieves the effect of dose-dependent slow release of nitric oxide in the presence of a thiol-containing nucleophilic reagent in vitro. When applied to antifungal drugs, the above compound can inhibit fungal growth at low concentrations and has broad-spectrum antibacterial activity. It has a strong inhibitory effect on Candida albicans, Candida glabrata, and Cryptococcus neoformans, and is also effective against cryptococcal meningitis caused by invasive Cryptococcus neoformans.

Description

Technical Field

[0001] This invention relates to an antifungal compound, particularly to a NO donor-type antifungal compound, and also to the preparation and application of the above compound. Background Technology

[0002] Invasive fungal infections (IFIs) are increasingly becoming a significant factor affecting human health (Science, 2012, 336(6082):647). For example, Cryptococcus neoformans, a pathogenic fungus that is widely distributed globally, can cause numerous life-threatening diseases in the human body, such as sepsis, multiple organ failure, and fungal meningitis (Nat. Rev. Drug. Discov., 2017, 16(9):603-616; Fungal Genet. Biol., 2015, (78):16-48). One of the reasons for the high mortality rate of IFIs is the resistance of pathogenic fungi to antifungal drugs (Antimicrob. Agents Chemother., 2015, 59(8):4982-4989; Antimicrob. Agents Chemother., 1999, 43(8):1856-1861).

[0003] Among the many factors contributing to drug resistance, fungal biofilms are a significant factor. The formation of fungal biofilms provides a safe haven for fungi, preventing antibiotic penetration and degrading them. Cells within biofilms exhibit higher levels of resistance (Cell Chem. Biol., 2016, 23(11): 1383-1394). Due to their inherent resistance, biofilm infection is an emerging serious health problem. Biofilms are important virulence factors in fungal pathogenesis. Generally, biofilms are embedded in a self-produced extracellular matrix composed of polysaccharides, proteins, and trace amounts of eDNA (Antimicrob. Agents Chemother. 2001, 45(9), 2475-2479; Nat. Rev. Drug. Disc. 2003, 2(2), 114-122). The structure of biofilms facilitates the exchange of nutrients and gases. Therefore, fungal cells can resist invasion by the environment, immune cells, and drugs. In particular, cells in biofilms exhibit phenotypic characteristics that are distinctly different from those of freely floating planktonic cells. Therefore, biofilm-associated infections are inherently difficult to eradicate completely (Antimicrob. Agents Chemother. 2001, 45(9), 2475-2479; Nat. Rev. Drug. Disc. 2003, 2(2), 114-122). It has been reported that fungal biofilm formation can effectively defend against the invasion of classic antifungal drugs such as azoles and amphotericin B (Antimicrob. Agents Chemother. 2002, 46(11), 3634-3636; Rev. Iberoam. Micol. 2014, 31(1), 22-29). The widespread use and long-term treatment regimens of azole drugs have led to potential treatment failures due to resistance to biofilms (Cold Spring Harbor Perspect. Med. 2014, 5(7), a019752; FutureMicrobiol. 2014, 9(4), 523-542). Therefore, there is an urgent need to develop new treatment strategies for IFIs, especially to overcome resistance associated with biofilms.

[0004] Dispersing biofilms to allow fungal cells to return to a planktonic state has become a popular antifungal strategy in recent years. However, while the cells return to a free state after dispersal, there is also the possibility of recolonization and reformation of biofilms, which can lead to more serious secondary infections. How to safely disperse fungal cell membranes and effectively inhibit fungal growth is an urgent problem to be solved in the research of antifungal drugs. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a NO donor-type antifungal compound that releases NO while simultaneously exerting an azole fragment to achieve highly efficient inhibition of fungal growth, and to provide a method for preparing and applying the above compound.

[0006] Technical solution: A NO donor-type antifungal compound of the present invention is shown in general formula V:

[0007]

[0008] Where A is

[0009]

[0010] When A is in 4-Hydroxypiperidinyl Diethylamino D-prolyl N-methylpiperazinyl Tetrahydropyrrolidone or N-methylethanolamine

[0011] When A is Where n = 2, 3, 5;

[0012] When A is X is an aliphatic hydrocarbon or an aromatic hydrocarbon.

[0013] Preferably, when A is X is

[0014] Preferably, when A is X is or

[0015] Preferably, the compound is as follows:

[0016] 1a: 5-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amino-O)-2,4-dinitrophenoxy-1-(4-hydroxypiperidin-1-yl)diazacyclic-1-onthium-1,2-dihydroester;

[0017] 1b: 5-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amine-o)-2,4-dinitrophenoxy-1-(S)-2-hydroxymethylpyrrolidone-1-yl)diazacyclohexane-1-onium-1,2-dihydroester;

[0018] 1c: 5-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amino-O)-2,4-dinitrophenoxy-1-(pyrrolidone-1-yl)diazacyclohexane-1-onium-1,2-dihydroester;

[0019] 1d: 5-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl(methyl)amino-o)-2,4-dinitrophenyl;

[0020] 1e: 5-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amino-O)-2,4-dinitrophenoxy-1-(4-methylpiperazin-1-yl)diazacyclohexane-1-onthium-1,2-dihydroester;

[0021] 1f: 5-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl(methyl)amine o)-2,4-dinitrophenoxy-3-(2-hydroxyethyl)-3-methyldiazacyclopentene-1-onthium-1,2-dihydroester;

[0022] 2a: 4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amino)methyl)benzyl)amino)ethoxy)-3-(benzenesulfonyl)-1,2,5-oxadiazole 2-oxide;

[0023] 2b: 4-(3-(4-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amino)methyl)benzyl)aminopropoxy)-3-benzenesulfonyl-1,2,5-oxadiazole 2-oxide;

[0024] 2c: 4-((5-(4-((2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methylamino)methyl)benzyl)amino)pentyl)oxy)-3-(benzenesulfonyl)-1,2,5-oxadiazole 2-oxide;

[0025] 2d: 4-(4-(2-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propionyl)(methylamino)methyl)phenoxy)propoxy)phenyl)acetoxy)butoxy)-3-(benzenesulfonyl)-1,2,5-oxadiazole 2-oxide;

[0026] 2e: 4-(5-(2-(4-(3-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propionyl)(methylamino)methyl)phenoxy)propoxy)phenyl)acetoxy)pentyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazine-2-oxide;

[0027] 2f: 4-(2-(2-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propionyl(methyl)amino)methyl)phenoxy)propoxy)phenyl)acetamido)ethoxy)-3-(benzenesulfonylnylonyl)-1,2,5-oxadiazole 2-oxide;

[0028] 2g: 4-(2-(2-(4-(3-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propionyl(methylamino)methyl)phenoxy)propoxy)phenyl)acetamido)ethoxy)-3-(phenylsulfonate only)-1,2,5-oxadiazol-2-oxide;

[0029] 2h: 4-(4-(4-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propionyl(methyl)amino)methyl)phenoxy)propoxy)phenyl)acetoxy)but-2-ynyl-1-yl)oxy)-3-(PH-enylsulfonyl)-1,2,5-oxadiazole 2-oxide;

[0030] 2i: 4-(2-(2-(2-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amino)methyl)phenoxy)propoxy)phenyl)acetoxy)ethoxy)ethoxy)-3-(phenylalanine-nylonsulfonyl)-1,2,5-oxadiazole 2-oxide;

[0031] 3a: 2-(dihydroxyoxy)ethyl-2-(4-((2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amino)methyl)phenoxy)propoxy)phenyl)acetate;

[0032] 3b: 3-(nitoxy)propyl-2-(4-((2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amino)methyl)phenoxy)propoxy)phenyl)acetate;

[0033] 3c: 4-(nitoxy)butyl-2-(4-((2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amino)methyl)phenoxy)propoxy)phenyl)acetate;

[0034] 3d: 5-(nitoxy)pentyl-2-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazo-1-1-yl)propyl)(methyl)amino)methyl)phenoxy)propoxy)phenyl)acetate;

[0035] 3e: 4-((nitoxy)methyl)benzyl-2-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amino)methyl)phenoxy)propoxy)phenyl)acetate;

[0036] 3f: 2-((nitoxy)methyl)benzyl-2-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amino)methyl)phenoxy)propoxy)phenyl)acetate;

[0037] 3g: (3S, 3aR, 6R, 6aS)-6-(nitoxy)hexahydrofuran[3,2-b]furan-3-yl-2-(4-((2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl(methyl)amino)methyl-ylphenoxy)propoxy)hydroenyl)acetate;

[0038] 3h: 2-(2-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amino)methyl)phenoxy)propoxy)phenyl)acetyl)-L2-azayl)ethyl nitrate.

[0039] The preparation method of the above compounds is as follows: using secondary amine compounds 4a-f as raw materials, they react with NO gas under high pressure to obtain sodium azomonium glycol salt compounds 5a-f. Under the action of a weak base, they undergo a nucleophilic substitution reaction with 2,4-dinitrofluorobenzene to generate compounds 6a-f. At the same time, trimethyl sulfoxide is heated in a strongly alkaline environment to generate a carbanion intermediate C-H3, which then undergoes an epoxidation reaction with the raw material Az-1 to obtain Az-2. Az-2 is ring-opened by amine compounds under alkaline conditions to obtain Az-3. Finally, compounds 6a-f are subjected to alkaline attack by Az-3 to obtain the target compound.

[0040]

[0041] The preparation method of the above compound is as follows: Sodium thiophene 7 is used as a raw material and reacted with chloroacetic acid to obtain phenylthioacetic acid 8. Then, 8 is oxidized to obtain compound 9. After removing the solvent, it is reacted with fuming nitric acid at high temperature to obtain furazolidone nitrate 10. Furazolidone nitrate reacts with Boc-protected amino alcohol under alkaline conditions to obtain 11a-c. After deprotection, intermediate 12a-c is obtained. At the same time, intermediate Az-3 undergoes a nucleophilic addition reaction with 4-bromomethylbenzaldehyde to obtain Az-4. Finally, 12a-c and Az-4 are reduced and aminationed by the weak reducing agent sodium nitrile borohydride to obtain the target compound.

[0042]

[0043] The preparation method of the above compounds is as follows: Methyl p-hydroxyphenylacetate 14 is reacted with 1,3-dibromopropane under alkaline conditions via nucleophilic addition to give 15. 15 is further reacted with p-hydroxybenzaldehyde to give 16. After purification, methylamine hydrochloride is reacted with triethylamine under anhydrous conditions to give an imine intermediate, which is then directly reduced to give compound 17. Az-2 is dissolved in acetonitrile and excess triethylamine is added for release. Then, 17 is added and ring-opening is performed under alkaline conditions to give compound Az-5. The ethyl ester portion is removed to give intermediate Az-6. Simultaneously, 10 undergoes nucleophilic substitution with a diol under alkaline conditions to give 13d-h. Finally, Az-6 and 13d-h are condensed under the action of a catalyst to give the target compound.

[0044]

[0045] The preparation method of the above compound is as follows: using terminal bromool as raw material, it reacts with silver nitrate under high temperature and light-protected conditions to generate hydroxy nitrate ester compounds, which are then esterified with Az-6 in one step to obtain the target compound;

[0046]

[0047] The present invention also discloses a pharmaceutical composition comprising a compound of general formula V or a medically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

[0048] The above-mentioned compounds are used in the preparation of antifungal drugs.

[0049] Invention Principle: The NO donor-type antifungal compound of the present invention introduces a nitric oxide (NO) donor fragment into an active azole antibacterial fragment, achieving a dose-dependent slow release of NO in the presence of a thiol-containing nucleophilic reagent in vitro. NO can inhibit the formation of fungal biofilms and promote the dispersal of biofilms by reducing the level of the intracellular second messenger cyclic diguanylate (c-di-GMP), thereby affecting fungal growth. The compound has high stability. By replacing the ester bond in compound 3a with an amide bond, i.e., compound 3h, the stability of compound 3h in rat plasma and liver microparticles is significantly improved.

