Azole compound and its synthesis and pharmaceutical application

Abstract

The present invention relates to an azole compound and its synthesis and pharmaceutical application. The azole compound has a molecular structure shown in Formula I: wherein at least one R1 is present, and each R1 is independently selected from hydrogen, C1-C4 alkyl, cyclohexyl, halogen-substituted C1-C3 alkyl, C2-C4 alkenyl, halogen-substituted C2-C4 alkenyl, nitro, amino, halogen, methylamino, hydroxyl, carboxyl, hydroxyl-substituted C1-C3 alkyl, phenyl, methylphenyl, benzyl, C1-C3 alkoxy, C2-C4 alkenyloxy, sulfonic acid, nitrophenyl, formyl, acetyl, benzoyl, benzyloxycarbonyl, benzyloxy, phenoxy, formyl Oxy, acetoxy, benzoyloxy, mercapto, methylsulfonyl, sulfonic acid, C1-C3 alkylamino, di-C1-C3 alkyl substituted amino, tert-butyloxycarbonyl; a=0-4; b=0-4; at least one R2, each R2 is independently selected from C1-C3 alkyl, halogen, amino, methylamino, dimethylamino, hydroxyl, carboxyl, nitro, cyano, haloalkyl; X1 is ‑(CH2)n‑ or ‑(CH2)n‑O‑ or ‑O‑(CH2)n‑, n=0‑4; X2 is ‑(CH2)m‑ or ‑C(O)‑(CH2)m‑, m=0‑4; Y is C or N. #imgabs0#

Description

Technical Field

[0001] The invention relates to the field of heterocyclic compounds, in particular to azole antifungal compounds and preparation methods thereof, and belongs to the field of synthesis of pharmaceutical compounds. Background Art

[0002] Fungal infection is a disease common to humans and animals. Common pathogenic fungi in humans include Candida, ringworm, Cryptococcus, etc. Fungal skin diseases in fur animals are common animal diseases in clinical practice, and the main pathogens are Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, and Trichophyton verrucosum. After infection, it can cause skin ringworm in humans, cattle, horses, pigs, cats, dogs, etc., and the infection is relatively stubborn and difficult to cure. It is easy to cross-infect and spread. If not treated in time, it may induce systemic infection, which is very harmful.

[0003] The treatment of fungal infections requires a long cycle, a complex process, and high costs. Ketoconazole, itraconazole, and fluconazole are commonly used in human clinical practice, but obvious drug resistance has emerged; terbinafine, ketoconazole, and itraconazole are commonly used in veterinary clinical practice for treatment, and there is a lack of animal-specific antifungal compounds. On the one hand, it conflicts with Article 41 of the "Regulations on Veterinary Drug Administration" on "prohibiting the use of human drugs in animals". On the other hand, most antifungal drugs used in clinical practice have fungal resistance, adverse drug reactions, narrow antibacterial spectrum, low tissue concentration, and oral non-absorption. This has led to the inappropriate use of a large number of antifungal drugs, which have gradually shown drug abuse problems and seriously endangered the normal application and development of drugs.

[0004] Therefore, it is necessary to develop antifungal drugs with high safety, resistance to drug resistance, and convenience of use, especially new antifungal drugs for animals. In particular, how to achieve high antifungal efficiency and low toxicity is of great significance to animal husbandry. If a drug that can effectively treat animal fungal infections can be developed, it can greatly reduce the problems of slow growth and animal death caused by fungal infections in animals in the breeding industry, thereby reducing the cost of the breeding industry and improving economic benefits. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art, which lacks antifungal drugs specifically for animals, and the current situation that the existing human medicines used for antifungal treatment of animals also have drug resistance and adverse reactions, and to provide a new type of nitrogen azole compound. The compound can effectively inhibit Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes and Candida albicans, and has low toxicity, and can be used for antifungal applications, especially antifungal applications in veterinary clinics.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] An azole compound having a molecular structure shown in Formula I:

[0008]

[0009] Among them, there is at least one R1, and R1 is independently selected from hydrogen, C1-C4 alkyl, cyclohexyl, halogen-substituted C1-C3 alkyl, C2-C4 alkenyl, halogen-substituted C2-C4 alkenyl, nitro, cyano, amino, halogen, methylamino, hydroxyl, carboxyl, hydroxy-substituted C1-C3 alkyl, phenyl, methylphenyl, benzyl, C1-C3 alkoxy, C2-C4 alkenyloxy, sulfonic acid, nitrophenyl, formyl, acetyl, benzoyl, benzyloxycarbonyl, benzyloxy, phenoxy, formyloxy, acetoxy, benzoyloxy, mercapto, methylsulfonyl, sulfonic acid, C1-C3 alkylamino, di-C1-C3 alkyl-substituted amino, and tert-butoxycarbonyl.

[0010] a represents the number of R1, a=0-4; b represents the number of R2, b=0-4.

[0011] There is at least one R2, and each R2 is independently selected from C1-C3 alkyl, halogen, amino, methylamino, dimethylamino, hydroxyl, carboxyl, nitro, cyano, and halogenated alkyl.

[0012] X1 is -(CH2)n- or -(CH2)nO- or -O-(CH2)n-, and n=0-4.

[0013] X2 is -(CH2)m- or -C(O)-CH(CH3)-(CH2)m-, m=0-4.

[0014] Y is C or N.

[0015] Further, each of the R1s is independently selected from the group consisting of hydrogen, methyl, halogen-substituted methyl, ethyl, n-propyl, isopropyl, isobutyl, allyl, propynyl, vinyl, sec-butyl, cyclohexyl, benzyl, propenyl, tert-butyl, isopropenyl, ethynyl, phenyl, p-tolyl, m-tolyl, o-tolyl, o-nitrophenyl, formyl, acetyl, benzoyl, carboxyl, benzyloxycarbonyl, cyano, amino, methylamino, ethylamino, propylamino, dimethylamino, diethylamino, nitro, hydroxyl, methoxy, ethoxy, benzyloxy, phenoxy, formyloxy, mercapto, fluoro, chloro, bromo, iodo, tert-butoxycarbonyl, and fluoromethyl.

[0016] Preferably, the R1 is independently selected from the group consisting of hydrogen, hydroxyl, cyano, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, fluorine, chlorine, bromine, trifluoromethyl, perfluoroethyl, amino, tert-butyloxycarbonyl and the like.

[0017] Further, each of the R2 groups is independently selected from the group consisting of C1-C3 alkyl, halogen, amino, nitro, cyano, fluoro, chloro, bromo, iodo, hydroxyl, and perfluoro C1-C3 alkyl groups. Perfluoro C1-C3 alkyl groups include perfluoromethyl, perfluoroethyl, perfluoro-n-propyl, and perfluoro-isopropyl.

[0018] Furthermore, X1 and X2 are located at the ortho position or para position of the benzene ring. X1 and X2 are located at the ortho position or para position of the benzene ring on the commonly connected benzene ring.

[0019] Furthermore, a represents the number of R1, a=0-3. Preferably, a=0, 1, 2.

[0020] Furthermore, b represents the number of R2, b = 0-3. Preferably, b = 0, 1, 2.

[0021] Furthermore, X1 is selected from methylene, ethylene, methyleneoxy, and ethyleneoxy. It may be methylene or ethylene, or methyleneoxy or ethyleneoxy, and ethyleneoxy means -CH2-CH2-O-.

[0022] Furthermore, X2 is selected from -C(O)-CH(CH2)-, methylene, and ethylene.

[0023] Furthermore, n=0-3. Preferably, n=0, 1, 2.

[0024] Further, m = 0-3. Preferably, m = 0, 1, 2. For example, when m = 0, X2 can be -C(O)-CH(CH3)-.

