Aromatic ring substituted pyrazole derivative and use thereof

Abstract

The present invention relates to an aromatic ring substituted pyrazole derivative and the use thereof. The aromatic ring substituted pyrazole derivative has a structure as shown in formula I, wherein Ar1 is any one of phenyl, substituted phenyl, naphthyl, substituted naphthyl, substituted anthryl, phenanthryl, substituted phenanthryl, fluorenyl, substituted fluorenyl, pyrrolyl, substituted pyrrolyl, pyridyl and substituted pyridyl; Ar2 is any one of phenyl, substituted phenyl, naphthyl, substituted naphthyl, substituted anthryl, phenanthryl, substituted phenanthryl, pyrrolyl, substituted pyrrolyl, pyridyl and substituted pyridyl; R1 is selected from hydrogen, halogen atom, C1-C6 alkyl, halogen substituted C1-C5 alkyl, C1-C5 alkoxy, C2-C4 alkenyl, nitro and phenyl; and R2 is selected from hydrogen, halogen atom, carboxyl, carboxyl C1-C3 alkyl, hydroxyl, and C1-C6 alkyl. The compound can effectively inhibit Microsporum canis, Microsporum gypseum and Trichophyton mentagrophytes, has a low toxicity, and can be used in antifungal application, in particular in veterinary clinical antifungal application.

Description

Aromatic ring substituted pyrazole derivative and its application 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 infections are common diseases in both humans and animals. Common human pathogenic fungi include Candida, Ringworm, and Cryptococcus. Fungal skin diseases in fur-bearing animals are common clinical animal diseases, primarily caused by Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, and Trichophyton verrucosum. Infection can cause ringworm in humans, cattle, horses, pigs, cats, dogs, and other animals. These infections are often stubborn and difficult to cure, prone to cross-infection and transmission. If left untreated, they can lead to systemic infection, which is highly harmful.

[0003] Treatment for this disease is long, complex, and costly. Ketoconazole, itraconazole, and fluconazole are commonly used in human clinical practice, but resistance has emerged. Veterinary medicine typically uses the human antifungal drugs terbinafine, ketoconazole, and itraconazole, but there is a lack of animal-specific antifungal compounds. This conflicts with Article 41 of the Regulations on the Administration of Veterinary Drugs, which prohibits the use of human drugs in animals. Furthermore, many clinically used antifungal drugs suffer from fungal resistance, adverse reactions, narrow antimicrobial spectrum, low tissue concentrations, and poor oral absorption. Therefore, the development of safe, resistant, and convenient antifungal drugs, particularly novel antifungal drugs specifically for animals, is crucial. Summary of the Invention

[0004] The present invention aims to overcome the existing problem of a lack of antifungal chemicals specifically for animal use, which has led to the legal and regulatory issues of using human-grade drugs for animal treatment. The present invention provides an aromatic-ring-substituted pyrazole derivative. By synthesizing a series of aromatic-ring-substituted pyrazole derivatives, particularly azole benzoic acid compounds, compounds with low toxicity and high efficacy for animal-specific antifungal applications are screened, thereby meeting the demand for specific animal antifungal drugs. Furthermore, 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 the structure shown in Formula I:

[0007] in,

[0008] 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;

[0009] Ar 2 is any one of phenyl, substituted phenyl, naphthyl, substituted naphthyl, substituted anthracenyl, phenanthryl, substituted phenanthryl, pyrrolyl, substituted pyrrolyl, pyridinyl, and substituted pyridinyl;

[0010] R 1 Selected from: hydrogen, halogen atoms, C1-C6 alkyl, halogen-substituted C1-C5 alkyl, C1-C5 alkyloxy, C2-C4 alkenyl, nitro, phenyl;

[0011] R 2 Selected from: hydrogen, halogen, carboxyl, carboxyl C1-C3 alkyl, hydroxyl, C1-C6 alkyl, halogen-substituted C1-C5 alkyl, amino, nitro, formamide, acetamido, aminoformamide, aminoacetamide, phenyl;

[0012] m=0-3, n=0-3.