[0050] NO donor-type antifungal compounds 3a-3h penetrate fungal cells, disrupting the integrity of the fungal cell membrane, and slowly release effective concentrations of NO in vivo, exerting significant antifungal activity and effectively inhibiting the growth of Cryptococcus neoformans. They also exhibit broad-spectrum antibacterial activity. Compound 3a, due to its smaller nitric oxide donor portion, is more conducive to binding with CYP51 (sterol 14α-demethylase), effectively inhibiting Cryptococcus neoformans growth even at low concentrations. It significantly outperforms the positive control drug fluconazole (FLC) by inhibiting Cryptococcus neoformans biofilm formation and also inhibits the biosynthesis of ergosterol in Cryptococcus neoformans, thus exerting its antibacterial function. Compound 3b effectively inhibits Cryptococcus neoformans growth, superior to FLC; compounds 3e and 3g effectively inhibit Candida albicans growth. Simultaneously, nasal administration of compounds 3a and 3h significantly reduced bacterial load in mouse brain and lung tissues, and significantly alleviated the degree of pathological damage in mouse brain tissue.

[0051] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0052] (1) The NO donor type azole derivative of the present invention exerts the antifungal effect of azole fragments while releasing NO, thereby achieving the effect of synergistic effect between multiple targets and avoiding the problems of narrowing therapeutic window and difficulty in setting dosage caused by complex pharmacokinetic properties when single-target drugs are used in combination.

[0053] (2) Through in vitro antifungal activity tests, highly active nitrate azole derivatives 3a and 3e were screened out. They have excellent in vitro inhibitory activity against Cryptococcus neoformans H99 and can achieve extremely high anti-cryptococcal activity at a low concentration of <1μg / mL, which is significantly better than existing antifungal drugs.

[0054] (3) Nitrate ester NO donor type azole derivatives can also inhibit the production of ergosterol on the cell membrane of Cryptococcus neoformans, thereby exerting antifungal activity;

[0055] (4) Due to the superior in vitro and in vivo anti-cryptococcal activity of the compounds in this application, they can serve as a new antifungal framework for the treatment of cryptococcal meningitis and to combat invasive fungal infections. Attached Figure Description

[0056] Figure 1 V is the general formula for the NO donor-type antifungal compound of this invention;

[0057] Figure 2 The results of in vitro nitric oxide release experiments for compounds 1f, 2i, 3a, and 3h are presented.

[0058] Figure 3 For the in vitro antifungal activity studies of compounds 2a-2i and 3a-3f, and for the plate diffusion experiments of compounds 3a and 3e;

[0059] Figure 4 The antifungal biofilm formation effects of compounds 3a and 3e in vitro;

[0060] Figure 5 Experiments on NO release from compounds 3a and 3e in vivo;

[0061] Figure 6 The effects of compounds 3a and 3e on ergosterol biosynthesis and microstructure in Cryptococcus neoformans cells;

[0062] Figure 7 To study the in vivo antifungal activity of compounds 3a and 3h. Detailed Implementation

[0063] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0064] Example 1

[0065] Preparation of -(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amino-O)-2,4-dinitrophenoxy-1-(4-hydroxypiperidin-1-yl)diazacyclic-1-onthium-1,2-dihydroester (1a)

[0066]

[0067] (a) 4-hydroxypiperidine (0.72 g, 5 mmol), freshly prepared sodium methoxide / methanol solution (30% w / w, 100 mL), and anhydrous diethyl ether (300 mL) were successively added to a specially prepared high-pressure reactor. After the air was replaced with nitrogen, NO gas was introduced and the mixture was mechanically stirred at room temperature. After 72 h, a large amount of diethyl ether was added to precipitate the solid. The solid was filtered, washed with anhydrous diethyl ether, and dried under vacuum at room temperature to obtain product 5a, which was directly added to the next step of the reaction without purification.

[0068] (b) A solution of 1,5-difluoro-2,4-dinitrobenzene (204 mg, 1 mmol) in 15 mL of acetone was cooled to 0 °C under nitrogen protection. A solution of sodium azomonium glycol 5a (1.1 mmol) in 15 mL of 5% sodium bicarbonate aqueous solution was added dropwise. After the reaction was complete, the acetone was removed by concentration under reduced pressure. The residue was absorbed in CH2Cl2 and washed with water. The organic solution was dried over sodium sulfate and evaporated under vacuum to give the desired product, which was recrystallized from ethanol to give compound 6a, a pale yellow solid, in 35% yield. 1H NMR(500MHz,Chloroform-d)δ8.91(d,J=7.5Hz,1H,ArH),7.43(d,J=11.7Hz,1H,ArH),4.06-4.00(m,1 H,CH),3.92-3.86(m,2H,CH2),3.69-3.63(m,2H,CH2),2.09-2.02(m,2H,CH2),1.88-1.81(m,2H,CH2).

[0069] (c) 2,4-Difluoro-2-[1-(1H-1,2,4-triazolyl)]acetophenone (7.30 g, 32.7 mmol) was added to a suitable amount of toluene (150 mL), followed by the addition of trimethyl sulfoxide (8.64 g, 39.30 mmol), and then 20% sodium hydroxide solution (8 mL) was slowly added. The reaction mixture was then heated at 45-50 °C for 3 hours. After the reaction was complete, EA (50 mL) was added for dilution, and the organic layer was washed with water (2 × 200 mL) and saturated sodium chloride solution (200 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure to give a light brown oily substance; the oily substance was added to EA (100 mL), and methanesulfonic acid was slowly added dropwise, resulting in the formation of a white solid. After reacting for 3 hours, the mixture was quickly filtered and washed with EA, and then rapidly evaporated to dryness to give a white solid, Az-2, in 60% yield. 1H NMR(300MHz,Chloroform-d)δ8.05(s,1H,ArH),7.86(s,1H,ArH),7.22-7.11(m,1H,ArH),6.87-6.75(m ,2H,ArH),4.81(d,J=14.8Hz,1H,CH2),4.50(d,J=14.9Hz,1H,CH2),2.90(dd,J=18.7,4.6Hz,2H,CH2).

[0070] (d) Take Az-2 (500 mg, 0.156 mmol), add ethanol, then add triethylamine and stir until dissolved. Add 130 mg of 25% methylamine ethanol solution, then add 426 mg of triethylamine. Reflux for 4 h. After the reaction is complete as detected by TLC, remove the solvent by vacuum distillation. Purify by silica gel column chromatography (CH2Cl2 / MeOH, 15 / 1, v / v). Dry to obtain a yellow oily liquid, namely Az-3, with a yield of 30%. 1H-NMR(300MHz,Chloroform-d)8.22(s,1H,ArH),8.17(s,1H,ArH),7.70-7.82(m,1H,ArH),6.95-6.97(m, 2H,ArH),4.50-4.55,4.53-4.68(dd,2H,CH2),2.76-2.80,3.11-3.15(dd,2H,CH2),2.28-2.30(s,3H,CH3).

[0071] (e) 6a (57 mg, 0.311 mmol) was added to THF (10 mL) and stirred until dissolved. Az-3 (92 mg, 0.343 mmol) was added and mixed thoroughly with 6a. Then Na2CO3 (66 mg, 0.622 mmol) was added and the mixture was reacted at room temperature for 6 h. After the reaction was confirmed to be complete by TLC, the mixture was dissolved completely by adding ethanol under reduced pressure. After filtering out sodium carbonate, column chromatography (CH2Cl2 / MeOH, 10 / 1, v / v) was performed to obtain a yellow solid, i.e., 1a, with a yield of 40%.1H-NMR(CDCl3, 300 MHz) δ 8.61 (s, 1H, ArH), 7.92 (s, 1H, ArH), 7.83 (s, 1H, ArH), 7.43 - 7.51 (m, 1H, ArH), 7.10 (s, 1H, ArH), 6.69 - 6.82 (m, 2H, ArH), 4.59 - 4.63, 4.71 - 4.75 (dd, 2H, CH2), 4.00 - 4.04 (m, 1H, CH), 3.92 (s, 2H, NCH2), 3.81 - 3.86 (m, 2H, CH2), 3.57 - 3.64 (m, 2H, CH2), 2.92 (s, 3H, NCH3), 2.83 (s, 1H, OH), 2.02 - 2.09 (m, 2H, CH2), 1.81 - 1.86 (m, 2H, CH2). 13C NMR(126 MHz, CDCl3) δ 164.24 - 164.14, 162.23 - 162.13 (dd, J = 247.5, 12 Hz), 159.53 - 159.44, 157.59 - 157.49 (dd, J = 247.5, 12 Hz), 153.05, 151.90, 151.69, 150.42, 150.14, 149.97, 144.59, 144.45, 133.07, 131.05, 131.01, 130.32 - 130.20 (dd, J = 9, 6 Hz), 130.10 - 129.98 (dd, J = 9, 6 Hz), 129.35, 127.87, 127.12, 122.98 - 122.88 (d, J = 12 Hz), 112.32 - 112.02 (d, J = 20.25 Hz), 108.75, 105.99, 104.44 - 104.02 (t, J = 26.25 Hz), 66.19, 64.79, 59.65, 54.71, 54.67, 48.26, 47.59 - 47.21 (m, 2C), 45.06, 44.17, 31.94 - 31.74 (m, 2C). HRMS(ESI-MS m / z) calculated for C23H25F2N9O8 [M+Na]+ 616.1669, Found 616.1675 ppm (parts per million) error: 0.9.

[0072] Example 2

[0073] Preparation of 5-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amine-o)-2,4-dinitrophenoxy-1-(S)-2-hydroxymethylpyrrolidine-1-yl)diazacyclohexane-1-onthium-1,2-dihydrogen ester (1b)

[0074]

[0075] The synthesis method was followed as described in Example 1. ¹H NMR (CDCl₃, 500MHz) 8.57 (d, J = 7.6Hz, ¹H, ArH), 7.92 (s, ¹H, ArH), 7.83 (s, ¹H, ArH), 7.46 (d, J = 11.8Hz, ¹H, ArH), 7.09-7.12 (m, ¹H, ArH), 6.69-6.82 (m, 2H, ArH), 4.59-4.63, 4.71-4.75 (dd) ,2H,-CH2-),3.93-3.95(m,2H,NCH2-),3.88-3.91(m,2H,prolinol),3.84-3.86(m,1H,proli nol),3.73-3.79(m,2H,prolinol),2.84-2.86(s,3H,NCH3),2.03-2.17(m,4H,prolinol).13C NMR (126MHz, CDCl3) δ164.19-164.09, 162.19-162.09 (dd, J=247.5, 12Hz), 159.58-159.48, 157.63-157.53 (dd, J= 247.5,12Hz),153.25,153.19,151.76,150.34,144.54,132.51,130.32-130.21(dd,J=9,6Hz),127.14(s,2C),123. 03-122.95(d,J=12Hz),112.22-112.06(d,J=20.25Hz),105.03,104.40-103.98(t,J=26.25Hz),64.20(s,3C),59.5 2,54.85-54.81(d,1C),52.53-52.47(d,1C),45.04(s,2C),27.34-27.21(d,1C),23.21-23.14(d,1C).HRMS(ESI-MS m / z)calculated for C23H25F2N9O8[M+Na]+616.1669,Found 616.1673ppm error:0.8.

[0076] Example 3

[0077] Preparation of 5-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amino-O)-2,4-dinitrophenoxy-1-(pyrrolidone-1-yl)diazacyclohexane-1-onthium-1,2-dihydroester (1c)

[0078]

[0079] The synthesis method is the same as in Example 1. 1H NMR(DMSO-d6,300MHz)8.57(d,J=7.6Hz,1H,ArH),7.92(s,1H,ArH),7.83(s,1H,ArH), 7.46(d,J=11.8Hz,1H,ArH),7.09-7.12(m,1H,ArH),6.69-6.82(m,2H,ArH),4.59-4.63 ,4.71-4.75(dd,2H,-CH2-),3.78-3.83(m,4H,pyrrolidine),3.77-3.82,4.06-4.18(d d,2H,-NCH2-),2.84-2.86(s,3H,NCH3),2.05-2.12(m,4H,pyrrolidine).HRMS(ESI-MS m / z) calculated for C22H23F2N9O7[M+H]+563.1689, found 564.1668ppm error:-0.5.

