Aromatic ring substituted pyrazole derivative and its application

Abstract

The present invention relates to an aromatic ring substituted pyrazole derivative and its application, wherein the aromatic ring substituted pyrazole derivative has a structure shown in formula I: wherein Ar 1 is any one of phenyl, substituted phenyl, naphthyl, substituted naphthyl, substituted anthracenyl, phenanthryl, substituted phenanthryl, fluorenyl, substituted fluorenyl, pyrrolyl, substituted pyrrolyl, pyridinyl, and substituted pyridinyl; Ar 2 is any one of phenyl, substituted phenyl, naphthyl, substituted naphthyl, substituted anthracenyl, phenanthryl, substituted phenanthryl, pyrrolyl, substituted pyrrolyl, pyridyl, and substituted pyridyl; R 1 is selected from the group consisting of: hydrogen, halogen atoms, C1-C6 alkyl, halogen-substituted C1-C5 alkyl, C1-C5 alkyloxy, C2-C4 alkenyl, nitro, and phenyl; R 2 Selected from: hydrogen, halogen atoms, carboxyl, carboxyl C1-C3 alkyl, hydroxyl, C1-C6 alkyl... This type of compound can effectively inhibit Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, and has low toxicity, and can be used in antifungal applications, especially antifungal applications in veterinary clinics.

Description

Technical Field

[0001] The present invention relates to the field of synthesis of pharmaceutical compounds, in particular to the synthesis of azole antifungal compounds and their pharmaceutical uses, in particular their application in animal medicines, and belongs to the field of biomedicine. 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 this disease requires a long period of time, 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 the Administration of Veterinary Drugs" 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. 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. Summary of the invention

[0004] The purpose of the present invention is to overcome the problem that there is a lack of antifungal chemical drugs specifically for animals in the prior art, and that the use of human drugs for animal treatment is not in compliance with laws and regulations, and to provide an aromatic ring substituted pyrazole derivative. A series of aromatic ring substituted pyrazole derivatives, especially azole benzoic acid compounds, are prepared by synthesis, and low-toxic and highly effective animal-specific antifungal compounds are screened therefrom to meet the application requirements of special animal antifungal drugs. At the same time, these antifungal drugs also have the potential to be developed as human drugs.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] An aromatic ring substituted pyrazole derivative having a structure shown in Formula I:

[0007]

[0008] in,

[0009] Ar 1 is any one of phenyl, substituted phenyl, naphthyl, substituted naphthyl, substituted anthracenyl, phenanthryl, substituted phenanthryl, fluorenyl, substituted fluorenyl, pyrrolyl, substituted pyrrolyl, pyridinyl, and substituted pyridinyl;

[0010] Ar 2 is any one of phenyl, substituted phenyl, naphthyl, substituted naphthyl, substituted anthracenyl, phenanthryl, substituted phenanthryl, pyrrolyl, substituted pyrrolyl, pyridyl, and substituted pyridyl;

[0011] R 1 Selected from: hydrogen, halogen atoms, C1-C6 alkyl, halogen-substituted C1-C5 alkyl, C1-C5 alkyloxy, C2-C4 alkenyl, nitro, phenyl;

[0012] R 2 Selected from: hydrogen, halogen atoms, carboxyl, carboxyl C1-C3 alkyl, hydroxyl, C1-C6 alkyl, halogen-substituted C1-C5 alkyl, amino, nitro, formamide, acetamido, aminoformamide, aminoacetamide, phenyl;

[0013] m=0-3, n=0-3.

[0014] Further, Ar 1 is any one of phenyl, substituted phenyl, naphthyl, substituted naphthyl, substituted anthracenyl, 2-phenanthryl, substituted phenanthryl, 2-fluorenyl, and substituted fluorenyl. Preferably, Ar 1 It is any one of phenyl, substituted phenyl, naphthyl, substituted naphthyl, 2-phenanthrenyl and 2-fluorenyl.

[0015] Further, Ar 2 It is any one of phenyl, substituted phenyl, 1-naphthyl, 2-naphthyl, substituted-1-naphthyl, substituted-2-naphthyl, anthracenyl, and substituted anthracenyl.

[0016] Preferably, when Ar 1 When it is a substituted phenyl group and m=1, R 1 The substitution position is 4; when Ar 2 is substituted-1-naphthyl or substituted-2-naphthyl, and n=1, R 2 The substitution position is 4; when Ar 2 When it is a substituted-2-fluorenyl group and n=1, R 2 The substitution position is at the 4th, 5th, 6th, 7th or 8th position.

[0017] Preferably, when Ar 2 When it is a substituted phenyl group and n=1, R 2 The substitution position is 4; when Ar 2When it is a substituted-1-naphthyl group and n=1, R 2 The substitution position is 4.

[0018] Further, R 1 Selected from: hydrogen, halogen atom, C1-C6 alkyl, halogen-substituted C1-C5 alkyl, C1-C5 alkyloxy, amino, nitro, formamide, acetylamino, aminoformamide, aminoacetamide, and phenyl.

[0019] Further, R 2 Selected from: hydrogen, halogen atoms, carboxyl, carboxyl C1-C3 alkyl, hydroxyl, C1-C6 alkyl, halogen-substituted C1-C5 alkyl, amino, nitro, formamide, acetylamino, aminoformamide, aminoacetamide.

[0020] Further, Ar 1 and Ar 2 It is an aromatic group.

[0021] Furthermore, the aromatic ring substituted pyrazole derivative has a structure shown in Formula II, Formula III or Formula IV:

[0022]

[0023] Further, R 1 Selected from: hydrogen, halogen atom, C1-C6 alkyl, halogen-substituted C1-C5 alkyl, C1-C5 alkyloxy, amino, nitro, formamide, acetylamino, aminoformamide, aminoacetamide, and phenyl.

[0024] Further, R 2 Selected from: hydrogen, halogen atoms, carboxyl, carboxyl C1-C3 alkyl, hydroxyl, C1-C6 alkyl, halogen-substituted C1-C5 alkyl, amino, nitro, formamide, acetylamino, aminoformamide, aminoacetamide.

[0025] Further, m=0-2. Preferably, m=0-1.

[0026] Further, n=0-2. Preferably, n=0-2.

[0027] Further, R 1 Selected from: hydrogen, C1-C4 alkyl, halogen-substituted C1-C3 alkyl, C1-C3 alkyloxy, halogen atom, amino, nitro.

[0028] Further, R 2 Selected from: carboxyl group, halogen atom, aminoacetylamino group, nitro group, nitro group, amino group.

[0029] Further, R 2 Substitution is at the 4-position of the benzene ring or the naphthalene ring.

[0030] Further, C1-C6 alkyl refers to an alkyl group having 1-6 carbon atoms; preferably, C1-C4 alkyl refers to an alkyl group having 1-4 carbon atoms. Specifically, C1-C6 alkyl is selected from: methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl, tert-butyl. C1-C4 alkyl is selected from: methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl.

[0031] Furthermore, the carboxyl C1-C3 alkyl refers to a carboxymethyl, 1-carboxy-ethyl, 2-carboxy-ethyl, 1-methyl-2-carboxy-ethyl, 1-carboxypropyl, 2-carboxypropyl, 3-carboxypropyl group, and the carboxyl group may be at any position of a, β, γ...ω, in particular the carboxyl group at the ω position of the alkyl group.