[0013] Further, Ar 1 is any one of phenyl, substituted phenyl, naphthyl, substituted naphthyl, substituted anthracenyl, 2-phenanthrenyl, substituted phenanthrenyl, 2-fluorenyl, and substituted fluorenyl. 1 It is any one of phenyl, substituted phenyl, naphthyl, substituted naphthyl, 2-phenanthrenyl, and 2-fluorenyl.

[0014] 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.

[0015] Preferably, when Ar 1 When it is a substituted phenyl group and m=1, R 1 The substitution position is 4; when Ar 2 When it 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 position 4, 5, 6, 7 or 8.

[0016] 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 substituted-1-naphthyl and n=1, R 2 The substitution position is 4.

[0017] 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, phenyl.

[0018] 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.

[0019] Further, Ar 1 and Ar 2 It is an aromatic group.

[0020] Furthermore, the aromatic ring-substituted pyrazole derivative has a structure shown in Formula II, Formula III or Formula IV:

[0021] 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, phenyl.

[0022] 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.

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

[0024] Furthermore, n = 0-2. Preferably, n = 0-2.

[0025] Further, R 1 Selected from: hydrogen, C1-C4 alkyl, halogen-substituted C1-C3 alkyl, C1-C3 alkyloxy, halogen atom, amino, nitro.

[0026] Further, R 2 Selected from: carboxyl group, halogen atom, aminoacetylamino group, nitro group, nitro group, amino group.

[0027] Further, R 2 Substitution is at the 4-position of the benzene ring or the naphthalene ring.

[0028] Furthermore, a C1-C6 alkyl group refers to an alkyl group having 1 to 6 carbon atoms; preferably, a C1-C4 alkyl group refers to an alkyl group having 1 to 4 carbon atoms. Specifically, the C1-C6 alkyl group is selected from the group consisting of: methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl, and tert-butyl. The C1-C4 alkyl group is selected from the group consisting of: methyl, ethyl, propyl, isopropyl, isobutyl, and n-butyl.

[0029] Furthermore, the carboxyl C1-C3 alkyl group refers to a carboxylmethyl group, 1-carboxyl-ethyl group, 2-carboxyl-ethyl group, 1-methyl-2-carboxyl-ethyl group, 1-carboxylpropyl group, 2-carboxylpropyl group, or 3-carboxylpropyl group, and can be a carboxyl group at any position of a, β, γ...ω, in particular a carboxyl group at the ω position of the alkyl group.

[0030] 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.

[0031] Preferably, the halogen-substituted C1-C5 alkyl group is a C1-C5 alkyl group in which some or all of the hydrogen atoms are replaced by halogen, for example, the following alkyl groups substituted by halogen: methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl, tert-butyl.

[0032] 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 group, an ethyl group, an n-propyl group or an isopropyl group are substituted by halogen atoms.

[0033] Furthermore, the C1-C5 alkyloxy group refers to an oxy group connected to an alkyl group having 1-5 carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butyloxy, isobutyloxy, and tert-butyloxy. Preferably, the C1-C4 alkyl group is methyl, ethyl, propyl, or isopropyl.

[0034] Furthermore, the C1-C5 alkyloxy group is preferably a C1-C3 alkyloxy group, specifically a methoxy group, an ethoxy group or a propyloxy group.

[0035] Further, R 1 It is isopropyl.

[0036] Further, R 2 Selected from carboxyl, methyl, amino. For example, R 2 Preferred is a carboxyl group.

[0037] Furthermore, the halogen atom or halogen in the present invention is chlorine, fluorine, bromine or iodine, and is particularly preferably chlorine, fluorine or bromine. "Halo" means chloro, fluoro, bromo or iodo, and can be substituted by one or more halogen atoms.

[0038] Furthermore, when m=1, R 1 Substituted 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;

[0039] When n=1, R 2 Substituted at the 4-position of the aromatic ring or the 4-position of the naphthalene ring;.

[0040] Furthermore, the above-mentioned halogen or halogen atom is a fluorine atom and / or a halogen atom.

[0041] 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.