[0080] Example 4

[0081] Preparation of 5-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl(methyl)amino-o)-2,4-dinitrobenzene (1d)

[0082]

[0083] The synthesis method was followed as described in Example 1. ¹H NMR (CDCl₃, 500MHz): 8.57 (s, ¹H, ArH), 7.97 (s, ¹H, ArH), 7.82 (s, ¹H, ArH), 7.46 (m, ¹H, ArH), 7.09 (s, ¹H, ArH), 6.69-6.77 (m, 2H, ArH), 4.59-4.63, 4.71-4.75 (dd, 2H, -CH₂-), 3.93-3.95 (m, 2H, -NCH₂-), 3.50-3.51 (d, 4H, -NCH₂CH₃), 2.93 (s, 3H, NCH₃), 1.22-1.24 (t, 6H, -NCH₂CH₃). ¹³C NMR(75MHz,DMSO-d6)δ160.12,155.34,148.40,147.36,145.38,139.57,128.24,125.50(dd,J=15.0Hz),123.16,122.39,11 8.07(dd,J=15.0Hz),107.59,107.31,101.16,99.41(t,J=105.0Hz),54.80,49.75,48.76,42.39,40.28,6.81.HRMS(ESI-MS m / z) calculated for C22H25F2N9O7[M+Na]+588.1732, found 588.1737ppm error:0.59.

[0084] Example 5

[0085] Preparation of 5-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amino-O)-2,4-dinitrophenoxy-1-(4-methylpiperazin-1-yl)diazacyclohexane-1-onthium-1,2-dihydroester (1e)

[0086]

[0087] The synthesis method was followed as described in Example 1. ¹H NMR (CDCl₃, 500MHz): 8.61 (s, ¹H, ArH), 7.90 (s, ¹H, ArH), 7.80 (s, ¹H, ArH), 7.46 (m, ¹H, ArH), 7.08 (s, ¹H, ArH), 6.69-6.80 (m, 2H, ArH), 4.59-4.63, 4.71-4.75 (dd, 2H, -CH₂-), 3.88-3.96 (m, 2H, -NCH₂-), 3.65-3.67 (m, 4H, piperazine), 2.93 (s, 3H, -NCH₃), 2.64-2.66 (m, 4H, piperazine), 2.37 (s, 3H, piperazine). ¹³C NMR (75MHz, DMSO-d6) δ152.42, 151.35, 150.49, 150.16, 145.58 (d, J = 6.0Hz), 145.05 (d, J = 6.0Hz), 132.45, 127.31 (d, J = 9. 0Hz),126.87,111.69,111.42,106.23,104.22,76.56(d,J=9.0Hz),60.17,55.78,53.29,50.32,45.39,44.09.HRMS(ESI-MS m / z)calculated for C23H26F2N10O7[M+Na]+615.1836,found615.1846ppm error:1.56.

[0088] Example 6

[0089] Preparation of 5-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl(methyl)amine o)-2,4-dinitrophenoxy-3-(2-hydroxyethyl)-3-methyldiazacyclopentene-1-onthium-1,2-dihydroester (1f)

[0090]

[0091] The synthesis method was followed as described in Example 1. ¹H NMR (CDCl₃, 500MHz): 8.61 (s, ¹H, ArH), 7.90 (s, ¹H, ArH), 7.81 (s, ¹H, ArH), 7.46 (m, ¹H, ArH), 7.08 (s, ¹H, ArH), 6.69-6.77 (m, 2H, ArH), 3.93-3.95 (m, 2H, -NHCH₂⁻), 3.88-3.90 (t, 2H, -NHCH₂CH₂OH), 3.72-3.80 (m, 2H, -NHCH₂CH₂OH), 3.34 (s, 3H, -NHCH₃), 2.84 (s, 3H, -NHCH₃). ¹³C NMR(75MHz, CDCl3)δ181.62,153.25,151.78(d,J=21.0Hz),150.23,144.51,13 2.80,130.09,127.62,127.17,122.93(d,J=69.0Hz),112.42(d,J=12.0Hz),11 2.14(d,J=12.0Hz),105.50,104.26(t,J=105.0Hz),54.96,59.34(d,J=15.0Hz ),55.89,54.70(d,J=27.0Hz),54.39(d,J=24.0Hz),44.99,40.77.HRMS(ESI-MS m / z)calculated for C21H23F2N9O8[M+Na]+590.1523,found590.1529ppm error:0.91.

[0092] Example 7:

[0093] Preparation of 4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amino)methyl)benzyl)amino)ethoxy)-3-(benzenesulfonyl)-1,2,5-oxadiazole 2-oxide (2a)

[0094]

[0095] (a, b, c) Sodium thiophene 7 was dissolved in ethanol and heated until completely dissolved. Chloroacetic acid and potassium carbonate were then added and refluxed. The reaction was monitored by TLC. After the reaction was completed, the mixture was distilled under reduced pressure and acidified with 1N HCl. When the pH value was <2, a white solid precipitated. The white solid was filtered to obtain compound 8. Compound 8 was dissolved in glacial acetic acid and an excess of 30% H2O2 was added. The mixture was refluxed overnight, and most of the solvent was removed. A small amount of glacial acetic acid was added again, and the temperature was raised to 90°C. Fuming nitric acid was added, and the reaction temperature was further raised to 120°C. After reacting for 2 hours, ice water was poured into the reaction solution, and a white solid precipitated. The solid was filtered and washed to obtain compound 10, a white solid with a yield of 36%. 1H NMR (500MHz, Chloroform-d) δ8.17 (d, J=8.5Hz, 4H, ArH), 7.80 (d, J=7.4Hz, 2H, ArH), 7.70-7.63 (m, 4H, ArH).

[0096] (d) Compound 10 (500 mg, 1.366 mmol) was dissolved in THF. After the solution was cleared, N-(tert-butoxycarbonyl)amino alcohol (2.732 mmol) was added, followed by slow dropwise addition of 50% NaOH solution (109 mg, 2.732 mmol, based on NaOH). The reaction was detected by TLC (PE / EA, 5 / 1, v / v). After the reaction was completed, the solvent was evaporated and purified by silica gel column chromatography (PE / EA, 10 / 1, v / v) to give a white solid compound 11a.

[0097] (e) 11a-c (100 mg) was dissolved in DCM. The reaction solution was cooled to 0 °C and TFA was added. The reaction was detected by TLC (PE / EA, 3 / 1, v / v). After the reaction was complete, the solution was evaporated to dryness and EA (20 mL) was added. The solution was washed with saturated sodium bicarbonate, pure water, and saturated sodium chloride. The organic layer was collected, dried over Na2SO4, filtered, and the solvent was removed to obtain compound 12a as a white solid with a yield of 47%. ¹H NMR (500 MHz, DMSO-d6) δ 8.04 (d, J = 7.7 Hz, 2H, ArH), 7.90 (d, J = 7.5 Hz, 1H, ArH), 7.75 (d, J = 7.8 Hz, 2H, ArH), 4.34 (d, J = 5.5 Hz, 2H, CH2), 2.92 (d, J = 5.6 Hz, 2H, CH2).

[0098] (f) Az-3 (536 mg, 2 mmol) was dissolved in ACN (30 mL) and stirred until dissolved. K2CO3 (828 mg, 6 mmol) was then added and reacted at room temperature for 4 h. The reaction solution became turbid. The reaction was detected by TLC (PE / EA, 5 / 1, v / v). After the reaction was complete, column chromatography was used to obtain a white solid, namely compound Az-4, with a yield of 74%. 1H NMR(CDCl3,300MHz)8.22(s,1H,ArH),8.17(s,1H,ArH),7.70-7.82(m,1H,ArH),7.30-7.38(d,2H,ArH),7.14-7.19(d,2H,ArH),6.95-6 .97(m,2H,ArH),4.50-4.55,4.53-4.68(dd,2H,-CH2-),3.76(s,2H,-NCH2-),2.76-2.80,3.11-3.15(dd,2H,NCH2-),2.25(s,3H,NCH3).

[0099] (g) Az-4 (100 mg, 0.373 mmol) and 12a (0.410 mmol) were dissolved together in methanol (15 mL) and reacted at room temperature for 2 h. The reaction was monitored by TLC (CH2Cl2 / MeOH, 20 / 1, v / v). After the reaction was complete, sodium cyanoborohydride (118 mg, 1.865 mmol) was slowly added in portions and the reaction was allowed to proceed overnight. After TLC (CH2Cl2 / MeOH / Et3N, 10 / 1 / 1 drop, v / v / v), the solvent was evaporated and the solution was purified by alkaline alumina column chromatography (CH2Cl2 / MeOH / Et3N, 10 / 1 / 1%, v / v / v) to give a white solid, namely the target compound 2a, in a yield of 27%. NMR(CDCl3,500MHz)8.04-8.07(m,2H,ArH),7.75-7.78(m,2H,ArH),7.65-7.70(m,1H,ArH),7.60-7.63(m, 2H,ArH),7.34-7.35(m,2H,ArH),7.14-7.15(m,2H,ArH),6.81-6.89(m,2H,ArH),4.68(s,J=6Hz,2H,NCH2CH 2O-),4.32-4.35,4.51-4.55(dd,2H,-CH2-),4.05(s,2H,Ar-CH2-),3.33-3.36,3.54-3.56(dd,2H,NCH2-A r),3.36(t,J=7Hz,2H,NCH2CH2O-),2.64-2.67,3.01-3.04(dd,2H,NCH2-),2.06(s,3H,NCH3).HRMS(ESI-MS m / z)calculated for C30H31F2N7O6S[M+H]+656.2097,found656.2102ppm error:-0.77.

[0100] Example 8

[0101] Preparation of 4-(3-(4-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amino)methyl)benzyl)aminopropoxy)-3-benzenesulfonyl-1,2,5-oxadiazole 2-oxide (2b)

[0102]

[0103] The synthesis method was followed as described in Example 7. ¹H NMR (CDCl₃, 500MHz): 8.06-8.07 (m, 2H, ArH), 7.75-7.78 (m, 2H, ArH), 7.62-7.65 (m, 3H, ArH), 7.34-7.35 (m, 2H, ArH), 7.14-7.15 (m, 2H, ArH), 6.80-6.86 (m, 2H, ArH), 4.57-4.60 (t, J = 6Hz, 2H, CH₂O⁻), 4.32-4.35, 4.51-4 .55(dd,2H,-CH2-),4.05(s,2H,Ar-CH2-),3.33-3.36,3.54-3.56(dd,2H,NCH2-Ar),3.36(t,J=7Hz,2H,NCH2 -),2.64-2.67,3.01-3.04(dd,2H,NCH2-),2.09-2.16(m,2H,-CH2CH2CH2-),2.06(s,3H,NCH3).HRMS(ESI-MS m / z)calculated for C31H34F2N7O6S[M+H]+670.2253,found670.2257ppm error:-0.44.

[0104] Example 9

[0105] Preparation of 4-((5-(4-((2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methylamino)methyl)benzyl)amino)pentyl)oxy)-3-(benzenesulfonyl)-1,2,5-oxadiazole 2-oxide (2c)

[0106]

[0107] The synthesis method was followed as described in Example 7. ¹H NMR (CDCl₃, 500MHz) 8.09-8.11 (m, 2H, ArH), 7.88 (m, 1H, ArH), 7.75-7.78 (m, 2H, ArH), 7.73 (m, 1H, ArH), 7.62-7.65 (m, 2H, ArH), 7.34-7.35 (m, 2H, ArH), 7.14-7.15 (m, 2H, ArH), 6.96-6.99 (m, 1H, ArH), 6.85-6.88 (m, 1H, ArH), 4.57-4.60 (t, J = 6Hz, 2H, CH₂O⁻), 4.32-4.35. 4.51-4.55(dd,2H,-CH2-),4.05(s,2H,Ar-CH2-),3.33-3.36,3.54-3.56(dd,2H,NCH2-Ar),3.36(t,J=7Hz,2H,CH2O-),2.64-2.67,3.0 1-3.04(dd,2H,NCH2-),2.09-2.16(m,2H,-CH2-),2.06(s,3H,NCH3),1.99-2.02(m,2H,-CH2-),1.73-1.77(m,2H,-CH2-).HRMS(ESI-MS m / z)calculated for C33H37F2N7O6S[M+H]+698.2528, found 698.2439ppm error:1.27.