[0025] Furthermore, the azole compound is a compound having a molecular structure shown in Formula II or Formula III:

[0026]

[0027] Furthermore, the substituent connected to the azole ring and the substituent connected to the left benzene ring are connected at the ortho position or the para position on the middle benzene ring. That is, the azole compound is a compound having a molecular structure shown in Formula IV or Formula V:

[0028]

[0029] Further, wherein n=0-3;

[0030] Y is C or N;

[0031] R1 is at least one, and R1 is independently selected from hydrogen, C1-C4 alkyl, cyclohexyl, halogen-substituted C1-C3 alkyl, C2-C4 alkenyl, halogen-substituted C2-C4 alkenyl, nitro, cyano, amino, halogen, methylamino, hydroxy, carboxyl, hydroxy-substituted C1-C3 alkyl, phenyl, methylphenyl, benzyl, C1-C3 alkoxy, C2-C4 alkenyloxy, sulfonic acid, nitrophenyl, formyl, acetyl, benzoyl, benzyloxycarbonyl, benzyloxy, phenoxy, formyloxy, acetoxy, benzoyloxy, mercapto, methylsulfonyl, sulfonic acid, C1-C3 alkylamino, di-C1-C3 alkyl-substituted amino, and tert-butoxycarbonyl.

[0032] a represents the number of R1, a=0-4; b represents the number of R2, b=0-4.

[0033] There is at least one R2, and each R2 is independently selected from C1-C3 alkyl, halogen, amino, methylamino, dimethylamino, hydroxyl, carboxyl, nitro, cyano, and halogenated alkyl.

[0034] Further, each of the R1s is independently selected from the group consisting of hydrogen, methyl, halogen-substituted methyl, ethyl, n-propyl, isopropyl, isobutyl, allyl, propynyl, vinyl, sec-butyl, cyclohexyl, benzyl, propenyl, tert-butyl, isopropenyl, ethynyl, phenyl, p-tolyl, m-tolyl, o-tolyl, o-nitrophenyl, formyl, acetyl, benzoyl, carboxyl, benzyloxycarbonyl, cyano, amino, methylamino, ethylamino, propylamino, dimethylamino, diethylamino, nitro, hydroxyl, methoxy, ethoxy, benzyloxy, phenoxy, formyloxy, mercapto, fluoro, chloro, bromo, iodo, tert-butoxycarbonyl, fluoromethyl, and fluoroethyl.

[0035] Among them, the fluoromethyl group includes perfluoromethyl group, and the fluoroethyl group includes perfluoroethyl group.

[0036] Preferably, the R1 is independently selected from the group consisting of hydrogen, hydroxyl, cyano, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, fluorine, chlorine, bromine, trifluoromethyl, amino, tert-butyloxycarbonyl and the like.

[0037] Furthermore, each of the R2 groups is independently selected from the group consisting of C1-C3 alkyl, halogen, amino, nitro, cyano, fluoro, chloro, bromo, iodo, hydroxyl, and perfluoro C1-C3 alkyl.

[0038] Furthermore, a represents the number of R1, a=0-3. Preferably, a=0, 1, 2.

[0039] Furthermore, b represents the number of R2, b = 0-3. Preferably, b = 0, 1, 2. For example, b = 0, there is no substitution on the middle benzene ring.

[0040] Furthermore, n = 0, 1, 2. For example, n = 1.

[0041] Further, Y is C.

[0042] Further, Y is N.

[0043] The substituents of the compounds of the above formula I, formula II, formula III, formula IV or formula V may be replaced with each other.

[0044] Further, the azole compound is one of the following compounds:

[0045]

[0046] Another object of the present invention is to use the above azole compounds in the preparation of medicines.

[0047] The above-mentioned azole compounds are used in the preparation of drugs, especially in the preparation of animal antifungal drugs, have excellent antibacterial activity, can effectively treat fungal skin diseases of fur animals, and have the advantages of low toxicity and high efficiency.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] 1. The azole compounds of the present invention can effectively solve the problem of the lack of antifungal chemical drugs for animals in the prior art. The nitrogen azole compounds screened by the present invention have the characteristics of low toxicity and high efficiency, and are more suitable for use as active ingredients of antifungal drugs for animals.

[0050] 2. Animal experiments have shown that the azole compounds of the present invention have better antibacterial activity than the clinical first-line drugs fluconazole, itraconazole and ketoconazole, and have good effects on fungal infections in fur animals, especially the mycelial growth inhibition of Microsporum canis, Trichophyton mentagrophytes, Microsporum gypseum and Candida albicans.

[0051] 3. The azole compounds of the present invention have less toxicity to normal cells and are better than ketoconazole. Animal experiments show that the azole compounds of the present invention have excellent selectivity, and have lower cytotoxicity while effectively inhibiting bacteria. They are applied to animal skin diseases without affecting the normal growth of animals, and have significant economic benefits for concentrated breeding farms. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is the docking diagram of compound 4 and 1EQP.

[0053] Figure 2 This is the docking diagram of compound 4 and 1CZ1.

[0054] Figure 3 This is the docking diagram of compound 4 and 1AI9.

[0055] Figure 4 This is the docking diagram of compound 4 and 4IIB. DETAILED DESCRIPTION

[0056] Another object of the present invention is to provide a method for synthesizing the above-mentioned azole compounds, comprising the following steps: substituted phenylmethyl chloride or substituted phenylmethyl bromide, and imidazole or triazole, reacting under the catalytic action of sodium hydroxide in an acetonitrile solution to obtain an azole compound.

[0057] Wherein, the substituents of substituted phenylmethyl chloride or substituted phenylmethyl bromide are: benzyl, phenyl, tert-butyloxycarbonylphenyl, benzyloxy,

[0058] The halogen mentioned in the present invention refers to fluorine, chlorine, bromine and iodine, and the halogen substitution refers to halogen substitution. Specifically, the halogen refers to fluorine substitution, chlorine substitution, bromine substitution and iodine substitution. It can be single substitution or multiple substitutions, and can be one halogen substitution or a mixture of multiple halogen substitutions.

[0059] The C1-C3 alkyl group refers to: methyl, ethyl, n-propyl, isopropyl.

[0060] The halogen-substituted C1-C3 alkyl group refers to a C1-C3 alkyl group substituted with a halogen, such as fluorine-substituted, chlorine-substituted, bromine-substituted, or iodine-substituted.

[0061] The C1-C3 alkoxy group refers to: methoxy group, ethoxy group, n-propyloxy group, isopropyloxy group.

[0062] The C2-C4 alkenyl group refers to: vinyl, propenyl, butenyl, 1-butenyl, and 2-butenyl.

[0063] The C2-C4 alkenyloxy group refers to vinyloxy, propenyloxy, butenyloxy and the like.

[0064] The present invention is further described in detail below in conjunction with test examples and specific implementation methods. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0065] Example 1

[0066] Synthesis of Imidazole Compounds 1-4

[0067] The basic process of the reaction is shown below.

[0068]

[0069] Synthesis route of imidazole compounds

[0070] (1) Add 1.2 eq (reaction equivalent, abbreviated as equivalent, the same below) of imidazole to a microwave reaction tube, dissolve it in an appropriate amount of acetonitrile, and then add 1 eq of sodium hydroxide to activate it for 0.5 h. Then add 1 eq of 1-benzyl-2-chloromethylbenzene to the reaction tube, react at room temperature for 5 h, dilute with an appropriate amount of distilled water, extract with ethyl acetate three times, wash with water, dry, and separate the crude product by column chromatography to obtain a benzyl imidazole derivative (1), i.e., compound 1. The structure of compound 1 is 1 H NMR, 13 C NMR and HR-MS confirmed.

[0071] (2) Add 1.2 eq of imidazole to a microwave reaction tube, dissolve it in an appropriate amount of acetonitrile, and then add 1 eq of sodium hydroxide to activate it for 0.5 h. Then add 1 eq of 2-bromomethyl-1,1'-biphenyl to the reaction tube, react at room temperature for 5 h, dilute with an appropriate amount of distilled water, extract with ethyl acetate three times, wash with water, dry, and separate the crude product by column chromatography to obtain a benzyl imidazole derivative (2), namely compound 2. The structure of compound 2 is 1 H NMR, 13 C NMR and HR-MS confirmed.

[0072] (3) Add 1.2 eq of imidazole to a microwave reaction tube, dissolve it in an appropriate amount of acetonitrile, and then add 1 eq of sodium hydroxide to activate it for 0.5 h. Then add 1 eq of 4'-bromomethyl-[1,1'-biphenyl]-2-carboxylic acid tert-butyl ester to the reaction tube, react at room temperature for 5 h, dilute with an appropriate amount of distilled water, extract with ethyl acetate three times, wash with water, dry, and separate the crude product by column chromatography to obtain a benzyl imidazole derivative (3), i.e., compound 3. The structure of the compound is 1 H NMR, 13 C NMR and HR-MS confirmed.

