An antifungal compound with light-responsive activity and its application

Abstract

The present invention discloses an antifungal compound or a pharmaceutical salt thereof having photoresponsive activity, wherein the general structural formula is selected from one of the following structures: The compound of the present invention is a photoresponsive azole compound, and the azole drug fluconazole is modified by photocaging and photo-switching, so that the antifungal activity is lost in the non-activated state, and the antifungal effect is exerted in the photoactivated state. The photocaging modification strategy can completely shield the antifungal activity of fluconazole, and release fluconazole efficiently under ultraviolet light activation, and can regulate fungal hyphae, proliferation, and in vitro and in vivo efficacy in a precise manner in time and space.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to an antifungal compound with light-responsive activity and application thereof. Background Art

[0002] In the past decade, invasive fungal infections (IFIs) have accounted for a large proportion of deaths related to infections. It is estimated that about 1 million people die from IFIs each year. The increase in immunocompromised people (such as AIDS patients) and the eukaryotic nature of pathogenic fungi make fungal infections more difficult to eradicate. In recent years, IFIs have become an important complication in a large number of severe COVID-19 patients, resulting in high mortality rates. COVID-19-related IFIs have received widespread attention worldwide, and the development of new antifungal drugs and treatment strategies is imperative. However, compared with the development of other anti-infective drugs, the types and quantities of antifungal drugs are quite limited. At the same time, the clinical use of these antifungal drugs is also hindered by drug toxicity, drug interactions and drug resistance. In addition, the evolution and development of fungal multidrug resistance has seriously threatened the effectiveness of current antifungal drugs in clinical treatment. The lag in the development of new antifungal drugs has resulted in few drugs that can effectively solve these problems. In the past decade, only one antifungal drug, isavuconazole (a triazole derivative), has been approved for clinical treatment. Acquired drug resistance in fungi is mainly due to the overuse of antifungal drugs and their accumulation in the environment. For example, the widespread use of antifungal drugs in the medical and health fields leads to their accumulation in the environment, exposing fungi to non-lethal concentrations of drugs for a long time, which puts drug pressure on fungi to evolve into drug resistance. In this way, fungi acquire drug resistance through mutations and changes in gene expression. Therefore, it is very necessary to develop new strategies for precision antifungal treatment.

[0003] Photopharmacology uses chemically synthesized light-responsive ligands to achieve light-dependent pharmacological activity. This emerging strategy often has the characteristics of reduced systemic toxicity and drug resistance. Light, as an external controller, is efficient, harmless, and has high spatiotemporal precision. Combining drug therapeutic effects with optical control can produce the advantages of spatiotemporal precision therapy, providing a promising new concept for future antifungal clinical treatment. Although photopharmacological drugs have made great progress in a wide range of targets, chemical strategies that can light-responsively regulate antifungal drug targets have not yet been realized in living fungal cells.

[0004] Triazole drugs, such as fluconazole (FLC), are the first-line antifungal drugs for the clinical treatment of IFIs. They exert their antifungal effects by inhibiting lanosterol 14α-demethylase (CYP51). CYP51 is an important enzyme in the biosynthesis pathway of fungal ergosterol and is involved in catalyzing the demethylation of fungal lanosterol at the 14α position. The catalytic product of CYP51 continues to participate in the biosynthesis of ergosterol on the fungal cell membrane. The inhibition of CYP51 by triazole drugs leads to the accumulation of lanosterol and the inhibition of ergosterol synthesis, which in turn damages the fungal cell membrane and inhibits the growth of fungal cells. However, CYP51 belongs to the cytochrome P450 protein family, and the use of triazole antifungal drugs is often limited by their hepatotoxicity and renal toxicity. The frequent use of azole drugs can also lead to the emergence of drug-resistant fungi. Therefore, the development of new chemical tools to precisely control CYP51 in time and space will help improve the efficacy and safety of antifungal treatment. Summary of the invention

[0005] The first object of the present invention is to provide an antifungal compound having photoresponsive activity.

[0006] The second object of the present invention is to provide an application of the antifungal compound having photoresponsive activity in the preparation of antifungal drugs.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides an antifungal compound having photoresponsive activity or a pharmaceutically acceptable salt thereof, the general structural formula of which is selected from one of the following structures:

[0009]

[0010] Wherein, X is selected from CH2,

[0011] R1 is selected from hydrogen, nitro, C1-C10 alkyl, C1-C10 alkoxy;

[0012] R2 is selected from hydrogen, nitro, C1-C10 alkyl, C1-C10 alkoxy;

[0013] R3 is selected from hydrogen, nitro, C1-C10 alkyl, C1-C10 alkoxy;

[0014] R4 is selected from hydrogen, nitro, C1-C10 alkyl, C1-C10 alkoxy;

[0015] R5 is selected from hydrogen, nitro, C1-C10 alkyl, C1-C10 alkoxy;

[0016] Or, R3, R4, carbon and at least one oxygen atom form a five-membered ring;

[0017] Y1 is selected from -(CH2)n-; n is selected from an integer of 1 to 10;

[0018] Y2 is selected from -(CH2)n-; n is selected from an integer of 1 to 10;

[0019] R6 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine);

[0020] R7 is selected from hydrogen, nitro, C1-C10 alkyl, C1-C10 alkoxy;

[0021] R8 is selected from hydrogen, nitro, C1-C10 alkyl, C1-C10 alkoxy;

[0022] R9 is selected from hydrogen, nitro, C1-C10 alkyl, C1-C10 alkoxy;

[0023] R 10 is selected from hydrogen, nitro, C1-C10 alkyl, C1-C10 alkoxy;

[0024] R 11 is selected from hydrogen, nitro, C1-C10 alkyl, C1-C10 alkoxy;

[0025] Y3 is selected from -(CH2)n-; n is selected from an integer of 1 to 10;

[0026] R 12 Selected from hydrogen, C1-C10 alkyl;

[0027] R 13 Selected from hydrogen, C1-C10 alkyl;

[0028] R 14 Selected from hydrogen, C1-C10 alkyl, C1-C10 alkoxy;

[0029] R 15 Selected from hydrogen, C1-C10 alkyl, C1-C10 alkoxy;

[0030] R 16 Selected from hydrogen, C1-C10 alkyl, C1-C10 alkoxy;

[0031] R 17 Selected from hydrogen, C1-C10 alkyl, C1-C10 alkoxy;

[0032] R 18 Selected from hydrogen, C1-C10 alkyl, C1-C10 alkoxy;

[0033] R 19 Selected from hydrogen, C1-C10 alkyl, C1-C10 alkoxy;

[0034] R 20Selected from hydrogen, C1-C10 alkyl, C1-C10 alkoxy;

[0035] R 21 Selected from hydrogen, C1-C10 alkyl, C1-C10 alkoxy;

[0036] R 22 Selected from hydrogen, C1-C10 alkyl, C1-C10 alkoxy;

[0037] R 23 Selected from hydrogen, C1-C10 alkyl, and C1-C10 alkoxy.