[0108] Example 10

[0109] Preparation of 4-(4-(2-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propionyl)(methylamino)methyl)phenoxy)propoxy)phenyl)acetoxy)butoxy)-3-(benzenesulfonyl)-1,2,5-oxadiazole 2-oxide (2d)

[0110]

[0111] (a) Methyl p-hydroxyphenylacetate 14 (10 g, 60.2 mmol), 1,3-dibromopropane (12.1 g, 90.3 mmol), and K2CO3 (24.88 g, 180.3 mmol) were dissolved in anhydrous ethanol (250 mL) and reacted overnight at 85 °C. The reaction was detected by TLC (PE / EA, 8 / 1, v / v). After the reaction was complete, potassium carbonate was removed by filtration, most of the solvent was removed by rotary evaporation, and then ethyl acetate (100 mL) and water (100 mL) were added for extraction three times. The extracts were washed with saturated sodium chloride solution, and the organic layers were combined. The mixture was dried over anhydrous Na2SO4, filtered, and subjected to silica gel stirred column chromatography (PE / EA, 15 / 1, v / v) to obtain compound 15 as a pale yellow liquid with a yield of 53%. 1H NMR(500MHz,Chloroform-d)δ7.19(d,J=8.4Hz,2H,ArH),6.86(d,J=8.4Hz,2H,ArH),4.09(d,J=5.5 Hz,2H,CH2),3.69(s,3H,CH3),3.60(d,J=6.4Hz,2H,CH2),3.57(s,2H,CH2),2.34-2.28(m,2H,CH2).

[0112] (b) Compound 15 (3 g, 10.4 mmol) was dissolved in anhydrous ethanol (30 mL), p-hydroxybenzaldehyde (1.9 g, 15.6 mmol) and potassium carbonate (4.3 g, 31.2 mmol) were added, and the mixture was refluxed at 85 °C for 6 h. After evaporation to dryness, EA (100 mL) was added and the solid was removed by filtration. The mixture was extracted with water three times and washed once with saturated brine. The organic layers were combined, dried with anhydrous Na2SO4 for 1 h, filtered, and the solvent was removed by rotary evaporation. After mixing with silica gel, the mixture was purified by silica gel column chromatography with (PE / EA, 15 / 1, v / v) as the mobile phase to obtain compound 16, a white solid with a yield of 42%. 1H NMR(500MHz,Chloroform-d)δ9.88(s,1H,CHO),7.83(d,J=8.7Hz,2H,ArH),7.19(d,J=8.7Hz,2H,ArH),7.01(d,J=8.6Hz,2H,ArH),6. 87(d,J=8.7Hz,2H,ArH),4.25(t,J=6.2Hz,2H,CH2),4.15(d,J=6.0Hz,2H,CH2),3.68(s,3H,CH3),3.56(s,2H,CH2),2.29(p,2H,CH2).

[0113] (c) Compound 16 (1 g, 2.9 mmol) was dissolved in anhydrous methanol (20 mL) by ultrasonic stirring. Methylamine hydrochloride (398 mg, 5.8 mmol), triethylamine (879 mg, 8.7 mmol), and anhydrous Na₂SO₄ were added, and the mixture was stirred overnight at room temperature. The reaction was monitored by TLC (CH₂Cl₂ / MeOH, 15 / 1, v / v). Sodium borohydride (812 mg, 11.6 mmol) was added, and the reaction was continued for 1 h. The reaction was monitored by TLC (CH₂Cl₂ / MeOH / Et₃N, 10 / 1 / 1%, v / v / v). The mixture was evaporated to dryness, ethyl acetate was added, and the mixture was filtered. The filter cake was washed with ethyl acetate, and the filtrate was collected. The organic layer was extracted three times with water, washed once with saturated brine, dried over anhydrous Na₂SO₄, and filtered. The organic solvent was removed from the filtrate under reduced pressure. The residue was compound 17, a white solid, with a yield of 93%. 1H NMR(500MHz,Chloroform-d)δ7.22(d,J=8.6Hz,2H,ArH),7.18(d,J=8.6Hz,2H,ArH),6.87(d,J=1.3Hz,2H,ArH),6.86(d,J =1.3Hz,2H,ArH),4.14(d,J=6.1Hz,4H,CH2),3.68(s,5H),3.56(s,2H,CH2),2.43(s,3H,CH3),2.24(p,J=6.1Hz,2H,CH2).

[0114] (d, e) Compound Az-2 (1.12 g, 3.4 mmol) was dissolved in acetonitrile (20 mL), and triethylamine (565 mg, 5.6 mmol) was added. After complete dissolution, 17 (1 g, 2.8 mmol) was added, and the mixture was reacted at 50 °C for 4 h. The solution turned yellow, and TLC (CH2Cl2 / MeOH, 10 / 1, v / v) was used for detection. After the reaction was completed, the solution was evaporated to dryness, and 1,4-dioxane was added to dilute it again (20 mL). Then, 1N NaOH solution was added and stirred to remove the ethyl ester. The solution became turbid, and TLC (CH2Cl2 / MeOH, 10 / 1, v / v) was used for detection. After the reaction was completed, the solution was evaporated to dryness, and water (30 mL) was added and 1N NaOH solution was used for further processing. The pH was adjusted to 7 with HCl, resulting in the precipitation of a large amount of solid. The mixture was extracted three times with ethyl acetate (100 mL), and the organic layers were combined. The extract was washed once with saturated brine, dried over anhydrous Na₂SO₄, filtered, and the organic solvent was removed from the filtrate under reduced pressure to obtain a white solid. The solid was then slurried with diethyl ether, filtered, and subjected to column chromatography to obtain intermediate Az-6, a white solid, with a yield of 25%. 1H NMR (500MHz, DMSO-d6) δ8.27(s,1H,ArH),7.73(s,1H,ArH),7.41(td,J=9.0,6.8Hz,1H,ArH),7.15(d,J=8.4Hz,2H,ArH),7.13 -7.10(m,1H,ArH),6.99(d,J=8.5Hz,2H,ArH),6.95(td,J=8.5,2.6Hz,1H,ArH),6.88(d,J=8.7Hz,2H,ArH),6.82(d,J=8.6Hz, 2H,ArH),4.56-4.44(m,2H,ArCH2),4.09(d,J=5.3Hz,4H,OCH2),3.47(s,2H,ArCH2),3.45(d,J=13.1Hz,1H,NCH2),3.33(d,J= 13.2Hz,1H,CH2),2.96(dd,J=14.1,1.4Hz,1H,CH2),2.75(d,J=13.7Hz,1H,CH2),2.13(d,J=6.2Hz,2H,CH2),2.04(s,3H,CH3).

[0115] (f) Compound 10 (500 mg, 136.6 mmol) was dissolved in THF, and diol (273.2 mmol) was added, followed by 50% NaOH (273.3 mmol). The reaction was carried out at room temperature for 3-5 h. The reaction was detected by TLC (PE / EA, 2 / 1, v / v). After the reaction was completed, the solvent was evaporated, the sample was mixed with silica gel, and column chromatography (PE / EA, 2 / 1, v / v) was performed to obtain compound 13d as a white solid with a yield of 48%. 1H NMR(500MHz,DMSO-d6)δ8.01(dd,J=8.6,0.9Hz,2H,ArH),7.90(t,J=7.5Hz,1H,ArH),7.75(t,J=8.1Hz,2H,Ar H), 4.61 (s, 1H, OH), 4.46 (t, J = 6.3Hz, 2H, CH2), 3.53 (q, J = 5.4Hz, 2H, CH2), 1.90 (p, J = 12.3, 6.0Hz, 2H, CH2).

[0116] (g) Compound Az-6 (100 mg, 0.176 mmol) was dissolved in anhydrous THF, and TBTU (68 mg, 0.212 mmol) and DMAP (43 mg, 0.352 mmol) were added. After the starting material was dissolved, the air in the system was displaced. After stirring for 0.5 h, 13d-h and 12a (0.212 mmol) were added. The reaction was carried out under nitrogen protection for 6 h. The reaction was detected by TLC (CH2Cl2 / MeOH, 20 / 1, v / v). After the reaction was completed, the solvent was evaporated, the silica gel was stirred, and column chromatography (CH2Cl2 / MeOH, 30 / 1, v / v) was performed to obtain the target compound 2d, a colorless oil with a yield of 36%. 1H NMR(CDCl3,500MHz)8.02-8.08(m,3H,ArH),7.75-7.76(m,2H,ArH),7.57-7.64(m,3H,ArH),7.17-7.18(d,J=8.2Hz 2H,ArH),7.05-7.07(d,J=8.2Hz,2H,ArH),6.78-6.89(m,6H,ArH),4.46-4.48(m,3H,-OCH2-,-CH2- ),4.28-4.31(t,J=6Hz,2H,-CH2O-),4.11-4.18(m,5H,-OCH2-,-CH2-),3.59(s,2H,ArCH2-),3.31- 3.34,3.42-3.45(dd,J=13.7Hz,2H,-NCH2-),2.82-2.85,3.07-3.09(dd,J=13.7Hz,2H,-CH2N-),2. 22-2.26(m,2H,-CH2-),2.04(s,3H,NCH3),1.88-1.92(m,2H,-CH2-),1.77-1.82(m,2H,-CH2-).13C NMR(126MHz, CDCl3)δ171.72,163.77-163.67,161.78-161.69(dd,J=247.5,12Hz),159.98-159.89 158.02-157.93(dd,J=247.5,12Hz),158.82,158.36,158.11,151.02,144.6 6,138.11,135.66,133.86,130.25(s,2C),130.22(s,2C),129.88,129.75(s, 2C),129.64-129.59,129.56-129.52(dd,J=9,6Hz),129.32,128.56(s,2C), 127.89,125.98,114.66(s,2C),114.51(s,2C),111.63-111.60,111.46-111.44(dd,J=20.25,3Hz),110.46,104.45-104.24,104.23-104.03(dd,J=25.5Hz),72.09-72.05(d,1C),67.85,67.03,64.50 -64.49(d,2C),62.71,60.51,56.54,56.50,43.65,40.48,31.97,29.74,29.34,28.28,27.85,22.73,14.16.HRMS(ESI-MS m / z)calculated for C42H44F2N6O10S[M+H]+863.2880,found863.2875ppm Error: 0.38.

[0117] Example 11

[0118] Preparation of 4-(5-(2-(4-(3-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propionyl)(methylamino)methyl)phenoxy)propoxy)phenyl)acetoxy)pentyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazine-2-oxide (2e)

[0119]

[0120] The synthesis method was followed as described in Example 10. ¹H NMR (CDCl₃, 500MHz) 8.11 (s, ¹H, ArH), 8.05-8.07 (d, J = 6.5Hz, 2H, ArH), 7.75-7.76 (m, 2H, ArH), 7.57-7.64 (m, 3H, ArH), 7.17-7.18 (d, J = 8.2Hz, 2H, ArH), 7.05-7.07 (d, J = 8.2Hz). 2H,ArH),6.78-6.89(m,6H,ArH),4.44-4.50(dd,J=13.7Hz,-CH2-),4.40-4.43(t,J=6Hz,2H,-C H2O-),4.11-4.18(m,6H,-OCH2-),3.59(s,2H,ArCH2-),3.31-3.34,3.42-3.45(dd,J=13.7Hz,2 H,-NCH2-),2.82-2.85,3.07-3.09(dd,J=13.7Hz,2H,-CH2N-),2.22-2.26(m,2H,-CH2-),2.04( s,3H,NCH3),1.84-1.92(m,2H,-CH2-),1.71-1.77(m,2H,-CH2-),1.49-1.56(m,2H,-CH2-).13C NMR (126MHz, CDCl3) δ171.99,163.76-163.67,161.78-161.69(dd,J=247.5,12Hz),159.98-59.89,158.02-157.93(dd,J=247.5,12Hz),159.03, 158.36,158.06,151.02,144.66,138.19,135.63,130.33(s,2C),130.21 (s,2C),129.88,129.69(s,2C),129.64-129.60,129.57-129.52(dd,J=8 .75,6.25Hz),128.56,126.29,114.66(s,2C),114.50(s,2C),111.62-1 11.60,111.46-111.43(dd,J=20,3.75Hz),110.52,104.44-104.03(t,J= 26.5Hz),72.10-72.05(d,1C),71.32,64.50(s,2C),64.37,62.71,61.14 ,56.54-56.50(d,1C),43.65,40.56,29.74,29.35,28.14,28.05,22.20.HRMS(ESI-MS m / z)calculated for C43H46F2N6O10S[M+H]+877.2964,found 863.3034ppm error:0.79.