[0032] Furthermore, the halogen-substituted C1-C5 alkyl group refers to a group in which at least one hydrogen atom in an alkyl group having 1 to 5 carbon atoms is replaced by a halogen atom; preferably, the halogen-substituted C1-C5 alkyl group is a halogen-substituted C1-C3 alkyl group, which is a group in which at least one hydrogen atom in an alkyl group having 1 to 3 carbon atoms is replaced by a halogen atom.

[0033] Preferably, the halogen-substituted C1-C5 alkyl group is a C1-C5 alkyl group in which some or all of the hydrogen atoms are substituted by halogen. For example, the following alkyl groups are substituted by halogen: methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl, tert-butyl.

[0034] Preferably, the halogen-substituted C1-C3 alkyl group is a C1-C3 alkyl group in which some or all of the hydrogen atoms are substituted by halogen atoms, that is, some or all of the hydrogen atoms in a methyl, ethyl, n-propyl or isopropyl group are substituted by halogen atoms.

[0035] Furthermore, the C1-C5 alkyloxy group refers to an oxy group connected to an alkyl group having 1 to 5 carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butyloxy, isobutyloxy, tert-butyloxy. Preferably, the C1-C4 alkyl group is methyl, ethyl, propyl, isopropyl.

[0036] Furthermore, the C1-C5 alkyloxy group is preferably a C1-C3 alkyloxy group, specifically a methoxy group, an ethoxy group or a propyloxy group.

[0037] Further, R 1 It is isopropyl.

[0038] Further, R 2 Selected from carboxyl, methyl, and amino. 2 Preferred is a carboxyl group.

[0039] Furthermore, the halogen atom or halogen in the present invention is chlorine, fluorine, bromine or iodine, and chlorine, fluorine and bromine are particularly preferred. "Halo" means chloro, fluoro, bromo or iodo, and may be substituted by one halogen atom or more than one halogen atom.

[0040] Furthermore, when m = 1, R 1 Substitute at the 4-position of the benzene ring; when m = 2, the two R 1 The substituents may be different, and the two R 1 The substitution positions on the benzene ring are one of the following combinations: 2-position and 4-position, 3-position and 5-position, 3-position and 4-position;

[0041] When n = 1, R 2 Substituted at the 4-position of the aromatic ring or the 4-position of the naphthalene ring;.

[0042] Furthermore, the above-mentioned halogen or halogen atom is a fluorine atom and / or a halogen atom.

[0043] Furthermore, the aromatic ring substituted pyrazole derivative does not include 4-(5-p-tolyl-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzoic acid; that is, 4-(3-(trifluoromethyl)-5-p-tolyl-1H-pyrazol-1-yl)benzoic acid.

[0044] Furthermore, the aromatic ring substituted pyrazole derivative is any one of the following compounds:

[0045]

[0046] Furthermore, the aromatic ring substituted pyrazole derivative is 1-5, 7-30 among the above compounds.

[0047] Furthermore, the above aromatic ring substituted pyrazole derivatives are used in the preparation of animal antifungal drugs.

[0048] Preferably, the drug is a drug for treating fungal infection in fur animals.

[0049] Preferably, the application is the application for preparing a drug for treating Microsporum canis, Microsporum gypseum, and Trichophyton mentagrophytes.

[0050] Furthermore, the use of compound 3 and compound 4 among the above compounds in drugs for treating Trichophyton mentagrophytes and / or Microsporum gypseum.

[0051] An animal antifungal drug, comprising the above aromatic ring substituted pyrazole derivative. Preferably, the drug is an animal drug. Preferably, the drug is a drug for treating diseases caused by Microsporum canis, Microsporum gypseum and / or Trichophyton mentagrophytes.

[0052] Preferably, the pharmaceutical use of compounds 2, 4, 5, 6, 7, 9, 11, 12, 14, 17, 18, 26, and 27 is a drug for treating Microsporum canis.

[0053] Preferably, the pharmaceutical use of compounds 5, 6, 7, 8, 9, 10, 11, 17, 18, 20, 26, and 27 is a drug for treating Trichophyton mentagrophytes.

[0054] Preferably, the pharmaceutical use of compounds 2, 4, 12, 13, 15, 22, and 25 is a drug for treating Trichophyton mentagrophytes.

[0055] Preferably, the pharmaceutical use of compounds 3-12, 17, 18, 23, 26, and 27 is a drug for treating Microsporum gypseum.

[0056] Preferably, the pharmaceutical use of compounds 1, 2, 14, 15, 17, 18, 22, 24, and 25 is a drug for treating Microsporum gypseum.

[0057] In the above pharmaceutical application, the drug for treating Microsporum canis, Trichophyton mentagrophytes and / or Microsporum gypseum is a drug that has a killing effect on the corresponding fungi. That is, the corresponding drug has the therapeutic effect of inhibiting or killing the corresponding fungi.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] The aromatic ring-substituted pyrazole derivative of the present invention can effectively inhibit Microsporum canis, Microsporum gypseum and Trichophyton mentagrophytes, and has low toxicity, and can be used in antifungal applications, especially in antifungal applications in veterinary clinics.

[0060] In particular, some of the preferred compounds have good antibacterial activity against pathogenic fungi, which is stronger than the current first-line clinical drugs fluconazole, itraconazole and ketoconazole, and have great potential for drug development.

[0061] The aromatic ring-substituted pyrazole derivatives of the present invention have a lower selectivity to normal cells and are superior to ketoconazole in that they are less toxic to normal cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 This is a graph showing the inhibitory effects of compounds 3, 4, 7, 9, 11, and 12 on three bacteria at a concentration of 6 μmol / mL.

[0063] Figure 2 Molecular docking diagram of compound 1 and 1PN2.

[0064] Figure 3 This is the molecular docking diagram of compound 3 and 1PN2.

[0065] Figure 4 This is the molecular docking diagram of compound 4 and 1PN4. DETAILED DESCRIPTION

[0066] In order to more clearly describe the invention purpose, technical scheme and technical effect advantages in the specific implementation case of the present invention, the scheme in the specific embodiment will be described in detail in combination with the drawings of the specification of the present invention. The specific technical scheme involved in the following specific embodiments is only for a clear and complete description of the innovative technical scheme of the present invention. It is only a part of the specific implementation scheme that can be adopted by the present invention, not all embodiments, and should not be understood as a limitation on the innovative scheme of the present invention. Any scheme adopting the same inventive concept of the present invention should be included in the protection scope of the present invention.

[0067] Molar concentration, M, refers to the amount of solute contained in a unit volume of solution, and the common unit is mol / L. It is often expressed as M (molarity, the amount of substance concentration).

[0068] Normal concentration, N. The concentration of a solution expressed as the number of gram equivalents of solute contained in 1 liter of solution is called normal concentration, represented by the symbol N.

[0069] Reaction equivalent, eq, reaction equivalent refers to the equivalent amount of action played by a chemical substance in a reaction. Equivalent chemical reaction refers to a chemical reaction in which chemical substances act according to the stoichiometric relationship and consume or generate chemical equivalents.

[0070] Example 1 Synthesis of azole benzoic acid compounds

[0071] The basic process of the reaction is as follows: Synthesis route of azole benzoic acid compounds.

[0072]

[0073] R2=COOH

[0074] R 1 =2-phenanthrene; 2-F, 4-F; 3-CF3, 5-CF3; 4-iPr; 2-F, 4-Cl; 4-C2H5O; 3-MeO, 4-MeO; 2-fluorene;

[0075] 2-Cl,4-Cl;4-BP;4-Me;4-Et

[0076] Wherein, the reaction solvents THF = tetrahydrofuran, and EtOH = ethanol.