[0038] Preferably, among the antifungal compounds having light-responsive activity,

[0039] X is selected from CH2, R1 is selected from hydrogen, nitro, methyl, ethyl, methoxy, ethoxy, n-propoxy, tert-butoxy; R2 is selected from hydrogen, nitro, methyl, ethyl, methoxy, ethoxy, n-propoxy, tert-butoxy; R3 is selected from hydrogen, nitro, methyl, ethyl, methoxy, ethoxy, n-propoxy, tert-butoxy; R4 is selected from hydrogen, nitro, methyl, ethyl, methoxy, ethoxy, n-propoxy, tert-butoxy; R5 is selected from hydrogen, nitro, methyl, ethyl, methoxy, ethoxy, n-propoxy, tert-butoxy; or, R3, R4, carbon and two oxygen atoms form a five-membered ring (such as );

[0040] Y1 is selected from -(CH2)n-; n is selected from 1, 2, 3, 4, 5, 6;

[0041] Y2 is selected from -(CH2)n-; n is selected from 1, 2, 3, 4, 5, 6;

[0042] R6 is selected from hydrogen, fluorine, iodine;

[0043] R7 is selected from hydrogen, nitro, methyl, ethyl, methoxy, ethoxy, n-propoxy, tert-butoxy; R8 is selected from hydrogen, nitro, methyl, ethyl, methoxy, ethoxy, n-propoxy, tert-butoxy; R9 is selected from hydrogen, nitro, methyl, ethyl, methoxy, ethoxy, n-propoxy, tert-butoxy; R 10 is selected from hydrogen, nitro, methyl, ethyl, methoxy, ethoxy, n-propoxy, tert-butoxy; R 11 is selected from hydrogen, nitro, methyl, ethyl, methoxy, ethoxy, n-propoxy, tert-butoxy; Y3 is selected from -(CH2)n-; n is selected from 1, 2, 3, 4, 5, 6;

[0044] R 12 is selected from hydrogen, methyl, ethyl, n-propyl;

[0045] R 13is selected from hydrogen, methyl, ethyl, n-propyl;

[0046] R 14 Selected from hydrogen, methyl, ethyl, methoxy, ethoxy, n-propoxy, tert-butoxy;

[0047] R 15 Selected from hydrogen, methyl, ethyl, methoxy, ethoxy, n-propoxy, tert-butoxy;

[0048] R 16 Selected from hydrogen, methyl, ethyl, methoxy, ethoxy, n-propoxy, tert-butoxy;

[0049] R 17 Selected from hydrogen, methyl, ethyl, methoxy, ethoxy, n-propoxy, tert-butoxy;

[0050] R 18 Selected from hydrogen, methyl, ethyl, methoxy, ethoxy, n-propoxy, tert-butoxy;

[0051] R 19 Selected from hydrogen, methyl, ethyl, methoxy, ethoxy, n-propoxy, tert-butoxy;

[0052] R 20 Selected from hydrogen, methyl, ethyl, methoxy, ethoxy, n-propoxy, tert-butoxy;

[0053] R 21 Selected from hydrogen, methyl, ethyl, methoxy, ethoxy, n-propoxy, tert-butoxy;

[0054] R 22 Selected from hydrogen, methyl, ethyl, methoxy, ethoxy, n-propoxy, tert-butoxy;

[0055] R 23 Selected from hydrogen, methyl, ethyl, methoxy, ethoxy, n-propoxy, tert-butoxy.

[0056] Most preferably, the antifungal compound having photoresponsive activity is selected from one of the following structures:

[0057]

[0058] The second aspect of the present invention provides a use of the antifungal compound having photoresponsive activity or a pharmaceutically acceptable salt thereof in the preparation of an antifungal drug.

[0059] The fungus is selected from Cryptococcus neoformans and Candida albicans.

[0060] The antifungal compound or its pharmaceutical salt with photoresponsive activity is converted into an azole antifungal drug under ultraviolet light irradiation, and the azole drug modified by photocage undergoes a dephotocage reaction under ultraviolet light conditions, photolyzes and releases triazole active antifungal drugs. By inhibiting the fungal CYP51 protein, the biosynthesis of ergosterol on the fungal cell membrane is hindered, the fungal cell membrane is damaged, and thus the growth of the fungus is inhibited.

[0061] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0062] The compound of the present invention is a light-responsive azole compound, which loses its antifungal activity in an inactive state and exerts its antifungal effect in a light-activated state by photocaging and photo-switching the azole drug fluconazole. The photocaging modification strategy can completely shield the antifungal activity of fluconazole, and release fluconazole efficiently under ultraviolet light activation, and can regulate fungal hyphae, proliferation, and in vitro and in vivo efficacy in a precise manner in time and space.

[0063] The antifungal compound with photoresponsive activity provided by the present invention has excellent photoconversion efficiency and exerts antifungal activity in in vitro and in vivo antifungal activity tests (Examples 12 and 13), can be rapidly converted into fluconazole within 2 minutes under ultraviolet light, with a conversion rate of 93.2% (Example 14), and can regulate the growth of fungi with precise time accuracy (Example 15), and effectively inhibits the morphological transformation of fungi in a photoactivated state (Example 16), thereby achieving precise treatment and preventing the development of acquired drug resistance of fungi.

[0064] The present invention develops a class of light-responsive antifungal lead compounds by designing antifungal compounds with photopharmacological activity. Two photoactivation strategies, irreversible (photocaged compounds A1–A4, B1–B5) and reversible (photoswitch compounds C1–C4), were used to transform azole antifungal drugs. After bioactivity screening of the compounds of the two photoactivation strategies, it was found that photocaged triazole compounds can completely shield the inhibitory effect and antifungal activity of fluconazole (FLC) on fungal CYP51, and can effectively restore the antifungal efficacy of FLC under light conditions. The antifungal compounds with photoresponsive activity provided by the present invention broaden the scope of chemical tools used in the antifungal field, and realize external control of fungal CYP51 function through light, thereby improving the therapeutic index and reducing the risk of acquired resistance. This proof-of-concept study also greatly expands the application of photopharmacology in the treatment of fungal diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 Schematic diagram of the preventive and post-infection therapeutic effects of Compound A1 in G. mellonella larvae.

[0066] Figure 2 Schematic diagram of the photolysis and photolysis pathway of compound A1 under ultraviolet light.

[0067] Figure 3 Schematic diagram of the time growth curve of compound A1 with different concentrations after light activation at different time points.

[0068] Figure 4 Schematic diagram of the effect of compound A1 on the hyphae formation of Candida albicans under illumination and non-illumination conditions. DETAILED DESCRIPTION

[0069] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0070]

[0071] Scheme 1.Reagents and conditions: (a) N2H4 / H2O, KOH, EtOH, glycol, reflux, yield 83.6%; (b) HNO3, DCM, 0℃, yield 66.7%; (c) Benzyltrimethylammoniumhydroxide (Triton B), CH2O; MeOH, reflux, yield 33.0%; (d) ⅰ. Triphosgene, Na2CO3, THF; ⅱ. FLC, NaH, DMF, -20°C, yield 10.0% two steps.

[0072]

[0073] Scheme 2.Reagents and conditions:(a)NaH,DMF,-20℃,yield 2.6–7

[0074]

[0075] Scheme 3.Reagents and conditions: (a) NaNO2, TFA, yield 43.1%; (b) NaBH4, THF / EtOH, yield 82.1%; (c) ⅰ. triphosgene, Na2CO3, DCM; ii. Fluconazole (1), NaH, DMF, -20℃, yield 2.7%

[0076] two steps.