[0121] Example 12

[0122] Preparation of 4-(2-(2-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propionyl(methyl)amino)methyl)phenoxy)propoxy)phenyl)acetamido)ethoxy)-3-(benzenesulfonylnylonyl)-1,2,5-oxadiazole 2-oxide (2f)

[0123]

[0124] The synthesis method was followed as described in Example 10. ¹H NMR (CDCl₃, 500MHz): 8.11 (s, ¹H, ArH), 8.05-8.07 (d, J = 6.5Hz, 2H, ArH), 7.75-7.76 (m, 2H, ArH), 7.57-7.64 (m, 3H, ArH), 7.17-7.18 (d, J = 8.2Hz, 2H, ArH), 7.05-7.07 (d, J = 8.2Hz, 2H, ArH), 6.78-6.89 (m, 6H, ArH), 4.44-4.50 (dd, J = 13.7Hz, -CH₂-), 4.40-4.43 (t, J = 6Hz, 2H, -CH₂O-), 4.11-4.18 (m, 4H, -OCH₂-), 4.12 -4.14(t,J=13.7Hz,2H,-OCH2-),3.59(s,2H,ArCH2-),3.31-3.34,3.42-3.45 (dd,J=13.7Hz,2H,-NCH2-),2.82-2.85,3.07-3.09(dd,J=13.7Hz,2H,-CH2N- ),2.22-2.26(m,2H,-CH2-),2.04(s,3H,NCH3),1.84-1.92(m,2H,-CH2-),1.8 5-1.91(m,2H,-CH2-),1.66-1.71(m,2H,-CH2-),1.39-1.52(m,4H,-CH2-).13C NMR (126MHz, CDCl3) δ171.99,163.77-163.67,161.78-161.69(dd,J=247.5,12Hz),159.98-159.89,158.02-157.93(dd,J=247.5,12Hz) ,159.07,158.36,158.04,151.01,144.66,138.21,135.62,130.32(s,2C),130.21(s,2C),129.87,129.68(s,2C),129.64-129.59,129. 57-129.52(dd,J=8.75,6.25Hz),128.57,126.36,114.65(s,2C),114.50(s,2C),111.62-111.60,111.46-111.43(dd,J=20,3.75Hz),11 0.52,104.44-104.24,104.23-104.03(dd,J=26.5Hz),72.09-72.05(d,1C),71.45,64.60,64.51,64.49,62.71,61.14,61.11,56.54-56.51(d,1C),43.64,40.59,29.74,29.35,28.49,28.36,25.46,25.28.HRMS(ESI-MS m / z)calculatedfor C44H48F2N6O10S[M+H]+891.3193,found 891.3183ppm error:1.27.

[0125] Example 13

[0126] Preparation of 4-(2-(2-(4-(3-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propionyl(methylamino)methyl)phenoxy)propoxy)phenyl)acetamido)ethoxy)-3-(phenylsulfonate only)-1,2,5-oxadiazol-2-oxide (2g)

[0127]

[0128] The synthesis method was followed as described in Example 10. ¹H NMR (CDCl₃, 500MHz) 8.11 (s, ¹H, ArH), 8.05-8.07 (d, J = 6.5Hz, 2H, ArH), 7.75-7.76 (m, 2H, ArH), 7.57-7.64 (m, 3H, ArH), 7.17-7.18 (d, J = 8.2Hz, 2H, ArH), 7.05-7.07 (d, J = 8.2Hz, 2H, ArH), 6.78-6.89 (m, 6H, ArH), 4.44-4.50 (dd, J = 13.7Hz, -CH₂-), 4.11-4.18 ( m,4H,-OCH2-),4.12-4.14(t,J=13.7Hz,2H,-OCH2-),3.67-3.68(m,3H,CONHCH2-),3.59(s,2H,ArCH2-),3.31-3.34,3.42-3.45 (dd,J=13.7Hz,2H,-NCH2-),2.82-2.85,3.07-3.09(dd,J=13.7Hz,2H,-CH2N-),2.22-2.26(m,2H,-CH2-),2.04(s,3H,NCH3).13C NMR (126MHz, CDCl3) δ171.92, 163.82 (d, J = 60.0Hz), 159.97 (d, J = 50.0Hz), 158.75, 158. 36,157.91(d,J=50.0Hz),151.03,144.69,137.91,135.72,130.55,130.23,129.74,129 .61,128.61,126.53,115.18,114.54,111.63(d,J=70.0Hz),110.45,104.25(t,J=105.0 Hz),72.11,70.08,64.48,62.71,61.05,56.52,43.63,42.87,38.40,29.30.HRMS(ESI-MS m / z)calculated for C40H41F2N7O9S[M+H]+834.2727,found834.2708ppm error:2.21.

[0129] Example 14

[0130] Preparation of 4-(4-(4-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propionyl(methyl)amino)methyl)phenoxy)propoxy)phenyl)acetoxy)but-2-ynyl-1-yl)oxy)-3-(PH-enylsulfonyl)-1,2,5-oxadiazole 2-oxide (2h)

[0131]

[0132] The synthesis method was followed as described in Example 10. ¹H NMR (500 MHz, Chloroform-d) δ 8.08 (s, ¹H, ArH), 8.06 (dd, J = 8.5, 1.3 Hz, 2H, ArH), 7.77-7.71 (m, 2H, ArH), 7.64-7.53 (m, 3H, ArH), 7.19 (d, J = 8.6 Hz, 2H, ArH), 7.03 (d, J = 7.8 Hz, 2H, ArH), 6.88 (d, J = 8.6 Hz, 2H, ArH), 6.83-6.74 (m, 4H, ArH), 5.08 (t, J = 1.8 Hz, 2H, OCH₂). 4.74(t,J=1.8Hz,2H,CH2),4.48-4.41(m,2H,CH2),4.14(q,J=6.0Hz,4H,CH2),3.61(s,2H,ArCH2),3.40(d,J=12.8Hz,1H,NCH2),3.3 0(d,J=12.6Hz,1H,NCH2),3.05(d,J=13.3Hz,1H,CH2N),2.79(d,J=13.0Hz,1H,CH2N),2.28-2.20(m,2H,CH2),2.01(s,3H,NCH3).13C NMR (126MHz, CDCl3) δ171.01,158.38,158.36,158.22,158.01,157.97,157.9 2,151.03,144.67,137.94,135.73,130.37,130.22,129.74,128.69,125.47,1 14.76,114.52,111.60,111.47,104.45,104.24,104.03,83.99,78.65,72.09 ,64.49,62.72,61.11,58.64,56.50,52.21,43.64,40.07,29.34.HRMS(ESI-MS m / z)calculated for C42H40F2N6O10S[M+H]+859.2567, found 859.2547ppm error:1.48.

[0133] Example 15

[0134] Preparation of 4-(2-(2-(2-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amino)methyl)phenoxy)propoxy)phenyl)acetoxy)ethoxy)ethoxy)-3-(phenylalanine-nylonsulfonyl)-1,2,5-oxadiazole 2-oxide (2i)

[0135]

[0136] The synthesis method was followed as described in Example 10. ¹H NMR (CDCl₃, 500MHz): 8.11 (s, ¹H, ArH), 8.05-8.07 (d, J = 6.5Hz, 2H, ArH), 7.75-7.76 (m, 2H, ArH), 7.57-7.64 (m, 3H, ArH), 7.17-7.18 (d, J = 8.2Hz, 2H, ArH), 7.05-7.07 (d, J = 8.2Hz, 2H, ArH), 6.78-6.89 (m, 6H, ArH), 4.44-4.50 (dd, J = 13.7Hz, -CH₂-), 4.40-4.43 (t, J = 6Hz, 2H, -CH₂O-), 4.54-4.57 (t, J = 8Hz, 2H, -CH₂O-). O-),4.30-4.33(t,J=8Hz,2H,-CH2O-),4.11-4.18(m,4H,-OCH2-),3.87-3.9 0(t,J=8Hz,2H,-OCH2-),3.79-3.82(t,J=8Hz,2H,-CH2O-),3.59(s,2H,ArCH2 -),3.31-3.34,3.42-3.45(dd,J=13.7Hz,2H,-NCH2-),2.82-2.85,3.07-3.09 (dd,J=13.7Hz,2H,-CH2N-),2.22-2.26(m,2H,-CH2-),2.04(s,3H,NCH3).13C NMR (75MHz, CDCl3) δ171.89,164.39-164.23,161.09-160.92(dd,J=247.5,12Hz),160.60-160.44,157.33-157.18(dd,J=247.5,12Hz12Hz ),158.91,158.29,158.01,150.96,144.63,138.03,135.65,133.83,130.35(s,2C),130.19(s,2C),129.79,129.67,129.64(s,2C),129.5 6-129.51,129.43-129.24(dd,J=8.75,6.25Hz),128.57(s,2C),127.92,126.00,114.56(s,2C),114.42(s,2C),111.65-111.61,111.38-1 11.34(dd,J=20,3.75Hz),104.55-103.86(t,J=26.5Hz),72.01-71.94(d,1C),70.54,69.37,69.12,68.56,68.32,65.86,64.39,63.81,62.64,61.03,60.98,56.49-56.43(d,1C),43.59,40.28,29.27,15.31.HRMS(ESI-MS m / z)calculated forC42H44F2N6NaO11S[M+Na]+901.2649,found 901.2613ppm error:-4.99.

[0137] Example 16

[0138] Preparation of 2-(dihydroxyoxy)ethyl-2-(4-((2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amino)methyl)phenoxy)propoxy)phenyl)acetate (3a)

[0139]

[0140] (a) 10 mmol of 2-bromoethanol 18a was dissolved in acetonitrile, and 25 mmol of silver nitrate was added. The mixture was reacted overnight under reflux and detected by TLC (PE / EA, 10 / 1, v / v). After the reaction was completed and cooled to room temperature, excess saturated brine was added, resulting in a precipitate. The precipitate was filtered through diatomaceous earth, and the collected filtrate was extracted three times with EA (75 mL). The combined organic layers were washed with saturated brine, dried over anhydrous Na2SO4, and filtered. The organic solvent was removed from the filtrate under reduced pressure, and the residue was compound 19a, a colorless liquid with a yield of 92%. ¹H NMR (500 MHz, Chloroform-d) δ 4.59 (t, J = 4.6 Hz, 2H, CH2), 3.96–3.90 (m, 2H, CH2).

[0141] (b) At 5°C, 9.5 mL (0.22 mol, 13.9 g) of fuming nitric acid was slowly added dropwise to 20 mL of acetic anhydride. After the addition was complete, the mixture was stirred at -5 to 0°C for 30 min to form a nitric acid-acetic anhydride mixture. 6.0 mL (0.10 mol, 6.1 g) of ethanolamine was added to a three-necked flask equipped with a mechanical stirrer, thermometer, constant pressure funnel, anhydrous CaCl₂, and a desiccant tube. The mixture was then cooled to [temperature missing].

[0142] 6.7 mL (0.12 mol) of glacial acetic acid was added dropwise at approximately 10 °C. After the addition was complete, the mixture was stirred for 15 min until the solid dissolved. The solution was then transferred to a constant pressure funnel and added dropwise to a nitric acid-acetic anhydride mixture at 0–5 °C. After the addition was complete, the mixture was stirred for another 15 min at room temperature for 4–5 h. The solution was filtered, washed with diethyl ether, dried, and recrystallized from isopropanol. The solution was decolorized with activated carbon to obtain white flaky crystals, with a yield of 87.1%. ¹H NMR (500 MHz, DMSO-d⁶) δ 7.98 (s, 2H, NH₂), 4.72 (t, J = 5.1 Hz, 2H, CH₂), 3.23 (t, J = 5.2 Hz, 2H, CH₂).