[0077] R 2 is the 4-position carboxyl group.

[0078] R 1=2-phenanthrene, i.e. R 1 -phenyl group was replaced by 2-phenanthryl group.

[0079] R 1 =2-F,4-F; that is, R 1 There are two, namely 2 F atoms and 4 F atoms.

[0080] R 1 =3-CF3,5-CF3; that is, R 1 There are two, namely 3-digit CF3 and 5-digit CF3.

[0081] R 1 =4-iPr, i.e. 4-isopropyl.

[0082] R 1 =4-Me, i.e. methyl group at the 4th position.

[0083] R 1 =4-Et, i.e. 4-ethyl.

[0084] R 1 =2-F,4-Cl, i.e. R 1 There are two, namely the 2-position F atom and the 4-position chlorine atom.

[0085] R 1 =4-C2H5O, i.e. ethoxy group at the 4th position.

[0086] R 1 =3-MeO,4-MeO, that is, R 1 There are two, namely the 3-methoxy group and the 4-methoxy group.

[0087] R 1 =2-fluorene, that is, "R 1 -phenyl" was replaced by 2-fluorenyl.

[0088] R 1 =2-Cl,4-Cl, that is, R 1 There are two, namely the 2-position chlorine atom and the 4-position chlorine atom.

[0089] R 1 =BP, that is, R 1 =4-phenyl, R 1 Together with the attached phenyl groups, they form a biphenyl group.

[0090] (1) 1 eq of acetophenone derivative (A) and 2 eq of NaH were added to a tetrahydrofuran solution, and the mixture was stirred for 5 min. Then, 2.5 eq of ethyl trifluoroacetate was added. The reaction mixture was stirred at room temperature for 4 h and neutralized with 2N HCl. The precipitate formed was collected, washed with methanol and dried, and used directly in the next step without further purification.

[0091] (2) 1 eq of 1,3-diketone adduct (B) and 1.2 eq of substituted phenylhydrazine derivative were mixed in ethanol, refluxed for 8 hours, cooled to room temperature, added with distilled water, and the resulting precipitate was collected and dried to obtain a crude product (C). The crude product was separated by column chromatography, and the target compound was identified by TLC, 1 H-NMR, 13 C-NMR and HR-MS examination.

[0092] According to the above reaction route, the following compounds were synthesized:

[0093]

[0094] Compound 1, HR-MS m / z: calculated for C 25 H 14 F3N2O2 433.1164,found 433.1160[M+H] + ; 1 HNMR(400MHz,Chloroform-d)δ8.64(dd,J=8.5,4.8Hz,2H),8.16-8.01(m,2H),7.92(dd,J=7.8,1.5Hz,1H),7.86(d, J=1.9Hz,1H),7.80(d,J=8.8Hz,1H),7.73-7.60(m,3H),7.51-7.45(m,2H),7.40(dd,J=8.6,1.9Hz,1H),6.93(s,1H). 13 C NMR (101MHz, Chloroform-d) δ170.11, 144.80, 144.07 (d, J = 39.0Hz), 143.34, 132.36, 131.90, 131.17 (×2), 130.56, 129.62, 128. 84,128.70,128.69,128.20,127.36,127.08,126.74,126.40,126.39,124.98(×2),123.43,122.76,120.23(q,J=265Hz),106.72.

[0095] Compound 2, HR-MS m / z: calculated for C 17 H 10 F5N2O2 369.0662, found 369.0657 [M+H] + ; 1 1H NMR (400 MHz, Chloroform-d) δ 8.20 - 8.05 (m, 2H), 7.48 - 7.35 (m, 2H), 7.26 - 7.22 (m, 1H), 6.95 (td, J = 8.1, 2.4 Hz, 1H), 6.90 - 6.83 (m, 1H), 6.82 (s, 1H). 13 13C NMR (101 MHz, Chloroform-d) δ 170.34, 163.94 (dd, J = 253.9, 11.3 Hz), 159.68 (dd, J = 254.0, 11.9 Hz), 144.11 (d, J = 39.0 Hz), 143.32, 137.97, 132.13 (dd, J = 9.7, 3.6 Hz), 131.31 (×2), 129.05, 124.16, 120.94 (q, J = 269.1 Hz), 113.49 (d, J = 15.1 Hz), 112.35 (dd, J = 21.7, 3.5 Hz), 108.00, 105.12, 105.12 (d, J = 50.7 Hz).

[0096] Compound 3, HR-MS m / z: calculated for C 19 H 10 F9N2O2 469.0599, found 469.0595 [M+H] + ; 1 1H NMR (400 MHz, Chloroform-d) δ 8.23 - 8.05 (m, 2H), 7.91 (s, 1H), 7.73 - 7.59 (m, 2H), 7.51 - 7.35 (m, 2H), 6.95 (s, 1H). 13 13C NMR (101 MHz, Methanol-d4) δ 168.12, 144.85 (q, J = 38.5 Hz), 143.44, 143.08, 131.90 (×2, q, J = 100.8 Hz), 133.35 (×2, d, J = 28.5 Hz), 131.81 (×2), 129.28, 139.25, 126.75 (×2), 122.95 (d, J = 269 Hz), 122.44 (×2, m), 119.81 (d, J = 94.0 Hz), 108.06.

[0097] Compound 4, HR-MS m / z: calculated for C 20 H 18 F3N2O2 375.1320, found 375.1317 [M+H] + ; 1 1H NMR (400 MHz, Chloroform-d) δ 8.12 - 8.06 (m, 2H), 7.48 - 7.43 (m, 2H), 7.24 - 7.19 (m, 2H), 7.17 - 7.12 (m, 2H), 6.74 (s, 1H), 2.92 (q, J = 6.9 Hz, 1H), 1.26 (d, J = 6.9 Hz, 6H). 13 13C NMR (101 MHz, Methanol-d4) δ 167.18, 150.45, 145.65, 143.57 (d, J = 58.5 Hz), 142.77, 130.74, 130.36 (×2), 128.73 (×2), 126.63 (×2), 126.25, 125.21 (×2), 120.11 (q, J = 271.65 Hz), 105.41, 33.82, 22.79 (×2).

[0098] Compound 5, HR-MS m / z: calculated for C 19 H 15 F3N2O2, 361.1164, found 361.1123 [M+H] + ; 1 1H NMR (400 MHz, Chloroform-d) δ 8.06 - 8.00 (m, 2H), 7.80 - 7.74 (m, 2H), 7.65 - 7.59 (m, 2H), 7.33 - 7.26 (m, 2H), 7.22 (s, 1H), 2.69 (qt, J = 7.3, 1.1 Hz, 2H), 1.23 (t, J = 7.2 Hz, 3H). 13 13C NMR (101 MHz, Chloroform-d) δ 168.82, 144.95, 143.28, 142.93, 142.41, 131.72, 131.38, 129.53, 128.81, 128.72, 121.87, 117.98, 105.10, 28.92, 15.61.