[0073] (4) Add 1.2 eq of imidazole to a microwave reaction tube, dissolve it in an appropriate amount of acetonitrile, and then add 1 eq of sodium hydroxide to activate it for 0.5 h. Then add 1 eq of 1-(4-benzyloxy)phenyl-2-bromo-1-propanone to the reaction tube, react at room temperature for 5 h, dilute with an appropriate amount of distilled water, extract with ethyl acetate three times, wash with water, dry, and separate the crude product by column chromatography to obtain a benzyl imidazole derivative (4), i.e., compound 4. The structure of compound 4 is 1 H NMR, 13 C NMR and HR-MS confirmed.

[0074] 1 H NMR, 13 C NMR and HR-MS, the results of test compounds 1-4 are as follows:

[0075] Compound 1, HR-MS m / z: calculated for C17 H 16 N2Na 271.1211, found 271.1206 [M+Na] + ; 1 H NMR (600 MHz, Chloroform-d) δ 7.58 (d, J=1.2 Hz, 1 H), 7.55–7.40 (m, 6H), 7.30–7.24 (m, 3H), 7.19–7.15 (m, 1 H), 6.95 (t, J=1.3 Hz, 1 H), 5.21 (s, 2H), 4.17 (s, 2H). 13 C NMR (151 MHz, Chloroform-d) δ 139.36, 138.24, 137.38, 134.24, 131.09, 129.53, 128.64, 128.58, 128.52, 128.42, 127.25, 126.42, 119.21, 48.31, 38.73.

[0076] Compound 2, HR-MS m / z: calculated for C 16 H 14 N2Na 257.1055, found 257.1059 [M+Na] + ; 1 H NMR (600 MHz, Chloroform-d) δ 7.50–7.38 (m, 5H), 7.35 (dd, J=7.3, 1.7 Hz, 1 H), 7.29 (s, 1 H), 7.27–7.23 (m, 2H), 7.20 (dd, J=7.6, 1.5 Hz, 1 H), 7.05 (d, J=1.1 Hz, 1 H), 6.75 (d, J=1.3 Hz, 1 H), 5.09 (s, 2H). 13 C NMR (151 MHz, Chloroform-d) δ 141.81, 139.89, 137.15, 133.26, 130.33, 129.25, 128.77, 128.45, 128.38, 128.19, 127.91, 127.50, 118.92, 48.62.

[0077] Compound 3, HR-MS m / z: calculated for C2 1 H 22N2NaO2 357.1579, found 357.1579 [M+Na] + ; 1 H NMR (600 MHz, Chloroform-d) δ 7.78 (dd, J=7.7, 1.4 Hz, 1 H), 7.58 (d, J=1.2 Hz, 1 H), 7.48 (td, J=7.6, 1.5 Hz, 1 H), 7.39 (td, J=7.6, 1.3 Hz, 1 H), 7.31–7.26 (m, 2H), 7.18 (m, 2H), 7.08 (m, 2H), 6.94 (d, J=1.3 Hz, 1 H), 5.16 (s, 2H), 1.24 (s, 9H). 13 C NMR (151 MHz, Chloroform-d) δ 168.08, 142.37, 141.56, 137.66, 135.26, 133.08, 131.13, 130.78, 130.09, 130.07, 129.55, 127.73, 127.28, 119.59, 81.70, 50.96, 27.95.

[0078] Compound 4, HR-MS m / z: calculated for C 19 H 18 N2NaO2 329.1267, found 329.1266 [M+Na] + ; 1 H NMR (600 MHz, Chloroform-d) δ 7.94–7.89 (m, 2H), 7.63 (s, 1 H), 7.44–7.37 (m, 4H), 7.35 (ddt, J=8.5, 5.6, 2.1 Hz, 1 H), 7.08 (s, 1 H), 7.05–6.99 (m, 3H), 5.76 (q, J=7.1 Hz, 1 H), 5.13 (s, 2H), 1.75 (d, J=7.1 Hz, 3H). 13C NMR(151MHz,Chloroform-d)δ193.56,193.52,163.51,163.46,136.49,136.45,135.93,135.89,131.03,130.99,129.61,129.57,128.85,128.81, 128.48,128.44,127.57,127.53,127.24,127.21,118.30,118.26,115.21 ,115.18,77.34,77.12,76.91,70.37,70.34,55.88,55.84,19.34,19.30.

[0079] Example 2

[0080] Synthesis of 1,2,4-triazole derivatives (Compound 5-8)

[0081] The basic process of the elementary reaction is shown below.

[0082]

[0083] Triazole synthesis route

[0084] 1.2 eq 1,2,4-triazole was added to a microwave reaction tube and dissolved in an appropriate amount of acetonitrile. Then 1 eq sodium hydride was added to activate for 0.5 h. Then 1 eq 1-benzyl-2-chloromethylbenzene was added to the reaction tube, and the mixture was reacted at 50° C. for 5 h. The mixture was diluted with an appropriate amount of distilled water, extracted with ethyl acetate three times, washed with water, dried, and the crude product was separated by column chromatography to obtain a benzyltriazole derivative (5), i.e., compound 5. The structure of compound 5 is 1 H NMR, 13 C NMR and HR-MS confirmed.

[0085] 1.2 eq 1,2,4-triazole was added to a microwave reaction tube and dissolved in an appropriate amount of acetonitrile. Then 1 eq sodium hydride was added to activate for 0.5 h. Then 1 eq 2-bromomethyl-1,1'-biphenyl was added to the reaction tube, reacted at 50° C. for 5 h, diluted with an appropriate amount of distilled water, extracted with ethyl acetate three times, washed with water, dried, and the crude product was separated by column chromatography to obtain a benzyltriazole derivative (6), namely compound 6. The structure of compound 6 is 1 H NMR, 13 C NMR and HR-MS confirmed.

[0086] 1.2 eq 1,2,4-triazole was added to a microwave reaction tube and dissolved in an appropriate amount of acetonitrile. Then 1 eq sodium hydride was added to activate for 0.5 h. Then 1 eq 4'-bromomethyl-[1,1'-biphenyl]-2-carboxylic acid tert-butyl ester was added to the reaction tube, and the mixture was reacted at 50° C. for 5 h. The mixture was diluted with an appropriate amount of distilled water, extracted with ethyl acetate three times, washed with water, dried, and the crude product was separated by column chromatography to obtain a benzyltriazole derivative (7), i.e., compound 7. The structure of compound 7 is shown in FIG. 1 H NMR, 13 C NMR and HR-MS confirmed.

[0087] 1.2 eq 1,2,4-triazole was added to a microwave reaction tube and dissolved in an appropriate amount of acetonitrile. Then 1 eq sodium hydride was added to activate for 0.5 h. Then 1 eq 1-(4-benzyloxy)phenyl-2-bromo-1-propanone was added to the reaction tube. The reaction was carried out at 50° C. for 5 h. The mixture was diluted with an appropriate amount of distilled water, extracted with ethyl acetate three times, washed with water, dried, and the crude product was separated by column chromatography to obtain a benzyltriazole derivative (8), i.e., compound 8. The structure of compound 8 is shown in FIG. 1 H NMR, 13 C NMR and HR-MS confirmed.

[0088] 1 H NMR, 13 C NMR and HR-MS, the results of test compounds 5-8 are as follows:

[0089] Compound 5, HR-MS m / z: calculated for C 16 H 16 N3 250.1340,found 250.1344[M+H] + ; 1 H NMR(600MHz,Chloroform-d)δ7.93(s, 1 H),7.68(s, 1 H),7.34(td,J=7.5,1.4Hz, 1 H),7.31–7.18(m,5H),7.15(dd,J=7.6,1.4Hz, 1 H),7.10–7.04(m,2H),5.26(s,2H),4.03(s,2H). 13 C NMR (151MHz, Chloroform-d) δ172.45,145.03,125.82,116.51,112.21,109.71,109.39,108.42,106.74,96.37,51.33,42.15.