[0077]

[0078] Scheme 4.Reagents and conditions:(a)(3-Iodophenyl)methanamine or 4-Phenylbutan-1-amine or 3-Phenyl-1-propylamine,TEA,EtOH,HCl,reflux,yield 31.1–84.1%; (b) 1-(bromomethyl)-4,5-dimethoxy-2-nitrobenzene, K2CO3, KI, MeCN, 50℃, yield 28.4–43.4%; (c) ⅰ.7-(diethylamino)-4-(hydroxymethyl)-2H-chromen-2-one, diphosgene, MeCN, RT; ⅱ.TEA, DMAP, DMF, 90℃, yield 15.0–17.0% two steps.

[0079]

[0080] Scheme 5.Reagents and conditions: (a) methoxybenzene or 1,3-dimethoxybenzene or 1,3,5-methoxybenzene, NaNO2, H2SO4, 0–25℃, 24.8–31.4%; (b). Compound 12, K2CO3, DMF, 80℃, yield 5.0–18.5%.

[0081] Example 1

[0082] Preparation of 2-(2,4-difluorophenyl)-1,3-di(1H-1,2,4-triazol-1-yl)propan-2-yl(2-(6-nitrobenzo[d][1,3])dioxolan-5-yl)propyl)carbonate (A1)

[0083] (1) Preparation of Intermediate 5

[0084] Hydrazine hydrate (2.15 g, 50%, 21.5 mmol), starting material 4 (1.00 g, 6.09 mmol) and EtOH (20 mL) were added to a 50 mL eggplant-shaped bottle and refluxed for 6 h. After the EtOH was evaporated to dryness under reduced pressure, ethylene glycol (8 mL) and KOH (1.02 g, 18.39 mmol) were added and refluxed (160 ° C) for 8 h under N2 protection. Cooled to room temperature, water (10 mL) was added, and the oily product was separated. The aqueous phase was extracted with ether (2×20 mL), and the combined organic phase was washed with 1N HCl, dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and combined with the oily substance in the previous step. The distillate was collected at 170 ° C to obtain 764 mg of the target product, with a yield of 83.6%. 1 H NMR (600MHz, CDCl3) δ6.72(d,J=7.9Hz,1H),6.69(d,J=1.3Hz,1H),6.64(dd,J=7.9,1.6Hz,1H),5.91(s,2H),2.57(q,J=7.6Hz,2H),1.20(t,J=7.6Hz,3H).

[0085] (2) Preparation of Intermediate 6

[0086] Add intermediate 5 (1.50 g, 10 mmol) and DCM (20 mL) into a 50 mL eggplant-shaped bottle and cool to 0°C in an ice bath. Then slowly add nitric acid (40%, 10 mL) into the above solution, and continue stirring for 1 h after naturally warming to room temperature. Add H2O (30 mL) to the reaction solution, extract with DCM (2×20 mL), wash the combined organic phase with NaHCO3, dry with anhydrous Na2SO4, filter, concentrate under reduced pressure, and separate and purify using silica gel column chromatography (PE / EA=5 / 1). Obtain 1.30 g of the target product, yield: 66.7%. 1 HNMR (600MHz, DMSO-d6) δ7.56(s,1H),7.08(s,1H),6.21(s,2H),2.80(q,J=7.4Hz,2H),1.20(t,J=7.4Hz,3H).

[0087] (3) Preparation of Intermediate 7

[0088] Intermediate 6 (1.26 g, 6.46 mmol), Triton B (2.7 g, 40% in MeOH, 6.46 mmol), paraformaldehyde (196 mg, 6.53 mmol) and MeOH (20 mL) were added to a 50 mL eggplant-shaped bottle. The mixture was heated to reflux for 6 h, concentrated under reduced pressure, and neutralized with 5% HCl aqueous solution. After adding H2O (20 mL), it was extracted with EtOAc (2×30 mL), and the combined organic phase was dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and separated and purified using silica gel column chromatography (PE / EA=4 / 1). 1 H NMR (300MHz, DMSO-d6) δ7.43(s,1H),7.13(s,1H),6.17(s,2H),3.54–3.43(m,2H),3.30–3.17(m,1H),1.18(d,J=6.9Hz,3H).

[0089] (4) Preparation of target product A1

[0090] Intermediate 7 (122 mg, 0.54 mmol) and THF (15 mL) were added to a 25 mL eggplant-shaped bottle and cooled to 0°C. Triphosgene (158 mg, 0.54 mmol) and Na2CO3 (57.2 mg, 0.54 mmol) were then added, and the mixture was stirred and reacted for 16 h after the mixture naturally warmed to room temperature. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure at room temperature and freeze-dried in vacuo to obtain a crude product, which was used directly in the next step without further purification.

[0091] NaH (17.2 mg, 60% in oil, 0.43 mmol) was dissolved in dry DMF (2 mL) and ice-bathed to 0°C. FLC (compound 1) (110 mg, 0.36 mmol) was then dissolved in dry DMF (3 mL) and slowly added to the above NaH solution under ice-bath conditions. After stirring at room temperature for 2 h, the reaction temperature was lowered to -20°C. The acyl chloride product obtained in the previous step was dissolved in DMF and added to the above reaction solution using a dropping funnel (30 min to drip). The reaction was stirred at room temperature for 24 h, and NaHCO3 aqueous solution was added to the reaction solution. It was extracted with EtOAc (3×20 mL), the organic phases were combined and dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure, and the crude product was separated and purified using silica gel column chromatography (DCM / MeOH=100 / 1–100 / 3) to obtain 20 mg of the target product, with a yield of 10.0%. 1H NMR(600MHz, CDCl3)δ8.03(s,1H),7.96(s,1H),7.89(s,1H),7.88(s,1H),7.38(s,1H),6.92–6 .88(m,1H),6.88(s,1H),6.88–6.85(m,1H),6.82–6.78(m,1H),6.14(d,J=1.2Hz,1H),6.11(d,J =1.2Hz,1H),5.14(d,J=14.7Hz,1H),5.09(d,J=14.3Hz,2H),5.02(d,J=14.7Hz,1H),4.36(dd, J=10.7,6.9Hz,1H),4.31(dd,J=10.8,6.1Hz,1H),3.89(h,J=6.9Hz,1H),1.35(d,J=6.9Hz,3H). 13 C NMR (151MHz, CDCl3) δ163.30 (dd, J=253.2, 12.8Hz), 159.25 (dd, J=249.3, 11.6Hz), 152.27 (s), 152.08 (s), 152. 06(s),151.71(s),146.64(s),144.78(s),144.69(s),143.90(s),133.37(s),128.36(dd,J=9.4,4.9Hz),119.2 9(dd,J=11.2,3.6Hz),112.43(dd,J=21.1,1.7Hz),106.85(s),105.61(s),105.18(t,J=26.5Hz),102.94(s),82 .01(d,J=4.7Hz),72.13(s),51.38(d,J=5.1Hz),51.05(d,J=6.1Hz),33.42(s),17.72(s).ESI-HRMS:Calcd.for C 24 H 21 F2N7O7 m / z:[M+H] + =558.1544; found[M+H] + =558.1558.