[0099] Compound 6, HR-MS m / z: calculated for C 18 H 13F3N2O2, 347.1007, found 347.0996 [M+H] + ; 1 1H NMR (400 MHz, Chloroform-d) δ 8.06 - 8.00 (m, 2H), 7.78 - 7.72 (m, 2H), 7.65 - 7.59 (m, 2H), 7.34 - 7.27 (m, 2H), 7.20 (s, 1H), 2.41 (s, 3H). 13 13C NMR (101 MHz, Chloroform-d) δ 168.82, 144.90, 143.28, 142.41, 138.76, 131.38, 130.74, 130.46, 128.74 (d, J = 6.0 Hz), 121.87, 117.98, 105.10, 21.23。

[0100] Compound 7, HR-MS m / z: calculated for C 17 H 10 ClF4N2O2 385.0367, found 385.0361 [M+H] + ; 1 1H NMR (400 MHz, Chloroform-d) δ 8.12 (d, J = 8.3 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 7.23 - 7.17 (m, 2H), 7.14 (d, J = 9.4 Hz, 1H), 6.84 (s, 1H). 13 13C NMR (101 MHz, Methanol-d4) δ 167.08, 159.95 (d, J = 252.7 Hz), 144.28 (q, J = 38.6 Hz), 143.26, 139.04, 137.67 (d, J = 10.3 Hz), 133.04 (d, J = 2.9 Hz), 131.64, 131.21 (×2), 125.83 (d, J = 3.8 Hz), 124.88 (×2), 121.14 (d, J = 257 Hz), 117.54, 117.29, 116.62 (d, J = 15.0 Hz), 108.21。

[0101] Compound 8, HR-MS m / z: calculated for C 19 H 16F3N2O3 377.1113, found 377.1116 [M+H]+; 1H NMR (400 MHz, Dimethyl Sulfoxide-d6) δ 8.03 - 7.97 (m, 2H), 7.54 - 7.42 (m, 2H), 7.24 - 7.18 (m, 2H), 7.16 (s, 1H), 7.01 - 6.86 (m, 2H), 4.03 (q, J = 7.0 Hz, 2H), 1.31 (t, J = 7.0 Hz, 3H). 13 13C NMR (101 MHz, Methanol-d4) δ 167.08, 169.12, 162.19, 147.51, 144.48, 144.28 (d, J = 38.6 Hz), 132.46, 132.13 (×2), 131.90 (×2), 126.97 (×2), 121.14 (d, J = 257 Hz), 116.27 (×2), 106.84, 65.11, 16.67。

[0102] Compound 9, HR-MS m / z: calculated for C 19 H 16 F3N2O4 393.1062, found 393.1058 [M+H] + ; 1 1H NMR (400 MHz, Chloroform-d) δ 8.16 - 7.93 (m, 2H), 7.51 - 7.38 (m, 2H), 6.87 - 6.66 (m, 4H), 3.90 (s, 3H), 3.71 (s, 3H). 13 13C NMR (101 MHz, Methanol-d4) δ 167.16, 150.20, 149.16, 145.53, 143.28 (q, J = 38.4 Hz), 142.82, 130.70, 130.34 (×2), 125.30 (×2), 121.84, 121.16, 121.38 (d, J = 266 Hz), 112.33, 111.49, 105.11, 55.04, 55.01。

[0103] Compound 10, HR-MS m / z: calculated for C 24 H 16 F3N2O2 421.1164, found 421.1158 [M+H] + ; 1HNMR(400MHz,Chloroform-d)δ8.14 - 8.05(m,2H),7.82 - 7.71(m,2H),7.56(dt,J=7.5,1.0Hz,1H),7.52 - 7.43(m,3H),7.43 - 7.38(m,1H),7.35(td,J=7.4,1.3Hz,1H),7.24 - 7.17(m,1H),6.83(s,1H),3.89(s,2H). 13 C NMR(101MHz,Methanol-d4)δ167.24,145.92,143.25(d,J=98Hz),143.60,142.75,140.45,130.74,130.41,130.40(×2),127.64,127.25,126.90,126.69,125.34,125.13(×2),124.78,124.52,123.42(d,J=233Hz),119.97,119.73,105.63,36.22。

[0104] Compound 11, HR-MS m / z: calculated for C 17 H 10 Cl2F3N2O2 401.0071, found 401.0067[M + H] + ; 1 H NMR(400MHz,Dimethyl Sulfoxide-d6)δ8.06 - 7.96(m,2H),7.56(d,J=1.9Hz,1H),7.49 - 7.42(m,2H),7.41 - 7.35(m,2H),6.96(s,1H). 13 C NMR(101MHz,Methanol-d4)δ167.09,143.28(d,J=54.0Hz),142.46,141.15,136.65,134.55,133.21,130.68,130.67(×2),129.61,127.59,127.14,123.91(×2),121.23(d,J=266Hz),107.62。

[0105] Compound 12, HR-MS m / z: calculated for C 23 H 16 F3N2O2 409.1164, found 409.1158[M + H] + ; 1HNMR(400MHz,Chloroform-d)δ8.17-8.10(m,2H),7.63-7.55(m,4H),7.53-7.43(m,4H),7.41-7.34(m,1H),7.35-7.28(m,2H),6.82(s,1H). 13 C NMR(101MHz, Methanol-d4)δ167.16,145.20,143.46(d,J=38.3Hz),142.69,142.11,139.74,130.84,130.47(× 2),129.18(×2),128.64(×2),127.60(×2),127.02(×2),126.63(×2),125.24(×2),122.54(d,J=255Hz),105.70.

[0106] Example 2 Synthesis of derivatives of compounds 4 and 11

[0107] The basic process of the reaction is shown below: The synthetic route of the derivatives of compounds 4 and 11.

[0108]

[0109] R 2 =4-COOH; 4-Me; 4-F; 4-Cl; 4-Br; 4-NH2; 4-NO2; 2-Naphthylhydrazine; 4-(2-amino-N-methylacetamide)

[0110] R 1 =4-iPr; 2-Cl, 4-Cl

[0111] Among them, according to different R 2 According to the group situation, select the corresponding raw materials.

[0112] R 2 =4-COOH, i.e., carboxyl group at position 4.

[0113] R 2 =4-Me, i.e. methyl group at the 4th position.

[0114] R 2 =4-F, that is, 4 F atoms.

[0115] R 2 =4-Cl, that is, 4 Cl atoms.

[0116] R 2 =4-Br, that is, 4 Br atoms.

[0117] R2 =4-NH2, i.e. amino group at position 4.

[0118] R 2 =4-NO2, that is, the 4-nitro group.

[0119] R 2 =2-Naphthylhydrazine, that is, R in (F) 2 -phenyl- is entirely replaced by 2-naphthyl. Specifically, 2-naphthylhydrazine is used as the starting material instead of phenylhydrazine.

[0120] R 2 =4-(2-amino-N-methylacetamide), that is, 2-amino-N-acetamide is substituted at position 4. Specifically, 2-amino-N-(4-phenylhydrazine)acetamide is used as the raw material instead of phenylhydrazine.

[0121] R 1 =4-iPr, i.e. 4-isopropyl.

[0122] R 1 =2-Cl,4-Cl, that is, R 2 There are two, namely the 2-position chlorine atom and the 4-position chlorine atom.

[0123] (1) 1 eq of acetophenone derivative (D) and 2 eq of NaH were added to a tetrahydrofuran solution, and the mixture was stirred for 5 min. Then 2.5 eq of ethyl trifluoroacetate was added. The reaction mixture was stirred at room temperature for 4 h and neutralized with 2N HCl. The precipitate formed was collected, washed with methanol and dried, and used directly in the next step without further purification.

[0124] (2) 1 eq of 1,3-diketone adduct (E) and 1.2 eq of substituted phenylhydrazine derivative were mixed in ethanol, refluxed for 8 hours, cooled to room temperature, added with distilled water, and the resulting precipitate was collected and dried to obtain a crude product (F). The crude product was separated by column chromatography, and the target compound was identified by TLC, 1 H-NMR, 13 C-NMR and HR-MS examination.