[0090] Compound 6, HR-MS m / z: calculated for C 15 H 13 N3Na 258.0990, found 258.1007 [M+Na] + ; 1 1H NMR (600 MHz, Chloroform-d) δ 7.89 (s, 1 H), 7.61 (s, 1 H), 7.49–7.34 (m, 5H), 7.34–7.28 (m, 2H), 7.25–7.19 (m, 2H), 5.29 (s, 2H). 13 13C NMR (151 MHz, Chloroform-d) δ 151.89, 143.01, 142.04, 139.85, 131.83, 130.42, 129.34, 128.83, 128.64, 128.52, 128.04, 127.66, 51.33.

[0091] Compound 7, HR-MS m / z: calculated for C 20 H 21 N3NaO2 358.1540, found 358.1531 [M+Na] + ; 1 1H NMR (600 MHz, Chloroform-d) δ 8.17 (s, 1 H), 8.13 (s, 1 H), 7.99 (s, 1 H), 7.78 (dd, J=7.7, 1.4 Hz, 1 H), 7.48 (td, J=7.5, 1.4 Hz, 1 H), 7.39 (td, J=7.6, 1.3 Hz, 1 H), 7.35–7.27 (m, 4H), 5.39 (s, 2H), 1.23 (s, 9H). 13 13C NMR (151 MHz, Chloroform-d) δ 167.88, 152.11, 143.07, 142.55, 141.30, 133.43, 132.80, 130.96, 130.56, 129.89, 129.41, 127.82, 127.56, 81.57, 53.55, 29.78, 27.71.

[0092] Compound 8, HR-MS m / z: calculated for C 18 H17 N3NaO2 330.1218,found 330.1215[M+Na] + ; 1 H NMR(600MHz,Chloroform-d)δ8.35(s, 1 H),8.21(s, 1 H),8.07–7.88(m,3H),7.49–7.32(m,4H),7.17–6.92(m,2H),6.14(q,J=7.3Hz, 1 H),5.15(s,2H),1.83(d,J=7.3Hz,3H). 13 C NMR (151MHz, Chloroform-d) δ193.14,163.95,151.68,142.95,136.15,131.54,129.11,128.74,127.83,127.21,115.51,70.65,59.50,18.89.

[0093] Example 3

[0094] Preparation of derivatives of compound 4

[0095] 1. Fluorine-substituted derivatives of compound 4

[0096] The basic process of the reaction is as follows:

[0097]

[0098] Fluorine substituted derivatives

[0099] 1.2 eq N-fluoropyridine trifluoromethanesulfonate was added to a microwave reaction tube and dissolved in an appropriate amount of anhydrous dichloromethane. Then 1 eq of compound 4 was added to the reaction tube, and the mixture was reacted at 40°C for 20 h. The mixture was diluted with an appropriate amount of saturated sodium chloride, extracted with ethyl acetate three times, washed with water, dried, and the crude product was separated by column chromatography to obtain fluorine-substituted compounds 9 and 10 of compound 4, i.e., compounds 9 and 10, whose structures are shown in Table 1. 1 H NMR, 13 C NMR and HR-MS confirmed.

[0100] 1 H NMR, 13 C NMR and HR-MS, the results of test compounds 9-10 are as follows:

[0101] Compound 9, HR-MS m / z: calculated for C 19 H 16F2N2NaO2 365.1078,found 365.1083[M+Na] + ; 1 H NMR(500MHz,Chloroform-d)δ8.01–7.73(m,2H),7.66(t,J=1.6Hz, 1 H),7.42(dd,J=4.0,1.6Hz, 1 H),7.34(dt,J=7.7,1.0Hz, 1 H),7.19–7.02(m,3H),6.98–6.84(m,2H),5.74(q,J=5.3Hz, 1 H),5.22(d,J=1.1Hz,2H),1.60(s,3H); 13 C NMR(125MHz,Chloroform-d)δ194.03,162.82,161.64,160.74,137.27,131.30,130 .51,129.19,128.90,120.95,120.24,115.34,111.72,104.81,63.80,56.84,20.59.

[0102] Compound 10, HR-MS m / z: calculated for C 19 H 17 FN2NaO2 347.1172,found 347.1175[M+Na] + ; 1 H NMR(500MHz,Chloroform-d)δ7.92–7.87(m,2H),7.66(t,J=1.6Hz, 1 H),7.51–7.37(m,3H),7.09–7.01(m,4H),5.74(q,J=5.3Hz, 1 H), 5.07 (t, J = 1.0Hz, 2H), 1.60 (s, 3H).; 13 C NMR(125MHz,Chloroform-d)δ194.03,163.06,162.71,137.27,133.69,131.30,130.11,129.19,128.90,120.95,115.40,115.33,70.02,56.84,20.59.

[0103] 2. Chloro-substituted derivatives of compound 4

[0104] The basic process of the reaction is as follows:

[0105]

[0106] Chlorine substituted derivatives

[0107] Add 0.039 mol of calcium hypochlorite, 100 mL of water, and 10 mL of acetic acid to a flask equipped with a stirrer and a condenser. Then immerse the flask in an ice water bath (0°C) and stir until the contents are completely dissolved and a light yellow solution is obtained. Then add 0.039 mol of an acetone solution of compound 4 over three minutes and stir at 0°C. 1 h. The reaction mixture was then diluted with water, and an insoluble oily substance was found at the bottom of the bottle. The mixture was then extracted with ether, washed with saturated sodium bicarbonate, and dried. The crude product was separated by column chromatography to obtain the chlorine-substituted compounds 11 and 12 of compound 4, namely compounds 11 and 12, whose structures are 1 H NMR, 13 C NMR and HR-MS confirmed.

[0108] 1 H NMR, 13 C NMR and HR-MS, the test results of compounds 11 and 12 are as follows:

[0109] Compound 11, HR-MS m / z: calculated for C 19 H 16 Cl2N2NaO2 397.0487,found397.0483[M+Na]+; 1 H NMR(500MHz,Chloroform-d)δ7.96–7.86(m,2H),7.66(t,J=1.6Hz, 1 H),7.48(dt,J=8.3,1.1Hz, 1 H),7.45–7.37(m,2H),7.33(dd,J=8.3,2.1Hz, 1 H),7.09–7.03(m,3H),5.74(q,J=5.3Hz, 1 H), 5.20 (t, J = 0.8Hz, 2H), 1.60 (s, 3H).; 13 C NMR(125MHz,Chloroform-d)δ194.03,162.82,137.27,135.03,133.92,131.33,131 .30,131.21,129.19,128.90,128.05,127.41,120.95,115.33,68.73,56.84,20.59.

[0110] Compound 12, HR-MS m / z: calculated for C 19 H 17 ClN2NaO2 363.0876,found363.0874[M+Na]+; 1 H NMR(500MHz,Chloroform-d)δ7.95–7.85(m,2H),7.66(t,J=1.6Hz, 1 H),7.47–7.32(m,5H),7.11–6.93(m,3H),5.75–5.70(m, 1 H), 5.07 (t, J = 1.0Hz, 2H), 1.60 (s, 3H).; 13 C NMR(125MHz,Chloroform-d)δ194.03,163.06,137.27,136.11,134.36,131.30,129.87,129.19,128.90,128.64,120.95,115.33,70.02,56.84,20.59.

[0111] 3. Bromine substitution of compound 4

[0112] The basic process of the reaction is as follows

[0113]

[0114] Bromobutyrimide (NBS) and anhydrous FeCl3 (molar ratio: 1:0.1) were placed in a flask equipped with a stirrer, compound 4 was added at a molar ratio (NBS: compound 4 = 3:1), and then acetonitrile was added at 20% of the reaction volume, heated to 90°C, reacted for 7 hours, and extracted with ether after the reaction was completed, and dried over anhydrous sodium sulfate. The crude product was separated by column chromatography to obtain bromine-substituted compounds 13 and 14 of compound 4, namely compounds 13 and 14, whose structures are 1 H NMR, 13 C NMR and HR-MS confirmed.