[0042] Furthermore, the aromatic ring substituted pyrazole derivative is any one of the following compounds:

[0043] Furthermore, the aromatic ring substituted pyrazole derivative is 1-5, 7-30 among the above compounds.

[0044] Furthermore, the application of the above aromatic ring substituted pyrazole derivatives in the preparation of animal antifungal drugs.

[0045] Preferably, the drug is a drug for treating fungal infections in fur animals.

[0046] Preferably, the application is the application for preparing a drug for treating Microsporum canis, Microsporum gypseum, and Trichophyton mentagrophytes.

[0047] Furthermore, the use of compound 3 and compound 4 among the above compounds in drugs for treating Trichophyton mentagrophytes and / or Microsporum gypseum.

[0048] An animal antifungal drug comprising the aforementioned 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.

[0049] 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.

[0050] 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.

[0051] Preferably, the pharmaceutical use of compounds 2, 4, 12, 13, 15, 22, and 25 is a drug for treating Trichophyton mentagrophytes.

[0052] Preferably, the pharmaceutical use of compounds 3-12, 17, 18, 23, 26, and 27 is a drug for treating Microsporum gypseum.

[0053] Preferably, the pharmaceutical use of compounds 1, 2, 14, 15, 17, 18, 22, 24, and 25 is a drug for treating Microsporum gypseum.

[0054] In the above-mentioned pharmaceutical applications, the drugs for treating Microsporum canis, Trichophyton mentagrophytes and / or Microsporum gypseum are drugs that have a killing effect on the corresponding fungi. That is, the corresponding drugs have the therapeutic effect of inhibiting or killing the corresponding fungi.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] The aromatic ring-substituted pyrazole derivatives of the present invention can effectively inhibit Microsporum canis, Microsporum gypseum, and Trichophyton mentagrophytes, and have low toxicity, and can be used in antifungal applications, especially in veterinary clinical antifungal applications.

[0057] 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.

[0058] The aromatic ring-substituted pyrazole derivatives of the present invention have selectivity for normal cells and less toxicity to normal cells, and are superior to ketoconazole. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 shows the inhibitory effects of compounds 3, 4, 7, 9, 11, and 12 on three bacteria at a concentration of 6 μmol / mL.

[0060] Figure 2 is the molecular docking diagram of compound 1 and 1PN2.

[0061] Figure 3 is the molecular docking diagram of compound 3 and 1PN2.

[0062] Figure 4 is a molecular docking diagram of compound 4 and 1PN4. DETAILED DESCRIPTION

[0063] In order to more clearly describe the invention purpose, technical solutions and technical effect advantages in the specific implementation cases of the present invention, the solutions in the specific embodiments will be described in detail in conjunction with the drawings of the specification of the present invention. The specific technical solutions involved in the following specific embodiments are only for the purpose of clearly and completely describing the innovative technical solutions of the present invention. They themselves are only part of the specific implementation plans that can be adopted by the present invention, not all embodiments, and should not be understood as limiting the innovative solutions of the present invention. Any solution that adopts the same inventive concept of the present invention should be included in the scope of protection of the present invention.

[0064] Molarity, M, refers to the amount of solute per unit volume of solution, usually expressed in mol / L. It is often expressed as M (molarity).

[0065] Equivalent concentration, N, the concentration of a solution expressed in gram equivalents of solute contained in 1 liter of solution is called equivalent concentration, represented by the symbol N.

[0066] Reaction equivalent, eq, refers to the equivalent amount of action of a chemical substance in a reaction. An equivalent chemical reaction is one in which chemical substances act according to stoichiometric relationships, consuming or generating a chemical equivalent.

[0067] Example 1 Synthesis of azole benzoic acid compounds

[0068] The basic process of the reaction is as follows: Synthesis route of azole benzoic acid compounds.

[0069] R 2 =COOH

[0070] 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;

[0071] 2-Cl, 4-Cl; 4-BP; 4-Me; 4-Et

[0072] The reaction solvents are THF = tetrahydrofuran and EtOH = ethanol.

[0073] R 2 is the 4-position carboxyl group.

[0074] R 1 =2-phenanthrene, i.e. R in (A) 1 -phenyl group is replaced by 2-phenanthryl group.