[0125] According to the above reaction route, the following compounds were synthesized:

[0126]

[0127] Compound 13: C 23 H 19 F3N2, 11H NMR (400 MHz, Chloroform-d) δ 8.13 (t, J = 1.9 Hz, 1H), 7.90 - 7.82 (m, 5H), 7.79 (dd, J = 7.7, 2.0 Hz, 1H), 7.51 (dd, J = 6.1, 3.4 Hz, 2H), 7.29 - 7.25 (m, 2H), 7.23 (s, 1H), 2.88 (qd, J = 6.8, 6.1 Hz, 1H), 1.28 (d, J = 6.7 Hz, 6H). 13 13C NMR (101 MHz, Chloroform-d) δ 148.52, 145.02, 143.28, 136.66, 134.34, 134.09, 132.37, 128.97 (×2), 128.50, 128.17, 127.83, 127.55, 127.11 (×2), 127.02, 123.00, 121.63, 117.98, 105.07, 34.22, 23.93 (×2).

[0128] Compound 14: C 20 H 19 F3N2, 1 1H NMR (400 MHz, Chloroform-d) δ 7.87 - 7.80 (m, 1H), 7.53 - 7.46 (m, 1H), 7.30 - 7.20 (m, 3H), 2.88 (qd, J = 6.8, 6.1 Hz, 1H), 2.40 (d, J = 0.9 Hz, 2H), 1.28 (d, J = 6.7 Hz, 3H). 13 13C NMR (101 MHz, Chloroform-d) δ 148.52, 145.03, 143.27, 136.71, 133.79, 132.33, 130.09 (×2), 128.97 (×2), 127.11 (×2), 123.02 (×2), 117.98, 105.10, 34.22, 23.93 (×2), 21.09.

[0129] Compound 15: C 19 H 16 ClF3N2, 1 1H NMR (400 MHz, Chloroform-d) δ 7.87 - 7.79 (m, 2H), 7.61 - 7.52 (m, 2H), 7.52 - 7.44 (m, 2H), 7.31 - 7.24 (m, 2H), 7.22 (s, 1H), 2.88 (qd, J = 6.8, 6.1 Hz, 1H), 1.28 (d, J = 6.7 Hz, 6H). 1313C NMR (101 MHz, Chloroform-d) δ 148.52, 145.03, 143.28, 137.96, 134.26, 132.34, 129.32 (×2), 128.97 (×2), 127.11 (×2), 124.61 (×2), 117.98, 105.10, 34.22, 23.93 (×2).

[0130] Compound 16: C 21 H 21 F3N4O, 1 1H NMR (400 MHz, Chloroform-d) δ 9.77 (s, 1H), 7.89 - 7.81 (m, 2H), 7.81 - 7.70 (m, 2H), 7.70 - 7.60 (m, 2H), 7.29 - 7.25 (m, 2H), 7.22 (s, 1H), 3.79 (t, J = 6.2 Hz, 2H), 2.88 (qd, J = 6.8, 6.1 Hz, 1H), 2.23 - 2.03 (m, 2H), 1.28 (d, J = 6.7 Hz, 6H). 13 13C NMR (101 MHz, Chloroform-d) δ 169.06, 148.52, 144.67, 143.28, 138.67, 133.96, 132.34, 128.97 (×2), 127.11 (×2), 124.21 (×2), 121.82 (×2), 117.98, 105.10, 43.38, 34.22, 23.93 (×2).

[0131] Compound 17: C 24 H 19 F3N2O2, 1 1H NMR (400 MHz, Chloroform-d) δ 8.43 - 8.30 (m, 1H), 8.25 - 8.15 (m, 1H), 8.05 (d, J = 8.6 Hz, 1H), 7.88 - 7.81 (m, 2H), 7.78 (d, J = 8.6 Hz, 1H), 7.58 (td, J = 7.8, 1.3 Hz, 1H), 7.47 (td, J = 7.8, 1.2 Hz, 1H), 7.32 (s, 1H), 7.30 - 7.25 (m, 2H), 2.88 (qd, J = 6.8, 6.1 Hz, 1H), 1.28 (d, J = 6.7 Hz, 6H). 13C NMR(101MHz,Chloroform-d)δ169.72,148.52,146.07,144.20,136.90,132.81,132.73,131.18,130.75,129 .37,128.95(×2),127.93,127.11(×2),127.04,125.58,124.88,118.83,118.38,104.59,34.22,23.93(×2).

[0132] Compound 18: C 19 H 16 F4N2, 1 H NMR(400MHz,Chloroform-d)δ7.87-7.80(m,2H),7.74-7.67(m,2H),7.30-7.24(m,2H ),7.22(s,1H),7.18-7.11(m,2H),2.88(qd,J=6.8,6.1Hz,1H),1.28(d,J=6.7Hz,6H). 13 C NMR(101MHz,Chloroform-d)δ161.87,148.52,144.65,143.27,136.05,132.35,128.9 7(×2),127.11(×2),125.37(×2),117.98(×2),116.26,105.10(×2),34.22,23.93(×2).

[0133] Compound 19: C 19 H 18 F3N3, 1 H NMR(400MHz,Chloroform-d)δ7.88-7.78(m,2H),7.57-7.49(m,2H),7.31-7.24(m,2H),7.22 (s,1H),6.93-6.83(m,2H),3.93(s,2H),2.88(qd,J=6.8,6.1Hz,1H),1.28(d,J=6.7Hz,6H). 13 C NMR(101MHz,Chloroform-d)δ148.52,147.90,145.03,143.28,132.34,130.64,128 .97(×2),127.11(×2),123.20(×2),117.98,115.48(×2),105.10,34.22,23.93(×2).

[0134] Compound 20: C 19H 16 BrF3N2, 1 H NMR(400 MHz, Chloroform-d) δ 7.89 - 7.79(m, 2H), 7.70 - 7.60(m, 4H), 7.32 - 7.25(m, 2H), 7.22(s, 1H), 2.88(qd, J = 6.8, 6.1 Hz, 1H), 1.28(d, J = 6.7 Hz, 6H). 13 C NMR(101 MHz, Chloroform-d) δ 148.52, 145.03, 143.28, 138.53, 132.74(×2), 132.34, 128.97(×2), 127.11(×2), 124.57(×2), 121.59, 117.98, 105.10, 34.22, 23.93(×2).

[0135] Compound 21: C 20 H 11 Cl2F3N2, 1 H NMR(400 MHz, Chloroform-d) δ 8.37 - 8.25(m, 2H), 7.90 - 7.72(m, 4H), 7.34 - 7.24(m, 2H), 7.22(s, 1H), 2.88(qd, J = 6.8, 6.1 Hz, 1H), 1.28(d, J = 6.7 Hz, 6H). 13 C NMR(101 MHz, Chloroform-d) δ 148.52, 146.19, 144.86, 144.37, 143.27, 132.35, 128.97(×2), 127.11(×2), 125.21(×2), 123.53(×2), 117.98, 105.09, 34.22, 23.93(×2).

[0136] Compound 22: C 19 H 16 F3N3O2, 1 H NMR(400 MHz, Chloroform-d) δ 8.14(t, J = 1.9 Hz, 1H), 7.91 - 7.82(m, 3H), 7.81 - 7.76(m, 2H), 7.57(d, J = 2.3 Hz, 1H), 7.51(dd, J = 6.1, 3.4 Hz, 2H), 7.43(dd, J = 8.1, 2.2 Hz, 1H), 7.26(s, 1H). 13C NMR(101MHz,Chloroform-d)δ143.84,140.79,137.51,134.34,134.09,134.02,133.65,131.06,1 30.20,129.25,128.92,128.50,128.17,127.83,127.55,127.02,122.79,121.61,118.38,105.19.