[0143] (c) Compound Az-6 (100 mg, 0.176 mmol) was dissolved in anhydrous THF, and TBTU (68 mg, 0.212 mmol) and DMAP (43 mg, 0.352 mmol) were added. After the starting material was dissolved, the air in the system was displaced. After stirring for 10 min, 19a (0.212 mmol) was added. The reaction was carried out under nitrogen protection for 6 h. The reaction was detected by TLC (CH2Cl2 / MeOH, 20 / 1, v / v). After the reaction was completed, the solvent was evaporated and EA (30 mL) was added. The mixture was sonicated to dissolve the solvent completely. The mixture was extracted five times with saturated saline solution. The organic layer was collected, dried over anhydrous Na2SO4, and filtered. The filtrate was subjected to reduced pressure to remove the organic solvent. The mixture was mixed with silica gel and subjected to column chromatography (DCM / MeOH, 50 / 1, v / v) to obtain the target compound 3a, a white viscous solid with a yield of 34%.1H NMR(CDCl3,500MHz) 2.05(s,3H, -NCH3), 2.27 - 2.31(m,2H, -CH2-), 2.83 - 2.86, 3.08 - 3.11(dd,J = 13.7Hz,2H, -CH2N-), 3.33 - 3.35, 3.44 - 3.46(dd,J = 13.7Hz,2H, -NCH2-), 3.62(s,2H, ArCH2-), 4.17 - 4.20(m,4H, -OCH2-), 4.39 - 4.41(t,J = 5Hz,2H, -COOCH2-), 4.45 - 4.52(dd,J = 14.5Hz,2H, -CH2-), 4.68 - 4.69(t,J = 5Hz,2H, -CH2ONO2), 6.79 - 6.87(m,4H, ArH), 6.90 - 6.92(d,J = 8.5Hz,2H, ArH), 7.06 - 7.08(d,J = 8.5Hz,2H, ArH), 7.20 - 7.22(d,J = 8.5Hz,2H, ArH), 7.58 - 7.63(m,1H, ArH), 7.77(s,1H, ArH), 8.12(s,1H, ArH). 13C NMR(75MHz,CDCl3) δ171.39, 164.42 - 164.26, 161.12 - 160.96(dd,J = 247.5,12Hz), 160.64 - 160.48, 157.30 - 157.21(dd,J = 247.5,12Hz), 158.35(s,2C), 158.18(s,2C), 150.88, 144.58, 130.24(s,2C), 130.13(s,2C), 129.85, 129.63 - 129.55, 129.51 - 129.43(dd,J = 9,6Hz), 126.46 - 126.30(d,J = 12Hz), 125.53, 114.76(s,2C), 114.52(s,2C), 111.56 - 111.29(d,J = 20.25Hz), 104.48 - 103.78(t,J = 26.25Hz), 74.38, 72.17 - 72.10(d,1C),, 70.28, 64.52, 62.67, 61.19, 60.48, 56.53 - 56.48(d,1C), 43.59, 40.04, 32.29, 29.31, 0.95. HRMS(ESI - MS m / z) calculated for C32H35F2N5O8[M + H]+ 656.2548, Found 656.2542 PPM error: -0.91.

[0144] Example 17

[0145] Preparation of 3-(nitoxy)propyl-2-(4-((2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amino)methyl)phenoxy)propoxy)phenyl)acetate (3b)

[0146]

[0147] The synthesis method was followed as described in Example 16. ¹H NMR (CDCl₃, 500MHz): 2.00 (s, 3H, -NCH₃), 2.02-2.04 (m, 2H, -CH₂-), 2.27-2.31 (m, 2H, -CH₂-), 2.83-2.86, 3.08-3.11 (dd, J = 13.7Hz, 2H, -CH₂N-), 3.33-3.35, 3.44-3.46 (dd, J = 13.7Hz, 2H, -NCH₂-), 3.62 (s, 2H, ArCH₂-), 4.17-4.20 (m, 4H, -OCH₂-), 4.13-4. 15(t,J=5Hz,2H,-COOCH2-),4.43-4.46(m,4H,-CH2-,-CH2ONO2),6.79-6.87(m,4H,ArH),6.90-6.92(d,J=8.5Hz,2H,ArH),7.0 6-7.08(d,J=8.5Hz,2H,ArH),7.20-7.22(d,J=8.5Hz,2H,ArH),7.58-7.63(m,1H,ArH),7.77(s,1H,ArH),8.12(s,1H,ArH).13C NMR(75MHz, CDCl3)δ171.52,164.40-164.24,161.10-160.94(dd,J=247.5,12Hz),160.64-160.49,157.37-157.21(dd,J=245.75,12Hz),15 8.34,158.12,156.14,150.84,144.57,130.19(s,2C),130.13(s,2C),129.86,129.64-129.56,129.52-129.44(dd,J=9,6Hz),126.48-126. 30(d,J=12Hz),125.95,114.72(s,2C),114.52(s,2C),111.55-111.51,111.28-111.23(dd,J=20.25,3Hz),104.45-103.76(t,J=25.5Hz),7 2.21-72.14(d,1C),70.50,69.78,64.53,62.65,61.23,60.62,56.52-56.44(d,1C),53.39,43.59,40.33,29.31,26.40,0.95.HRMS (ESI-MS m / z)calculated forC33H37F2N5O8[M+H]+670.2670,Found 670.2683PPM error:1.94.

[0148] Example 18

[0149] Preparation of 4-(nitoxy)butyl-2-(4-((2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amino)methyl)phenoxy)propoxy)phenyl)acetate (3c)

[0150]

[0151] The synthesis method is described in Example 16. 1 H NMR(CDCl3,500MHz)1.72-1.73(m,4H,-CH2-),2.00(s,3H,-NCH3),2.27-2.31(m,2H,-CH2-),2.83-2.86,3.08-3.11(d d,J=13.7Hz,2H,-CH2N-),3.33-3.35,3.44-3.46(dd,J=13.7Hz,2H,-NCH2-),3.62(s,2H,ArCH2-),4.11-4.15(m,6H,-O CH2-,-COOCH2-),4.41-4.47(m,4H,-CH2-,-CH2ONO2),6.79-6.87(m,4H,ArH),6.90-6.92(d,J=8.5Hz,2H,ArH),7.06-7 .08(d,J=8.5Hz,2H,ArH),7.20-7.22(d,J=8.5Hz,2H,ArH),7.58-7.63(m,1H,ArH),7.77(s,1H,ArH),8.12(s,1H,ArH). 13C NMR (125MHz, CDCl3) δ171.78,163.75-163.65,161.76-161.67(dd,J=247.5,12Hz),159.97-159.87,158.00-157.91(dd,J=245.5,12Hz),158.35 ,158.10,150.94,144.62,130.24(s,2C),130.17(s,2C),129.86,129.6 1-129.57,129.54-129.49(dd,J=9,6Hz),126.50-126.47,126.40-126.3 7(dd,J=12.5,3.75Hz),126.11,114.68(s,2C),114.50(s,2C),111.57- 111.55,111.41-111.38(dd,J=20.25,3Hz),104.39-103.98(t,J=25.5Hz ),73.15,(s,1C),72.56,72.10-72.05(d,1C),64.50,63.78,62.68,61.8 1,61.14-61.10(d,1C),43.61,40.49,28.66,24.93,23.62.HRMS(ESI-MS m / z)calculated for C 34 H 39 F2N5O8[M+H] + 684.2828, Found 684.2823PPM error:-0.73.

[0152] Example 19

[0153] Preparation method of 5-(nitoxy)pentyl-2-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazo-1-1-yl)propyl)(methyl)amino)methyl)phenoxy)propoxy)phenyl)acetate (3d)

[0154]

[0155] The synthesis method is described in Example 16. 1H NMR:(CDCl3,500MHz)1.42-1.48(m,2H,-CH2-),1.66-1.70(m,2H,-CH2-),1.73-1.77(m,2H,-CH2-),2.05(s,3H,-NCH3),2.27-2.31(m,2H,-CH2-),2.83-2.86,3.08-3.11(dd,J=13.7Hz,2H,-CH2N-),3.33-3.35,3.44-3.46(dd,J=13.7Hz,2H,-NCH2-),3.62(s,2H,ArCH2-),4.11-4.14(t,J=6.5Hz,2H,-COOCH2-),4.17-4.19(m,4H,-OCH2-),4.42-4.45(t,J=6.5Hz,2H,-CH2ONO2),4.48-4.51(dd,J=13.7Hz,2H,-CH2-),6.79-6.87(m,4H,ArH),6.90-6.92(d,J=8.5Hz,2H,ArH),7.06-7.08(d,J=8.5Hz,2H,ArH),7.20-7.22(d,J=8.5Hz,2H,ArH),7.58-7.63(m,1H,ArH),7.77(s,1H,ArH),8.12(s,1H,ArH). 13 CNMR(75MHz,CDCl3)δ171.76,164.42-164.26,161.32-161.13(dd,J=232.5,12Hz),160.72-160.50,158.36-158.06(dd,J=251.5,12Hz),157.37,157.27,150.92,144.58,130.22(s,2C),130.13(s,2C),129.89,129.64-129.44(dd,J=9,6Hz),126.53-126.30(dd,J=12.5,3.75Hz),114.67(s,2C),114.53(s,2C),111.57-111.30(d,J=20.25,3Hz),104.48-103.78(t,J=25.5Hz),73.19,72.94,72.18,64.17,64.55,62.69,62.22,61.23,43.60,40.53,29.34,28.07,26.39,22.20.HRMS(ESI-MS m / z)calculatedfor C 35 H 41 F2N5O8[M+H] +698.2984, Found 698.2981PPM error:-0.43.

[0156] Example 20

[0157] Synthetic method of 4-((nitoxy)methyl)benzyl-2-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amino)methyl)phenoxy)propoxy)phenyl)acetate (3e)

[0158]

[0159] Refer to the synthesis method of Example 16. 1H NMR (CDCl3, 500 MHz): 2.05 (s, 3H, -NCH3), 2.27 - 2.31 (m, 2H, -CH2-), 2.83 - 2.86, 3.08 - 3.11 (dd, J = 13.7 Hz, 2H, -CH2N-), 3.33 - 3.35, 3.44 - 3.46 (dd, J = 13.7 Hz, 2H, -NCH2-), 3.62 (s, 2H, ArCH2-), 4.17 - 4.19 (m, 4H, -OCH2-), 4.48 - 4.51 (dd, J = 13.7 Hz, 2H, -CH2-), 5.16 (s, 2H, -COOCH2-), 5.45 (s, 2H, -CH2ONO2), 6.79 - 6.87 (m, 4H, ArH), 6.90 - 6.92 (d, J = 8.5 Hz, 2H, ArH), 7.06 - 7.08 (d, J = 8.5 Hz, 2H, ArH), 7.20 - 7.22 (d, J = 8.5 Hz, 2H, ArH), 7.36 - 7.37 (d, J = 8 Hz, 2H, ArH), 7.40 - 7.41 (d, J = 8 Hz, 2H, ArH), 7.58 - 7.63 (m, 1H, ArH), 7.77 (s, 1H, ArH), 8.12 (s, 1H, ArH). 13C NMR (75 MHz, CDCl3), δ, 171.32, 164.42 - 164.26, 161.11 - 160.97 (dd, J = 247.5, 12 Hz), 160.65 - 160.50, 157.38 - 157.12 (dd, J = 247.5, 12 Hz), 158.37, 158.05, 150.90, 144.59, 135.17, 130.99, 130.26 (s, 6C), 130.16 (s, 2C), 129.81, 129.66 - 129.46 (dd, J = 9, 6 Hz), 128.94, 126.49 - 126.24 (dd, J = 12.5, 3.75 Hz), 125.87, 114.70 (s, 2C), 114.55 (s, 2C), 111.56 - 111.30 (dd, J = 20.25, 3 Hz), 104.49 - 103.79 (t, J = 25.5 Hz), 72.19, 64.55, 63.91, 62.68, 61.22, 56.48, 51.84, 43.61, 40.34, 29.33, 0.99. HRMS (ESI-MS m / z) calculated for C38H39F2N5O8 [M + H]+ 732.2834, Found 732.2839 PPM error: 0.68