[0115] 1 H NMR, 13 C NMR and HR-MS, the test results of compounds 13 and 14 are as follows:

[0116] Compound 13, HR-MS m / z: calculated for C 19 H 16 Br2N2NaO2 484.9476,found484.9491[M+Na]+; 1H NMR(500MHz,Chloroform-d)δ7.95–7.88(m,2H),7.66(t,J=1.6Hz, 1 H),7.59(d,J=1.9Hz, 1 H),7.48(dd,J=8.2,1.8Hz, 1 H),7.45–7.37(m,2H),7.13–6.88(m,3H),5.74(q,J=5.3Hz, 1 H), 5.23 (d, J = 1.0Hz, 2H), 1.60 (s, 3H).; 13 C NMR(125MHz,Chloroform-d)δ194.03,162.82,137.27,134.46,132.34,131.30,131 .03,130.91,129.19,128.90,124.97,122.15,120.95,115.33,71.39,56.84,20.59.

[0117] Compound 14, HR-MS m / z: calculated for C 19 H 17 BrN2NaO2 407.0371,found407.0365[M+Na]+; 1 H NMR(500MHz,Chloroform-d)δ7.94–7.88(m,2H),7.66(t,J=1.6Hz, 1 H),7.57–7.47(m,2H),7.47–7.35(m,3H),7.12–7.02(m,3H),5.74(q,J=5.3Hz, 1 H), 5.07 (t, J = 1.0Hz, 2H), 1.60 (s, 3H).; 13 C NMR(125MHz,Chloroform-d)δ194.03,163.06,137.27,136.68,131.92,131.30,130.14,129.19,128.90,123.33,120.95,115.33,70.02,56.84,20.59.

[0118] 4. Amino Substitution of Compound 4

[0119] The basic process of the reaction is as follows:

[0120]

[0121] Amino substituted derivatives

[0122] Compound 4 (0.20 mmol), trimethylsilyl azide (TMSN3, 0.24 mmol), CHCl3 (500 μL) and trifluoromethanesulfonic acid (TfOH, 1.8 mmol) were added to a reaction tube with a stirrer, and then sealed in a vial, and then reacted at 60°C for 20 min. After the reaction, the vial was cooled in an ice bath, and 500 μL of methanol was slowly added under stirring. Wash with an appropriate amount of saturated sodium bicarbonate solution, then extract with dichloromethane, and dry with anhydrous sodium sulfate. The crude product was separated by column chromatography to obtain the amino-substituted compound 15 of compound 4, i.e., compound 15, the structure of which is 1 H NMR, 13 C NMR and HR-MS confirmed.

[0123] 1 H NMR, 13 C NMR and HR-MS, the results of test compound 15 are as follows:

[0124] Compound 15, HR-MS m / z: calculated for C 19 H 19 BrN3NaO2 344.1375,found344.1359[M+Na]+; 1 H NMR(500MHz,Chloroform-d)δ8.02–7.83(m,2H),7.66(t,J=1.6Hz, 1 H),7.42(dd,J=4.0,1.6Hz, 1 H),7.36–7.26(m,2H),7.14–6.98(m,3H),6.61–6.48(m,2H),5.74(q,J=5.3Hz, 1 H),5.07(t,J=1.0Hz,2H),4.17(d,J=5.7Hz, 1 H), 4.07(d, J=5.7Hz, 1 H), 1.60(s,3H).; 13 C NMR(125MHz,Chloroform-d)δ194.03,163.06,147.95,137.27,131.30,129.92,129.19,128.90,128.10,120.95,115.61,115.33,70.02,56.84,20.59.

[0125] 5. Trifluoromethyl Substitution of Compound 4

[0126] The basic process of the synthesis of trifluoromethyl substituted derivatives is as follows:

[0127]

[0128] Compound 4 (100 μL), dimethyl sulfoxide (2.0 ml), 1N dimethyl sulfoxide sulfuric acid solution (2.0 ml), 3.0 mol / l trifluoromethyl iodide dimethyl sulfoxide solution (1.0 ml), 30% hydrogen peroxide aqueous solution (0.2 ml) and 1.0 mol / l ferrous sulfate (II) aqueous solution (0.3 ml) were placed in a double-necked flask, and the mixture was stirred for 20 min under an argon environment. During the stirring process, the temperature of the reaction system was between 40°C and 50°C. Thereafter, the resulting solution was cooled to room temperature. Diluted with an appropriate amount of saturated sodium chloride, extracted three times with ethyl acetate, washed three times with water, dried, and the crude product was separated by column chromatography to obtain trifluoromethyl substituted compounds 16 and 17 of compound 4, namely compounds 16 and 17, the structures of which are shown in Table 1. 1 H NMR, 13 C NMR and HR-MS confirmed.

[0129] 1 H NMR, 13 C NMR and HR-MS, the test results of compounds 16 and 17 are as follows:

[0130] Compound 16, HR-MS m / z: calculated for C 20 H 17 F3N2NaO2 397.1140,found397.1144[M+Na]+; 1 H NMR(500MHz,Chloroform-d)δ7.96–7.85(m,2H),7.72–7.58(m,3H),7.53–7.35(m,3H),7.12–6.98(m,3H),5.74(q,J=5.3Hz, 1 H), 5.07 (t, J = 1.0Hz, 2H), 1.60 (s, 3H).; 13 C NMR(125MHz,Chloroform-d)δ194.03,163.06,137.27,137.10,131.30,130.64, 129.19,128.90,128.86,126.17,124.13,120.95,115.33,70.12,56.84,20.59.

[0131] Compound 17, HR-MS m / z: calculated for C2 1 H 16F6N2NaO2 465.1014,found465.1017[M+Na]+; 1 H NMR(500MHz,Chloroform-d)δ7.94–7.86(m,3H),7.66(t,J=1.6Hz, 1 H),7.51(dd,J=6.4,1.8Hz, 1 H),7.44–7.35(m,2H),7.10–6.99(m,3H),5.74(q,J=5.3Hz, 1 H), 5.28 (t, J = 0.9Hz, 2H), 1.60 (s, 3H).; 13 C NMR(125MHz,Chloroform-d)δ194.03,162.82,137.27,133.06,131.30,130.09,130.06,129 .64,129.19,128.90,125.35,124.16,123.89,123.43,120.95,115.33,69.38,56.84,20.59.

[0132] 6. Cyano substitution of compound 4

[0133] The basic process of the synthesis of cyano substituted derivatives is as follows:

[0134]

[0135] Pd(OAc)2 (4.5 mg, 0.02 mmol, 10 mol%), 3-(trifluoromethyl)quinoline (7.9 mg, 0.040 mmol, 20 mol%), N-acetylglycine (7.0 mg, 0.060 mmol, 30 mol%), AgF (101.5 mg, 0.80 mmol, 4 eq), aromatic hydrocarbons (0.20 mmol, 1 eq), CuCN (35.8 mg, 0.40 mmol, 2 eq) and HFIP (2 mL) were added. The reaction vessel was sealed, and the mixture was stirred at room temperature for 2 minutes, then heated to 90°C, and the reaction mixture was stirred at 1000 rpm for 18 hours at this temperature. The reaction mixture was cooled to room temperature, and the crude product was separated by column chromatography to obtain the cyano-substituted compound 18 of compound 4, i.e., compound 18, the structure of which is 1 H NMR, 13 C NMR and HR-MS confirmed.

[0136] 1 H NMR, 13 C NMR and HR-MS, the results of test compound 18 are as follows:

[0137] Compound 18, HR-MS m / z: calculated for C 20 H 17 N3NaO2 354.1218,found 354.1222[M+Na]+;

[0138] 1 H NMR(500MHz,Chloroform-d)δ7.96–7.85(m,2H),7.69–7.60(m,3H),7.54–7.45(m,2H),7.42(dd,J=4.0,1.6Hz, 1 H),7.10–7.00(m,3H),5.74(q,J=5.3Hz, 1 H), 5.07 (t, J = 1.0Hz, 2H), 1.60 (s, 3H).;

[0139] 13 C NMR(125MHz,Chloroform-d)δ194.03,163.06,139.40,137.27,132.22,131.30, 129.19,129.09,128.90,120.95,118.50,115.33,110.20,70.02,56.84,20.59.

[0140] 7. Hydroxyl Substitution of Compound 4

[0141] The basic process of the reaction of hydroxyl substituted derivatives is as follows.