[0092] Example 2

[0093] Preparation of target product (A2)

[0094] NaH (48 mg, 60% in oil, 1.2 mmol) was dissolved in DMF (3 mL) and ice-bathed to 0°C. FLC (compound 1) (306 mg, 1 mmol) was dissolved in DMF (6 mL) and slowly added dropwise to the above NaH solution. The reaction solution was stirred at room temperature for 2 h and then placed at -20°C. Compound 8 (303 mg, 1.1 mmol) was dissolved in DMF and then added dropwise to the above reaction solution using a dropping funnel (addition was completed in 30 min). The reaction mixture was further stirred at room temperature for 3 h. After adding saturated NaCl aqueous solution, it was extracted with EtOAc (3×20 mL), the organic phases were combined, dried with anhydrous Na2SO4, filtered, concentrated under reduced pressure, and separated and purified using silica gel column chromatography (DCM / MeOH=100 / 1–100 / 3) to obtain 36 mg of the target product with a yield of 7.2%. 1 H NMR (600MHz, CDCl3) δ8.09(s,2H),7.89(s,2H),7.72(s,1H),7.23(s,1H),7.10(td,J=8.8,6.1Hz,1H),6.90(ddd,J=12.3, 8.3, 2.5Hz, 1H), 6.87–6.82 (m, 1H), 5.20 (s, 2H), 4.99 (d, J = 14.8Hz, 2H), 4.88 (d, J = 14.9Hz, 2H), 3.97 (s, 3H), 3.96 (s, 3H). 13 C NMR (151MHz, CDCl3) δ163.49 (dd, J=255.0, 13.9Hz), 160.45 (dd, J=250.1, 11.2Hz), 153.90 (s), 151.92(s,2C),147.91(s),144.82(s,2C),139.05(s),129.56(dd,J=9.2,5.4Hz),128.72(s),11 9.41(dd,J=11.3,3.7Hz),112.66(dd,J=21.1,1.9Hz),109.64(s),107.95(s),105.57(t,J=26. 6Hz),80.08(d,J=4.4Hz),62.46(s),56.43(s),56.40(s),51.36(s),51.32(s).ESI-HRMS:calcd forC 22 H 21 F2N7O5 m / z:[M+H] + =502.1645; found[M+H] + =502.1647.

[0095] Example 3

[0096] Preparation of target product (A3)

[0097] NaH (24 mg, 60% in oil, 0.6 mmol) was dissolved in DMF (2 mL), and FLC (compound 1) (153 mg, 0.5 mmol) dissolved in DMF (4 mL) was slowly added dropwise to the above NaH solution under ice bath conditions. The reaction solution was stirred at room temperature for 2 h and then cooled to -20 °C. Compound 9 (206 mg, 0.75 mmol) was dissolved in DMF and added dropwise to the above reaction solution using a dropping funnel (30 min for the addition). The reaction mixture was further stirred at room temperature for 3 h. After adding saturated NaCl aqueous solution, it was extracted with EtOAc (3×20 mL), the organic phases were combined, dried with anhydrous Na2SO4, filtered, concentrated under reduced pressure, and separated and purified using silica gel column chromatography (DCM / MeOH=100 / 1–100 / 3) to obtain 34 mg of the target product with a yield of 2.6%. 1 H NMR (600MHz, CDCl3) δ8.04(s,2H),7.89(s,2H),7.75(s,1H),7.03(s,1H),6.94(td,J=8.9,6.0Hz,1H),6.90(ddd,J=12.3, 8.2,2.5Hz,1H),6.83–6.79(m,1H),5.61(s,2H),5.17(d,J=14.7Hz,2H),5.11(d,J=14.7Hz,2H),4.02(s,3H),3.98(s,3H). 13 C NMR (151MHz, CDCl3) δ163.40 (dd, J=253.5, 12.8Hz), 159.31 (dd, J=249.2, 11.5Hz), 153.68 (s), 152 .11(s,2C),152.07(s),148.86(s),144.81(s,2C),140.14(s),128.32(dd,J=9.3,4.7Hz),125.23( s),119.14(dd,J=11.1,3.4Hz),112.52(dd,J=21.3,1.9Hz),110.97(s),108.40(s),105.31(t,J=2 6.3Hz),82.36(d,J=4.6Hz),67.25(s),56.66(s),56.48(s),51.20(s),51.17(s).ESI-HRMS:calcd for C 23 H 21 F2N7O7 m / z:[M+H] + =546.1544; found[M+H]+ =546.1544.

[0098] Example 4

[0099] Preparation of target product (A4)

[0100] (1) Preparation of intermediate 10

[0101] The starting material 4 (2.00 g, 25.5 mmol) and TFA (20 mL) were added to a 50 mL eggplant-shaped bottle, and NaNO2 (2.00 g, 28.99 mmol) was added to the above solution in batches. The reaction mixture was stirred at room temperature for 4 h, H2O was added to the reaction solution, and DCM (3×30 mL) was used for extraction. The combined organic phase was washed with NaHCO3, separated and purified by silica gel column chromatography (PE / EA=16 / 1–8 / 1), and 2.30 g of the target product was obtained, with a yield of 43.1%. 1 H NMR (600MHz, DMSO-d6) δ7.69(s,1H),7.30(s,1H),6.30(s,2H),2.49(s,3H). 13 C NMR(151MHz,DMSO-d6)δ199.16(s),152.52(s),149.33(s),140.71(s),133.75(s),107.16(s),105.09(s),104.52(s),30.24(s).ESI-HRMS:calcd for C9H7NO5 m / z:[M+H] + =210.0397; found [M+H] + =210.0399.

[0102] (2) Preparation of intermediate 11

[0103] Intermediate 10 (525 mg, 2.51 mmol) was dissolved in 20 mL of a mixed solvent of THF / EtOH (1 / 1), and NaBH4 (191 mg, 5.02 mmol) was added to the above solution in batches. The reaction solution was stirred at room temperature overnight, quenched with 2N HCl, and concentrated under reduced pressure. H2O was added to the concentrate, and extracted with DCM (3×20 mL). The combined organic phase was washed with a saturated aqueous NaCl solution, dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and separated and purified using a silica gel column chromatography (PE / EA=2 / 1–1 / 1). 435 mg of the target product was obtained with a yield of 82.1%. 1H NMR(600MHz,DMSO-d6)δ7.51(s,1H),7.27(s,1H),6.22(d,J=0.9Hz,1H),6.1 9(d,J=0.9Hz,1H),5.46(s,1H),5.15(q,J=6.3Hz,1H),1.34(d,J=6.3Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ152.30(s),146.79(s),141.12(s),140.67(s),106.52(s),104.70(s),103.60(s),64.45(s),25.53(s).ESI-HRMS:calcd forC9H9NO5 m / z:[M+Na] + =234.0373; found [M+H] + =234.0367.

[0104] (4) Preparation of target product A4

[0105] Intermediate 11 (500 mg, 2.36 mmol), Na2CO3 (247 mg, 2.36 mmol) and THF (5 mL) were added to a 25 mL eggplant-shaped bottle and cooled to 0° C. Triphosgene (701 mg, 2.36 mmol) was then added and reacted at room temperature for 12 h. The crude product was filtered and concentrated under reduced pressure to obtain a crude product, which was directly used in the next step without further purification after drying.