[0160] Example 21

[0161] Preparation method of 2-((nitoxy)methyl)benzyl-2-(4-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amino)methyl)phenoxy)propoxy)phenyl)acetate (3f)

[0162]

[0163] The synthesis method according to Example 16 was as follows: ¹H NMR (CDCl₃, 500MHz) 2.05 (s, 3H, -NCH₃), 2.27-2.31 (m, 2H, -CH₂-), 2.83-2.86, 3.08-3.11 (dd, J = 13.7Hz, 2H, -CH₂N-), 3.33-3.35, 3.44-3.46 (dd, J = 13.7Hz, 2H, -NCH₂-), 3.62 (s, 2H, ArCH₂-), 4.17-4.19 (m, 4H, -OCH₂-), 4.48-4.51 (dd, J = 13.7Hz, 2H, -CH₂-), 5 .20(s,2H,-COOCH2-),5.44(s,2H,-CH2ONO2),6.79-6.87(m,4H,ArH),6.90-6.92(d,J=8.5Hz,2H,ArH),7.06-7.08(d,J=8.5Hz,2 H,ArH),7.20-7.22(d,J=8.5Hz,2H,ArH),7.36-7.38(m,4H,ArH),7.58-7.63(m,1H,ArH),7.77(s,1H,ArH),8.12(s,1H,ArH).13C NMR(75MHz, CDCl3)δ171.52,164.42-164.26,161.11-160.96(dd,J=247.5,12Hz),160.65-160.49,158.11-158.04(dd,J=245.5,12Hz),158 .34,157.16,150.85,144.57,137.41,132.22,130.29(s,6C),130.25(s,2C),130.14,129.90,129.64-129.56,129.52-129.44(dd,J=9,6Hz ),129.17,129.13,128.36-128.25,127.16-127.06(dd,J=12.5,3.75Hz),126.22,120.02,114.70(s,2C),114.54(s,2C),111.58-111.29(d d, J=20.25, 3Hz), 104.49-103.79 (t, J=25.5Hz), 74.61, 61.19, 74.31, 72.19, 56.52, 65.83, 64.55, 62.68, 43.62, 40.25, 29.33. HRMS (ESI-MS m / z)calculated for C38H39F2N5O8[M+H]+732.2835,Found 732.2839PPM error:0.55.

[0164] Example 22

[0165] Preparation of (3S,3aR,6R,6aS)-6-(nitoxy)hexahydrofuran[3,2-b]furan-3-yl-2-(4-((2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl(methyl)amino)methyl-ylphenoxy)propoxy)hydroenyl)acetate (3g)

[0166]

[0167] The synthesis method was followed as described in Example 16. ¹H NMR (CDCl₃, 500MHz): 8.12 (s, ¹H, ArH), 7.77 (s, ¹H, ArH), 7.58-7.63 (m, ¹H, ArH), 7.20-7.22 (d, J = 8.5Hz, 2H, ArH), 7.06-7.08 (d, J = 8.5Hz, 2H, ArH), 6.90-6.92 (d, J = 8.5Hz, 2H, ArH), 6.79-6.87 (m, 4H, ArH), 4.91-4.93 (m, ¹H, ISMN), 5.31-5.32 (m, ¹H, ISMN), 4.39-4.46 (m, 3H, -CH₂-, ISMN). MN),4.12-4.14(m,4H,-OCH2-),3.95-4.00(m,3H,ISMN),3.91-3.92(m,1H,ISMN),3.84-3.88(m,1H,ISMN),3.54(s,2H,ArCH2-),3.28-3.30, 3.39-3.41(dd,J=13.7Hz,2H,-NCH2-),2.78-2.81,3.03-3.05(dd,J=13.7Hz,2H,-CH2N-),2.22-2.26(m,2H,-CH2-),2.05(s,3H,-NCH3).13C NMR (75MHz, CDCl3) δ170.86, 164.40-164.24, 161.10-160.94 (dd, J=247.5, 12Hz), 160.61-160.45, 157.34-157.18 (dd, J=247.5, 12Hz), 158.29, 158. 14,150.84,144.59,130.21(s,2C),130.17(s,2C),129.83,129.61-129.5 4,129.49-129.42(dd,J=9,6Hz),126.49-126.45,126.32-126.28(dd,J=12 .5,3.75Hz),125.45,114.69(s,2C),114.44(s,2C),111.63-111.35(dd,J =20.25,3Hz),104.54-103.84(t,J=25.5Hz),89.02,86.54,81.60-81.50,8 1.27-81.14(dd,1C,J=24.75,7.5Hz),77.62,75.88,75.41,73.51,72.02-7 1.95(d,1C),,69.22,69.07,64.41(s,2C),62.63,61.02,60.38,56.51-56.44(d,1C),43.60,40.20,29.67,29.27,21.02,1.01.HRMS(ESI-MS m / z)calculated for C38H39F2N5O8[M+H]+740.2726,Found 740.2737PPM error:2.32.

[0168] Example 23

[0169] 2-(2-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)(methyl)amino)methyl)phenoxy)propoxy)phenyl)acetyl)-L2-azayl)ethyl nitrate (3h)

[0170]

[0171] The synthesis method was followed according to Example 16. ¹H NMR (300MHz, CDCl₃) δ 8.08 (s, ¹H, ArH), 7.72 (s, ¹H, ArH), 7.56 (td, J = 9.2, 6.6Hz, ¹H, ArH), 7.14 (d, J = 8.6Hz, 2H, ArH), 7.04 (d, J = 8.0Hz, 2H, ArH), 6.90 (d, J = 8.6Hz, 2H, ArH), 6.85-6.73 (m, 4H, ArH), 4.50 (t, J = 5.1Hz, 2H, CH₂ONO₂), 4 .43(s,2H,CH2),4.20-4.10(m,4H,OCH2),3.54(t,J=6.0Hz,2H,COOCH2),3.52(s,2H,ArCH2),3.35(dd,J=36.1,10. 7Hz,2H,CH2),2.92(dd,J=78.0,15.0Hz,2H,CH2),2.27(p,J=6.1Hz,2H,CH2),2.01(s,3H,CH3),1.65(s,1H,NH).13C NMR(126MHz, CDCl3) δ171.94,163.86-163.76,161.87-161.76(dd,J=250.7,12.6Hz),159.94-159.85,157.98-1 57.89(dd,J=247.0,11.3Hz),158.38,151.08,144.73,130.55,130.10,129.67,129.62-129.55(t,J=6.3Hz),12 6.40,115.21,114.69,114.62,111.73-111.59(d,J=17.6Hz),104.58-104.50(t,J=26.5Hz),72.15-72.11(d,J= 5.0Hz),71.55,67.73,64.51,64.43,62.65,60.85,56.41,43.61,42.73,37.17,33.88,29.31,1.06.HRMS (ESI-MS m / z)calculated forC32H37F2N6O7[M+H]+655.2686,Found 655.2670PPM error:2.37.

[0172] Example 24

[0173] In vitro nitric oxide release experiment

[0174] Experimental Methods: Weigh compound 3a or 3h and dissolve it in 40 μL of DMSO solution to prepare a 50 mM DMSO stock solution. Absorb 40 μL of the DMSO stock solution and add 3960 μL of rat plasma. The final concentration of the compound is 500 μM. Incubate the solution at 37℃. Absorb 200 μL of the solution at 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h, respectively. Add 200 μL of methanol, centrifuge, absorb 300 μL of the supernatant, add 100 μL of Leriess reagent, centrifuge, absorb the supernatant, and filter. Add 200 μL of the filtrate to a 96-well plate and measure the absorbance at 540 nm. Weigh NaNO2 and prepare rat plasma solutions with different concentration gradients. Measure the absorbance of sodium nitrite solutions of different concentrations according to the above procedure and plot a concentration-absorbance standard curve. Substitute the absorbance data of the compound into the standard curve to calculate the NO release rate at different time points. For compounds 1a and 2i, weigh out the compounds and dissolve them in 1×PBS solution to a final concentration of 100 μM. Add different concentrations of glutathione, mix well, incubate at 37°C, absorb 100 μL of the solution at a given time, add 100 μL of Griess reagent to a 96-well plate, and measure the absorbance at 540 nm.

[0175] Experimental results: The results are attached. Figure 2 As shown, under GSH conditions, Figure 2 a and Figure 2 Compounds 1a and 2i in b can release NO well, and the release rate increases with increasing GSH concentration. After 24 hours of incubation, the release rates reached over 60% and 14%, respectively. Figure 2 In c, compounds 3a and 3h, when incubated in rat plasma at 37°C for 24 hours, both showed significant NO release levels.

[0176] Example 25

[0177] In vitro antifungal activity analysis

[0178] Experimental method: MIC 80 The assays were performed using the micro-liquid-based dilution method recommended by the Clinical and Laboratory Standards Institute (CLSI) documents M27-A3 and M38-A2. Fungal growth was evaluated using the optical density (OD) value of the culture to assess the in vitro inhibitory activity of the compound against fungi. 1×10⁻⁶ ppm of the compound was prepared using RPMI 1640 medium. 3A bacterial suspension of cells / mL was prepared, and then vortexed to mix the mixture before adding 100 μL to each well of a 96-well cell culture plate. The compound was serially diluted 0.5 times, starting at a concentration of 64 μg / mL, to achieve a final concentration of 64–0.125 μg / mL. A bacterial suspension without any added drug was used as a negative control, and RPMI 1640 medium was used as a blank control. The 96-well cell culture plates were incubated statically at 30°C (72 h for Cryptococcus, 48 ​​h for other strains). After incubation, the optical density (OD) of the fungus at 630 nm was measured using a microplate reader. 630 The assay was performed in triplicate. The lowest concentration at which the inhibition rate was ≥80% was taken as the minimum inhibitory concentration (MIC) of the compound. 80 ).

[0179] Experimental results: as attached Figure 3 As shown in a and Table 1, compounds 2a-2i exhibit moderate antibacterial activity (MIC) against Candida albicans, Candida glabrata, and Cryptococcus neoformans. 80 =1~16μg / mL). Compound 3a-3h exhibits good antibacterial activity (MIC). 80 =0.125~4μg / mL).

[0180] Table 1. In vitro antifungal activity (MIC) of NO donor compounds and FLC 80 (μg / mL)

[0181]

[0182]

[0183] a Abbreviations:C.alb.,Candida albicans;C.gla.,Candida glabrata;C.neo.,Cryptococcus neoformans;FLC,fluconazole.

[0184] Example 26

[0185] Time-Growth Curve Experiment

[0186] Experimental method: The pre-counted fungal cell stock solution was sequentially diluted 5 / 6 times in 96-well cell culture plates, and the OD of each well was measured using a microplate reader. 630 According to different OD 630 A standard curve was obtained by plotting the values ​​(x) and their corresponding bacterial concentrations (y). A bacterial suspension was prepared using RPMI 1640 medium to a concentration of 1 × 10⁻⁶. 5cells / mL. 5 mL of the prepared bacterial suspension was pipetted into a 15 mL centrifuge tube, along with different concentrations of the compound. FLC was used as a positive control, and DMSO as a blank control. The 15 mL centrifuge tubes were then placed in a 30°C air bath incubator with shaking at 200 rpm. At the designed culture time points (0 h, 3 h, 6 h, 9 h, 12 h, 24 h, 48 h, 72 h), 100 μL of fungal cell culture medium was aspirated from the centrifuge tubes into 96-well cell culture plates (triple-well). The OD630 value of each well was measured using a microplate reader. The bacterial concentration of each group was calculated using the equation fitted to the standard curve, and graphs were plotted using Graphpad Prism 8.

[0187] Experimental results: The results are attached. Figure 3 As shown in b. Compounds 3a, 3b, and 3e inhibited the growth of Cryptococcus neoformans, and even at concentrations as low as 2 μg / mL, they completely inhibited cell growth, which was significantly better than fluconazole (FLC).