[0142]

[0143] Benzene (1 mmol) and catalyst Cu-Au@g-C3N4 (30 mg) in acetonitrile (5 mL) were added to a 25 mL round-bottom flask equipped with a magnetic stirrer. Subsequently, 30% H2O2 (1 mmol) was added to the reaction mixture and irradiated with a 20 W household cold LED lamp, and the progress of the reaction was monitored by TLC. After the reaction was completed, the catalyst was separated from the reaction mixture and then centrifuged. It was extracted with ethyl acetate and dried under reduced pressure to obtain the hydroxyl-substituted compound 19 of compound 4, i.e., compound 19, the structure of which is 1 H NMR, 13 C NMR and HR-MS confirmed.

[0144] 1 H NMR, 13 C NMR and HR-MS, the test results of compound 19 are as follows:

[0145] Compound 19, HR-MS m / z: calculated for C 19 H 18 N2NaO3 345.1215,found 345.1221[M+Na]+;

[0146] 1 H NMR(500MHz,Chloroform-d)δ8.79(s, 1 H),7.96–7.83(m,2H),7.66(t,J=1.6Hz, 1 H),7.42(dd,J=4.0,1.6Hz, 1 H),7.36–7.22(m,2H),7.09–7.01(m,3H),6.82–6.60(m,2H),5.74(q,J=5.3Hz, 1 H), 5.07 (t, J = 1.0Hz, 2H), 1.60 (s, 3H).;

[0147] 13 C NMR(125MHz,Chloroform-d)δ194.03,163.06,157.08,137.27,131.30,129.97,129.65,129.19,128.90,120.95,115.90,115.33,70.02,56.84,20.59.

[0148] 8. Alkyl Substitution of Compound 4

[0149] Alkyl substitution process, the basic process of the reaction is as follows,

[0150]

[0151] The reaction was carried out in a continuous flow jet bubbling reactor with a glass tube (8 mm inner diameter) at atmospheric pressure. The catalyst: H-mordenite (H-MOR, 0.5 g) was in situ heat treated from ambient temperature to 673 K (10 K / min). Then a mixture of compound 4:methanol (1:1) was added to the reactor (WHSV = 2.0 HR-1), reacted for 3 h, and cooled to room temperature. The alkyl substituted compound 20 of compound 4 was obtained, i.e., compound 20, the structure of which is 1 H NMR, 13 C NMR and HR-MS confirmed.

[0152] 1 H NMR, 13 C NMR and HR-MS, the results of test compound 20 are as follows:

[0153] Compound 20, HR-MS m / z: calculated for C 20 H 20 N2NaO2 3431422,found 3431427[M+Na]+;

[0154] 1 H NMR(500MHz,Chloroform-d)δ7.96–7.89(m,2H),7.66(t,J=1.6Hz, 1 H),7.42(dd,J=4.0,1.7Hz, 1 H),7.34–7.24(m,2H),7.20–7.12(m,2H),7.12–6.98(m,3H),5.74(q,J=5.3Hz, 1 H),5.06(t,J=1.0Hz,2H),1.59(d,J=5.3Hz,3H).;

[0155] 13 C NMR(125MHz,Chloroform-d)δ194.03,163.06,137.88,137.27,135.07,131.30 ,129.23,129.19,128.90,128.51,120.95,115.33,70.02,56.84,21.04,20.59.

[0156] Example 4

[0157] In vitro antifungal activity assay of synthetic compounds

[0158] 1. Experimental strains

[0159] Standard strain of Microsporum gypseum (M873.579), standard strain of Trichophyton mentagrophytes (M3.859), standard strain of Microsporum canis (ATCC8137), and Candida albicans (Robin) Berkhout (ATCC10231).

[0160] 2 Experimental methods

[0161] 2.1 Preparation of drug solution

[0162] Fluconazole, ketoconazole, itraconazole and the synthesized compound were dried under reduced pressure at 25°C for 12h, the dried samples were accurately weighed, and an appropriate amount of 20% DMSO was added to dissolve, and the volume was fixed to prepare 4mL of stock solutions with drug molar concentrations of 6μmol / mL, 3μmol / mL, 1.5μmol / mL, 0.75μmol / mL, and 0.375μmol / mL, respectively. After filtering with a 0.22μm microporous filter membrane, the samples were stored in a refrigerator at 4°C for later use.

[0163] 2.2 Culture medium preparation

[0164] (1) Preparation of potato agar medium (PDA): Take fresh peeled potatoes, cut into small pieces, weigh 200g, put into 1L distilled water and boil for 30min, filter with three layers of gauze, and make up the distilled water reduced by evaporation to 1L. Add 20g of glucose and 20g of agar to the filtrate, heat it, and when the agar is completely melted, divide it into 250mL conical flasks while hot and plug it with cotton plugs, wrap it with newspaper, and sterilize it in a high-pressure steam sterilizer at 121℃ for 30min. After sterilization, transfer it to a sterile operating table and wait until the temperature of the culture medium drops to about 50℃. Pour the sterile plate and shake it evenly, lay it flat, and make a PDA plate. After solidification, store it at 4℃ for later use.

[0165] (2) Preparation of drug-containing plates: 1 mL of the different drug solutions prepared above and 9 mL of the PDA medium cooled to about 50°C were respectively poured into sterilized culture dishes to prepare drug-containing plates with concentrations of 0.6 μmol / mL, 0.3 μmol / mL, 0.15 μmol / mL, 0.075 μmol / mL, and 0.0375 μmol / mL. After the culture medium was cooled to 50°C, 1 mL of 30% DMSO and 9 mL of PDA medium were added to prepare control plates of the drug-containing plates, which were stored at 4°C after solidification for later use.

[0166] 2.3 Preparation of bacterial suspension

[0167] Take out the required Microsporum gypseum, Trichophyton mentagrophytes, Microsporum canis, and Candida albicans from the refrigerator, use a sterile inoculation loop to inoculate the three fungi onto the SDA plate, and culture at 27°C for 2 weeks to activate the fungi so that the colonies are evenly covered on the culture medium. Pipette 2mL of sterile saline to rinse the hyphae and spores on the surface of the colony, count the washing liquid containing hyphae and spores with a hemocytometer, adjust the concentration to 105-106 CFU / mL, and obtain a bacterial suspension for use.

[0168] 2.4 Determination of mycelial growth rate and antibacterial rate

[0169] The test fungi were inoculated on PDA plates and cultured at 27°C for 5 days. A sterilized punch was used to cut a 1 cm diameter bacterial cake from the edge of the PDA plate inoculated with the test fungi, and the cake was inoculated on the drug-containing culture medium, with the mycelium side facing down, and one bacterial cake was inoculated in each culture dish. At the same time, a blank control, a negative control, and a positive control (fluconazole, itraconazole, ketoconazole) were set up. Three repeated tests were performed and cultured in a constant temperature incubator at 27°C. After 7 days of culture, the colony diameter of the test fungi on the drug-containing culture medium was measured by the cross method, and the inhibition rate of each drug solution treatment on the linear growth of mycelium was calculated by comparing with the control. The mycelium growth rate and the inhibition rate were calculated according to the following formula: Mycelium growth rate (cm / d) = (average measured diameter - 1.0) / number of days; Inhibition rate (%) = (blank control colony diameter - treated colony diameter) ÷ blank control colony diameter × 100.

[0170] 2.5 MIC determination

[0171] Take a sterile 96-well plate, first add 100 μL of PDB medium to each well, then add 100 μL of drug solution to each well in the 1st and 2nd columns, and mix well; perform serial dilutions in columns 2 to 10: take 100 μL of liquid from the second column to the third column, mix well; take 100 μL of liquid from the third column to the fourth column, and so on to the 10th column (concentration range 6000 μmol / L-11.78 μmol / L). Add 100 μL of DMSO to the 11th column, take out 100 μL of liquid after mixing columns 10 and 11, and discard; finally, add 100 μL of bacterial suspension to each well in the 12th column. The 1st column is a blank control, the 11th column is a negative control, and the 12th column is a growth control. The 96-well plate was placed in a 27°C constant temperature incubator for 48 hours, and the minimum drug concentration of the well with clear and transparent solution was the MIC. Three groups of replicates were set for each.

[0172] 3. Antibacterial test results

[0173] 3.1 Effects of compounds 1-8 on mycelial growth rate

[0174] The test results of mycelium growth rate method are shown in Tables 1-3.