[0106] Dissolve NaH (75.6 mg, 60% in oil, 1.89 mmol) in DMF (2 mL) and cool to 0°C in an ice bath. Dissolve FLC (compound 1) (480 mg, 1.57 mmol) in dry DMF (3 mL) and slowly add it to the above NaH solution under ice bath conditions. Stir at room temperature for 2 h and then lower the temperature to -20°C again. Dissolve the acyl chloride product obtained in the previous step in DMF and add it to the above reaction solution using a dropping funnel (30 min to drip). Stir the reaction at room temperature for 24 h, add NaHCO3 aqueous solution to the reaction solution, extract with EtOAc (3×20 mL), combine the organic phases, dry with anhydrous Na2SO4, filter, concentrate under reduced pressure, and separate and purify using silica gel column chromatography (DCM / MeOH=100 / 1–100 / 3) to obtain 23 mg of the target product with a yield of 2.7%. 1H NMR(600MHz,DMSO-d6)δ8.22(s,1H),8.21(s,1H),7.90(s,1H),7.90(s,1H),7.61(s,1H),7.24( ddd,J=11.9,9.0,2.5Hz,1H),7.20(s,1H),7.02(td,J=8.9,6.6Hz,1H),6.95(td,J=8.5,2.6Hz, 1H),6.31(d,J=0.8Hz,1H),6.27(d,J=0.8Hz,1H),6.10(q,J=6.4Hz,1H),5.16(d,J=15.0Hz,1H) ,5.11(d,J=14.9Hz,1H), 4.99(d,J=15.5Hz,1H), 4.97(d,J=15.4Hz,1H), 1.62(d,J=6.5Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ162.37(dd,J=248.6,12.8Hz),159.06(dd,J=250.0,12.2Hz),152.35(s), 151.62(s,2C),151.02(s),147.46(s),145.57(s),145.52(s),141.49(s),133.15(s),128.98(dd, J=9.2, 4.0Hz), 119.81 (d, J=7.4Hz), 111.69 (d, J=20.5Hz), 106.30 (s), 104.95 (t, J=26.8Hz), 104. 65(s),103.72(s),82.36(d,J=3.6Hz),72.04(s),50.97(s),50.71(s),21.24(s).ESI-HRMS:calcd forC 23 H 19 F2N7O7 m / z:[M+H] + =544.1387; found[M+H] + =544.1390.

[0107] Example 5

[0108] Preparation of target product B1.

[0109] (1) Preparation of intermediate b1

[0110] The starting material 12 (0.10 g, 0.3 mmol), 3-iodobenzylamine hydrochloride (80.7 mg, 0.3 mmol) and EtOH (10 mL) were added to a 25 mL eggplant-shaped bottle, and Et3N (60.6 mg, 0.6 mmol) was added to the reaction solution, and the reaction was refluxed for 6 h. The product was concentrated under reduced pressure and redissolved in EtOAc (4 mL), and a 1 M HCl solution in EtOAc was slowly added dropwise, and the precipitated white solid was filtered to obtain 73 mg of the target product, with a yield of 31.1%. 1 H NMR(600MHz,DMSO-d6)δ8.28(s,1H),7.73(s,1H),7.61(s,1H),7.58(d,J=7.9Hz,1H) ,7.42–7.36(m,1H),7.25(d,J=7.6Hz,1H),7.16–7.11(m,1H),7.10(t,J=7.7Hz,1H),6 .98–6.93(m,1H),5.79(s,1H),4.59(d,J=14.3Hz,1H),4.55(d,J=14.3Hz,1H),3.64(d ,J=14.2Hz,1H),3.61(d,J=14.1Hz,1H),2.92(d,J=12.5Hz,1H),2.85(d,J=12.4,1H). 13 C NMR (151MHz, DMSO-d6) δ162.97 (dd, J=246.9, 12.6Hz), 159.63 (dd, J=247.2, 12.6Hz) ,150.43(s),145.52(s),139.34(s),138.11(s),134.17(s),131.03(s,2C),130.33( s),123.02(d,J=10.3Hz),111.77(d,J=20.8Hz),104.89(t,J=26.9Hz),95.32(s),72 .56(d,J=4.0Hz),55.48(d,J=3.2Hz),52.08(d,J=4.7Hz),50.43(s).ESI-HRMS:calcd for C 18 H 17 F2IN4O m / z:[M+H] + =471.0488; found [M+H] + =471.0500.

[0111] (2) Preparation of target product (B1)

[0112] Intermediate b1 (65 mg, 0.148 mmol), 1-(bromomethyl)-4,5-dimethoxy-2-nitrobenzene (82 mg, 0.3 mmol), K2CO3 (69 mg, 0.5 mmol), KI (14 mg, 0.083 mmol) and MeCN (15 mL) were added to a 25 mL eggplant-shaped bottle. The mixture was stirred at 50°C for 10 h, the reaction solution was filtered, the filtrate was concentrated under reduced pressure, and the target product was obtained by separation and purification using silica gel column chromatography (DCM / MeOH=100 / 1-100 / 3) to obtain 28 mg of the target product with a yield of 28.4%. 1 H NMR(600MHz,DMSO-d6)δ8.23(s,1H),7.75(s,1H),7.57–7.53(m,1H),7.45(s,1H),7.41–7.35(m,1H),7.3 3–7.31(m,1H),7.09–7.06(m,1H),7.04–7.01(m,1H),7.00–6.92(m,2H),6.80(s,1H),5.91(s,1H),4.46( d,J=14.3Hz,1H),4.39(d,J=14.3Hz,1H),4.08(d,J=15.7Hz,1H),3.81(s,3H),3.76(d,J=15.6Hz,1H),3. 71(s,3H),3.58(d,J=13.8Hz,1H),3.32(d,J=14.9Hz,1H),3.20(d,J=14.4Hz,1H),2.83(d,J=13.9Hz,1H). 13 C NMR(151MHz,DMSO-d6)δ162.06(dd,J=245.9,12.3Hz),159.02(dd,J=247.2,12.1Hz),152.82(s),151.01(s),14 7.56(s),145.33(s),141.69(s),141.63(s),137.74(s),136.09(s),130.55(s),130.50–130.33(m),129.80(s) ,128.66(s),126.30–125.93(m),112.34(s),111.11(d,J=19.8Hz),108.03(s),104.24(t,J=26.7Hz),95.15(s) ,76.25(d,J=5.0Hz),60.23(s),59.58(s),56.44(s),56.34(d,J=4.4Hz),56.24(s),55.84(s).ESI-HRMS:calcd for C 27 H 26F2IN5O5 m / z:[M+H] + =666.1020; found[M+H] + =666.1020.

[0113] Example 6

[0114] The 3-iodobenzylamine hydrochloride in step (1) of Example 5 was replaced by 4-phenyl-1-butylamine, and the rest was the same as in Example 5 to obtain compound B2.

[0115] Example 7

[0116] The 3-iodobenzylamine hydrochloride in step (1) of Example 5 was replaced by 3-phenyl-1-propylamine, and the rest was the same as in Example 5 to obtain compound B3.

[0117] Example 8

[0118] Preparation of target product B5.