[0075] R 1 =2-F,4-F; that is, R 1 There are two, namely 2 F atoms and 4 F atoms.

[0076] R 1 =3-CF3,5-CF3; that is, R 1 There are two, namely 3-digit CF3 and 5-digit CF3.

[0077] R 1 =4-iPr, i.e. 4-isopropyl.

[0078] R 1 =4-Me, i.e. methyl group at the 4th position.

[0079] R 1 =4-Et, i.e. 4-ethyl group.

[0080] R 1 =2-F,4-Cl, i.e. R 1 There are two, namely 2 F atoms and 4 chlorine atoms.

[0081] R 1 =4-C2H5O, i.e. ethoxy group at position 4.

[0082] R 1 =3-MeO,4-MeO, i.e. R 1 There are two, namely methoxy at position 3 and methoxy at position 4.

[0083] R 1 =2-fluorene, that is, "R 1 -phenyl" is replaced by 2-fluorenyl.

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

[0085] R 1 =BP, that is, R 1 =4-phenyl, R 1 Together with the attached phenyl groups, they form a biphenyl group.

[0086] (1) To a tetrahydrofuran solution, 1 eq of acetophenone derivative (A) and 2 eq of NaH were added, 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 resulting precipitate was collected, washed with methanol, dried, and used directly in the next step without further purification.

[0087] (2) 1eq 1,3-diketone adduct (B) and 1.2eq 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.

[0088] According to the above reaction route, the following compounds were synthesized:

[0089] Compound 1, HR-MS m / z: calculated for C 25 H 14 F3N2O2433.1164, found 433.1160[M+H] + ; 1 H NMR (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.

[0090] Compound 2, HR-MS m / z: calculated for C 17 H 10 F5N2O2369.0662 found 369.0657[M+H] + ; 11H 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).

[0091] 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.

[0092] 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).

[0093] 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.

[0094] Compound 6, HR-MS m / z: calculated for C 18 H 13 F3N2O2, 347.1007, found 347.0996 [M+H] + ; 11H 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。

[0095] 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。

[0096] 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。

[0097] 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。

[0098] Compound 10, HR-MS m / z: calculated for C 24 H 16 F3N2O2 421.1164, found 421.1158 [M+H] + ; 11H NMR (400 MHz, Chloroform-d) δ 8.14 - 8.05 (m, 2H), 7.82 - 7.71 (m, 2H), 7.56 (dt, J = 7.5, 1.0 Hz, 1H), 7.52 - 7.43 (m, 3H), 7.43 - 7.38 (m, 1H), 7.35 (td, J = 7.4, 1.3 Hz, 1H), 7.24 - 7.17 (m, 1H), 6.83 (s, 1H), 3.89 (s, 2H). 13 13C NMR (101 MHz, Methanol-d4) δ 167.24, 145.92, 143.25 (d, J = 98 Hz), 143.60, 142.75, 140.​​​​​​​​​​​​​​​​​​​​​​​H NMR (400MHz, Chloroform-d) δ 8.17-8.10 (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.

[0101] Example 2 Synthesis of derivatives of compounds 4 and 11

[0102] The basic process of the reaction is shown below: Synthesis route of derivatives of compounds 4 and 11.

[0103] R 2 =4-COOH; 4-Me; 4-F; 4-Cl; 4-Br; 4-NH2; 4-N02; 2-Naphthylhydrazine; 4-(2-amino-N-methylacetamide)

[0104] R 1 =4-iPr; 2-Cl, 4-Cl

[0105] Among them, according to different R 2 According to the group situation, select the corresponding raw materials.

[0106] R 2 =4-COOH, i.e., carboxyl group at position 4.

[0107] R 2 =4-Me, i.e. methyl group at the 4th position.

[0108] R 2 =4-F, i.e. 4 F atoms.

[0109] R 2 =4-Cl, that is, 4 Cl atoms.

[0110] R 2 =4-Br, that is, 4 Br atoms.

[0111] R 2 =4-NH2, i.e. amino group at position 4.

[0112] R 2 =4-NO2, i.e. nitro group at position 4.

[0113] 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.