[0137] Compound 23: C 17 H 11 Cl2F3N2, 1 H NMR(400MHz,Chloroform-d)δ7.78(d,J=8.0Hz,1H),7.57(d,J=2.3Hz,1H),7.51(d,J=1.7H z,1H),7.50(d,J=1.6Hz,1H),7.43(dd,J=8.1,2.2Hz,1H),7.25(s,1H),7.25-7.21(m,2H). 13 C NMR(101MHz,Chloroform-d)δ143.71,140.77,136.92,134.02,133.79,133.51,1 31.06(×2),130.20,130.09,129.23,128.92,123.00(×2),118.38,105.20,21.09.

[0138] Compound 24: C 16 H8Cl3F3N2, 1 H NMR (400MHz, Chloroform-d) δ7.78(d,J=8.1Hz,1H),7.60-7.54(m,3H),7.50-7.45(m,2H),7.43(dd,J=8.1,2.2Hz,1H),7.25(s,1H). 13 C NMR(101MHz,Chloroform-d)δ143.70,140.77,138.14,134.26,134.02,133.5 1,131.06,130.20,129.32(×2),129.23,128.92,124.61(×2),118.38,105.20.

[0139] Compound 25: C 18 H 13 Cl2F3N4O, 11H NMR (400 MHz, Chloroform-d) δ 9.77 (s, 1H), 7.85 - 7.70 (m, 3H), 7.70 - 7.62 (m, 2H), 7.57 (d, J = 2.2 Hz, 1H), 7.43 (dd, J = 8.1, 2.2 Hz, 1H), 7.25 (s, 1H), 3.79 (t, J = 6.2 Hz, 2H), 2.17 (dt, J = 7.5, 6.1 Hz, 1H), 2.12 - 2.02 (m, 1H). 13 13C NMR (101 MHz, Chloroform-d) δ 169.06, 143.70, 140.77, 138.67, 134.02 (×2), 133.51, 131.06, 130.20, 129.23, 128.92, 124.20 (×2), 121.82 (×2), 118.38, 105.20, 43.38。

[0140] Compound 26: C 21 H 11 Cl2F3N2O2, 1 1H NMR (400 MHz, Chloroform-d) δ 8.43 - 8.38 (m, 1H), 8.23 - 8.17 (m, 1H), 8.06 (d, J = 8.6 Hz, 1H), 7.79 (dd, J = 8.3, 1.2 Hz, 2H), 7.62 - 7.55 (m, 2H), 7.50 - 7.40 (m, 2H), 7.31 (s, 1H). 13 13C NMR (101 MHz, Chloroform-d) δ 169.72, 144.97, 140.67, 137.10, 134.02, 133.69, 132.81, 131.18, 131.11, 130.75, 130.20, 129.60, 129.40, 128.92, 127.93, 127.04, 125.58, 124.88, 118.80 (d, J = 1.9 Hz), 104.54。

[0141] Compound 27: C 16 H8Cl2F4N2, 1 1H NMR (400 MHz, Chloroform-d) δ 7.78 (d, J = 8.1 Hz, 1H), 7.74 - 7.67 (m, 2H), 7.57 (d, J = 2.3 Hz, 1H), 7.43 (dd, J = 8.1, 2.2 Hz, 1H), 7.25 (s, 1H), 7.17 - 7.12 (m, 2H). 13C NMR(101MHz,Chloroform-d)δ161.87,143.72,140.77,136.37,134.02,133.5 1,131.06,130.20,129.23,128.92,125.32(×2),118.38,116.26(×2),105.20.

[0142] Compound 28: C 16 H 10 Cl2F3N3, 1 H NMR(400MHz,Chloroform-d)δ7.78(d,J=8.1Hz,1H),7.57(d,J=2.3Hz,1H),7.56-7. 51(m,2H),7.43(dd,J=8.1,2.2Hz,1H),7.25(s,1H),6.93-6.84(m,2H),3.93(s,2H). 13 C NMR(101MHz,Chloroform-d)δ147.90,143.70,140.77,134.02,133.51,131.0 6,130.36,130.20,129.23,128.92,123.19(×2),118.38,115.48(×2),105.20.

[0143] Compound 29: C 16 H8BrCl2F3N2, 1 H NMR (400MHz, Chloroform-d) δ7.78(d,J=8.0Hz,1H),7.68-7.62(m,4H),7.57(d,J=2.3Hz,1H),7.43(dd,J=8.1,2.2Hz,1H),7.25(s,1H). 13 C NMR(101MHz,Chloroform-d)δ143.70,140.77,138.80,134.02,133.51,132.7 4(×2),131.06,130.20,129.23,128.92,124.54(×2),121.59,118.38,105.20.

[0144] Compound 30: C 16 H8Cl2F3N3O2, 1H NMR (400MHz, Chloroform-d) δ8.34-8.29(m,2H),7.85-7.80(m,2H),7.78(d,J=8.1Hz,1H),7.57(d,J=2.3Hz,1H),7.43(dd,J=8.1,2.2Hz,1H),7.25(s,1H). 13 C NMR(101MHz,Chloroform-d)δ146.19,145.30,143.43,140.77,134.02,133.5 1,131.06,130.20,129.23,128.92,125.21(×2),123.53(×2),118.38,105.20.

[0145] Example 3 Determination of in vitro antifungal activity of synthetic compounds

[0146] In order to better study the antifungal activity of Compounds 1 to 30 synthesized in Examples 1 and 2 above, the following in vitro antifungal experiment was designed, and the specific experimental method is as follows.

[0147] 1. Experimental strains

[0148] Standard strain of Microsporum gypseum (M873.579), standard strain of Trichophyton mentagrophytes (M3.859), standard strain of Microsporum canis (ATCC8137).

[0149] 2 Experimental methods

[0150] 2.1 Preparation of drug solution

[0151] Fluconazole, ketoconazole, itraconazole and the synthesized compound were dried under reduced pressure at 25°C for 12 h, the dried samples were accurately weighed, and an appropriate amount of 20% DMSO was added to dissolve and the volume was fixed to prepare the drug molar concentrations of 6 μmol / mL, 3 μmol / mL,

[0152] 4 mL of each stock solution of 1.5 μmol / mL, 0.75 μmol / mL, and 0.375 μmol / mL was filtered through a 0.22 μm microporous filter membrane and stored in a refrigerator at 4°C for later use.

[0153] 2.2 Culture medium preparation

[0154] (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.

[0155] (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.

[0156] 2.3 Preparation of bacterial suspension

[0157] Take out the required Microsporum gypseum, Trichophyton mentagrophytes, and Microsporum canis 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 and make the colonies evenly cover 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, and adjust the concentration to 10 5 ~10 6 CFU / mL, and obtain bacterial suspension for later use.