[0119] 7-(Diethylamino)-4-(Hydroxymethyl)-2H-benzopyran-2-one (50 mg, 0.202 mmol), diphosgene (39.6 mg, 0.202 mmol) and anhydrous MeCN (10 mL) were added to a 25 mL eggplant-shaped bottle. The reaction mixture was stirred at room temperature for 2 h. After the solvent was removed by concentration under reduced pressure, the resulting acid chloride product was redissolved in dry DMF. Intermediate b3 (85 mg, 0.202 mmol) and TEA (81.6 mg, 0.808 mmol) were dissolved in DMF (5 mL) and slowly added dropwise to the above acid chloride solution. The reaction was stirred at 90 ° C overnight. Saturated aqueous NaCl solution was added to the reaction solution and extracted with EtOAc (3×20 mL). The combined organic phase was washed with saturated aqueous NaCl solution (5×20 mL), dried, filtered, concentrated under reduced pressure, and separated and purified using silica gel column chromatography (DCM / MeOH=100 / 1–100 / 2) to obtain 20 mg of the target product in a yield of 15.0%. 1H NMR(600MHz,CDCl3)δ8.10(s,1H),7.80(s,1H),7.59–7.53(m,1H),7.24(d,J=7.5Hz,2H),7.20(d,J=9.0Hz,1H),7.15(t,J=7.4Hz,1H),7.11(d,J=7.3Hz,2H),6.87–6.83(m,1H),6.81–6.75(m,1H),6.53(dd,J=9.0,2.4Hz,1H),6.50(d,J=2.4Hz,1H),5.92(s,1H),5.88(s,1H),5.21(d,J=14.8Hz,1H),5.12(d,J=14.8Hz,1H),4.68(d,J=14.2Hz,1H),4.57(d,J=14.2Hz,1H),3.85(d,J=14.9Hz,1H),3.59(d,J=15.0Hz,1H),3.39(q,J=7.1Hz,4H),3.33–3.26(m,1H),3.04–2.97(m,1H),2.54(t,J=7.1Hz,2H),1.55–1.44(m,4H),1.19(t,J=7.1Hz,6H). 13 C NMR(151MHz,CDCl3)δ163.06(dd,J=250.4,12.4Hz),161.77(s),158.82(dd,J=245.4,11.7Hz),158.10(s),156.42(s),151.51(s),150.84(s),149.59(s),144.78(s),141.79(s),130.58(dd,J=7.7,5.9Hz),128.47(s,2C),128.41(s,2C),126.00(s),124.42(s),123.74(d,J=11.3Hz),112.02(d,J=20.3Hz),108.73(s),106.40(s),105.96(s),104.16(t,J=26.5Hz),97.98(s),63.34(s),56.06(d,J=4.7Hz),55.21(s),49.89(s),44.85(s,2C),35.50(s),29.80(s),28.45(s),27.76(s),12.52(s,2C).ESI-HRMS:calcd for C 36 H 39 F2N5O5 m / z:[M+H] + =660.2993;found[M+H] +=660.3007.

[0120] Example 9

[0121] The intermediate b3 in Example 8 was replaced by b2, and the rest was the same as in Example 8 to obtain compound B4.

[0122] Example 10

[0123] Preparation of target products C1 and C2.

[0124] The initial raw material 19 (1.00 g, 11.9 mmol) and H2SO4 (30%, 10 mL) were added to a 50 mL eggplant-shaped bottle, and an aqueous solution of NaNO2 (0.98 g, 14.28 mmol) was slowly added dropwise to the above acid solution under ice bath conditions. The reaction solution was stirred and reacted for 15 min at 0°C. Anisole (1.54 g, 14.28 mmol) was added to the above reaction solution and stirred at room temperature for 20 h. Water and EtOAc were added, the organic phase was separated, the aqueous phase was extracted with EtOAc (2×30 mL), the organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain 600 mg of the product, which was directly used in the next step without purification, with a yield of 24.8%. The above product 20 (100 mg, 0.49 mmol) and the starting material 12 (180 mg, 0.54 mmol) were dissolved in DMF (20 mL), and K2CO3 (135 mg, 0.98 mmol) was added thereto. The reaction mixture was stirred at 80 ° C for 12 h. The reaction solution was cooled to room temperature and filtered, and the filtrate was diluted with H2O (20 mL) and extracted with EtOAc (3×30 mL). The combined organic phase was washed with saturated NaCl (3×20 mL), dried with Na2SO4, filtered and concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography to obtain the target products C1 (40 mg, yield: 18.5%) and C2 (27 mg, yield: 12.5%) which are isomers of each other. 1 H NMR(C1,600MHz,DMSO-d6)δ8.35(s,1H),8.05(s,1H),7.88(d,J=9.1Hz,2H),7 .83(s,1H),7.20(d,J=9.1Hz,2H),7.15(td,J=9.0,6.9Hz,1H),6.95–6.90(m, 1H),6.82(td,J=8.5,2.6Hz,1H),6.25(s,1H),5.10(d,J=14.2Hz,1H),4.94(d ,J=14.2Hz,1H),4.87(d,J=14.5Hz,1H),4.77(d,J=14.5Hz,1H),3.93(s,3H). 13C NMR(151MHz,DMSO-d6)δ164.18(s),162.34(dd,J=246.7,12.6Hz),159.59(dd,J=247.4,12.4Hz),159.22(s),151.25(s),151.24(s),146.94(s),145.71(s),130.24(dd,J=8.6,5.8Hz),126.25(s,2C),124.07–123.72(m),115.32(s,2C),111.35(d,J=20.4Hz),104.21(t,J=26.8Hz),74.57(d,J=4.5Hz),56.38(s),55.14(d,J=5.9Hz),54.09(s).ESI-HRMS:calcd for C 20 H 18 F2N8O2 m / z:[M+H] + =441.1594;found[M+H] + =441.1608. 1 H NMR(C2,600MHz,DMSO-d6)δ8.44(s,1H),8.33(s,1H),7.88(d,J=9.0Hz,2H),7.80(s,1H),7.30–7.25(m,1H),7.25–7.20(m,1H),7.14(d,J=9.1Hz,2H),6.91(td,J=8.5,2.6Hz,1H),6.48(s,1H),4.81(d,J=14.5Hz,2H),4.71(d,J=14.5Hz,1H),4.64(d,J=14.5Hz,1H),3.88(s,3H). 13 C NMR(C3,151MHz,DMSO-d6)δ168.75(s),163.27(s),162.59(dd,J=246.5,12.5Hz),159.56(dd,J=247.2,12.7Hz),151.32(s),147.13(s),146.84(s),145.64(s),130.28(s),125.44(s,2C),123.78(d,J=10.2Hz),115.25(s,2C),111.55(d,J=20.5Hz),104.59(t,J=26.9Hz),74.12(d,J=4.5Hz),56.21(s,2C),55.30(d,J=4.12).ESI-HRMS:calcd for C 20 H 18F2N8O2 m / z:[M+H] + =441.1594; found[M+H] + =441.1603.

[0125] Embodiment 11

[0126] The anisole in Example 10 was replaced by 1,3-dimethoxybenzene, and the other steps were the same as in Example 10 to obtain compounds C3 and C4.