[0175] Among them, itraconazole and fluconazole in the positive control group had no obvious inhibitory effect on the hyphal growth of Microsporum canis, Trichophyton mentagrophytes, and Microsporum gypseum, while ketoconazole had a significant inhibitory effect and a better effect, especially on the hyphal growth rate of Trichophyton mentagrophytes. Compounds 1-8 had different degrees of influence on the hyphal growth of these three fungi, and were better than itraconazole and fluconazole. At 6 μmol / mL, compound 5 had a very obvious inhibitory effect on the hyphal growth of Microsporum canis, and compounds 3 and 4 had the greatest influence on the hyphal growth rate of Trichophyton mentagrophytes and Microsporum gypseum, both less than 1 mm / d.

[0176] Table 1 Effects of different concentrations of compounds on the mycelial growth rate of Microsporum canis

[0177]

[0178] Table 2 Effects of different concentrations of compounds on the hyphae growth rate of Trichophyton mentagrophytes

[0179]

[0180] Table 3 Effects of different concentrations of compounds on the mycelial growth rate of Microsporum gypseum

[0181]

[0182]

[0183] 4.1.2 Antibacterial rate of compounds 1-8 against three bacteria

[0184] The results of the inhibition rate determination are shown in Tables 4-6.

[0185] In the positive control group, ketoconazole had the highest antibacterial rate, and its antibacterial effect was better than that of itraconazole and fluconazole. Compounds 1-8 had different degrees of antibacterial effect on the three fungi. At a concentration of 6 μmol / mL, the antibacterial rates of compounds 3 and 4 on the three fungi were greater than 60%, with good antibacterial effects. The antibacterial rate of compound 2 on Microsporum canis and Trichophyton mentagrophytes was greater than 60%, with good antibacterial effects, but the antibacterial rate for Microsporum gypseum was greater than 45%, with good antibacterial effects. Compound 1 had a good antibacterial effect on Trichophyton mentagrophytes, and had a good antibacterial effect on Microsporum canis and Microsporum gypseum. At a concentration of 3 μmol / mL, compound 4 still had a good antibacterial effect on the three fungi, compound 3 had a good antibacterial effect on Trichophyton mentagrophytes and Microsporum gypseum, but had a poor antibacterial effect on Microsporum canis, and compound 1 had a good antibacterial effect.

[0186] Table 4 Inhibition rate of compounds at different concentrations against Microsporum canis

[0187]

[0188] Table 5 Inhibition rate of compounds at different concentrations against Trichophyton mentagrophytes

[0189]

[0190]

[0191] Table 6 Inhibition rate of compounds at different concentrations against Microsporum gypseum

[0192]

[0193] 4.1.3 MIC results of compounds 1-20 against four fungi

[0194] The results of MIC determination are shown in Table 7. Compounds 1-20 have different degrees of inhibitory effects on the four fungi. Compared with the positive control group (itraconazole, fluconazole and ketoconazole), the MICs of compound 4 and its derivatives compounds 9, 10, 13, 14, 16, 17 against Microsporum canis, Trichophyton mentagrophytes, Microsporum gypseum and Candida albicans are better than the positive control; compounds 11, 12, 18 are second; compounds 1-3, 15, 19, 20 are better than itraconazole and fluconazole.

[0195] Table 7 MIC of compounds 1-20 against four bacteria

[0196]

[0197]

[0198] The antifungal test showed that, among the 20 compounds, except for compounds 5-8, the other compounds had antifungal activities superior to or equivalent to the positive control against the four pathogenic fungi.

[0199] Example 5

[0200] The compounds 1-20 synthesized in the above examples were subjected to normal cell cytotoxicity assay.

[0201] 1. Cell lines

[0202] NIH3T3 cells

[0203] 2. Experimental Methods

[0204] 2.1 Cell inoculation

[0205] When NIH3T3 cells grow well and are in the logarithmic growth phase, follow the subculture procedure and resuspend the cells in 1 mL of complete culture medium. Add an appropriate amount of complete culture medium to dilute and count 5 × 10 4 The prepared cell suspension was inoculated into a 96-well cell culture plate, 100 μL was added to each well, and after sealing, the plate was transferred to a 37°C, 5% CO2 constant temperature incubator for 24 h.

[0206] 2.2 Preparation of drug solution and administration

[0207] The compound was dissolved in DMSO, and the corresponding solvent was used as the negative control group. 100 μL of complete medium was added to the control group, and the complete medium was supplemented to 200 μL in the experimental group, so that the final concentration of the drug solution in each well was 20 μM, and 5 groups were paralleled. Derivatives that showed good inhibitory activity at a concentration of 20 μM were rescreened for active drugs using NIH3T3 cells according to a certain concentration gradient.

[0208] 2.3 MTS kit to detect cell viability

[0209] After the cells were treated with drugs for 48 hours, the MTS solution and complete culture medium were mixed at a ratio of 1:10, the medium containing the drug solution in the 96-well plate was discarded, 200 μL of the mixed solution was added to each well, and 5 blank groups of the mixed solution without cells were set up and placed in a constant temperature box for reaction for 2-4 hours. The absorbance of each well at an absorption wavelength of 490 nm was detected by an ELISA instrument, the data were exported and the inhibition rate was calculated, and the IC was calculated using the Bliss method using GraphPad Prism 8 software. 50 .

[0210] 3. Experimental results

[0211] IC of compounds against NIH3T3 cells 50 The results are shown in Table 8.

[0212] Among the tested compounds, compound 3 had an IC 50 The smallest, the cytotoxicity is the greatest, and the remaining compounds are all less cytotoxic. It can be seen that the toxicity of these compounds to normal tissues should be lower than that of ketoconazole.

[0213] Table 8 IC of compounds 1-20 on NIH3T3 cells 50

[0214]

[0215] in conclusion

[0216] In summary, the positive controls fluconazole and itraconazole have no obvious inhibitory effect on Microsporum canis, Trichophyton mentagrophytes, and Microsporum gypseum, while ketoconazole has a significant inhibitory effect on these three bacteria. The three positive controls all have a certain inhibitory effect on Candida albicans. The synthesized compounds 1-20 have different degrees of antibacterial effects on Microsporum canis, Trichophyton mentagrophytes, Microsporum gypseum, and Candida albicans. Among them, compounds 4, 9, 10, 13, 14, 16, and 17 have obvious inhibitory effects on these four bacteria, and are better than fluconazole, itraconazole, and ketoconazole (partially equivalent); compounds 11, 12, and 18 are second; compounds 1-3, 15, 19, and 20 are third. And compounds 1-20 show low cytotoxicity to normal cells and have high safety. The above compounds all have potential antifungal application value.

[0217] Example 6

[0218] Prediction of antifungal targets of representative compounds

[0219] Representative compounds 1-4 were selected, and the molecular docking method was used to examine the binding and action mode of the representative compounds with common antifungal target proteins, and to predict the possible targets of the active compounds in this application.

[0220] 1. Molecular docking method

[0221] From the RCSB database (http: / / www.rcsb.org / ), we found the following: sterol 14-α demethylase (CYP51, PDB ID: 5FSA) of pathogenic yeast Candida albicans; sterol 14-α demethylase (CYP51, PDB ID: 5TZ1) of Candida albicans; exo-β(1,3)-glucanase (XOG1, PDB ID: 1EQP) of Candida albicans; exo-β(1,3)-glucanase (XOG1, PDB ID: 1CZ1) of Candida albicans; chitin synthase 2 (CHS2, PDB ID: 7STL) from Candida albicans; dihydrofolate reductase (DFR1, PDB ID: 1AI9) of Candida albicans; N-myristoyltransferase (NMT1, PDB ID: 1IYK) of Candida albicans; phosphomannose isomerase (PMI, PDB ID: 1IYK) of Candida albicans. ID: 1PMI); β-(1,4)-galactosidase from Aspergillus (GAL1, PDB ID: 1FOB); β-glucosidase 1 from Aspergillus (BGL1, PDB ID: 4IIB); crystal structure of rhamnogalacturonase A from Aspergillus (RHGA, PDB ID: 1RMG).