[0158] 2.4 Determination of mycelial growth rate and antibacterial rate

[0159] Inoculate the test fungus on a PDA plate and culture it at 27°C for 5 days for later use. Use a sterilized hole puncher to cut a 1 cm diameter cake from the edge of the PDA plate inoculated with the test fungus, and inoculate the cake onto the drug-containing culture medium with the mycelium side facing down. Inoculate one cake into each culture dish. At the same time, set up a blank control, a negative control, and a positive control (fluconazole, itraconazole, ketoconazole). Repeat the test three times and culture it in a constant temperature incubator at 27°C. After 7 days of culture, measure the colony diameter of the test fungus on the drug-containing culture medium using the cross method, and calculate the inhibition rate of the linear growth of mycelium by each drug solution treatment compared with the control. The mycelium growth rate and inhibition rate were tested using the methods of references 1 and 2. References [1]: Lu H, Zou W, Meng JC, et al. New bioactive metabolites produced by Colletotrichum, sp. anendophytic fungus in Artemisia annua [J]. Plant Science, 2000, 151 (1): 67-73; Reference [2] Chen Yiling. Optimization of extraction process and antibacterial activity of effective components from Scutellaria baicalensis and Gastrodia elata [D]. Jilin University, 2017.

[0160] The mycelium growth rate and antibacterial rate were calculated as follows:

[0161] Mycelial growth rate (cm / d) = (average measured diameter - 1.0) / days;

[0162] Inhibition rate (%) = (blank control colony diameter - treated colony diameter) ÷ blank control colony diameter × 100.

[0163] 2.5 Minimum inhibitory concentration (MIC) determination

[0164] 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.

[0165] 3. Antibacterial test results

[0166] 3.1 Effects of compounds 1-30 on mycelial growth rate

[0167] The test results of mycelium growth rate method are shown in Tables 1 to 3.

[0168] Among them, the positive control group itraconazole and fluconazole 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 1-30 had different degrees of influence on the hyphal growth of these three fungi, and were all better than itraconazole and fluconazole. At 6μmol / mL, compound 5 had a very obvious inhibitory effect on the hyphal growth of Microsporum canis, and compounds 3 and 4 had the greatest effect on the hyphal growth rate of Trichophyton mentagrophytes and Microsporum gypseum, both less than 1mm / d.

[0169] Table 1 Effects of different concentrations of compounds on the mycelial growth rate of Microsporum canis

[0170]

[0171]

[0172] Table 2 Effects of different concentrations of compounds on the hyphae growth rate of Trichophyton mentagrophytes

[0173]

[0174]

[0175] Table 3 Effects of different concentrations of compounds on the mycelial growth rate of Microsporum gypseum

[0176]

[0177]

[0178] 4.1.2 Inhibitory effects of compounds 1-30 on three bacteria

[0179] The results of the determination of the inhibition rate are shown in Tables 4 to 6.

[0180] In the positive control group, ketoconazole had the highest antibacterial rate, and its antibacterial effect was better than that of itraconazole and fluconazole. Compounds 1-30 had different degrees of antibacterial effect on the three fungi.

[0181] For Microsporum canis, when the concentration was 6 μmol / mL, the inhibition rates of compounds 2, 4, 5, 6, 7, 9, 11, 12, 14, 17, 18, 26, and 27 were all greater than 60%. However, at low concentrations (0.375 μmol / mL), the inhibition rates of these compounds were lower and the antibacterial effects were poor. Only compound 12 had a higher inhibition rate (>50%) at the measured concentrations.

[0182] For Trichophyton mentagrophytes, at a concentration of 6 μmol / mL, the inhibition rates of compounds 5, 6, 7, 8, 9, 10, 11, 17, 18, 20, 26, and 27 were all greater than 60%, and the inhibition rates of compounds 2, 4, 12, 13, 15, 22, and 25 were all greater than 50%.

[0183] For Microsporum gypseum, at a concentration of 6 μmol / mL, the inhibition rates of compounds 3-12, 17, 18, 23, 26, and 27 against Microsporum gypseum were all greater than 60%, and the inhibition rates of compounds 1, 2, 14, 15, 17, 18, 22, 24, and 25 against Microsporum gypseum were all greater than 50%.

[0184] Table 4 Inhibition rate of compounds at different concentrations against Microsporum canis

[0185]

[0186]

[0187] Table 5 Inhibition rate of compounds at different concentrations against Trichophyton mentagrophytes

[0188]

[0189]

[0190] Table 6 Inhibition rate of compounds at different concentrations against Microsporum gypseum

[0191]

[0192]

[0193] 4.1.3 MIC results of compounds 1-30 against four fungi

[0194] The results of MIC determination are shown in Table 7. Compounds 1-30 have different degrees of inhibitory effects on the three fungi. Compounds 4, 17, 20, 26, and 27 have the best overall effect. Among them, compounds 11, 12, 17, 18, 26, and 27 have the lowest MIC against Microsporum canis, which is 187.5 μmol / L, followed by compound 14, with a MIC of 375 μmol / L. Compounds 17 and 26 have the lowest MIC against Trichophyton mentagrophytes, which is 187.5 μmol / L, followed by compounds 4, 5, 6, 12, 13, 18, and 27, which is 375 μmol / L. Compounds 26 and 27 have the lowest MIC against Microsporum gypseum, which is 187.5 μmol / L, followed by compounds 4, 11, 17, and 18, with a MIC of 375 μmol / L. In the positive control group, the minimum MIC of ketoconazole against Trichophyton mentagrophytes and Microsporum gypseum was 187.5 μmol / L, and compounds 20 and 26 had comparable effects. In addition, the MIC of compound 26 against Microsporum canis was lower than that of ketoconazole.

[0195] Table 7 MIC of compounds 1-30 against four bacteria

[0196]

[0197]

[0198] The antibacterial test showed that among the 30 compounds, compounds 4, 17, 20, 26, and 27 had inhibitory effects on the three bacteria, among which compounds 17, 26, and 27 had the best effects.

[0199] Example 4 Determination of cytotoxicity of synthetic compounds on normal cells

[0200] 1. Cell line: NIH3T3 cells

[0201] 2. Experimental Methods

[0202] 2.1 Cell inoculation

[0203] 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.

[0204] 2.2 Preparation of drug solution and administration

[0205] 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.

[0206] 2.3 MTS kit to detect cell viability

[0207] 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 .

[0208] 3. Experimental results

[0209] IC of compounds against NIH3T3 cells 50 The results are shown in Table 8. In general, the compounds have little cytotoxicity to normal cells. All compounds have less cytotoxicity than ketoconazole, which shows that the toxicity of these compounds to normal tissues should be lower than that of ketoconazole. 50 The smallest, the most cytotoxic, IC of compounds 2, 7, 8, 9, 20, 27, 30 50 The values ​​were all >200 μmol / L, indicating low cytotoxicity, and IC 50 The largest one was compound 7, which was 399.7 μmol / L.

[0210] Table 8 IC of compounds 1-30 on NIH3T3 cells 50

[0211]

[0212] In summary, the positive controls fluconazole and itraconazole have no obvious inhibitory effect on Microsporum canis, Trichophyton mentagrophytes, and Microsporum gypseum, while ketoconazole has obvious inhibitory effect on these three bacteria. The synthesized compounds 1-30 have different degrees of antibacterial effect on Microsporum canis, Trichophyton mentagrophytes, and Microsporum gypseum, among which compounds 4, 14, and 19 have obvious inhibitory effect on these three bacteria, and are all better than fluconazole, itraconazole and ketoconazole (partially equivalent); compounds 15, 16, 17, 19, 21, 22, 23, 24, 26, and 28 are second. And compounds 1-30 show low cytotoxicity to normal cells and have high safety. The above compounds all have potential antifungal application value.

[0213] Example 5 Prediction of antifungal targets of representative compounds

[0214] Representative compounds 4, 9, and 10 were selected, and the molecular docking method was used to investigate 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.