[0127] Table 1. Structural formula and NMR mass spectrometry data of the compounds of the present invention

[0128]

[0129]

[0130]

[0131]

[0132]

[0133] Example 12

[0134] Antifungal activity of the compounds prepared in Examples 1 to 11 under illumination and non-illumination conditions

[0135] Ideally, light-responsive antifungal compounds would only exert antifungal effects when activated by light, and would be inactive under non-light conditions. Therefore, the antifungal activities of the designed compounds under light and non-light conditions were tested to investigate the effectiveness of the two photomodification strategies.

[0136] (1) Test compound: Prepare a solution with a concentration of 2 mg / mL using DMSO.

[0137] (2) Test strains: Candida albicans (SC5314), Cryptococcus neoformans (H99).

[0138] (3) Activation of the test strain: Take out the frozen test strain from the -80℃ refrigerator, and pipette 10μL into 1mL YEPD culture medium. Place in a 30℃ incubator and shake culture at 200rpm. After 24h, pipette 10μL from the YEPD bacterial suspension and add it to a new 1mL YEPD culture medium. Shake culture at 30℃ for 16h, and activation is complete. At this time, the fungus is in the late exponential growth stage.

[0139] (4) Preparation of bacterial suspension: Place the activated strain to be tested in a 1.5 mL centrifuge tube, centrifuge (3000 r, 1 min), discard the supernatant, wash the strain with 1 mL PBS buffer, centrifuge, discard the supernatant, and repeat 3 times. Count the strains using a hemocytometer and adjust the bacterial concentration to 1×10 using RPMI 1640 culture medium. 3 cells / mL.

[0140] (5) Drug effect and detection: The micro-liquid dilution method recommended by the Clinical and Laboratory Standards Institute (CLSI) M27-A3 and M38-A2 documents was used, and the fungal growth was evaluated by the optical density (OD) of the culture. The prepared bacterial suspension was added to a 96-well plate (columns 1-11, rows BG), with 200 μL in the first column and 100 μL in columns 2-11. 6.4 μL of the prepared drug solution was aspirated into the first column and triplicate wells were made to make the drug concentration 64 μg / mL. From the first column to the tenth column, half dilution was performed in sequence, that is, the concentration gradient was 0.12-64 μg / mL. No drug was added to the 11th column, which was used as the negative control group. Blank 1640 culture medium was added to the outermost periphery as a blank control. The antifungal activity of the compounds under light conditions was determined by diluting the compounds to be tested by half the above operation and placing the 96-well plate under ultraviolet light of 365 nm wavelength and 25 mW / cm 2 The 96-well plate to be tested was then placed in a 30°C constant temperature incubator for 48 hours and the OD was measured using an enzyme reader. 630 The optical density (OD) value of the negative control group was set as 100%, and the lowest drug concentration that decreased the optical density by more than 80% compared with the negative control group was taken as the minimum inhibitory concentration (MIC). 80 ).

[0141] The antifungal activities of the photocage type compounds before and after illumination are shown in Table 1. Candida albicans and Cryptococcus neoformans are two clinically important fungal pathogens and are used for antifungal activity determination in the present invention. The results show that under 365nm ultraviolet light conditions, the photoactive protective group can shield the antifungal activity of FLC and restore its antifungal effect. The antifungal activity of the compounds (A1, A2, A4) with the introduction of photocage groups on the hydroxyl oxygen atoms of FLC is significantly reduced. Compound A1 exhibits better shielding activity and more effective photolysis efficiency than other compounds (A2 and A4). Compound A1 is almost inactive in inhibiting the growth of Candida albicans (MIC>64μg / mL) and Cryptococcus neoformans (MIC=64μg / mL). When irradiated with 365nm ultraviolet light, the antifungal activity is restored to a similar level of FLC, with MIC values ​​of 0.5 and 2μg / mL for Candida albicans and Cryptococcus neoformans, respectively. However, the photocage type compound A3 cannot be photolyzed and release FLC under 365nm ultraviolet light, and has no antifungal activity. When the photocage group is connected to the side chain nitrogen atom of the idiconazole derivatives (compounds b1-b3) (B1-B5), its shielding effect on antifungal activity is weak (Table 1). Compounds b1-b3 are idiconazole derivatives, among which compound b1 is the photolysis product of B1, b2 is the photolysis product of B2 and B4, b3 is the photolysis product of B3 and B5, and FLC is the theoretical photolysis product of A1-A4.

[0142] The antifungal activities of the photoswitchable compounds C1–C4 under illumination and non-illumination conditions are shown in Table 2. The compounds containing azo structures are in a heat-stable trans configuration under non-illumination conditions, and are excited to a cis configuration after irradiation with 365nm ultraviolet light. Since this configuration conversion is reversible, low light intensity and long illumination time are used to maintain the configuration throughout the fungal culture process. However, there is no significant difference in the antifungal activity of compounds C1–C4 under illumination and non-illumination conditions, which may be due to the fact that the substrate entry channel of the CYP51 protein can accommodate more flexible side chain groups. Therefore, based on the above results, the photocage modification (A1, A2, A4) at the hydroxyl position of FLC can achieve the desired goal.

[0143] Table 1. Antifungal activities (MICs) of target compounds A1–A4 and B1–B5 under illumination and non-illumination conditions 80 , μg / mL)

[0144]

[0145] Light condition:365nm, 25mW / cm 2 , 3min.

[0146] Table 2. Antifungal activities (MICs) of target compounds C1–C4 under illumination and non-illumination conditions 80 ,μg / mL)

[0147]

[0148] a Light conditions:365nm, 1mW / cm 2 , 48h for C.albians; 72h for C.neoformans.

[0149] Example 13

[0150] Efficacy test of the preferred compound A1 in the larvae of G. mellonella under light activation

[0151] Based on the excellent photoconversion efficiency of the above-mentioned photocage compounds and the antifungal activity exhibited after the release of FLC, the larvae of G. mellonella were used as experimental animals to explore the photolysis and therapeutic effects of the compounds in vivo. G. mellonella larvae in good condition, weighing about 0.5 g, and without obvious gray or black spots on the body surface were selected for the experiment. Each group had 15 larvae, and each larva was inoculated with 6×10 cells of Candida albicans (SC5314). 5 cells for infection. A dose of 8 mg / kg was administered before or after inoculation for prophylactic treatment or post-infection treatment. The specific operation was as follows: Candida albicans cells (SC5314) in the late exponential growth period were centrifuged, washed with PBS (3 × 1 mL), and resuspended in PBS to a concentration of 6.25 × 10 7 or 7.5×10 7 The fungal suspension (8 μL, 7.5×10 7 cells mL) was injected into the larvae from the left hind leg through a Hamilton syringe to complete the inoculation. Larvae infected with Candida albicans were placed in a 12-well plate (1 per well) in the order of inoculation. 30 minutes after the completion of the inoculation, a PBS solution of compound A1 (0.5 mg / kg, 8 μL, containing 25% DMSO) was injected into the larvae from the right hind leg. The group that needed light was illuminated with 365 nm ultraviolet light (50 mW / cm 2 ) for 10 minutes, the wax moth larvae were incubated at 37°C, and the survival rate of the larvae was observed and recorded every 12 hours. For preventive treatment, the larvae were treated with ultraviolet light (as described above) or non-ultraviolet light 30 minutes after administration, and then infected with Candida albicans (as described above).