[0114] R 2 =4-(2-amino-N-methylacetamide), i.e., 2-amino-N-acetamido is substituted at position 4. Specifically, 2-amino-N-(4-phenylhydrazine)acetamide is used as the raw material instead of phenylhydrazine.

[0115] R 1 =4-iPr, i.e. 4-isopropyl.

[0116] R 1 =2-Cl, 4-Cl, i.e. R 2 There are two, namely the 2-position chlorine atom and the 4-position chlorine atom.

[0117] (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 resulting precipitate was collected, washed with methanol, dried, and used directly in the next step without further purification.

[0118] (2) 1 eq of 1,3-diketone adduct (E) was mixed with 1.2 eq of substituted phenylhydrazine derivative 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.

[0119] According to the above reaction route, the following compounds were synthesized:

[0120] 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).

[0121] 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.

[0122] 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).

[0123] 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).

[0124] 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).

[0125] 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).

[0126] 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).

[0127] 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).

[0128] 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).

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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, 1,27.93, 127.04, 125.58, 124.88, 118.80 (d, J = 1.9 Hz), 104.54.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] Compound 30: C 16 H8Cl2F3N3O2, 1H NMR (400MHz, Chloroform-d) δ 8.34-8.29 (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.

[0138] Example 3 In vitro antifungal activity assay of the synthesized compounds

[0139] 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 experiments were designed. The specific experimental methods are as follows.

[0140] 1. Experimental strains

[0141] Standard strains of Microsporum gypseum (M873.579), Trichophyton mentagrophytes (M3.859), and Microsporum canis (ATCC8137).

[0142] 2 Experimental methods

[0143] 2.1 Preparation of drug solution

[0144] 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 dissolved in 20% DMSO. The resulting solutions were fixed to volume to obtain 4 mL of stock solutions with drug concentrations of 6 μmol / mL, 3 μmol / mL, 1.5 μmol / mL, 0.75 μmol / mL, and 0.375 μmol / mL, respectively. The solutions were filtered through a 0.22 μm microporous membrane and stored in a refrigerator at 4°C until use.

[0145] 2.2 Culture medium preparation

[0146] (1) Preparation of potato agar medium (PDA): Take fresh peeled potatoes, cut them into small pieces, weigh 200g, put them into 1L distilled water and boil for 30min, filter them through 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 and heat it. After the agar is completely melted, divide it into 250mL conical flasks while it is hot and plug them with cotton plugs. Wrap them with newspaper and sterilize them in a high-pressure steam sterilizer at 121℃ for 30min. After sterilization, transfer them to a sterile operating table and wait until the temperature of the culture medium drops to about 50℃. Pour the sterile plate into a shaker and lay it flat to make a PDA plate. After solidification, store it at 4℃ for later use.

[0147] (2) Preparation of drug-containing plates: 1 mL of each of the above-prepared drug solutions and 9 mL of the PDA medium cooled to approximately 50°C were 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 cooled to 50°C, 1 mL of 30% DMSO and 9 mL of PDA medium were added to prepare control plates for the drug-containing plates. After solidification, the plates were stored at 4°C for future use.

[0148] 2.3 Preparation of bacterial suspension

[0149] Take out the required Microsporum gypseum, Trichophyton mentagrophytes, and Microsporum canis from the refrigerator and inoculate the three fungi onto SDA plates using a sterile inoculating loop. Incubate at 27°C for 2 weeks to activate the fungi and allow the colonies to evenly cover the culture medium. Pipette 2 mL of sterile saline to rinse the hyphae and spores on the surface of the colonies. Count the rinse containing hyphae and spores using a hemocytometer and adjust the concentration to 10 5 ~10 6 CFU / mL, and obtain bacterial suspension for later use.