[0222] The SDF files of compound 1-4 and positive control fluconazole, ketoconazole, and itraconazole structures were downloaded from the Pub Chem database (https: / / pubchem.ncbi.nlm.nih.gov / ) and imported into the Discovery software to obtain their 3D structures. The Discovery software was used to remove ligands and non-protein molecules (such as water molecules) in the target protein and perform hydrogenation treatment. Then, the compound and target protein were molecularly docked and visualized in the software.

[0223] 2. Molecular docking results and analysis

[0224] The Lib Dock Score indicates the degree of binding between the ligand and the target protein crystal. The higher the Lib Dock Score, the higher the activity of the small molecule binding to the receptor. Compounds 1-4 were docked with the proteins in "1.1" respectively and visualized. The representative binding diagram is shown in Figures 1 to 4 As shown in Table 9, the spatial configuration of the molecular docking of compound 4 with 1EQP, 1CZ1, 1AI9, and 4IIB, as well as the binding of the active groups of the compounds are shown. The Lib Dock Score is shown in Table 9.

[0225] Sterol 14-α demethylase is a potential antifungal target for most azole compounds, but molecular docking revealed that, compared with the most active ketoconazole, compounds 1-4 had weak binding ability with sterol 14-α demethylase (CYP51, PDB ID: 5FSA and CYP51, PDB ID: 5TZ1), but showed higher binding ability with glucanase (XOG1, PDB ID: 1EQP, 1CZ1 and XOG1, PDB ID: 1CZ1), dihydrofolate reductase (DFR1, PDB ID: 1AI9) and β-glucosidase 1 (BGL1, PDB ID: 4IIB).

[0226] Table 9 Lib Dock Score of each compound and target protein

[0227]

[0228] The results of molecular docking showed that, unlike the targets of traditional azole compounds, the synthesized active compounds did not act on sterol 14-α demethylase, but may achieve antifungal effects through multiple targets. Their antifungal effects may be highly correlated with glucanase, dihydrofolate reductase and β-glucosidase 1.

[0229] Example 8

[0230] Preparation example (spray)

[0231] A fur animal topical spray (10 mg / mL) is prepared from the following raw materials by weight percentage: 1% active compound, 40% ethanol, 5% glycerol, 0.2% sodium thiosulfate, 0.5% water-soluble azone, 0.2% sorbic acid, 0.5% PVA, 2% PVP, and 1% Tween 80. The active compound can be any one of the compounds 1-20 prepared in the above embodiments.

[0232] The preparation method is as follows: take various raw material ingredients according to the above raw material formula ratio, put alcohol in a beaker, add glycerol, sodium thiosulfate, water-soluble azone, sorbic acid, stir for 10 minutes, and mix evenly. Then slowly add PVA (polyvinyl alcohol, Kuraray PVA-217, the same below) and PVP (polyvinyl pyrrolidone, k30, the same below), stir well, and heat appropriately to promote dissolution. After PVA and PVP are fully dissolved, add Tween 80 and continue stirring for 20 minutes. Cool to below 50°C, add active compounds, stir until fully dissolved, filter, and bottle to obtain a fur animal external spray.

[0233] Example 9

[0234] Preparation example (spray)

[0235] A spray for fur animals (10 mg / mL) is prepared from the following raw materials by weight percentage: 1% active compound, 5% DMSO, 10% Tween 80, 20% PEG400, 20% ethanol, 5% glycerol, 0.2% sodium thiosulfate, 0.5% water-soluble azone, 0.2% sorbic acid, 0.5% PVA, and 2% PVP. The active compound can be any one of the compounds 1-20 prepared in the above embodiments.

[0236] Prepare the raw materials according to the above formula ratio, and then make the spray according to the following preparation method.

[0237] ① Weigh 1g of active compound, dissolve it in 5mL DMSO (dimethyl sulfoxide), add 10mL Tween 80, 20mL PEG400 (polyethylene glycol, molecular weight 380-420), 5mL glycerol, and 20mL ethanol and mix well.

[0238] ② Add 0.2g sodium thiosulfate, 0.2g sorbic acid, and 0.5mL water-soluble azone to 40mL water and dissolve them. Then add 2g PVP and 0.5g PVA little by little, heat until dissolved, and cool to room temperature.

[0239] ③ Slowly add the dissolved substance in ② into ①, gradually stir until dissolved, bottle, and obtain an external spray for fur animals.

Claims

1. An application of an azole compound in the preparation of an antifungal drug for fur animals, characterized in that: The azole compound is a compound having a molecular structure shown in Formula I: Formula I wherein R1 is independently selected from any one of hydrogen, C1-C4 alkyl, halogen-substituted C1-C3 alkyl, cyano, amino, halogen, methylamino, hydroxy, hydroxy-substituted C1-C3 alkyl, C1-C3 alkoxy, benzyloxycarbonyl, benzyloxy, phenoxy, formyloxy, acetoxy, benzoyloxy, C1-C3 alkylamino, di-C1-C3 alkyl-substituted amino, and tert-butyloxycarbonyl; a represents the number of R1, a=0-3; b represents the number of R2, b=0-3; R2 is independently selected from any one of C1-C3 alkyl, halogen, amino, methylamino, dimethylamino, hydroxyl, carboxyl, nitro, and haloalkyl; X1 is -(CH2)n- or -(CH2)nO- or -O-(CH2)n-, n=0-3; X2 is -(CH2)m- or -C(O)-CH(CH3)-(CH2)m-, m=0-3; Y is C or N.

2. The use of an azole compound according to claim 1 in the preparation of an antifungal drug for fur animals, characterized in that: The R1 is independently selected from any one of hydrogen, methyl, ethyl, n-propyl, isopropyl, isobutyl, sec-butyl, tert-butyl, benzyloxycarbonyl, amino, methylamino, ethylamino, propylamino, dimethylamino, diethylamino, hydroxyl, methoxy, ethoxy, benzyloxy, phenoxy, formyloxy, fluoro, chloro, bromo, iodo, tert-butoxycarbonyl, and fluoromethyl.

3. The use of an azole compound according to claim 1 in the preparation of an antifungal drug for fur animals, characterized in that: The R2 is independently selected from any one of C1-C3 alkyl, halogen, amino, fluoro, chloro, bromo, iodo, hydroxyl, and perfluoro C1-C3 alkyl; perfluoro C1-C3 alkyl refers to perfluoromethyl, perfluoroethyl, perfluoro-n-propyl, and perfluoro-isopropyl.

4. The use of an azole compound according to claim 1 in the preparation of an antifungal drug for fur animals, characterized in that: a represents the number of R1, a=0, 1, 2.

5. The use of an azole compound in the preparation of an antifungal drug for fur animals according to claim 1, characterized in that: b represents the number of R2, b=0, 1, 2.

6. The use of an azole compound in the preparation of an antifungal drug for fur animals according to claim 1, characterized in that: n=0、1、2。 7. The use of an azole compound in the preparation of an antifungal drug for fur animals according to claim 1, characterized in that: m=0、1、2。 8. The use of an azole compound in the preparation of an antifungal drug for fur animals according to claim 1, characterized in that: The azole compound is a compound having a molecular structure shown in Formula II or Formula III: Formula II or Formula III Where n=0-3; Y is C or N; At least one R1, each R1 is independently selected from any one of hydrogen, C1-C4 alkyl, halogen-substituted C1-C3 alkyl, cyano, amino, halogen, methylamino, hydroxyl, hydroxyl-substituted C1-C3 alkyl, C1-C3 alkoxy, benzyloxycarbonyl, benzyloxy, phenoxy, formyloxy, acetoxy, benzoyloxy, C1-C3 alkylamino, di-C1-C3 alkyl-substituted amino, and tert-butyloxycarbonyl; a represents the number of R1, a=0-3; b represents the number of R2, b=0-3; There is at least one R2, and each R2 is independently selected from any one of C1-C3 alkyl, halogen, amino, methylamino, dimethylamino, hydroxyl, carboxyl, nitro, and halogenated alkyl.

9. The use of an azole compound in the preparation of an antifungal drug for fur animals according to claim 8, characterized in that: The azole compound is one of the following compounds: 。 10. Use of an azole compound according to any one of claims 1 to 9 in the preparation of an antifungal drug for fur animals, characterized in that: The antifungal drug is an antifungal drug that inhibits at least one of Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, or Candida albicans.

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