[0188] Example 27

[0189] Time-sterilization curve experiment

[0190] Experimental methods: Cryptococcus neoformans H99 cells exhibiting exponential growth were washed with PBS and resuspended in RPMI 1640 medium to a concentration of 1.0 × 10⁻⁶. 5 / mL. Different concentrations of FLC and compounds 3a and 3e were added to the suspension of Cryptococcus neoformans H99. The addition of FLC and the selected compounds did not serve as a control group. Different concentrations of the compounds and FLC were added and incubated in a shaker (200 rpm) at 30°C. At set times (0, 6, 12, 24, 48, 72 h), the suspension was coated onto SDA plates and incubated at 30°C for 72 h. The number of monoclonal fungal colonies on the plates was counted, completing three independent experiments.

[0191] Experimental results: as attached Figure 3 As shown in c. At a concentration of 4 μg / mL, FLC showed no fungicidal activity, consistent with previous reports. Compounds 3a and 3e, however, exhibited significant fungicidal activity against Cryptococcus neoformans, with a concentration of 8 μg / mL completely killing the fungus.

[0192] Example 28

[0193] plate diffusion experiment

[0194] Experimental method: Approximately 1×10 5 Cells were placed on SDA medium containing different concentrations of FLC or compounds 3a and 3e. After incubation at 35°C for 72 hours, photographs were taken to visually reflect the antifungal activity of Cryptococcus neoformans strain H99.

[0195] Experimental results: as attached Figure 3 As shown in Figure d, compounds 3a and 3e completely inhibited the growth of Cryptococcus neoformans at a concentration of 0.125–1 μg / mL after 72 hours of colony culture. At the same concentration, FLC showed almost no inhibitory effect on the growth of Cryptococcus neoformans cells.

[0196] Example 29

[0197] In vitro biofilm formation inhibition assay

[0198] Experimental method: The bacterial suspension (in RPMI 1640 medium at 1.0 × 10⁻⁶) was prepared. 6 Cells / mL were added to 96-well plates (100 μL per well) and incubated at 37°C for 3 hours to allow adhesion. Adherent cells were washed three times with PBS to remove the supernatant culture medium and phytoplankton cells. Simultaneously, different concentrations of FLC and the compound were added to fresh RPMI 1640 medium, and the plates were incubated at 37°C for another 24 hours. The XTT reduction method was used to calculate a semi-quantitative determination of biofilm formation. The optical density (OD490) at 490 nm was measured using a microplate reader, and three independent experiments were performed.

[0199] Experimental results: as attached Figure 4 As shown. Compounds 3a, 3b, and 3e significantly inhibited the formation of Cryptococcus neoformans biofilm at a concentration of 0.25 μg / mL (P < 0.0001), with inhibition rates exceeding 50%, and their inhibitory activity was significantly superior to FLC (P < 0.01). Figure 4 a). To investigate the effect of NO release on biofilm formation, the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxy-3-oxide (PTIO) was added to the treatment group. The results showed that after adding PTIO, at a concentration of 0.25 μg / mL, the inhibitory effect of compound 3a on biofilm formation was significantly reduced (P < 0.01). Figure 4 b). We also investigated the disruption of pre-formed mature biofilms from compounds 3a and 3e. Figure 4 c and Figure 4 d) FLC showed a weak disruptive effect on established mature biofilms, while compounds 3a and 3e could disrupt approximately 25% of pre-established mature biofilms at a concentration of 0.125 μg / mL, and approximately 50% at a high concentration (64 μg / mL). Furthermore, the disruptive effect of compounds 3a and 3e was significantly weakened upon the addition of the NO scavenger PTIO. This indicates that compounds 3a and 3e have a significant inhibitory effect on biofilm formation of Cryptococcus neoformans strain H99, and this inhibitory effect is dependent on NO release.

[0200] Example 30

[0201] Nitric oxide release experiment

[0202] Experimental methods: Collect exponentially growing cells, wash three times with PBS, and then... 7 The cells were resuspended in RPMI 1640 medium at a concentration of 10 cells / mL. Then, different concentrations of the compound were gradually added to the suspension. After incubation at 37°C and 5% CO2 for 12 h, Cryptococcus neoformans H99 cells were collected by centrifugation and lysis buffer was added. Subsequently, the cells were resuspended in DAF-FM DA (1 mL, 10 μM) in the dark at 37°C for 40 min. The cells were washed three times with PBS and then analyzed using a laser scanning confocal microscope (Leica SP5, Germany) at an excitation wavelength of 495 nm and an emission wavelength of 515 nm.

[0203] Experimental results: Most fungal cells in the 3a and 3e treatment groups showed green fluorescence, indicating NO release. Simultaneously, most fungal cells also showed red fluorescence after treatment with 3a and 3e, indicating cell death. After merging, overlapping red and green fluorescence was observed in many cells. Figure 5 This indicates that both 3a and 3e exert their antifungal effects by penetrating fungal cells, releasing NO, disrupting the integrity of the fungal cell membrane, and thus causing fungal cell death.

[0204] Example 31

[0205] Fungal Ergosterol Content Experiment

[0206] Experimental Methods: Sterol composition analysis was performed using gas chromatography-mass spectrometry (GC-MS). Sterols were identified by matching molecular fragments from each peak in the GC-MS with corresponding sterol compounds in the reference database of the National Institute of Standards and Technology. Sample preparation steps were as follows: 50 μL of the bacterial strain was added to 50 mL of YEPD medium to prepare a suspension with a concentration of 1.0 × 10⁶ cells / mL. Simultaneously, the compound and FLC were added to the culture medium to treat Cryptococcus neoformans H99 cells. After incubation at 35 °C for 24 h, the cells were harvested by centrifugation and saponified with 10 mL of 20% NaOH (w / v) in 90% ethanol at 80 °C for 3 h. Then, 10 mL of n-hexane was added to the saponification mixture to extract the sterols. The solvent was then evaporated under reduced pressure, and the residue was dissolved using 400 μL of cyclohexane. Finally, GC-MS was used to analyze the sterol composition of each group.

[0207] Experimental results: Sterol content results ( Figure 6 a and Figure 6b) shows that the ergosterol content in the blank control group was 58.33%; after treatment with FLC (4 μg / mL), the ergosterol content decreased to 19.21%; nitrate azole derivatives 3a and 3e at 0.125 μg / mL reduced the ergosterol content to 23.68% and 10.66%, respectively. The lanosterol content in the blank control group was 6.24%, which increased to 53.85% after treatment with FLC (4 μg / mL), and increased to 44.52% and 50.94% after treatment with 0.125 μg / mL of 3a and 3e, respectively. Sterol content analysis indicates that compounds 3a and 3e exert their antifungal activity by disrupting the production of ergosterol on the fungal cell membrane and generating most of the lanosterol.

[0208] Example 32

[0209] Transmission electron microscopy experiments to observe capsule formation

[0210] Experimental method: Prepare bacterial suspension with YEPD culture medium to a concentration of 1×10⁻⁶. 6 Cells / mL: Take several 15mL centrifuge tubes, add 10mL of bacterial suspension to each, and add different concentrations of compounds for treatment. Use the untreated group as a blank control. Then place in a 35℃ constant temperature shaking incubator and culture at 220rpm for 8 hours. After culture, centrifuge, collect fungal cells, wash three times with PBS buffer, add 1mL of electron microscopy fixative, repeatedly pipette, vortex, and mix well, allowing to settle naturally, and fix overnight at 4℃. Observe and photograph the morphology of fungal cells using a transmission electron microscope.

[0211] Experimental results: The results are as follows Figure 6 As shown in Figure c, normal Cryptococcus neoformans H99 cells exhibit uniform density, clear outlines, and intact cell membranes, cell walls, and dense capsules. Treatment with 4 μg / mL FLC did not cause significant damage to Cryptococcus neoformans H99 cells. However, treatment with 1 μg / mL compound 3a completely ruptured the cell membrane of Cryptococcus neoformans H99, resulting in leakage of cell contents. These results indicate that compound 3a can disrupt the integrity of the Cryptococcus neoformans H99 cell membrane.

[0212] Example 33

[0213] In vivo anti-cryptococcal activity assay

[0214] Experimental methods: such as Figure 7 As shown in a, female ICR mice (weight: 18-22g) were selected, and fungal inoculation was performed via tail vein injection to establish the model (inoculation amount: 0.2mL / mouse, i.e., 2×10⁻⁶). 5(cells / mouse). Mice were inoculated with the fungus for 24 hours, then administered the compound via nasal drops (10 μL in each nostril) for five consecutive days, recording the mice's survival status. On the sixth day, mice were sacrificed, and brain or lung tissue was dissected (weighed and recorded). The tissue was placed in numbered 1.5 mL centrifuge tubes, with 1 mL of physiological saline and 2 steel balls added to each tube. The tissue was then homogenized using a tissue homogenizer and stored at 4°C. The brain or lung tissue fluid was diluted appropriately and plated onto SDA medium. The culture dishes were incubated at 30°C for 72 hours. Single colony counts were performed on the SDA culture dishes to calculate the bacterial load in the brain or lung tissue of each group. Statistical differences between groups were analyzed using ANOVA and post-hoc (Bonferroni and Student-Newman-Keuls') analysis with Graphpad Prism 8 software. Simultaneously, pathological section experiments were performed on mouse brain tissue. The dissected tissue was fixed with 4% paraformaldehyde solution, dehydrated, embedded in paraffin, prepared into slides, stained with PAS and H&E (hematoxylin-eosin), and observed and photographed under an optical microscope.

[0215] Experimental results: The results are as follows Figure 7 b and Figure 7 As shown in Figure c, after treatment with compounds 3a and 3h (at two doses: 1 mg / kg and 10 mg / kg, respectively), the bacterial load in mouse brain tissue was significantly lower than that in the control group, with a statistically significant difference (P < 0.001). At a dose of 1 mg / kg, the inhibitory effect of compound 3h on bacterial load in mouse brain tissue was superior to that of the FLC group, with a significant difference (P < 0.05). These results indicate that NO donor-type azole derivatives have a good inhibitory effect on bacterial load in mouse brain tissue, and compound 3h has significantly better in vivo activity than 3a. The bacterial load experiment results show that compounds 3a and 3h have excellent in vivo anti-Cryptococcus neoformans activity.

[0216] PAS (periodic acid-Schiff) staining results showed irregular cavities and abundant fungal cell colonization in the brain tissue of control mice, indicating a large-scale invasion of Cryptococcus neoformans into the brain tissue. In contrast, treatment with compounds 3a and 3h effectively reduced the amount of Cryptococcus neoformans invading the brain, further demonstrating the excellent in vivo antifungal activity of the NO donor derivative. Figure 7 d). H&E (hematoxylin-eosin) staining results showed that the brain tissue of the control group mice had typical pathological changes, including vascular congestion, neuronal necrosis, and irregular cavities in the brain. In addition, multiple instances of irregular neuronal arrangement and an increased number of glial cells were observed. Figure 7e). Treatment with compounds 3a and 3h significantly reduced pathological damage to mouse brain tissue. These findings indicate that NO-donor nitrate azole derivatives possess good in vivo anti-cryptococcal activity, thus providing a new direction for the treatment of cryptococcal meningitis.

Claims

1. A NO donor-type antifungal compound, characterized in that, The compound is represented by general formula V: ； Where A is , , or ; When A is ,in It is 4-hydroxypiperidinyl, diethylamino, D-prolyl, N-methylpiperazinyl, tetrahydropyrrolyl, or N-methylethanolamine; When A is , where n = 2, 3, 5; When A is X is , , , , or ; When A is X is , , , , , , or .

2. A NO donor-type antifungal compound, characterized in that, The specific compounds are as follows: ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； 。 3. A method for preparing the compound according to claim 1, characterized in that, The specific synthetic route of the preparation method is as follows: .

4. A method for preparing the compound according to claim 1, characterized in that, The specific synthetic route of the preparation method is as follows: .

5. A method for preparing the compound according to claim 1, characterized in that, The specific synthetic route of the preparation method is as follows: .

6. A method for preparing the compound of claim 1, characterized in that, The specific synthetic route of the preparation method is as follows: .

7. A pharmaceutical composition comprising a compound of general formula V as claimed in claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

8. Use of a compound of general formula V according to claim 1 in the preparation of an antifungal medicament.

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