[0150] 2.4 Determination of mycelial growth rate and inhibition rate

[0151] The test fungus was inoculated on a PDA plate and cultured at 27°C for 5 days. Use a sterilized puncher to cut a 1 cm diameter bacterial cake from the edge of the PDA plate inoculated with the test fungus, and inoculate the bacterial cake onto the drug-containing culture medium with the mycelium side facing down. One bacterial cake was inoculated into each culture dish. At the same time, a blank control, a negative control, and a positive control (fluconazole, itraconazole, ketoconazole) were set up. The test was repeated three times and cultured in a constant temperature incubator at 27°C. After culturing for 7 days, the colony diameter of the test fungus on the drug-containing culture medium was measured using the cross-cross method, and the inhibition rate of the linear growth of the mycelium was calculated by comparing 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. an endophytic fungus in Artemisia annua [J]. Plant Science, 2000, 151(1): 67-73; Reference [2] Chen Yiling. Optimization of extraction process and antibacterial activity of active ingredients from Scutellaria baicalensis and Gastrodia elata [D]. Jilin University, 2017.

[0152] The mycelial growth rate and inhibition rate were calculated as follows:

[0153] Mycelial growth rate (cm / d) = (average of measured diameters - 1.0) / days;

[0154] Inhibition rate (%) = (blank control colony diameter - treated colony diameter) ÷ blank control colony diameter × 100.

[0155] 2.5 Minimum inhibitory concentration (MIC) determination

[0156] In a sterile 96-well plate, first add 100 μL of PDB medium to each well. Then, add 100 μL of the drug solution to each well in columns 1 and 2, and mix thoroughly. Serial dilutions are performed in columns 2 through 10: 100 μL of solution from column 2 is transferred to column 3 and mixed thoroughly. 100 μL of solution from column 3 is transferred to column 4, and so on, through column 10 (concentration range 6000 μmol / L-11.78 μmol / L). 100 μL of DMSO is added to column 11. After mixing columns 10 and 11, 100 μL of solution is removed and discarded. Finally, 100 μL of bacterial suspension is added to each well in columns 12. Column 1 serves as a blank control, column 11 as a negative control, and column 12 as a growth control. The 96-well plate is incubated in a 27°C incubator for 48 hours. The minimum drug concentration in the wells where the solution remains clear is the MIC. Three replicates are performed for each well.

[0157] 3. Antibacterial test results

[0158] 3.1 Effects of compounds 1-30 on mycelial growth rate

[0159] The test results of the mycelium growth rate method are shown in Tables 1 to 3.

[0160] The positive control groups, itraconazole and fluconazole, had no significant inhibitory effect on the hyphal growth of M. canis, T. mentagrophytes, and M. gypseum. Ketoconazole exhibited a significant inhibitory effect with a superior effect, particularly on the hyphal growth rate of T. mentagrophytes. Compounds 1-30 had varying degrees of effects on the hyphal growth of these three fungi, and all were more effective than itraconazole and fluconazole. At 6 μmol / mL, compound 5 exhibited a very significant inhibitory effect on the hyphal growth of M. canis. Compounds 3 and 4 had the greatest effects on the hyphal growth rates of T. mentagrophytes and M. gypseum, with both rates being less than 1 mm / day.

[0161] Table 1 Effects of different concentrations of compounds on the mycelial growth rate of Microsporum canis

[0162] Table 2 Effects of different concentrations of compounds on the hyphal growth rate of Trichophyton mentagrophytes

[0163] Table 3 Effects of different concentrations of compounds on the mycelial growth rate of Microsporum gypseum

[0164] 4.1.2 Inhibitory effects of compounds 1-30 on three bacteria

[0165] The results of the determination of the inhibition rate are shown in Tables 4 to 6.

[0166] In the positive control group, ketoconazole had the highest inhibition rate and better inhibition effect than itraconazole and fluconazole.Compounds 1-30 had different degrees of inhibition effect on the three fungi.

[0167] 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 low and the antibacterial effects were poor. Only compound 12 had a high inhibition rate (>50%) at all tested concentrations.

[0168] 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%.

[0169] 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 were all greater than 50%.