[0152] The experimental results are as follows Figure 1 As shown, Figure 1 Schematic diagram of the preventive and post-infection therapeutic effects of Compound A1 in G. mellonella larvae. Figure 1 A in the middle is a schematic diagram of the effect of preventive treatment. When preventive treatment is given (administered 30 minutes before infection) Figure 1 (A) Compound A1 had no therapeutic effect without light, while the median survival of the wax larvae treated with Compound A1 under light conditions was extended to 14 days, which was similar to the therapeutic effect of FLC. Figure 1 B is a schematic diagram of the treatment effect after infection. Figure 1 In B), compared with the control group and the non-illumination group of compound A1, the illumination group and the FLC group significantly prolonged the survival period of the greater wax larvae. It is still a great challenge to achieve optical control effects at the molecular and cellular levels in animals. In the present invention, optical regulation of compound A1 in animals is achieved, and excellent antifungal treatment effects are shown.

[0153] Embodiment 14

[0154] Determination of the photolysis and release of FLC by the preferred compound A1 under 365 nm ultraviolet light

[0155] (1) Test compound: Compound A1 was prepared into a solution with a concentration of 0.25 mM using methanol.

[0156] (2) Test method: The solution of the compound to be tested is exposed to (25 mW / cm 2 , 365nm) under ultraviolet light, samples were taken at time points such as 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 120s, etc. HPLC (Eclipse Plus C18, 4.6×250mm, 5μm, MeOH / H2O=60 / 40–80 / 20, 0–10min, MeOH / H2O=80 / 20, 10–18min, 0.5mL / min, 254nm) was used to determine the content changes of each component in the compound solution at each time point. After calculating the concentration of compound A1 at the above time points, Graphpad Prism was used to draw a graph to calculate the photolysis rate of compound A1.

[0157] The results are as follows Figure 2 As shown, Figure 2The figure is a schematic diagram of the photolysis and photolysis pathway of compound A1 under ultraviolet light. A is the photolysis of compound A1 after different illumination times under HPLC monitoring, which is used to determine the photolysis efficiency of compound A1 under ultraviolet light. Compound A1 can be quickly converted into FLC (within 2 minutes); B is the concentration of unphotolyzed compound A1 after different illumination times; C is the natural logarithm of the concentration of compound A1 in Figure B and plotted against time, indicating that the photolysis of compound A1 conforms to the first-order reaction rate equation; D is an analysis of the possible photolysis process of compound A1, which is excited to a triplet state by light, then undergoes H transfer, and then releases FLC after β elimination and fragmentation.

[0158] Embodiment 15

[0159] Effect of the preferred compound A1 on the time growth curve under illumination and non-illumination conditions

[0160] (1) Test compound: Compound A1 was dissolved in DMSO to a solution with a concentration of 2 mg / mL.

[0161] (2) Test strain: Candida albicans (SC5314).

[0162] (3) Experimental method: The activation of the strain to be tested was the same as step (3) in Example 12. The activated Candida albicans cells SC5314 were centrifuged and washed three times with PBS buffer. RPMI 1640 culture medium was used to dilute the culture medium to a bacterial concentration of 1×10 5 or 5×10 4 cells / mL. Take several 15mL centrifuge tubes, add 5mL of bacterial suspension and a certain volume of the drug A1 to be tested (to make its concentration 4, 8, 16μg / mL) to each tube. The light-treated group was exposed to ultraviolet light (50mW / cm 2 The samples were shaken and cultured at 30°C (200 rpm). Part of the cell suspension (10 μL) was taken at 0, 4, 6, 8, 10, 12, 24, 36, and 48 h, and diluted to appropriate times. The cell number was detected, the concentration was calculated, and the logarithm was taken. GraphPad Prism was used to plot the graph.

[0163] The results are as follows Figure 3 As shown, Figure 3 Schematic diagram of the time growth curve of compound A1 with different concentrations after light activation at different time points. Figure 3 In Figure A, the time growth curves of fungi treated with light or without light were measured at different drug concentrations (4, 8, and 16 μg / mL) and the appropriate drug concentration (8 μg / mL) was determined. 5cells / mL, compound A1 showed a better inhibitory effect on fungal growth after illumination. Figure 3 The concentration of compound A1 in medium B was 8 μg / mL, and the initial bacterial concentration was reduced to 5×10 4 cells / mL, in order to optimize the antifungal effect of the drug. Light irradiation was performed at different time points (0, 4, 6, 8, 10, 12h) after administration, and the resulting time growth curve showed that the growth of the fungus before light irradiation was consistent with that of the control group. After 4-6h of light treatment, the fungal growth slowed down and separated from the growth of the control group. As light activation was delayed, the inhibitory effect of compound A1 on fungal growth gradually weakened, which was due to the increase in bacterial concentration caused by the proliferation of fungi before light treatment. This antibacterial effect that changes with the time point of light irradiation proves the temporal adjustability of the light cage photoprotection strategy.

[0164] Example 16

[0165] Effect of preferred compound A1 on fungal hyphae formation under illumination and non-illumination conditions

[0166] The transition from yeast state to hyphae state exists in the whole process of Candida albicans invading biological organisms and plays an important role in diffusion and colonization. Therefore, the effect of compound A1 on fungal hyphae generation was tested. Candida albicans cells SC5314 in the late exponential growth stage were resuspended in Spider medium at a concentration of 1×10 5 cells / mL. The bacterial suspension was transferred to a transparent 12-well plate, 1 mL per well. Compound A1 and FLC were then placed under ultraviolet light (50 mW / cm 2 , 365nm, 2min) or non-ultraviolet light. The samples were divided into six groups, namely Control (black), Control (hv), A1 (16μg / mL, hv), A1 (16μg / mL, black), FLC (16μg / mL, hv) and FLC (16μg / mL, black). The samples were cultured at 37℃ for 2h. The mycelial formation was observed using an inverted fluorescence microscope and photographed and recorded.

[0167] The experimental results are as follows Figure 4 As shown, Figure 4 Schematic diagram of the effect of compound A1 on hyphae formation of Candida albicans under illumination and non-illumination conditions. In the Spider medium that induces hyphae formation of Candida albicans, the morphological transformation from yeast state to hyphae state can still be observed in the group treated with photocaged compound A1, while FLC and light-activated compound A1 successfully blocked the morphological transformation process of Candida albicans. Therefore, the photocaged modification of FLC achieves precise external optical regulation of fungal morphological transformation.

[0168] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. An antifungal compound having photoresponsive activity or a pharmaceutically acceptable salt thereof, characterized in that: The general structural formula is shown below: in, Y3 is selected from -(CH2)n-; n is selected from 1, 2, 3, 4, 5, 6; R 12 is selected from hydrogen, methyl, ethyl, n-propyl; R 13 Selected from hydrogen, methyl, ethyl, n-propyl.

2. The antifungal compound having photoresponsive activity or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The antifungal compound having light-responsive activity is selected from one of the following structures:

3. Use of an antifungal compound having photoresponsive activity or a pharmaceutically acceptable salt thereof according to claim 1 or 2 in the preparation of an antifungal drug, characterized in that: The fungus is selected from Cryptococcus neoformans and Candida albicans.

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