[0215] From the RCSB database (http: / / www.rcsb.org / ), we found: PKh kinase domain from Candida albicans SC5314 (PDB ID: 4C0T); peroxisomal hydratase-dehydrogenase-epimerase from Candida tropicalis (PDB ID: 1PN2); peroxisomal hydratase-dehydrogenase-epimerase from Candida tropicalis (PDB ID: 1PN4); acetate kinase from Cryptococcus neoformans (PDB ID: 4H0P); acetyl-CoA acetyltransferase from Aspergillus fumigatus Af293 (PDB ID: 6ARE); endopeptidase from Cryptococcus neoformans (PDB ID: 6R61); DNA repair protein Rad8 from Cryptococcus neoformans variant grubii H99 (PDB ID: 7T02); acetyl-CoA synthetase from Cryptococcus neoformans (PDB ID: 8EPS); peptidyl-prolyl isomerase from Aspergillus fumigatus (PDB ID: 8EPS); ID: 7U0U); hydroxylamine oxidoreductase-like protein from Candida albicans (PDB ID: 6T5E). Download the SDF format files of compounds 1-28 and positive control fluconazole, ketoconazole, and itraconazole structures from the Pub Chem database (https: / / pubchem.ncbi.nlm.nih.gov / ), import them into the Discovery software, and obtain their 3D structures. Use the Discovery software to remove ligands and non-protein molecules (such as water molecules) in the target protein and perform hydrogenation treatment, then perform molecular docking of the compounds and target proteins in the software and perform visualization. Some of the results are shown in Figure 2. Figures 2 to 4 The results are shown in Table 9.

[0216] Table 9 Lib Dock Score of each compound and each protein

[0217]

[0218]

[0219] In the table, FCA stands for fluconazole, KET stands for ketoconazole, and ICZ stands for itraconazole.

[0220] The above proteins are directly or indirectly involved in the conversion of acetyl-CoA. It can be seen from Table 9 that most of the compounds 1-30 and proteins 1PN2, 1PN4, 4H0P, 6ARE, 6R61, 7T02, 7U0U, and 6T5E can be docked successfully. Proteins 1PN2 and 1PN4 are peroxisomal hydratase-dehydrogenase-epimerase of Candida tropicalis, which are respectively involved in the conversion of high-energy molecules such as fatty acids in cells; decomposing high-energy molecules such as fatty acids or converting them into acetyl-CoA. There are 16 compounds 1-30 and protein 1PN2 with LibDock Scores greater than 100, of which compounds 1 and 3 have LibDock Scores greater than 120.

[0221] There are 10 compounds with a Lib Dock Score greater than 100 for compound 1-30 and protein 1PN4, of which compounds 4, 8, and 10 have a Lib Dock Score greater than 120. Protein 4H0P is a new Cryptococcus acetate kinase protein, responsible for catalyzing the reversible reaction of acetyl phosphate and ADP generation and ATP, and there are 9 compounds with a Lib Dock Score greater than 100 docked with compound 1-30. Protein 6ARE ​​is an acetyl-CoA acetyltransferase of Aspergillus fumigatus Af293, and there are 6 compounds with a Lib Dock Score greater than 100 docked with compound 1-30. Protein 6R61 is an endopeptidase of Cryptococcus neoformans, and there are 15 compounds with a Lib Dock Score greater than 100 docked with compound 1-30. Protein 7U0U is an Aspergillus fumigatus peptidyl prolyl isomerase, and there are 8 compounds with a Lib Dock Score greater than 100 docked with compound 1-30, of which compounds 1, 3, and 12 have a Lib Dock Score greater than 120. For proteins 4C0T and 8EPS, most of the compounds can be successfully docked with them. Therefore, it can be predicted that the mechanism of action of these compounds may be to directly or indirectly affect the conversion of acetyl-CoA in fungal cells.

[0222] The above embodiments describe only the basic principles, main features and / or advantages of the present invention. For those skilled in the art, when understanding the scheme described in the specific embodiments of the present invention, they can refer to the conventional technical manuals in the field. At the same time, for the places where the above terms appear, they can make appropriate understandings or adjustments for reference, and deduce the implementation of the same or similar technical solutions without paying creative work. These changes and improvements all fall within the scope of protection required by the present invention.

Claims

1. Application of the aromatic ring substituted pyrazole derivatives of the structure shown in Formula I in the preparation of antifungal drugs for fur animals, in, Ar 1 is any one of phenyl, naphthyl, phenanthrenyl and fluorenyl; Ar 2 is any of phenyl and naphthyl; R 1 Selected from: any one of hydrogen, halogen atom, C1-C6 alkyl, halogen-substituted C1-C5 alkyl, C1-C5 alkyloxy, and phenyl; R 2 Any one selected from the group consisting of hydrogen, halogen atoms, carboxyl, carboxyl C1-C3 alkyl, hydroxyl, C1-C6 alkyl, halogen-substituted C1-C5 alkyl, amino, nitro, formamide, acetylamino, aminoformamide, and aminoacetamide; m=0-3, n=0-3.

2. The application according to claim 1, characterized in that: Ar 2 is any one of phenyl, 1-naphthyl and 2-naphthyl; Ar 1 It is any one of phenyl, naphthyl, 2-phenanthrenyl and 2-fluorenyl.

3. The application according to claim 1, characterized in that: When Ar 1 When it is phenyl and m=1, R 1 The substitution position is 4; When Ar 2 When it is -1-naphthyl or -2-naphthyl, and n=1, R 2 The substitution position is 4; When Ar 2 When it is -2-fluorenyl and n=1, R 2 The substitution position is at position 4, 5, 6, 7 or 8; When Ar 2 When it is phenyl and n=1, R 2 The substitution position is 4; When Ar 2 When it is -1-naphthyl and n=1, R 2 The substitution position is 4.

4. The use according to claim 1, characterized in that: The aromatic ring substituted pyrazole derivative has a structure shown in Formula II, Formula III or Formula IV: R 1 Selected from: hydrogen, halogen atoms, C1-C6 alkyl, halogen-substituted C1-C5 alkyl, C1-C5 alkyloxy, phenyl; R 2 Any one selected from the group consisting of hydrogen, halogen atoms, carboxyl, carboxyl C1-C3 alkyl, hydroxyl, C1-C6 alkyl, halogen-substituted C1-C5 alkyl, amino, nitro, aminoformamide, and aminoacetamide; m=0-2; n=0-2.

5. The use according to claim 4, characterized in that: R 1 Any one selected from: hydrogen, C1-C4 alkyl, halogen-substituted C1-C3 alkyl, C1-C3 alkyloxy, and halogen atom; R 2 Any one selected from: a carboxyl group, a halogen atom, an aminoacetylamino group, a nitro group, a nitro group, and an amino group.

6. The use according to claim 4, characterized in that: When m = 1, R 1 Substitute at the 4-position of the benzene ring; when m = 2, the two R 1 The substitution positions on the benzene ring are one of the following combinations: 2-position and 4-position, 3-position and 5-position, 3-position and 4-position; When n = 1, R 2 Substitution is at the 4-position of the phenyl group or the 4-position of the naphthyl group.

7. The use according to claim 4, characterized in that: The aromatic ring substituted pyrazole derivative is any one of the following compounds:

8. The use according to claim 1, characterized in that: The medicine is a medicine for treating Microsporum canis, Microsporum gypseum and Trichophyton mentagrophytes.

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