[0170] Table 4 Inhibitory rate of compounds at different concentrations against Microsporum canis

[0171] Table 5 Inhibitory effect of different concentrations of compounds on Trichophyton mentagrophytes

[0172] Table 6 Inhibition rate of compounds at different concentrations against Microsporum gypseum

[0173] 4.1.3 MIC determination results of compounds 1-30 against four fungi

[0174] The MIC determination results are shown in Table 7. Compounds 1-30 all showed varying degrees of inhibitory activity against the three fungi. Compounds 4, 17, 20, 26, and 27 had the best overall effect. Among them, compounds 11, 12, 17, 18, 26, and 27 had the lowest MIC against Microsporum canis, which was 187.5 μmol / L, followed by compound 14 with a MIC of 375 μmol / L. Compounds 17 and 26 had the lowest MIC against Trichophyton mentagrophytes, which was 187.5 μmol / L, followed by compounds 4, 5, 6, 12, 13, 18, and 27, which was 375 μmol / L. Compounds 26 and 27 had the lowest MIC against Microsporum gypseum, which was 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.

[0175] Table 7 MIC of compounds 1-30 against four bacteria

[0176] 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.

[0177] Example 4 Cytotoxicity assay of synthetic compounds on normal cells

[0178] 1. Cell line: NIH3T3 cells

[0179] 2. Experimental Methods

[0180] 2.1 Cell inoculation

[0181] When NIH3T3 cells grow well and are in the logarithmic growth phase, follow the subculture procedure, resuspend the cells in 1 mL of complete culture medium, add 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 the plate was sealed and transferred to a 37°C, 5% CO2 constant temperature incubator for 24 h.

[0182] 2.2 Preparation of drug solution and administration

[0183] Compounds were dissolved in DMSO and used as negative controls. 100 μL of complete medium was added to the control group, while the experimental group was supplemented with 200 μL of complete medium, resulting in a final concentration of 20 μM per well. Five replicates were performed. Derivatives that showed strong inhibitory activity at 20 μM were rescreened using NIH3T3 cells using a concentration gradient.

[0184] 2.3 MTS kit for detecting cell viability

[0185] 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, and 200 μL of the mixture was added to each well. Five blank groups of the mixture without cells were set up and placed in a constant temperature box for reaction for 2-4 hours. The absorbance of each well at a wavelength of 490 nm was measured using a microplate reader, and the data were exported and the inhibition rate was calculated. The IC was calculated using the Bliss method using GraphPad Prism 8 software. 50 .

[0186] 3. Experimental Results

[0187] IC values ​​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. It can be seen that the toxicity of these compounds to normal tissues should be lower than that of ketoconazole. 50The 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 concentration was compound 7, which was 399.7 μmol / L.

[0188] Table 8 IC of compounds 1-30 against NIH3T3 cells 50

[0189] In summary, the positive controls fluconazole and itraconazole showed no significant inhibitory effects against Microsporum canis, Trichophyton mentagrophytes, and Microsporum gypseum, while ketoconazole showed significant inhibitory effects against these three fungi. The synthesized compounds 1-30 all exhibited varying degrees of antibacterial activity against Microsporum canis, Trichophyton mentagrophytes, and Microsporum gypseum. Compounds 4, 14, and 19 all showed significant inhibitory effects against these three fungi, and were superior to fluconazole, itraconazole, and ketoconazole (partially equivalent). Compounds 15, 16, 17, 19, 21, 22, 23, 24, 26, and 28 performed second best. Compounds 1-30 also exhibited low cytotoxicity against normal cells and demonstrated a high safety profile. All of these compounds have potential antifungal applications.

[0190] Example 5 Prediction of antifungal targets of representative compounds

[0191] Representative compounds 4, 9, and 10 were selected, and the molecular docking method was used to investigate the binding and action modes of the representative compounds with common antifungal target proteins, and to predict the possible targets of the active compounds in this application.

[0192] From the RCSB database (http: / / www.rcsb.org / ), we found the following proteins: 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 var. grubii H99 (PDB ID: 7T02); acetyl-CoA synthetase from Cryptococcus neoformans (PDB ID: 8EPS); peptidyl-prolyl isomerase from Aspergillus fumigatus (PDB ID: 8EPS); Compounds 1-28 and the positive controls fluconazole, ketoconazole, and itraconazole were downloaded from the PubChem database (https: / / pubchem.ncbi.nlm,nih.gov / ) as SDF files and imported into Discovery software to obtain their 3D structures. Ligands and non-protein molecules (such as water molecules) were removed from the target protein and hydrogenated using Discovery software. Molecular docking of the compounds and target proteins was then performed in the software, and visualization was performed. Some of the results are shown in Figures 2 to 4. The results are shown in Table 9.

[0193] Table 9 Lib Dock Score of each compound and each protein

[0194] In the table, FCA stands for fluconazole, KET stands for ketoconazole, and ICZ stands for itraconazole.

[0195] All of the above proteins are directly or indirectly involved in the conversion of acetyl-CoA. As can be seen from Table 9, most of the compounds 1-30 and proteins 1PN2, 1PN4, 4H0P, 6ARE, 6R61, 7T02, 7U0U, and 6T5E can all be successfully docked. Proteins 1PN2 and 1PN4 are peroxisomal hydratases-dehydrogenases-epimerases of Candida tropicalis, respectively involved in the conversion of high-energy molecules such as fatty acids in the cell; they decompose high-energy molecules such as fatty acids or convert them into acetyl-CoA. There are 16 compounds with Lib Dock Scores greater than 100 for both compounds 1-30 and protein 1PN2, of which compounds 1 and 3 have Lib Dock Scores greater than 120.

[0196] Compound 1-30 docked with protein 1PN4 for 10 dockings with Lib Dock Scores greater than 100, with compounds 4, 8, and 10 having Lib Dock Scores greater than 120. Protein 4H0P, a Cryptococcus neoformans acetate kinase protein, catalyzes the reversible reaction of acetyl phosphate and ADP to produce ATP. Nine docked with compound 1-30 had Lib Dock Scores greater than 100. Protein 6ARE, an acetyl-CoA acetyltransferase from Aspergillus fumigatus Af293, docked with compound 1-30 for Lib Dock Scores greater than 100. Protein 6R61, an endopeptidase from Cryptococcus neoformans, docked with compound 1-30 for Lib Dock Scores greater than 100 in 15 dockings. Protein 7U0U, an Aspergillus fumigatus peptidylprolyl isomerase, docked with compound 1-30 for Lib Dock Scores greater than 100 in 8 dockings, with compounds 1, 3, and 12 having Lib Dock Scores greater than 120. Most compounds successfully docked with proteins 4C0T and 8EPS, suggesting that their mechanism of action might be to directly or indirectly affect the conversion of acetyl-CoA within fungal cells.

[0197] 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 solutions described in the specific embodiments of the present invention, they can refer to 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 any creative work. These changes and improvements fall within the scope of protection required by the present invention.

Claims

1. Application of 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 either phenyl or naphthyl; R 1 Any one selected from the group consisting of hydrogen, a halogen atom, a C1-C6 alkyl group, a halogen-substituted C1-C5 alkyl group, a C1-C5 alkyloxy group, and a phenyl group; R 2 any one selected from the group consisting of hydrogen, a halogen atom, a carboxyl group, a carboxyl C1-C3 alkyl group, a hydroxyl group, a C1-C6 alkyl group, a halogen-substituted C1-C5 alkyl group, an amino group, a nitro group, a formamide group, an acetamido group, a carbamyl group, and an aminoacetamido group; m=0-3, n=0-3.

2. The application according to claim 1, characterized in that Ar 2 is any 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 replacement position is 4; When Ar 2 When it is -1-naphthyl or -2-naphthyl, and n=1, R 2 The replacement 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 replacement position is 4; When Ar 2 When it is -1-naphthyl and n=1, R 2 The substitution position is 4.

4. The application 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, a halogen atom, a carboxyl group, a carboxyl C1-C3 alkyl group, a hydroxyl group, a C1-C6 alkyl group, a halogen-substituted C1-C5 alkyl group, an amino group, a nitro group, a carbamylamino group, and an aminoacetylamino group; m=0-2; n=0-2.

5. The application 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 application according to claim 4, characterized in that: When m=1, R 1 Substituted 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 application according to claim 1, characterized in that: The medicine is used for treating Microsporum canis, Microsporum gypseum and Trichophyton mentagrophytes.

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